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Jumat, 31 Juli 2009

Antioxidant Activities of Flavonoids (Translated)


Dr. Donald R. Buhler dan Dr. Cristobal Miranda
Department of Environmental and Molecular Toxicology Oregon State University

Translated by : Fahry Marewo7

Ringkasan: Flavonoids merupakan kandungan tang terdapat pada buah-buahan, sayur-sayuran, dan pangan tertentu yang mempunyai komposisi biokimia yang menguntungkan dan efek antioxidant. Niali gizi makanan dalam dietnya sangat tinggi dibandingkan makan antioxidants lain seperti vitamins C dan E. Aktivitas Antioxidant flavonoids tergantung pada struktur molekularnya, dan karakteristik struktural dari kandungan flavonoids tertentu pada hops dan bir mempunyai aktivitas antioxidant yang sangat kuat melebihi anggur merah, teh, atau kedelai.

Flavonoids adalah campuran polyphenolic yang ada dimana mana secara alami dan digolongkan, menurut struktur kimia, ke dalam flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins dan chalcones. Lebih dari 4,000 flavonoids telah dikenali, banyak terdapat dari buah-buahan, sayur-sayuran dan sari buah (teh, kopi, bir, anggur dan minuman sari buah). Flavonoids mendapat perhatian pada kurun waktu terakhir karena potensinya mempunyai efek yang menguntungkan pada kesehatan manusia dan telah dilaporkan mempunyai aktivitas antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor dan antioxidant.

Antioxidants adalah kandungan yang melindungi sel melawan terhadap efek kerusakan dari jenis oksigen reaktif, seperti oksigen singlet, superoxide, peroxyl radikal, hidroksil radikal dan peroxynitrite. Ketidakseimbangan antara antioxidants dan jenis oksigen reaktif mengakibatkan tekanan oxidative, mendorong ke arah kerusakan selular. Tekanan Oxidative berhubungan dengan kanker, aging, atherosclerosis, luka-luka ischemic, peradangan dan penyakit neurodegenerative (Penyakit parkinson dan Alzheimer'S). Flavonoids dapat membantu menyediakan perlindungan melawan penyakit ini dengan dukungan, bersama dengan vitamins antioxidant dan enzim, untuk antioxidant total sistem pertahanan tubuh manusia. Studi Epidemiologi menunjukkan asupan flavonoid itu berhubungan terbalik dengan kematian akibat dari penyakit serangan jantung dan pada timbulnya serangan jantung.

Berkenaan dengan Antioxidants diet yang dikenali adalah vitamin C, vitamin E, selenium, dan carotenoids. Studi terbaru mempertunjukkan bahwa flavonoids yang ditemukan buah-buahan dan sayur-sayuran dapat bertindak sebagai antioxidants. Seperti alpha-tocopherol (vitamin E), flavonoids yang mengandung unsur-unsur kimia struktural yang mungkin bertanggung jawab untuk aktivitas antioxidantnya. Studi terbaru oleh Dr. Van Acker dan para rekan kerjanya di Belanda menyatakan bahwa flavonoids dapat menggantikan vitamin E sebagai rantai pemecah anti- oxidants pada membrane microsomal hati. Kontribusi flavonoids untuk antioxidant sistem pertahanan mungkin menjadi pertimbangan substansiil bahwa asupan total masukan harian dari flavonoids dapat berkisar antara 50 sampai 800 mg. Asupan ini adalah tinggi dibandingkan untuk rata-rata Asupan harian dari diet antioxidants seperti vitamin C (70 mg), vitamin E (7-10 mg) atau carotenoids (2-3 mg). Masukan Flavonoid tergantung pada konsumsi buah-buahan, sayur-sayuran, dan hidangan tertentu, seperti anggur merah, teh, dan bir. Konsumsi yang tinggi teh dan anggur mungkin yang paling berpengaruh pada total masukan flavonoid pada kelompok orang tertentu.

Oksidasi low-density lipoprotein (LDL) telah diketahui berperan penting dalam atherosclerosis. Sel Sistem kekebalan yang disebut macrophages mengenali dan menelan LDL yang dioksidasi, suatu proses yang mendorong pada pembentukan isyarat atau tanda peringatan atherosclerotic pada dinding arterial. Oksidasi LDL dapat diinduksi oleh macrophages dan dapat juga dikatalisasi oleh ion logam seperti tembaga (Cu). Beberapa studi menunjukkan bahwa flavonoids tertentu dapat melindungi LDL dari oksidasi oleh dua mekanisme tersebut .

Antioxidant flavonoids
(listed in order of decreasing potency)
• Quercetin (flavonol dalam sayuran, kulit buah-buahan, bawang)
• Xanthohumol (prenylated chalcone dalam hops dan beer)
• Isoxanthohumol (prenylated flavanone dalam hops dan beer)
• Genistein (isoflavone dalam kadelei)

Pro-oxidant flavonoids
• Chalconaringenin (non-prenylated chalcone dalam sari buah jeruk)
• Naringenin (non-prenylated flavanone dalam sari buah jeruk)

Kapasitas flavonoids untuk bertindak sebagai antioxidants tergantung pada struktur molekularnya. Posisi kelompok hidroksil dan corak lain dalam struktur kimia flavonoids adalah penting untuk antioxidantnya dan aktivitas pembebasan radikal bebas. Quercetin, merupakan flavonol dalam diet yang paling babyak, berpotensi sebagai antioxidant kuat karena mempunyai semua corak struktur yang benar untuk aktivitas pembebasan radikal bebas.

Baru-baru ini, flavonoids chalcone dan flavanone dengan sisi rantai prenyl atau geranyl diketahui berada dalam hops dan bir oleh Dr. Fred Stevens dan Dr. Max Deinzer dari Oregon State Universitas. Hops digunakan dalam bir untuk member rasa. Xanthohumol (chalcone) dan isoxanthohumol dan 6-prenylnaringenin (flavanones) adalah prenyl-flavonoids yang utama ditemukan dalam bir. Walaupun aktivitas antioxidant dari kandungan ini belum dipelajari, flavonoids ini mungkin bertanggung jawab untuk aktivitas antioxidant lager bir, yang mana lebih tinggi dibandingkan dengan teh hijau, anggur merah, atau sari buah anggur seperti yang dilaporkan lebih awal oleh Dr. Joe A. Vinson dari Universitas Scranton di Pennsylvania. Xanthohumol ditemukan hanya dalam bir tetapi dalam konsentrasi kecil. Untuk menilai aktivitas antioxidant dari prenylated flavonoids, kita bekerja sama/kolaborasi dengan peneliti LPI yang mengevaluasi kapasitas dari flavonoids ini untuk menghalangi oksidasi LDL oleh tembaga. Unsur Antioxidant dari prenyl flavonoids dibandingkan pada quercetin (flavonol), genistein (isoflavone utama dalam kedelai), chalconaringenin (chalcone non-prenylated), naringenin (flavanone non-prenylated), dan vitamin E. Kemugkinan interaksi xanthohumol, prenylchalcone utama dalam bir, dengan vitamin E untuk menghalangi oksidasi LDL yang diinduksi oleh tembaga adalah juga diuji.

Hasilnya menunjukkan bahwa prenylchalcones dan prenylflavones adalah efektif mencegah oksidasi LDL yang diaktifkan oleh tembaga dan prenylchalcones biasanya mempunyai aktivitas antioxidant lebih besar dibanding prenylflavanones. Xanthohumol, prenylchalcone utama dalam hops dan bir, adalah suatu antioxidant yang lebih kuat dibanding vitamin E atau genistein.

Seperti yang dilaporkan dalam Journal of Agricultural and Food Chemistry, juga ditemukan kelompok prenyl berperan penting didalam aktivitas antioxidant dari flavonoids tertentu. Flavonoid chalcone (chalconaringenin) dan flavanone (naringenin) dengan tidak adanya kelompok prenyl yang bertindak sebagai pro-oxidants, yaitu mereka mendukung bukannya membatasi oksidasi LDL oleh tembaga. Penambahan kelompok prenyl pada molekul flavonoid ini menetralkan aktivitas pro-oxidantnya.

Penelitian ini mengungkapkan bahwa ada flavonoids yang unik dalam hops dan bir yang mungkin berpotensi untuk dimanfaatkan dalam pencegahan (preventionof) penyakit manusia yang berhubungan dengan kerusakan oleh radikal bebas. Pengamatan kelompok prenyl adalah penting untuk pemahaman aktivitas antioxidant pada flavonoids tertentu yang dapat mendorong kearah penemuan atau sintesa tentang flavonoids prenylated sebagai agen pengobatan atau pencegahan terhadap penyakit manusia berhubungan dengan radikal bebas. Harapannya, hasil dengan xanthohumol menyarankan bahwa prenylchalcone ini harus dipelajari lebih lanjut untuk aktifitas antioxidantnya dan efek perlindungannya terhadap kerusakan oleh radikal bebas pada hewan dan manusia. Studi awal menunjukkan bahwa xanthohumol tersebut dapat diserap dari saluran pencernaan pada tikus, dan studi lebih jauh diperlukan untuk mengevaluasi bioavailabilitas dari flavonoids menarik ini pada manusia.

Studi lebih lanjut juga diperlukan untuk menetapkan keamanan dan keselamatan penggunaan xanthohumol atau flavonoids lain sebagai bahan tambahan dalam diet makanan sampai dosis yang tinggi dari kandungan ini dapat menghasilkan efek yang kurang baik pada manusia, menurut penemuan terbaru oleh Dr. Martyn Smith, Profesor toxilogy, Universitas California di Berkeley.

Untuk informasi tambahan tentang flavonoids, lihat Linus Pauling Institut Micronutrient Information Center.

Last Update November, 2000

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SAPE DALAM PEMBANGUNAN SAAT INI

Berikut penghuni memperkenalkan kampung halaman Kecamatan Sape Kabupaten Bima Nusa Tenggara Barat dalam gambar terbaru yang dikutip dari berbagai sumber :


























(1) Puskesmas Plus
(2) Terminal Bus Antar Propinsi (AKAP)
(3) Gedung Serba Guna (GSG) Sape
(4) Tempat Pelelangan Ikan (TPI) Sape
(5) Torowamba Beach Lamere Sape
(6) Kapal Pesiar di Selat Sape

Dengan berbagai bentuk potensi tersebut maka kecamatan Sape berada pada bagian terdepan dalam menunjang perekonomian kabupaten bima secara luas.

Sebagai obat rindu yang mendalam pada kampung halaman tercinta yang terus berbenah diri untuk melangkah jauh kedepan. Daerah yang menjadi gerbang timur pulau Sumbawa dan Kabupaten Bima pada khususnya. Gambar-gambar ini kiranya bisa menggambarkan keadaan terakhir dari Sape. Perubahan wajah yang terus menerus ini, adalah profil dinamika pembangunan dibima yang terus intens digalakkan.

Semangat membangun ini, mesti dijaga. Spirit yang tidak boleh padam oleh hanya riak kecil gelombang ditengah samudera besar harapan. Potensi sumberdaya pembangunan terus ditelaah dan dibangkitkan sembari tetap menjaga arah tujuan yang lebih baik. Ada banyak yang bisa kita berikan untuk membaktikan diri pada negeri.

Dalam mimpi hari esok, semoga kota ini bisa menjadi pengharapan besar bagi banyak jiwa. Melahirkan banyak pejuang dan pengabdi. Dengan keyakinan yang sesungguhnya, bahwa matahari kehidupan akan terbit diufuk timur menerangi dan menyinari setiap wajah, menyemangati dengan energinya.

Akankah daerah penuh potensi ini -pada masanya nanti- menjadi daerah baru yang mandiri ?? pertanyaan yang saat ini mulai mengemuka dikhalayak publik daerah maja labo dahu ini. Kita tunggu progress yang menghadap. Salam Penghuni.
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Alkaloid

by Fahry Marewo5

From Wikipedia, the free encyclopedia

This article is about the chemical compounds alkaloids. For the pharmaceutical company in the Republic of Macedonia, see Alkaloid (company).

Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms.[1] The name derives from the word alkaline and was used to describe any nitrogen-containing base. Alkaloids are produced by a large variety of organisms, including bacteria, fungi, plants, and animals and are part of the group of natural products (also called secondary metabolites). Many alkaloids can be purified from crude extracts by acid-base extraction. Many alkaloids are toxic to other organisms. They often have pharmacological effects and are used as medications, as recreational drugs, or in entheogenic rituals. Examples are the local anesthetic and stimulant cocaine, the stimulant caffeine, nicotine, the analgesic morphine, or the antimalarial drug quinine. Some alkaloids have a bitter taste.

Alkaloid classifications
Generally speaking, alkaloids are categorized under three main categories,depending on their biogenic origin. For those containing at least a nitrogen atom in a ring system derived from amino acids (i.e. alkaloids derived from phenylalanine are not grouped in this category), they are true alkaloids. The alkaloids derived from phenylalanine are categorized as protoalkaloids. While the remaining ones, such as steroidal alkaloids and purine alkaloids, are classified as pseudoalkaloids.
Alkaloids are usually classified by their common molecular precursors, based on the metabolic pathway used to construct the molecule.[citation needed] When not much was known about the biosynthesis of alkaloids, they were grouped under the names of known compounds, even some non-nitrogenous ones (since those molecules' structures appear in the finished product; the opium alkaloids are sometimes called "phenanthrenes", for example), or by the plants or animals they were isolated from. When more is learned about a certain alkaloid, the grouping is changed to reflect the new knowledge, usually taking the name of a biologically-important amine that stands out in the synthesis process.[original research?]
• Pyridine group: piperine, coniine, trigonelline, arecoline, arecaidine, guvacine, cytisine, lobeline, nicotine, anabasine, sparteine, pelletierine.
• Pyrrolidine group: hygrine, cuscohygrine, nicotine
• Tropane group: atropine, cocaine, ecgonine, scopolamine, catuabine
• Indolizidine group: senecionine, swainsonine
• Quinoline group: quinine, quinidine, dihydroquinine, dihydroquinidine, strychnine, brucine, veratrine, cevadine
• Isoquinoline group: opium alkaloids (papaverine, narcotine, narceine, morphine, codeine, heroine), sanguinarine, hydrastine, berberine, emetine, berbamine, oxyacanthine
• Phenanthrene alkaloids: opium alkaloids (morphine, codeine, thebaine)
• Phenethylamine group: mescaline, ephedrine, dopamine
• Indole group:
o Tryptamines: serotonin, DMT, 5-MeO-DMT, bufotenine, psilocybin
o Ergolines (the ergot alkaloids): ergine, ergotamine, lysergic acid
o Beta-carbolines: harmine, harmaline, tetrahydroharmine
o Yohimbans: reserpine, yohimbine
o Vinca alkaloids: vinblastine, vincristine
o Kratom (Mitragyna speciosa) alkaloids: mitragynine, 7-hydroxymitragynine
o Tabernanthe iboga alkaloids: ibogaine, voacangine, coronaridine
o Strychnos nux-vomica alkaloids: strychnine, brucine
• Purine group:
o Xanthines: caffeine, theobromine, theophylline
• Terpenoid group:
o Aconitum alkaloids: aconitine
o Steroid alkaloids (containing a steroid skeleton in a nitrogen containing structure):
 Solanum (e.g. potato and tomato) alkaloids (solanidine, solanine, chaconine)
 Veratrum alkaloids (veratramine, cyclopamine, cycloposine, jervine, muldamine)[2]
 Fire Salamander alkaloids (samandarin)
 Others: conessine
• Quaternary ammonium compounds: muscarine, choline, neurine
• Miscellaneous: capsaicin, cynarin, phytolaccine, phytolaccotoxin

Physicochemical properties
Low-molecular weight alkaloids without hydrogen bond donors such as hydroxy groups are often liquid at room temperature, examples are nicotine, sparteine, coniine, and phenethylamine.
The basicity of alkaloids depends on the lone pairs of electrons on their nitrogen atoms. As organic bases, alkaloids form salts with mineral acids such as hydrochloric acid and sulfuric acid and organic acids such as tartaric acid or maleic acid. These salts are usually more water-soluble than their free base form.

See also
References
1. ^ International Union of Pure and Applied Chemistry (1995). "alkaloids". Compendium of Chemical Terminology Internet edition.
2. ^ http://www.ansci.cornell.edu/plants/toxicagents/steroid.html,
External links

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Secondary metabolite
From Wikipedia, the free encyclopedia

Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of organisms. Unlike primary metabolites, absence of secondary metabolities does not result in immediate death, but rather in long-term impairment of the organism's survivability, fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group.[1]
Contents
• 1 Categories
o 1.1 Small "small molecules"
o 1.2 Big "small molecules", produced by large, modular, "molecular factories"
o 1.3 Non-"small molecules" - DNA, RNA, ribosome, or polysaccharide "classical" biopolymers
• 2 See also
• 3 References

Categories
Most of the secondary metabolites of interest to humankind fit into categories which classify secondary metabolites based on their biosynthetic origin. Since secondary metabolites are often created by modified primary metabolite synthases, or "borrow" substrates of primary metabolite origin, these categories should not be interpreted as saying that all molecules in the category are secondary metabolites (for example the steroid category), but rather that there are secondary metabolites in these categories.
Small "small molecules"
• Alkaloids (usually a small, heavily derivatized amino acid):
o Hyoscyamine, present in Datura stramonium
o Atropine, present in Atropa belladonna, Deadly nightshade
o Cocaine, present in Erythroxylon coca the Coca plant
o Codeine and Morphine, present in Papaver somniferum, the opium poppy
o Tetrodotoxin, a microbial product in Fugu and some salamanders
o Vincristine & Vinblastine, mitotic inhibitors found in the Rosy Periwinkle
• Terpenoids (come from semiterpene oligomerization):
o Azadirachtin, (Neem tree)
o Artemisinin, present in Artemisia annua Chinese wormwood
o tetrahydrocannabinol, present in cannabis sativa
o Steroids (Terpenes with a particular ring structure)
 Saponins (plant steroids, often glycosylated)
• Glycosides (heavily modified sugar molecules):
o Nojirimycin
o Glucosinolates
• Phenols:
o Resveratrol
• Phenazines:
o Pyocyanin
o Phenazine-1-carboxylic acid (and derivatives)
Big "small molecules", produced by large, modular, "molecular factories"
• Polyketides:
o Erythromycin
o Discodermolide
• Fatty acid synthase products :
o FR-900848
o U-106305
o phloroglucinols
• Nonribosomal peptides:
o Vancomycin
o Thiostrepton
o Ramoplanin
o Teicoplanin
o Gramicidin
o Bacitracin
• Hybrids of the above three:
o Epothilone
Non-"small molecules" - DNA, RNA, ribosome, or polysaccharide "classical" biopolymers
• Ribosomal peptides:
o Microcin-J25
See also
Secondary metabolism
References
1. ^ "Chemical plants". http://www.open2.net/sciencetechnologynature/worldaroundus/chemicalplants.html. Retrieved on 2008-12-19.

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Flavonoid



Kutipan By : Fahry Marewo4
From Wikipedia, the free encyclopedia

Flavonoids (or bioflavonoids) are a class of plant secondary metabolites. According to the IUPAC nomenclature,[1] they can be classified into:
•flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure
•isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure
•neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure.
Flavonoids are most commonly known for their antioxidant activity. Flavonoids are also commonly referred to as bioflavonoids in the media – the terms are largely equivalent and interchangeable, for most flavonoids are biological in origin. Flavonoids are "the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants".[2]

Struktur molekular kerangka flavone ( 2-phenyl-1,4-benzopyrone)

Flavonoids (atau bioflavonoids) adalah kelas dari metabolit sekunder tumbuhan. Menurut nomenklatur IUPAC, flavonoids dapat digolongkan ke dalam :

1.Flavonoids, turunan dari struktur 2-phenylchromen-4-one ( 2-phenyl-1,4-benzopyrone)
2.Isoflavonoids, turunan dari struktur 3-phenylchromen-4-one ( 3-phenyl-1,4-benzopyrone)
3.Neoflavonoids, turunan dari struktur 4-phenylcoumarine ( 4-phenyl-1,2-benzopyrone).

Flavonoids paling umum dikenal karena aktivitas antioxidantnya. Flavonoids biasanya juga dikenal sebagai bioflavonoids pada media - istilah padanan umum dan dapat bertukar tempat. Flavonoids secara umum termasuk dalam kelompok polyphenolic pada diet manusia dan ditemukan pada sebagian besar dari tumbuhan (Spencer, Jp 2008).

Flavonoid merupakan komponen yang ditemukan dalam buah, sayuran, dan beberapa organisme yang mengandung senyawa biokimia penting dan memiliki efek antiosidan. Flavonoid merupakan komponen polifenolic yang dapat ditemukan secara alami dan dikategorisasi berdasarkan struktur kimia, kedalam flavonol, flavon, flavonones, isoflavon, catechin, anthocyanidine, dan chalcones. Lebih dari 4000 flavonoid telah diidentifkasi, kebanyakan ditemukan dalam buah, sayuran dan biji-bijian (teh, kopi, beer, wine, dan minuman buah). Flavonoid dianggap penting karena mempunyai konstribusi pada kesehatan manusia seperti yang telah dilaporkan bahwa flavonoid berpotensial sebagai antiviral, antiallergic, antiplatelet, anti-inflammatory, antitumor, dan antioksidan.

Biosynthesis
Flavonoids are synthesized by the phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA[3]. This can be combined with malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones, which contain two phenyl rings (see polyphenols). Conjugate ring-closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone. The metabolic pathway continues through a series of enzymatic modifications to yield flavanones → dihydroflavonols → anthocyanins. Along this pathway, many products can be formed, including the flavonols, flavan-3-ols, proanthocyanidins (tannins) and a host of other various polyphenolics.

Biosynthesis
Flavonoids disintesis oleh jalur metabolisme phenylpropanoid di mana asam amino phenylalanine digunakan untuk memproduksi 4-coumaroyl-CoA (Ververidis Filippos, et. al,. 2007). Ini dapat dikombinasikan dengan malonyl-CoA untuk menghasilkan kerangka yang sebenarnya dari flavonoids, suatu kelompok campuran yang disebut dengan chalcones, yang berisi dua cincin fenil. Konjugasi dari cincin penutup chalcones mengakibatkan format yang umum dikenal flavonoids, struktur tiga cincin dari flavone. Jalur metabolisme selanjutnya melalui suatu rangkaian dari modifikasi enzymatik untuk menghasilkan flavanones → dihydroflavonols → anthocyanins. Sepanjang jalur ini, banyak produk yang dapat dibentuk, termasuk flavonols, flavan-3-ols, proanthocyanidins (tannins) dan sejumlah besar jenis polyphenolics.

Biological effects
Flavonoids are widely distributed in plants fulfilling many functions including producing yellow or red/blue pigmentation in flowers and protection from attack by microbes and insects. The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Flavonoids have been referred to as "nature's biological response modifiers" because of strong experimental evidence of their inherent ability to modify the body's reaction to allergens, viruses, and carcinogens. They show anti-allergic, anti-inflammatory[4] , anti-microbial[5] and anti-cancer activity.
Consumers and food manufacturers have become interested in flavonoids for their medicinal properties, especially their potential role in the prevention of cancers and cardiovascular disease. The beneficial effects of fruit, vegetables, and tea or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins[6].

Efek Biologi
Flavonoids secara luas terbagi dalam banyak fungsi tumbuhan termasuk memproduksi pigmentasi kuning atau merah/biru pada bunga dan perlindungan dari serangan mikroba dan serangga. Distribusi yang luas ri dflavonoids, variasi dan hubunganya toksisitas yang rendah dibandingkan dengan kandungan aktifnya pada tumbuhan (contohnya alkaloids) rata-rata pada kebanyakan hewan, termasuk manusia, jumlah yang signifikan dalam pencernaan pada diet mereka. Flavonoids dikenal sebagai " modifikasi respon biologi alami " karena terbukti kuat secara eksperimental flavonoids tidak bisa dipisahkan dari kemampuan untuk memodifikasi reaksi tubuh pada penyebab alergi, virus, dan segala penyebab kanker (carcinogens). Mereka menunjukkan aktivitas anti-allergic, anti-inflammatory (Yamamoto and Gaynor, 2007), anti-microbial (Cushnie TPT, Lamb AJ 2005), dan anti-cancer.
Konsumen dan pabrik makanan olahan menjadi tertarik akan flavonoids untuk bahan obat, terutama potensi flavonoisd dalam pencegahan kanker dan penyakit kardiovasculer. Efek yang menguntungkan dari buah, sayur, dan teh atau bahkan anggur merah telah dihubungkan dengan kandungan flavonoid dibandingkan dengan gizi dan vitamins (Félicien Breton (2008).

Health benefits aside from antioxidant values
In 2007, research conducted at the Linus Pauling Institute and published in Free Radical Biology and Medicine indicates that inside the human body, flavonoids themselves are of little or no direct antioxidant value [7]. Body conditions prove to be unlike controlled test tube conditions, and the flavonoids are poorly absorbed (less than 5%), with most of what is absorbed being quickly metabolized and excreted.
The huge increase in antioxidant capacity of blood seen after the consumption of flavonoid-rich foods is not caused directly by the flavonoids themselves, but most likely is due to increased uric acid levels that result from expelling flavonoids from the body.[8] According to Frei, "we can now follow the activity of flavonoids in the body, and one thing that is clear is that the body sees them as foreign compounds and is trying to get rid of them.

Manfaat Kesehatan Selain antioxidant

Pada 2007, riset yang diselenggarakan di Linus Pauling Institut dan diterbitkan dalam Free Radical Biology and Medicine menunjukkan bahwa di dalam tubuh manusia, flavonoids hanya sedikit atau tidak ada nilai antioxidant langsung (Lotito SB, Frei B 2006). Kondisi tubuh membuktikan menjadi tidak sama dengan uji kondisi tabung dikendalikan, dan flavonoids sangat sedikit diserap (kurang dari 5%), dengan kebanyakan dari apa yang diserap dengan cepat dikeluarkan dan dimetabolis.
Peningkatan yang sangat besar dalam kapasitas dari antioxidant darah terlihat setelah konsumsi makanan yang kayak an flavonoid tidak disebabkan secara langsung oleh flavonoids tersebut, tetapi hampir bisa dipastikan adalah berkaitan dengan peningkatan level asam uric yang diakibatkan oleh terusirnya flavonoids dari tubuh. (David Stauth, 2006)

Cancer
The process of gearing up to get rid of unwanted compounds is inducing so-called Phase II enzymes that also help eliminate mutagens and carcinogens, and therefore may be of value in cancer prevention. Flavonoids could also induce mechanisms that help kill cancer cells and inhibit tumor invasion."[8] UCLA cancer researchers have found that study participants who ate foods containing certain flavonoids seemed to be protected from developing lung cancer. Dr. Zuo-Feng Zhang, of the UCLA's Jonsson Cancer Center and a professor of public health and epidemiology at the UCLA School of Public Health said the flavonoids that appeared to be the most protective included catechin, found in strawberries and green and black teas; kaempferol, found in brussel sprouts and apples; and quercetin, found in beans, onions and apples.[9]
Their research also indicated that only small amounts of flavonoids are necessary to see these medical benefits. Taking large dietary supplements provides no extra benefit and may pose some risks.[8]

Kanker
Proses dari pemasangan bahan yang masuk mendapatkan dari kandungan yang tak dikehendaki adalah berpengaruh, yang disebut enzim Tahap II yang juga membantu mengurangi mutagens dan segala penyebab kanker, dan oleh karena itu mungkin bermanfaat dalam pencegahan kanker. Flavonoids dapat juga mempengaruhi mekanisme yang membantu pembunuhan sel kanker dan menghalangi invasi tumor (David Stauth, 2006) Para peneliti kanker dari UCLA sudah menemukan study yang membantu bahwa makanan yang mengandung flavonoids tertentu menunjukan mampu melindungi dari perkembangan kanker paru-paru. Dr. Zuo-Feng Zhang, dari UCLA's Jonsson Cancer Center dan profesor kesehatan masyarakat dan epidemiologi di mengakatan bahwa flavonoids yang muncul tersebut yang kebanyakan bersifat melindungi termasuk catechin, yang ditemukan dalam strawberry dan teh hitam dan hijau; kaempferol, yang ditemukan dalam kecambah brussel dan buah apel; dan quercetin, yang ditemukan dalam kacang, bawang dan apel.(UCLA news May 2008).
Riset mereka juga menunjukkan bahwa hanya sejumlah kecil flavonoids yang diperlukan untuk melihat manfaat medis ini. Pengambilan suplemen makanan yang besar tidak menyediakan apapun manfaat ekstra dan dapat memperlihatkann beberapa resiko (David Stauth, 2006)

Diarrhea
A study done at Children's Hospital & Research Center Oakland, in collaboration with scientists at Heinrich Heine University in Germany, has shown that epicatechin, quercetin and luteolin can inhibit the development of fluids that result in diarrhea by targeting the intestinal cystic fibrosis transmembrane conductance regulator Cl– transport inhibiting cAMP-stimulated Cl– secretion in the intestine.[10]

Diarrhea
Suatu studi yang dilakukan pada Rumah sakit Anak-Anak & Pusat Riset Oakland, bekerjasama dengan ilmuwan pada Heinrich Heine University di Jerman, telah menunjukkan epicatechin, quercetin dan luteolin itu dapat menghalangi pengembangan cairan yang mengakibatkan diarrhea dengan mengarahkan fibrosis cystic transmembrane pengatur konduktans Cl- mengangkut menghambat cAMP- yang distimulasi - Pengeluaran Cl didalam usus.(Schuier M, Sies H, Illek B, Fischer H, 2005).

Important flavonoids
Quercetin
Main article: quercetin
Quercetin is a flavonoid and, to be more specific, a flavonol. It is the aglycone form of a number of other flavonoid glycosides, such as rutin and quercitrin, found in citrus fruit, buckwheat and onions. Quercetin forms the glycosides quercitrin and rutin together with rhamnose and rutinose, respectively. It may also help to prevent some types of cancer, however currently there is more research needed in this area.
Quercetin adalah suatu flavonoid dan, untuk lebih spesifik, suatu flavonol. Itu adalah bentuk aglycone sejumlah lain dari flavonoid glycosides, seperti rutin dan quercitrin, yang ditemukan dalam buah jeruk, gandum dan bawang. Bentuk quercetin yaitu glycosides quercitrin dan rutin bersama-sama dengan rhamnose dan rutinose, secara berturut-turut. Mungkin juga membantu untuk mencegah beberapa bentuk kanker, sekarang ini membutuhkan lebih banyak riset pada wilayah ini.

Epicatechin

Epicatechin improves blood flow and thus seems good for cardiac health. Cocoa, the major ingredient of dark chocolate, contains relatively high amounts of epicatechin and has been found to have nearly twice the antioxidant content of red wine and up to three times that of green tea in in-vitro tests.[11] [12] But in the test outlined above it now appears the beneficial antioxidant effects are minimal as the antioxidants are rapidly excreted from the body.
Epicatechin meningkatkan peredaran darah dan dengan begitu baik untuk kesehatan jantung. Cocoa, kandungan utama dari coklat gelap, berisi epicatechin yang tinggi dan telah ditemukan hampir dua kali lebih dari kandungan antioxidant anggur merah dan lebih dari] tiga kali dari teh hijau dalam uji in-vitro (Lee KW, Kim YJ, Lee HJ, Lee CY 2003). Tetapi pada uji yang diuraikan di atas sekarang terlihat efek antioxidant yang menguntungkan minimal seperti antioxidants yang dikeluarkan dengan cepat dari tubuh.

Oligomeric proanthocyanidins
Proanthocyanidins extracts demonstrate a wide range of pharmacological activity. Their effects include increasing intracellular vitamin C levels, decreasing capillary permeability and fragility, scavenging oxidants and
free radicals, and inhibiting destruction of collagen, the most abundant protein in the body.
Oligomeric Proanthocyanidins
Ekstrak Proanthocyanidins mempertunjukkan suatu cakupan luas dari aktivitas pharmakologis. Efeknya meliputi peningkatan jumlah intracellular vitamin C, mengurangi permeabilitas kapiler dan kerapuhan, pencarian oxidants dan radikal bebas, dan penghambatan kerusakan dari collagen, protein yang paling berlimpah di dalam tubuh.

Important dietary sources
Good sources of flavonoids include all citrus fruits, berries, ginkgo biloba, onions[13][14], parsley[15], pulses[16], tea (especially white and green tea), red wine, seabuckthorn, and dark chocolate (with a cocoa content of seventy percent or greater). (Tsushida T., Suzuki, M. 1996)

Sumber diet yang penting
Sumber flavonoids yang baik meliputi semua buah jeruk, biji, ginkgo biloba, bawang (Tsushida T., Suzuki, M. 1996), parsley (Justesen U, Knuthsen P 2001). pulses (Ewald C, Fjelkner-Modig S, Johansson K, Sjöholm I, Åkesson B 1999). teh (terutama teh putih dan hijau), anggur merah, seabuckthorn, dan coklat gelap (dengan isi cocoa tujuh puluh persen atau lebih besar).

Citrus

The citrus bioflavonoids include hesperidin (a glycoside of the flavanone hesperetin), quercitrin, rutin (two glycosides of the flavonol quercetin), and the flavone tangeritin. In addition to possessing antioxidant activity and an ability to increase intracellular levels of vitamin C, rutin and hesperidin exert beneficial effects on capillary permeability and blood flow. They also exhibit some of the anti-allergy and anti-inflammatory benefits of quercetin. Quercetin can also inhibit reverse transcriptase, part of the replication process of retroviruses.[17] The therapeutical relevance of this inhibition has not been established. Hydroxyethylrutosides (HER) have been used in the treatment of capillary permeability, easy bruising, hemorrhoids, and varicose veins.
Ginkgo
Leaf extract from the Ginkgo tree is widely marketed as an herbal supplement. The active ingredients are flavoglycosides.

Tea

Bai Hao Yinzhen from Fuding in Fujian Province, widely considered the best grade of white tea

Green tea flavonoids are potent antioxidant compounds, thought to reduce incidence of cancer and heart disease. The major flavonoids in green tea are the kaempferol and catechins (catechin, epicatechin, epicatechin gallate, and epigallocatechin gallate (EGCG)).
In producing teas such as oolong tea and black tea, the leaves are allowed to oxidize, during which enzymes present in the tea convert some or all of the catechins to larger molecules. However, green tea is produced by steaming the fresh-cut leaf, which inactivates these enzymes, and oxidation does not significantly occur. White tea is the least processed of teas and is shown to present the highest amount of catechins known to occur in camellia sinensis.

Wine
See also: Phenolic compounds in wine
Grape skins contain significant amounts of flavonoids as well as other polyphenols[18]. Both red and white wine contain flavonoids; however, since red wine is produced by fermentation in the presence of the grape skins, red wine has been observed to contain higher levels of flavonoids, and other polyphenolics such as resveratrol.
Dark chocolate
Flavonoids exist naturally in cacao, but because they can be bitter, they are often removed from chocolate, even the dark variety[19].
Subgroups
Over 5000 naturally occurring flavonoids have been characterized from various plants. They have been classified according to their chemical structure, and are usually subdivided into the following subgroups (for further reading see [3]):
Sub-Sub kelompok
Lebih dari 5000 flavonoids terjadi secara alami telah ditandai dari berbagai tumbuhan. Flavonoids telah digolongkan menurut struktur kimianya, dan pada umumnya dibagi lagi ke dalam sub-sub kelompok berikut :
Flavones
Flavones are divided into four groups:[20]
Flavones dibagi menjadi empat groups:[20]

Isoflavones
•Isoflavones
Isoflavones use the 3-phenylchromen-4-one skeleton (with no hydroxyl group substitution on carbon at position 2).
Examples: Genistein, Daidzein, Glycitein
Isoflavones menggunakan 3-phenylchromen-4-one rangka ( dengan tidak ada penggantian kelompok hidroksit atas karbon pada posisi 2).
Contoh: Genistein, Daidzein, Glycitein
Flavan-3-ols, Proanthocyanadins, and Anthocyanidins
•Flavan-3-ols (also known as Flavanols) and Proanthocyanidins
Flavan-3-ols use the 2-phenyl-3,4-dihydro-2H-chromen-3-ol skeleton.
Catechins (Catechin (C), Gallocatechin (GC), Catechin 3-gallate (Cg), Gallocatechin 3-gallate (GCg)), Epicatechins (Epicatechin (EC), Epigallocatechin (EGC), Epicatechin 3-gallate (ECg), Epigallocatechin 3-gallate (EGCg))
Proanthocyanidins are dimers, trimers, oligomers, or polymers of the flavanols.
•Anthocyanidins
Anthocyanidins are the aglycones of anthocyanins. Anthocyanidins use the flavylium (2-phenylchromenylium) ion skeleton
Examples: Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin

Availability through microorganisms
A number of recent research articles have demonstrated the efficient production of flavonoid molecules from genetically-engineered microorganisms[21][22].
See also

References
1.^ Flavonoids (isoflavonoids and neoflavonoids)., IUPAC Compendium of Chemical Terminology
2.^ Spencer, Jp (May 2008). "Flavonoids: modulators of brain function?". The British journal of nutrition 99 E Suppl 1: ES60–77. doi:10.1017/S0007114508965776. ISSN 0007-1145. PMID 18503736. edit
3.^ a b Ververidis Filippos; Trantas Emmanouil, Douglas Carl, Vollmer Guenter, Kretzschmar Georg, Panopoulos Nickolas (October 2007). "Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I: Chemical diversity, impacts on plant biology and human health". Biotechnology Journal 2 (10): 1214. doi:10.1002/biot.200700084.
4.^ "Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer". Yamamoto and Gaynor 107 (2): 135 -- Journal of Clinical Investigation. http://www.jci.org/cgi/content/full/107/2/135?ijkey=a1e09ce2dbca283cec170598f2410b15d5f4304f&keytype2=tf_ipsecsha.
5.^ Cushnie TPT, Lamb AJ (2005). "Antimicrobial activity of flavonoids". International Journal of Antimicrobial Agents 26 (5): 343–356. doi:10.1016/j.ijantimicag.2005.09.002. PMID 16323269.
6.^ Félicien Breton (2008). "Health benefits of oligomeric proanthocyanidins". http://www.frenchscout.com/polyphenols#procyanidins.
7.^ Lotito SB, Frei B (2006). "Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon?". Free Radic. Biol. Med. 41 (12): 1727–46. doi:10.1016/j.freeradbiomed.2006.04.033. PMID 1715775.
8.^ a b c "Studies force new view on biology of flavonoids", by David Stauth, EurekAlert!. Adapted from a news release issued by Oregon State University. URL accessed .
9.^ UCLA news May 2008 - Fruits, vegetables, teas may protect smokers from lung cancer
10.^ Schuier M, Sies H, Illek B, Fischer H (01 October 2005). "Cocoa-related flavonoids inhibit CFTR-mediated chloride transport across T84 human colon epithelia". J. Nutr. 135 (10): 2320–5. PMID 16177189. http://jn.nutrition.org/cgi/reprint/135/10/2320.
11.^ Lee KW, Kim YJ, Lee HJ, Lee CY (December 2003). "Cocoa has more phenolic phytochemicals and a higher antioxidant capacity than teas and red wine". J. Agric. Food Chem. 51 (25): 7292–5. doi:10.1021/jf0344385. PMID 14640573.
12.^ "Cocoa nutrient for 'lethal ills'". BBC News. http://news.bbc.co.uk/2/hi/health/.stm.
13.^ Tsushida T., Suzuki, M. (1996) Content of flavonol glucosides and some properties of enzymes metabolizing the glucosides in onion. J. Jap. Soc. Food Sci. Technol., 43, 642-649.
14.^ Slimestad R, Fossen T, Vågen IM (December 2007). "Onions: a source of unique dietary flavonoids". J. Agric. Food Chem. 55 (25): 10067–80. doi:10.1021/jf0712503. PMID 17997520.
15.^ Justesen U, Knuthsen P (2001). "Composition of flavonoids in fresh herbs and calculation of flavonoid intake by use of herbs in traditional Danish dishes". Food Chem. 73: 245–50. doi:10.1016/S0308-8146(01)00114-5.
16.^ Ewald C, Fjelkner-Modig S, Johansson K, Sjöholm I, Åkesson B (1999). "Effect of processing on major flavonoids in processed onions, green beans, and peas". Food Chem. 64: 231–5. doi:10.1016/S0308-8146(98)00136-8.
17.^ Spedding G, Ratty A, Middleton E (September 1989). "Inhibition of reverse transcriptases by flavonoids". Antiviral Res. 12 (2): 99–110. doi:10.1016/0166-3542(89)90073-9. PMID 2480745.
18.^ Kennedy JA, Matthews MA, Waterhouse AL (2002). "[Effect of Maturity and Vine Water Status on Grape Skin and Wine Flavonoids". Am. J. Enol. Vitic. 53 (4): 268–74. [http://www.ajevonline.org/cgi/content/abstract/53/4/268.
19.^ The Lancet, (December 2007). "The devil in the dark chocolate". Lancet 370 (9605): 2070. doi:10.1016/S0140-6736(07)61873-X. PMID 18156011. http://linkinghub.elsevier.com/retrieve/pii/S0140-6736(07)61873-X.
20.^ Phenolics:figure 4
21. ^ Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S (May 2003). "Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster". Appl. Environ. Microbiol. 69 (5): 2699–706. doi:10.1128/AEM.69.5.2699-2706.2003. PMID 12732539. PMC: 154558. http://aem.asm.org/cgi/pmidlookup?view=long&pmid=12732539.
22. ^ Ververidis Filippos; Trantas Emmanouil, Douglas Carl, Vollmer Guenter, Kretzschmar Georg, Panopoulos Nickolas (October 2007). "Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part II: Reconstruction of multienzyme pathways in plants and microbes". Biotechnology Journal 2 (10): 1235. doi:10.1002/biot.200700184.

External links
•Instant insight considering the effect of flavonoids on memory and learning from the Royal Society of Chemistry
•USDA Database of Flavonoid content of food (pdf)
•Flavonoids (chemistry)
•Cornell news on Cocoa
•A Dark Chocolate a Day Keeps the Doctor Away
•Antioxidant in Green Tea may fight Alzheimer's-EGCG
•Yamamoto Y, Gaynor RB (January 2001). "Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer". J. Clin. Invest. 107 (2): 135–42. doi:10.1172/JCI11914. PMID 11160126. PMC: 199180. http://www.jci.org/cgi/content/full/107/2/135.
•FLAVO - European Project on Flavonoid Research

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Commercial release of Genetically Modified Cotton



By. Fahry Marewo3

Commercial Release of Genetically Modified Cotton, Roundup Ready Cotton (MON 1445)
Technical Opinion no. 1598/2008
Proceedings: 01200.004487/2004-48
Applicant: Monsanto do Brasil Ltda.
CNPJ: 64.858.525/0001-45
Address: Avenida Nações Unidas, 12901, Torre Norte, 7º Andar, São Paulo, SP.
Matter: Commercial release of Genetically Modified Cotton.
Previous extract: 242/2004. Published in the Federal Official Gazette no. 195, of 10.08.2004, Section no. 3, page no. 06.
Meeting: 116th CTNBio Regular Meeting, held on 09.18.2008.
Decision: GRANTED.

CTNBio, following approval of an application for a Technical Opinion related to commercial release of genetically cotton (Roundup Ready Cotton, Event MON 1445) as well as all progenies originated from the MON 1445 transformation event and its derivatives obtained by crossing non transgenic cotton lineages and populations with lineages carrying event MON 1445, was favorable to the GRANTING under the terms of this technical opinion.

Monsanto do Brasil Ltda. requested a CTNBio Technical Opinion related to biosafety of the genetically modified cotton (Gossypium hirsute) tolerant to the herbicide glyphosate, namely Roundup Ready Cotton for the free registration, use in the environment, human and animal consumption, marketing and industrial use and any other use and activity related to this GMO including derivative lineages and cultivars as well as byproducts, in compliance with other regulations and requirements applicable to any use of cultivated species of the genus Gossypium effective in Brazil. Roundup Ready Cotton Event MON 1445 was produced by genetic transformation mediated by Agrobacterium tumefactions containing plasmid PV-GHGT07, using cotton variety Coker 312 as receiving plant. In this plasmid, genes cp4-epsps, nptll, aad and gox are present. The inserted gene cp4-epsps was obtained from a specific stretch of DNA of the Agrobacterium sp., CP4 strain, and codifies enzyme CP4-EPSPS (CP4 5-enolpyruvylshikimate-3-phosphate synthase) granting to cotton plants the attribute that enables the use, in post-emergence, of the glyphosate herbicide, for management of pest plants without injuring the cotton crop. Gene nptll codified the protein Neomycin Phosphotransferase type II that grants tolerance to antibiotics neomycin and kanamycin. Gene aad, which codifies protein AAD (3’(9)-O-aminoglycoside adenyltransferase – antibiotic resistance selection marker. Gene gox codifies enzyme GOX (glyphosate oxyreductase), responsible for metabolizing the glyphosate herbicide. Gene gox was not transferred to cotton and, consequently, the protein GOX was not detected in Roundup Ready Cotton Event MON1455. There is no evidence that the donor organisms of inserted genes are pathological to humans. Molecular and segregation analyses (Mendelian standard of 3:1) show that T-DNA was partially inserted in a single locus of the cotton genome. Genetic stability of MON 1445 was determined by hereditary stability standard, integrity of inserted DNA and stability phenotype in different environmental conditions determined in several generations of lineages obtained by retro-crossing with elite cultivars. This stability was additionally demonstrated by the integrity of inserted DNA and functionality of the protein CP4 EPSPS expressed in lineages obtained by crossing with a cultivar adapted for sowing in the Brazilian environment. Proteins EPSPS and NPTII, which have no history of toxicity or allergenicity, resulting from the expression of transgenes, showed to be equivalent to the ones present in nature. Gene epsps is present both in plants and in microorganisms, while nptll is present in several species of microorganisms, including intestinal bacteria and in genus Bacillus found in Brazilian soils. Studies in vitro demonstrated that in simulated intestinal fluids (pH 1.2 and pH 7.5) protein EPSPS is rapidly degraded, which is common in the digestive tract of mammals with proteins that pose a minimum risk of toxicity and allergenicity. Besides, studies of oral acute toxicity in mice showed that both EPSPS and NPTII fail to present toxic potential when orally administered at doses of 572mg/kg body weight and 5 mg/kg body weight, respectively. Both transgenic proteins, CP4 EPSPS and NPTII, are in the nature, widely distributed among microorganisms from where they are derived. The introgression of a transgene to wild cotton plants could only occur if a strong selective advantage was granted, higher than the disadvantages granted by the alleles that are genetically linked to the transgene. However, the herbicide-tolerant characteristic is recognized as being unable to grant receiving genotypes any adaptive advantage outside agricultural areas, since outside such areas the potentially receiving wild genotypes are not under the selective pressure from the herbicide and therefore any pollination of such genotypes would not result in genic introgression. Assessment of phenotypic and agronomic characteristics of Roundup Ready Cotton event MON 1445, cultivar DP50RR, conducted in Brazil reached results similar to the ones found in other regions of the world in experimental and commercial crops. Except for tolerance to the glyphosate herbicide, resulting from expression of gene cp4 epsps, Roundup Ready Cotton event MON 1445 demonstrates phenotype and agronomic characteristics equivalent to those of the standard conventional kindred lineages and commercial cultivars of conventional cotton. Glyphosate is a post-emergent herbicide, belonging to the chemical group of substituted glycines, classified as non-selective and possessing systemic action. It has a wide spectrum action, enabling the control of large and short-leaved annual and perennial weeds. The herbicide is registered with the Brazilian Ministry of Agriculture and Supply - MAPA for agricultural purposes, and with the Brazilian Environment and Renewable Resources Institute – IBAMA, of the Brazilian Ministry of the Environment for non-agricultural purposes, in addition to possessing a monograph approved by the Brazilian National Sanitary Surveillance Agency - ANVISA. Available information indicates that transgenic plants are not fundamentally different from the non-transformed cotton genotypes, except for the tolerance to glyphosate. Besides, there is no evidence of adverse reactions to the use of Roundup Ready Cotton. For these reasons, there are no restrictions to the use of this cotton or its derivatives both for human and animal feeding. Therefore, commercial release of Roundup Ready Cotton event MON 1445 is not a potential cause of harm to human and animal health and does not significantly degrades the environment. According to Article 1 of Law no. 11,460, of March 21 2007, “research and cultivation of genetically modified organisms may not be conducted in indigenous lands and areas of conservation units”. There are not creole varieties of cotton plants and the chains of special, conventional and transgenic cottons have lived together in a satisfactory fashion, without records of coexistence problems. According to Annex I of Regulating Resolution no. 5, of March 12, 2008, the applicant shall have a term of thirty (30) days from the publication date of this Technical Opinion to adjust its proposal to the post-commercial release monitoring plan. Under Article 14 of Law no. 11,105/2005, CTNBio found that the request complies with the applicable rules and legislation securing the biosafety of environment, agriculture, human and animal health.

TECHNICAL OPINION

I. GMO Identification
GMO name: Roundup Ready Corn, Event MON 1445.
Applicant: Monsanto do Brasil Ltda.
Species: Gossypium hirsutum L.
Inserted characteristics: Tolerance to glyphosate herbicide
Method of insertion: Co-culture with Agrobacterium tumefaciens
Prospective use: Production of fibers for the textile industry and grains for human and animal consumption of the GMO and its derivatives.

II. General Information
Cotton is a plant of genus Gossypium, Tribe Gossypiae, Family Malvaceae, Order Malvales and subdivides into four subgenera (Gossypium, Sturtia, Houzingenia and Karpas) that in turn subdivide into nine sections and a number of subsections(32). Genus Gossypium currently encompasses 50 species, coming from America, Africa, Asia and Australia(31). The centers of G. hirsutum origin are in Mexico and Guatemala, while G. barbadense originated in Peru and Bolivia(79).
There are worldwide four species of cotton that are important under the agronomic viewpoint, whose fibers are commercially worthwhile: two of them are diploid European species (G. arboretum and G. herbaceum) and other two are allotetraploid species found in America (G. barbadense and G. hirsutum). The two allotetraploid species answer for about 98% of the world cotton production. G. hirsutum is cultivated in 90% of the cotton-planted area(57). G. hirsutum cultivars were classified in four main types: Eastern, Acala, Delta and Plains(66).The Eastern type includes variety Coker 312 that was used in the genetic transformation, generating Roundup Ready Cotton event MON 1445.
Two cotton plants are primarily cultivated in Brazil: conventional cotton and caterpillar-resistant genetically modified cotton. These cotton plants answer for practically all cotton produced in the country. Besides, three other cotton plants featuring special genetic or ecologic characteristics are cultivated: the naturally colored fiber cotton, organic cotton and agroecologic cotton. Colored cotton may be found practically just in the State of Paraíba, where in 2007 about 300 hectares were sowed. Certified organic cotton is planted in the States of Paraná and Paraíba, and 250 hectares were sowed in 2007. Crops of agroecologic cotton were cultivated by 235 farmers in the semiarid biome of four states in the Brazilian Northeastern area and produce 42 tons(58).
The chains of special, conventional and transgenic cotton plants have lived together in a satisfactory fashion, without records of coexistence problems. The cotton planted area in Brazil during the 2007/2008 crop was about one million and one thousand hectares, of which 85% are concentrated in the Cerrado biome, especially in the States of Mato Grosso, Bahia, Goiás and Mato Grosso do Sul. The remaining crops are present in other States of the country, mainly in the Northeastern Semiarid, Paraná, Minas Gerais and São Paulo(49).
In addition to the herbaceous cotton plant, there are other three cottons in Brazil, all of them allotetraploid and sexually compatible with the cultivars. None of such species is deemed to be a pest plant in agricultural and natural environments.
The species G. barbadense has its domestication center in the Northern Peru and Southern Ecuador(16). In was introduced by pre-Columbian peoples and the cotton fiber was used in the production of textile craftsmanship by some indigenous ethnic groups before the arrival of Portuguese colonizers(63). Its use as a textile plant was disseminated among colonizers but suffered a diminishing driven by the dissemination of exotic G. hirsutum races. G. barbadense cannot be found in natural environments and is basically kept as a backyard plant. It is widely distributed, present in almost the whole country and the in situ conservation is directly linked to the maintenance of traditional use as a medicine plant(10).
G. mustelinum is a species indigenous to Brazil, being its natural distribution restricted to the Northeastern semiarid(31,51). The populations are known only in the States of Bahia and Rio Grande do Norte, in locations that do not produce herbaceous cotton. Two problems affect the in situ maintenance of G. mustelinum. The first and most severe is the destruction of non-perennial rivers and rivulets gallery forests, the natural habitat of the species. The second is the extensive cattle raising conducted in the region, especially goats. The animals feed on buds, leaves, fruits, seeds and bark, harming the development and, in some cases, killing adult plants. Renewal of populations is also jeopardized, since the grazing on young individuals destroys part of the plants(10). The distance between known populations of G. mustelinum and cotton producing regions prevents the crossing between this species and herbaceous cotton present in cultivars.
The third cotton plant is known as mocó cotton and belongs to a race different from the same species of herbaceous cotton (G. hirsutum var. marie galante (Watt) Hutch.). This cotton originated in the Antilles and its introduction to Brazil is uncertain. One conjectures that it may have been brought by the Dutch or Africans during colonial times(63). Mocó cotton was widely cultivated in the Northeastern semiarid until the end of the eighties, when different problems caused an abrupt interruption in planting(13).
A small amount of arboreal cotton plants, mainly inter-racial hybrids of colored and white fiber produced by the Embrapa improvement program are still cultivated. However, the planting of this material is in decline; 5,692 hectares were harvested during the 2004/2005 crop and just 1,326 hectares during the 2005/2006 crop(49). Tillage is cultivated with a minimum of external inputs and the most important is the insecticide to control insect pests. Control of weed is almost exclusively conducted through manual clearing. Transient populations of this race with high biologic importance, derived from forsaken farming, may be found in the high ridges of some municipalities of the Seridó area in the States of Paraíba and Rio Grande do Norte(10). These populations are geographically isolated from herbaceous cotton farms and well represented in the Embrapa germplasm banks.
The cotton plant is generally deemed to be an annual crop, although considered as perennial in some parts of the world, where species such as G. hirsutum are commercially cultivated. In Brazil, cotton may be cultivated during almost the whole year in regions featuring mild winters, enabling two harvests, although having an annual crop.
Cotton has one central branch and two other branch types (vegetative and fructification branches), reaching one 1.5 high. The leaves alternate in a spiral distribution around branches, are petiolated, and have from three to five clearly defined lobes, as well as bristles and cordate blade along 7.5 cm to 15.0 cm long by each lobe(27). At the base of each leaf of the main branch, at the angle between the leaf and the branch, there are two or three axillary buds that originate the vegetative and fructification branches. Fructification branches possess floral buds that develop in apples(62).
Flowering starts from 7 to 11 weeks after sowing, with an average of six to eight flowers for each fertile branch, all large, colored white or yellow, held by a reduced calyx and having three to four green, large, fringed bracts. The flowers possess five separated petals, as the other members of the Malvaceae family. The stamen column surrounds the stylet formed by 100 or more stamens that end in anthers and usually produce 45 thousand pollen grains per flower(86). Pollen grains are large, from 81 to 143 micra and covered by a sticky material that promotes adherence, causing the cotton pollen not to be transported by the wind. The petals have changing colors during the day, therefore attracting pollinators(61).
The higher ovary (apple), that develops within the capsule, is a dry structure that, when opened, shows a division in three to five carpels or locules, with five to ten ovules each. The fruit is a capsule with a size varying from 4.0 cm to 6.0 cm, spherical, soft textured, greenish, with few oily glands. Seeds are 1.0 cm long, egg-shaped, dark brown, in about 36 seeds for each fruit. The weight of 100 seeds varies from 10.0g to 13.0g, with long and short fibers in its epidermis(27). Long fiber seeds are inside the carpels. Long fibers are known as linter, while short ones are the wool. Most species of cotton do not have linter. Each fiber is, in reality, a single fiber cell that grows in the seed bark’s epidermis. The cell wall becomes thick with the addition of cellulose layers. Matured apples are or different forms and sizes, depending on the variety and environmental conditions, though apples that develop within the first three weeks of flowering are usually larger and have higher quality fibers.
Nectaries are normally found in five different parts of the plant (floral, internal, external, foliar and microscopic). Floral nectarines shall be associated to pollination and extra-floral nectarines to attraction of insects(61). Cotton has, in addition, special glands in different tissues (including kernel) producing a chemical compost, namely gossypol, a terpenoid substance biologically active that works as a defense against herbivores. Gossypol is an anti-oxidizing and inhibitor of pollination, and is toxic to monogastric animals such as pigs and rabbits. Main symptoms are constipation, loss of weight and appetite, as well as effects to the circulatory system. Acute toxicity of gossypol is low, though ingestion of small quantities over a long time may be lethal. Consensus is that pig and bird diets shall not contain over 100 mg of free gossypol/ kg of weight and that inclusion of cotton kernels in feeding shall range from 50kg/t to100kg/t of fodder. High temperature processing of cotton kernels is an efficient deactivator of gossypol(76).
Cotton is usually held as a partial crossed pollination culture, though several improvers consider the plant as fully auto-fertile and auto-pollinating, except for crossed pollination, caused by pollinating insects. Freire(31) maintains that the cotton plant displays a reproductive system that is intermediary between the one found in allogamous and autogamous plants, with cross pollinating rates between 5% and 95%. Crossed pollination is called natural crossing and maintains a degree of heterozygosis from F1(76).
Auto-pollinating is a form of hybridization that occurs preferably in cotton culture, though natural crossing may occur(66). Controlled pollination in cotton plants is simple and consists in the use of methods that prevent the flowers from opening. Crossing is also easily attained, by emasculation and protection of the stigma the day before and pollination the day after. Kernel production varies from 20 to 30 in each fruit when crossing and auto-pollination are well conducted(35).
The flowering term of the cotton plant may differ depending on the variety and environmental conditions, but begins in general 50 days after emergence and prolongs for 120 or more days, with the peak of the curve around 70 to 80 days. Auto-pollination and crossing procedures shall be conducted in the most propitious time, 30 to 40 days of flowering.
Combat to weeds is one of the main cultural handling conducted in cotton plants. Negative interactions of weeds with the cotton plant, especially competition, allelopathy and interference in agricultural activities cause reduced productivity and depreciation of cotton produced. Losses may be high in case control is not correctly and timely conducted(18). The main plants invading the cotton culture in Brazil are: River burr (Cenchus equinatus), alexandergrass (Brachiaria plantaginea), Jamaican crabgrass (Digitaria horizontalis), Coast cross grass (Cynodon dactilon), hairy beggarsticks (Bidens pilosa), hispid starbur (Acanthospermum hispidum) and moonvine (ipomea sp.). Management of weeds is conducted by cultural methods, either mechanical or chemical, and control is achieved by the use of herbicides, the main controlling method.
Roundup Ready Cotton is marketed in the United States of America (since 1995), Canada (1996), Japan (1997), Argentina (1999), Australia (2000), Mexico (2000), South Africa (2000), Korea (2003), Philippines (2003), China (2004) and the European Union (2005) (1).

III. Description of GMO and Proteins Expressed
Roundup Ready Cotton Event MON 1445 was genetically modified by transforming commercial variety Coker 312 with plasmid PVGHGT07, through a system mediated by A. tumefaciens. The transformation inserted genes cp4 epsps, nptll, gox and aad in the genome of this cotton variety(75). The lineage resulting from the transformation expresses enzyme CP4 EPSPS CP4 5-enolpyruvylshikimate-3-phosphate synthase) coming from Agrobacterium sp. strain CP4, which is naturally tolerant to glyphosate, the Roundup herbicide active ingredient. The lineage also expresses protein NPTII (neomycin phosphotransferase II), which grants resistance to aminoglycoside antibiotics, enabling selection of cells transformed by gene cp4 epsps in a culture containing antibiotic kanamycin in the in vitro phases of the transformation process. The third introduced gene, aad, codifies protein (3”(9)-O-aminoglycoside adeniltransferase). The gene is not expressed in plant tissue for being under the control of a prokaryotic promoter.
The methodology of genetic transformation used in the production of Roundup Ready Cotton Event MON 1445 was an indirect system mediated by bacterium A. tumefaciens. Several revisions on this plant transformation system, including details of the process molecular mechanism, were published in recent years(5, 15, 21, 44, 46, 83, 89).
A. tumefaciens is an aerobic gram-negative bacterium, commonly found in the soil and is responsible for causing a plant tumor known as crown gall(77). The tumor is associated to a natural process of specific DNA sequence transferences, (T-DNA, transferred DNA) present in the bacterium Ti (tumor inducible) plasmid, to the nuclear chromosome of infected plant cells, where the T-DNA is integrated and expressed in a stable way.
Physiologic changes caused by wild lineages of A. tumefaciens prevent their use in obtaining transgenic plants. In order to use this transformation methodology, the lineage of chloramphenicol-resistant A. tumefaciens (A208) was modified not to cause tumors in plants. To this end, hormone synthesis genes present in the Ti plasmid causing the tumor were removed and replaced by the disarmed plasmid pMP9ORK(77). The process created a lineage of disarmed A. tumefaciens (ABI), unable to cause tumors. Despite of disarming, plasmid pMP9ORK was constructed with all functions necessary for its self-replication with vir genes, which promote T-DNA transference to the plant chromosome. The system is able to transfer genes to plant cells and maintain regeneration feasibility of transformed cells in a plant without causing a tumor(46, 55, 56).
Plasmid pMP9ORK was constructed using genetic elements that enable replication and transference of plasmids between Escherichia coli and A. tumefaciens (RK2 mobilization and transfer factors) if the host organisms contain ancillary RK2 functions of replication and mobilization. vir genes and RK2 elements involved in mobilization between bacteria may act either in cis or in trans, and may be present in the same gene structure containing the T-DNA or in a different one(45).The only elements that must be present in the same gene structure containing the T-DNA are sequences of about 25 pb at the extremities of the T-DNA that bound their transfer. Using E. coli as host organism, the T-DNA of interest may be assembled in another binary plasmid (containing elements of ancillary RK2 replication and mobilization functions with specific signs for action of the vir genes) and transferred to A. tumefaciens by conjugation. Following, the co-cultivation of ABI A. tumefaciens containing the plasmid carrying the T-DNA of interest with the plant tissue may result in transferring the T-DNA to the nuclear genome of the plant cell through vir functions codified by the disarmed plasmid(52, 78). In these processes, the vir genes are not transferred to the plant cell and remain in the Agrobacterium. T-DNA transfer is initiated by the proteins codified by vir genes, which recognize the specific sequences in the T-DNA extremities. The extremities are necessary though not entirely transferred, resulting in an irreversible transfer of T-DNA(9, 48). Therefore the inserted DNA is no longer a T-DNA, since it lacks the extremities necessary to the transfer(9).
In order to generate Roundup Ready Cotton Event MON 1445, plasmid PV-GHGT07, containing the T-DNA with the genes of interest, was constructed in E. coli strain MM-294 (derived from E. coli strain K-12) and transferred to the ABI Agrobacterium lineage by means of a three-kindred conjugation. The three-kindred conjugation involved three actors: a disarmed ABI Agrobacterium lineage, the E. coli containing plasmid pRK2013 that embodies ancillary RK2 functions(26) and the E coli containing plasmid PV-GHGT07 including the T-DNA possessing the genes to be introduced.
The ABI Agrobacterium lineage containing plasmid PV-GHGT07 was selected and cultivated by means of a culture medium containing the antibiotics spectinomycin and streptomycin. Subsequently, the ABI Agrobacterium containing plasmid PV-GHGT07 was co-cultivated with cotton hypocotyl sections of the Coker 312 cultivar. Besides gene cp4 epsps, plasmid PV-GHGT07 contains gene nptll, which enables selecting of transformed cells in a medium containing the antibiotic kanamicyn. Later, residual cells of Agrobacterium in the culture medium were eliminated with the use of different antibiotics and the hypocotyls were transferred to a culture medium specific for inhibition of embryogenic calli containing the antibiotics carbenicillin and kanamicyn. Subsequently, the genetically transformed cells were stimulated for regeneration of sprouts and plantuls. The plantuls were cultivated in soil, assessed for tolerance to glyphosate and developed for the seed production according to a methodology described by Trolinder and Goodin(85).
Gene cp4 epsps, which is part of the chymeric construct above (P-FMV/CTP2/cp4 epsps / E9 3’), codifies the protein CP4 EPSPS, responsible for the tolerance of Roundup Ready Cotton Event MON 1445 to the herbicide glyphosate. The cp4 epsps codifying region derives from the soil bacterium Agrobacterium sp., strain CP4. The bacterium was identified by scanning microorganisms resistant to the glyphosate molecule action(68, 69). Agrobacterium sp., CP4 strain, as well as other bacteria and some soil fungi, are resistant to the action of glyphosate for possessing the EPSPS enzyme, little sensitive to the inhibition of the herbicide(74). The choice of the bacteria as the source of the resistance gene ensued mainly from the kinetic characteristics of the CP4 EPSPS enzyme for high tolerance to glyphosate, namely an inhibition constant (Ki) of about 2.7 mM, keeping high affinity for phosphoenolpyruvate (PEP) (Km = 12ƒÝM), one of its substrata. In other words, the concentration of glyphosate needed to inhibit the enzyme is about 2,000 times higher than the concentration of PEP, one of its substrata, indicating that it has very high affinity for this substratum and very low affinity for its inhibitor, the glyphosate. The parameters are inverse when the enzyme is susceptible to the glyphosate inhibitory effect.
Gene cp4 epsps was integrated to the codifying sequence of the transit peptide to the chloroplast derived from EPSPS of Arabidopsis thaliana, CTP2(53), complemented by the promoting and finalizing sequences that enable expression of the CP4 EPSPS enzyme and its transport to the chloroplast of plant cells(75).
Gene nptll codifies protein NPTII (neomycin phosphotransferase II) and is part of the chimeric construct described above (35S/nptll/NOS 3’). The codifying region of the nptll gene derives from prokaryotic transposon Tn5 of the E. coli bacterium(12), widely found in nature. The nptll gene sequence was not modified, but just complemented with promoting and finalizing sequences to enable expression of the NPTII protein in plants. Gene nptll works as a dominant selection marker in laboratory initial phases, identifying transformed plant cells(22, 47). Enzyme NPTII uses adenosine-triphosphate (ATP) to phosphorylate and inactivate aminoglycoside antibiotics (such as neomycin and kanamicyn), avoiding injuries to be caused to the NPTII-expressing cells when cultivated in a culture medium containing such selective agents. The only purpose of inserting the nptll gene in the Roundup Ready Cotton Event MON 1445 was, therefore, the selection of transformed cells containing gene cp4 epsps (since both genes are part of the same genic construct), without any other function(75).
The gene sequence aad was not changed away from its natural form. Since the promoting sequence of gene aad is of prokaryotic origin, expression of the AAD protein occurs only in microbian system and was not detected in Roundup Ready Cotton Event MON 1445. The gene was used as E. coli and A. tumefaciens bacterial selection maker, containing plasmid PV-GHGT07. Antibiotics spectinomycin and streptomycin were added to the culture medium as selective agents. The only purpose of inserting the aad gene in cotton is the selection of bacterial cells containing the plasmid of interest, with no other function(75).
Gene gox codified enzyme GOX (glyphosate oxidoreductase), responsible for metabolizing the glyphosate herbicide. The codifying region of gene gox derives from Ochrobactrum anthropi, strain LBAA, isolated in waste reservoirs and treatment of glyphosate industrial production effluents(11, 42). Enzyme GOX has the ability to degrade glyphosate into aminomethylphosphonic acid (AMPA). Conversion of glyphosate into is the degradation path of glyphosate in soil(37, 72, 84).
The primary purpose in using the gox gene was to obtain an increase in the herbicide tolerance through a general reduction in concentration of glyphosate in the plant and prevent collection of glyphosate toxic levels in the plant sensitive organs. The codifying region of gox gene was redesigned and re-synthesized for expression in dicotyledon plants, considering the same criteria described for gene cp4 epsps. The gene was bound to the codifying sequence of the transit peptide to the chloroplast of the Arabidopsis thaliana ribulose-1,5-biphosphate carboxylase small subunit, CTP1(54), complemented with the promoting and finalizing sequences to enable the GOX enzyme to be expressed and transported to the plant cell chloroplast. Despite being in the PV-GHGT07 plasmid, the gox gene was not transferred to the cotton and, therefore, protein GOX was not detected in the Roundup Ready Cotton Event MON 1445(75).

IV. Aspects Related to Human and Animal Health
Protein CP4 EPSPS 5-enolpyruvylshikimate-3-phosphate synthase belongs to the family of enzymes that is present in all plants (including cotton) and in a large number of microorganisms(29). The enzymes have been isolated from different sources, and their properties have been widely studied(48). Bacterial and plant EPSPS enzymes are monofunctional, having molecular mass from 44 to 48kD(30) and catalyze the enolpyruvil group transfer from phosphoenolpyruvate (PEP) to the 5-hidroxy group of shikimate-3-phosphate (S3P) producing inorganic phosphate and 5-enolpiruvylshikimate-3-phosphate(3).
Due to the rigorous specificity for substracts, EPSPS enzymes link just S3P, PEP and glyphosate. The only known resulting metabolic product is the 5-enolpyruvylshikimic-3-phosphate acid, corresponding to the penultimate product of the shikimic acid pathway. Shikimic acid is a precursor for the biosynthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan), in addition to several secondary metabolites, such as tetrahydrofolate, ubiquinone and vitamin K(39). Although the shikimic acid (or shikimate) pathway and EPSPS proteins do not occur in mammals, fish, birds, reptile and insects, they are important to plants. Estimates are that aromatic molecules, all derived from shikimic acid, represent not less than 35% of a plant dry weight(3,30).
In vitro studies with simulated digestive fluids are widely used tools as models for animal digestion. The simulated system was used to investigate the digestibility of plant proteins(60,65), animal proteins(88) and food additives(81), as well as assessing the allergenic potential and quality of a protein(2) through absorption of the protein by the digestive tract(6).
Besides, the knowledge on the way of action, specificity and background of safe use of the EPSPS and NPTII proteins, their potential toxic and allergenic effects in humans and other mammas was assessed through in vitro digestion studies. The studies used simulated gastric (pH 1.2) and intestinal (pH 7.5) fluids. The CP4 EPSPS protein degradation rate (mature protein, without the transit peptide) was assessed through Western Blot analyses. The studies showed that protein CP4 EPSPS and peptides degrade in no more than 15 seconds after being exposed to gastric fluid. In the simulated intestinal fluid, the CP4 EPSPS degradation occurred in a period not longer than 10 minutes(43).
Protein NPTII degradation rate was assessed through enzymatic activity. The study showed that NPTII protein was destructed after two minutes of incubation in simulated gastric fluid and after 15 minutes of incubation in simulated intestinal fluid(34). Based on the result of these studies, estimates are that proteins CP4 EPSPS and NPTII expressed in Roundup Ready Cotton Event MON 1445 are rapidly digested in the digestive tract of mammals. Rapidly digested exogenous proteins pose minimal toxicity and allergy risk when compared to the remaining safe dietetic proteins(6, 7).
Acute oral toxicity studies of proteins CP4 EPSPS and NPTII were conducted using albino mice as indicators(14, 34). He results showed no evidence of toxicity when the CP4 EPSPS protein was gavaged to mice in doses of 572, 154 and 49 mg of the protein by kg of body weight. In special, no abnormal clinic signs were observed in the animals during the study, and therefore there was not any statistically significant difference in body weight, aggregate body weight or food consumption between treatments and control groups. All animals were sacrificed and necropsied at the seventh post-dosage day, when no pathologic effect was found(14).
Based on the protein CP4 EPSPS expression data in kernels of Roundup Ready Cotton Event MON 1445, the doses of 572 mg/kg bodily weight is equivalent to a consumption ranging from 3.2 to7.1 kg of kernels by kg of bodily weight. In the study conducted with the NPTII protein, doses of 5,000, 1,000 and 100 mg of NPTII by kg of bodily weight were administered by gavage to mice. All animals were sacrificed in the seventh post-dosage day and submitted to necropsia and no pathological effect was found(34). Based on the NPTII protein expression data in the kernel of Roundup Ready Cotton Event MON 1445, the dose of 5,000 mg by kg of bodily weight is equivalent to a consumption of 700 kg of cotton kernel by kg of bodily weight.
Compositional analyses (proteins, lipids, fibers, carbohydrates, amino acids, minerals, caloric content, Ą-tocopherol, and gossypol) failed to evidence significant differences between event MON 1445 and the kindred non-GM or other commercial cotton varieties.
The results of the studies confirm that CP4 EPSPS and NPTII proteins are not acutely toxic for mammals. The results are in line with the absence of any similarity between amino acid sequences and known toxic proteins and with the rapid digestion of such proteins by gastric and intestinal fluids.

V. Environmental and Agronomic Aspects
Modern agriculture is an activity responsible for significant negative environmental impacts(8, 17, 40, 71, 82) and, therefore, the risk assessment of any GMO shall be conducted in relation to the impact that is inherent to conventional agriculture(13, 19, 64). Therefore, the analysis conducted by CTNBio intended to assess whether the impact caused by Roundup Ready Cotton Event MON 1445 is significantly higher than the one caused by conventional cotton varieties considering the practices associated to each system.
The majority of mammals, both wild and domesticated, avoid consuming cotton due to the presence of gossypol and other components of the plant tissue. For the same reason, the use of cotton kernels is also very limited in cattle raising. In addition, the cotton kern in the fields is covered with cotton plume, which makes it little attractive to birds. In both cases, it is little likely that wild animals feed on significant amounts of kernels or other parts of the cotton plant. In cattle raising, exposure to recombinant proteins shall be necessarily limited. The cotton seed is not used in fish farming. It is not expected that the cotton plants and pollen reach rivers and other water collections in amounts sufficient to cause any concern: in the management of this culture, retention of irrigation waters in fields is vital and drainage of rainwater to nearing rivers and rivulets shall be minimized for environmental reasons (carrying of herbicides and other agricultural pesticides to water collections). For the foregoing it is little likely that fish come to be regularly exposed to significant amounts of the recombinant proteins of Roundup Ready Cotton Event MON 1445, both in nature and in animal farms in dams or cisterns. Besides, protein EPSPS is deemed ubiquitous for its presence in all plants and large variety of microorganisms. For this reason, all living beings that feed on plants and microorganisms are already exposed to EPSPS proteins.
Invertebrates may enter in contact with recombinant proteins either by directly consuming the cotton plant or by predating insects that have fed on cotton. Exposure will be higher for those that feed directly on leaves and other cotton tissues. Pollinators and pollen consumers may be exposed to small amounts of recombinant proteins, since the expression of such proteins in pollen in much lower than in other plant tissues, a fact that is widely documented in the proceedings.
Since recombinant proteins are consumed, there seems to be no effect on insects: although there are not studies published on this matter, Roundup Ready Cotton Event MON 1445 was tested in both the U.S.A. and Brazil for susceptibility to different agricultural pests and there was no difference in susceptibility between the transgenic and the kindred lineage. One must keep in mind that protein EPSPS is present in all plants and in bacteria, algae and fungi, and that the structural or enzymatic difference between CP4 EPSPS and natural EPSPS is negligible. Protein NPTII, in turn, has no property that may distinguish it from the same enzyme in microorganisms(87). The two transgenic proteins are in nature, widely distributed among the microorganisms from where they derive. Thus, the expression of such proteins in cotton plants shall not have any toxic effect in invertebrates.
Though production of antibiotics by non-pathogenic soil bacteria has been associated to reduction of certain diseases in plants, there is no evidence of the involvement of either neomycin or kanamycin. Moreover, the antibiotics are not used in agriculture for controlling diseases originated in the soil. For this, it is unlikely that the presence of protein NPTII in the soil could cause any impact in the microbian population of such soil. In addition, the expression of such protein in different transgenic cultivars such as cotton, canola, maize and tomato is unrelated to the increase of plant diseases.
Horizontal gene transfer is a possible event, though extremely rare to occur between a eukaryote and a prokaryote, or even between two eukaryotes when between them there is no evolutionary proximity. Horizontal transfer may occur only among microorganisms and, even in this case, in very low rates. Microorganisms that, through horizontal transfer gained a copy of the cp4-epsps gene would be able to synthesize aromatic amino acids and other aromatic compounds, even in the presence of glyphosate. However, the ability to degrade glyphosate is widely distributed in the microbian community and therefore this new character would not contribute towards the maintenance of the transferred gene. As mentioned above, the soil microbian community does not seem to be affected by cultures expressing the cp4-epsps gene. Moreover, it must be emphasized that the epsps gene is ubiquitous in nature and the microbian flora is already glyphosate-tolerant.
Similarly, microorganisms that receive the ntp-II gene could become resistant to neomycin and other related antibiotics. However, as discussed above, there would not be selective pressure on such microorganisms, since the antibiotics are not employed in commercial cultivars. Also, there are no reports of change in the microbian soil flora associated to the expression of such genes. Transfer of other construct elements to the microbian flora, such as the CaMV promoter, could promote some effect, though none of this seems to happen. Recently, Lo et. al. (59) show that, in fact, this is unlikely for the npt-II gene in the soil of transgenic papaya cultivars.
The likelihood of gene transfer between phylogenetically unrelated higher organisms is extremely remote and the transfer from a plant to an animal is even more unlikely. For the cp4-epsps gene, the gain of herbicide tolerance would not grant any advantage or disadvantage to the animal, either a vertebrate or otherwise. The same occurs with gene npt-ll. The transfer of regulatory sequences could bring unpredictable effects, but such sequences are present in a vast range of foods (as the epsps gene).
The cotton plant (G. hirsutum) has not shown itself with any invading potential in Brazil, as well as in other parts of the world where it is regularly cultivated in large scale. At the side of highways where corn kernels are transported together with other cotton products, plants may appear that establish themselves only in places where there is, either naturally or otherwise, accumulated humidity. There is not observable, however, any advance of such plants towards other areas. Besides, within the Gossypium genus, there are no records of potential as an invading plant, except for the G. tomentosum, that does not exist in Brazil(25, 36).
Some authors suggest that the genic flow of GM plants to wild genotypes may result in reduction of biodiversity. However, reduction of genetic variability results from the phenomenon of genetic introgression, a process well more complex than simple hybridization(19, 23, 41, 80). Introgression of a gene to wild cotton plants could only take place in case it granted a strong competitive advantage, higher that the disadvantages granted by the alleles genetically bound to the transgene(38, 50, 80). However, the characteristic of tolerance to herbicide is recognized as not being capable to grant the receiving genotypes with any adaptive advantage outside agricultural areas(20, 80), since outside these areas potentially receiving wild genotypes are not under the selective pressure of the herbicide and, therefore, any pollination of the genotypes would not result in genic introgression. This way, transfer of the herbicide-tolerance characteristic of is extremely unlikely outside agricultural areas(20, 80).
One issue raised about herbicide-tolerant (HT) genetically modified plants is the possibility of crossing such GM plants with pests and consequent generation of invading HT plants with higher adaptability(28, 38). However, appearance of the so called “super pests” requires hybridization of a GM plant with an invading species and selective pressure (use of herbicide) in the physical area of the hybrid(80). Experimental data available from regions with large scale farming of genetically modified herbicide-tolerant plants confirm that the development of resistance to herbicides by pest plants is not related to genetic modification but to the management of cultures and herbicides used by farmers(73). Moreover, there are not in Brazil species sexually compatible with G. hirsutum with characteristics of invading plants. Thus, a conclusion is reached that it is extremely unlikely that transgene cp4 epsps of Event MON 1445 is transferred to pest plants making them more invasive. Customary care with management of cultures and herbicides, such as culture rotation and rotation of herbicides with different action mechanisms shall be the focus to mitigate the appearance of herbicide-tolerant pest plants.
Phenotypical assessment and evaluations of Roundup Ready Cotton Event MON 1445, cultivar DP50RR, and assessments of efficacy and tolerance to glyphosate were conducted in Brazil. The assessments were conducted in field studies on locations typical for cotton cultivation, simulating the Brazilian commercial production during the 1999/2000 and 2002/2003 crops.
Phenotype and agronomic performance of Roundup Ready Cotton Event MON 1445 cultivar DP50RR during the 2999/2000 crop were conducted in three place (Ituverava, SP; Rondonópolis, MT; and Capinópolis, MG). The experiment followed conventional agronomic practices, typically used in assessment of new cultivars, and assessed the following parameters: number of knots, height, position in the first branch, bolls per linear meter, susceptibility to diseases (Colletotrichum sp.; Ramularia sp.; Blue Disease; Red disease; Alternalia sp.) bolls per linear meter and productivity. There were no significant differences found in what regards phenotypic and morphologic aspects and agronomic performance between DP50 cultivars (conventional) and DP50RRR cultivars (Roundup Ready Cotton Event MON 1445) for the assessed parameters.
During the 2002/2003 crop, phenotypic, morphologic and agronomic parameters were again assessed and expanded. Experiments were conducted in two locations (Santa Cruz das Palmeiras, SP; and Santa Helena de Goiás, GO). The experiments compared the development of Roundup Ready Cotton Event MON 1445, cultivar DP50RR, the DP50 conventional cotton and eight commercial cultivars, four in each location. Plots planted with Roundup Ready Cotton Event MON 1445, cultivar DP50RR were treated with 1.5 kg/ha of the Roundup herbicide (formulation MON 14445) in foliar application in the V4 stadium and complemented with 1.0 kg/ha with application directed between rows of the same formulation 20 days after the first application. For the remaining cultivars, all plots were equally treated with disease and insect controlling fertilizers and agrochemicals, using the same products and doses and following traditional agronomic practices, typically used in each region. Comparisons between genetically modified cotton and conventional varieties were conducted during the crop for the following parameters: plant vigor, flowering cycle, plant height, maturation precocity, cycle up to harvesting, retention of plume by the boll after dehiscence, boll weight, average 100 seeds weight, percentage of fibers, susceptibility to diseases and plagues (Colletotrichum sp.; Ramularia sp.; Blue Disease; Red disease; Alternalia sp.), productivity and quality of fiber. There were found no significant differences between the genetically modified cultivar, with the use of glyphosate or otherwise, and its recurrent kindred conventional in the phenotypic, morphologic and agronomic parameters assessed.
Genes gox and epsps are under the control of the P-FMV Scrophularia mosaic virus(70) promoter, while gene npt-ll is under the control of RNA 35S of the cauliflower mosaic virus (CaMV)(67). The two promoters are functional in higher plants and, therefore, one would expect the presence of proteins C4 EPSPS, NPTII and GOX in plants transformed with the construct. Indeed, proteins C4 EPSPS and NPTII were detected by ELISA in several plant parts (0.02% and 0.028% of seeds total proteins; 7 to 170 ng of EPSPS by mg of fresh foliar tissue) in cultivated field conditions of the U.S.A., Spain and Brazil.
The presence of weeds reduces productivity and depreciation of the cotton produced(18) and may cause losses from 68 to 95%(24), and therefore the weed management is very important. The main invading plants in the cotton culture in Brazil are: Mossman River burr (Cenchrus equinatus), alexandergrass (Brachiaria plantaginea), Jamaican crabgrass (Digitaria horizontalis), Coast cross grass (Cynodon dactilon), hairy beggarsticks (Bidens pilosa), hispid starbur (Acanthospermum hispidum) and moonvine (ipomea sp.). Management of weeds is conducted by cultural methods, either mechanical or chemical, and control is achieved by the use of herbicides, the main controlling method. Chemical control is the most used in the country, mainly in large areas. Herbicides are applied before sowing, and in pre- and post-emergence.
Glyphosate is a post-emergence herbicide, belonging to the replaced glycines chemical group, classified as non-selective and having systemic action. It displays a large spectrum of action, enabling an excellent control of both large and small leaf annual and perennial weeds. The herbicide is duly registered with the Ministry of Agriculture and Supply – MARA for agricultural purposes and with the Brazilian Institute of Environment and Renewable Natural Resources - IBAMA, of the Brazilian Ministry of the Environment for non-agricultural purposes, in addition to possessing a monograph approved by the Brazilian National Sanitary Surveillance Agency – ANVISA(4). The glyphosate acts as a potent activity inhibitor of the 5-Enolpiruvylshikimate-3-Phosphate synthase (EPSPS), a catalyzer of one of the synthesis reactions of the aromatic amino acids phenylalanine, tyrosine and triptophan, and influences other processes, such as inhibition of chlorophyll synthesis, stimulates the production of ethylene, reduces the synthesis of proteins and increases concentration of indoleacetic acid. Some molecules of other herbicides, registered and used in cotton farming, have a residual action in soil and may cause phytotoxicity to cotton and other subsequent cultures, in addition to impacts to the environment.
For the foregoing, a conclusion is reached that the assessment of phenotypic and agronomic characteristics of the Roundup Ready Cotton Event MON 1445, cultivar DP50RR, conducted in Brazil reach results that are similar to those found in other regions of the world in experimental and commercial farming. With the exception of glyphosate-tolerance, resulting from expression of the cp4 epsps gene, Roundup Ready Cotton Event MON 1445 displays phenotypic and agronomic characteristics that are equivalent to the standard of conventional kindred lineages and commercial cultivars of conventional cotton.

VI. Restrictions to the Use of the GMO and its Derivatives
Technical opinions related to agronomic performance reached a conclusion that there is equivalence between transgenic and conventional plants. This way the information indicates that transgenic plants are not fundamentally different from the non-transformed cotton genotypes, except for the tolerance to glyphosate. In addition there is no evidence of adverse reactions to the use of Roundup Ready Cotton Event MON 1445. For the above reasons, there is no restriction to the use of this cotton and derivatives in human and animal food.
As established by Article 11 of Law no. 11,460, of March 21, 2007 “research and cultivation of genetically modified organisms may not be conducted in indigenous lands and areas of conservation units.”
VII. Consideration on the Particulars of Different Regions of the Country (Information to supervisory agencies)
The Roundup Ready Event MON 1445 technology revealed to be usable under all agricultural practices commonly used in different regions and conditions, including availability of inputs and labor, among others, used in the culture of cotton. In addition, the technology may enable greater success in the use of direct planting.
Studies reached a conclusion that the use of genetically modified varieties for the selective use of glyphosate does not restrict any procedure in cotton farming. There are not creole varieties of cotton plants and the chains of special cotton plants, both conventional and transgenic, have lived together in a satisfactory fashion, without any record of coexistence problems.

VII. Conclusion
Long experience with traditional plant improvement techniques, over three decades of experience in research and over one decade of marketing transgenic varieties over the world, in addition to knowledge advancements in the structure and dynamics of genomes, indicating whether a certain gene or characteristic is safe, give a sign that the genetic engineering process, by its own, leaves little room for appearance of unexpected consequences that would not be identified or eliminated during the process of development of commercial GM varieties(14).
Whereas Roundup Ready Cotton Event MON 1445 belongs to a well characterized species (Gossypium hirsutum) with a solid background of safety for human use and that the cp4 epsps gene introduced in this variety does not codify a toxic protein, being harmless to humans.
Whereas the genic construct used to insert the gene in cotton resulted in a stable insertion of a functional copy of the cp4 epsps gene, granting the plants tolerance to glyphosate.

Whereas:
1. Roundup Ready Cotton Event MON 1445 displays genetic stability in different environmental conditions determined in a number of generations of lineages obtained by retrocrossing with elite cultivars, including a variety adapted for cultivation in Brazil.
2. Proteins CP4 EPSPS and NPTII were expressed in different tissues and development phases of Roundup Ready Cotton Event MON 1445 in extremely low levels.
3. Security assessments of Roundup Ready Cotton Event MON 1445 and its products ensured that the genetically modified variety is safe for both the environment and use as food or food component for animals when compared to the risk posed by conventional cotton.
4. The new proteins expressed in the plants, CP4 EPSPS and NPTII revealed to be safe in all aspects studied.
5. Protein CP4 EPSPS is rapidly degraded in the gastrointestinal system of humans and animals for being a labile enzyme; and, in addition, its way of action, specificity and absence of homology with toxic sequences make this protein not risky to human health and to the environment.
6. Protein NPTII was assessed by the FDA in 1994 and was found safe for use in food. It is rapidly degraded in the human gastrointestinal system and its mode of action, specificity and absence of homology with toxic sequences lead to the conclusion that the protein is not a toxic risk.
7. Potential allergenicity assessment studies verified that proteins CP4 EPSPS and NPTII are not detectable in cotton products used as human and animal food and do not pose significant risk as allergenic and are not derived from allergenic forms.
8. Phenotypic and agronomic characteristic studies of Roundup Ready Cotton Event MON 1445 cultivar DP50RR conducted in Brazil have reached results comparable with experimental and commercial farming studies conducted in other regions of the world.
9. Field and farm experiments conducted with a number of generations and derivatives of Roundup Ready Cotton Event MON 1445 since 1993 show a stable phenotype of glyphosate tolerance occurring as a monogenic characteristic of high inheritability that is little affected by the environment in its expression.
10. Studies with animals show that a paste made of Roundup Ready Cotton Event MON 1445 kernels is as safe and nutritive as a paste of conventional cotton kernel.
11. It is little likely that wild animals feed on significant amounts of seeds or other parts of the cotton plant.
12. Protein EPSPS is present in all plants and also in bacteria, algae and fungi, being therefore widely distributed in nature. For this reason, all living beings that feed on plants and microorganisms are already exposed to EPSPS proteins.
13. The antibiotics neomycin and kanamycin are not used in agriculture to control plant diseases originated from the soil. Therefore, it is not likely that the presence of the NPTII protein in the soil may affect the microbial population of the soil.
14. The transfer of the herbicide-tolerant characteristic to outside agriculture areas is extremely unlikely.
15. It is unlikely that the MON 1445 event transgene cp4 epsps be transferred to weeds, making them more invasive. The customary care with management of culture and herbicides, such as rotation of cultures and rotation of herbicides with different action mechanisms shall be the focus to mitigate the appearance of herbicide-tolerant plants.
16. Data submitted, in addition to those existing in the literature, show that the impacts generated by the release in commercial scale to the field of the cotton event MON 1445 are similar to those presented by its kindred and other non-GM varieties currently cultivated in Brazil.
17. Event MON 1445 has already been approved by several countries, including U.S.A., Canada, Japan, Australia, Mexico, South Africa, Argentina, China, European Union, Philippines, Colombia and South Korea and has been cultivated in some of these countries without negative effects to human and animal health and to the environment.
Therefore, commercial release of Roundup Ready Cotton Event MON 1445 is not a potential cause of harm to human and animal health nor of significant degradation to the environment.
The CTNBio analysis considered the opinions issued by the Commission members; ad hoc consultants; documents delivered by the applicant to the CTNBio Executive Secretariat; results of planned releases to the environment; lectures, texts and discussions in public hearings held on 08.17.2007. Independent third party scientific studies and publications were also considered.

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14. SABHARWAL N.; ICOZ I.; SAXENA D.; STOTZKY G. 2007. Release of the recombinant proteins, human serum albumin, beta-glucuronidase, glycoprotein B from human cytomegalovirus, and green fluorescent protein, in root exudates from transgenic tobacco and their effects on microbes and enzymatic activities in soil. Plant Physiol Biochem. 45: 464-469.
15. SANCHES JR J.L.B.; MALERBO-SOUZA D.T. 2004. Freqüência dos insetos e produção de algodão. Acta Scient. Agron. 26: 461-465.
16. SAXENA D.; FLORES S.; STOTZKY G. 1999. Insecticidal toxin in root exudates from Bt corn. Nature 1999, 402: 480.
17. SHAN G.; EMBREY S.K.; HERMAN R.A.; MCCORMICK R. 2008. Cry1F protein not detected in soil after three years of transgenic Bt corn (1507 corn) use. Environ. Entomol. 37: 255-262.
18. UMBECK P.F.; BARTN K.A.; NORDHEIM E.V.; MCCARTY J.C.; PARROTT W.L.; JENKINS J.N. 1991. Degree of pollen dispersal by insects from a field test of genetically engineered cotton. J. Econ. Entomol. 84: 1943-1950.
19. WENDEL J.F.; ROWLEY R.; STEWART J.M. 1994. Genetic diversity in and phylogenetic-relationships of the Brazilian endemic cotton, Gossypium mustelinum (Malvaceae). Plant Syst Evol 192: 49-59.

Walter Colli
President of CTNBio

Dissenting Votes:
CTNBio member Doctor José Maria Gusman Ferraz (Permanent Sector Subcommission for the Environment) voted against the commercial release of Roundup Ready Cotton Event MON 1445. CTNBio members Doctors Graziela Almeida Silva (Permanent Sector Subcommission for Human Health), Kenny Bonfim (Permanent Sector Subcommission for Human Health), and Leonardo Melgarejo (Permanent Sector Subcommission for the Environment) abstained from voting in the matter of commercial release of Roundup Ready Cotton Event MON 1445.
Doctor Paulo Yoshio Kageyama (Permanent Sector Subcommission for the Environment) issued an opinion against the approval of the product based on:
1. The applicant failed to clearly demonstrate that glyphosate-based herbicides have properties favorable to the environment, do not move to ground waters, and possess low ecotoxicity and absence of residual effects to the soil.
2. A study of environmental impact, conducted under Brazilian legislation, would be necessary for applicant to reach a conclusion that “the use of Roundup Ready Cotton Event MON 1445 is safe and benefits the farmer and there is no evidence that it is a potential cause of harm to human and animal health nor of significant degradation to the environment.”
3. Absence of studies in the report of the application for commercial release does not enable assessing whether direct toxicity of glyphosate penetrating plant tissues on non-mammal organisms that feed on such cotton, when the product comes to be used sequentially several times during the year.
4. New studies shall be required of environmental impact using some key species, indicator species, of different orders of the animal and plant classification to enable an a posteriori qualitative and quantitative surveillance of adverse effects along time.
5. The process did not consider the presence of plant pests resistant to the glyphosate herbicide.
6. The visitation distance to cotton flowers by Bombus sp. bees was of 1,750 m from the nest. Therefore, the applicant shall consider the long flight distance of pollinating bumble bees.
7. Applicant shall consider the results of the studies by Sanches Jr. and Malerbo-Souza, giving emphasis to pollination by bumble bees.
8. Regarding effects of Roundup Ready Cotton Event MON 1445 on non-target organisms, the applicant states that “ecotoxicological risk assessment of ROUNDUP considers the direct effects of the herbicide and surfactant on non-target organisms and the environment”. In a later version, applicant submits data based on studies conducted outside Brazil and also do not consider the soil biota.
9. The studies mentioned do not answer adequately the issue of pollen dislocation and the question “what factors may affect the likelihood of genomic flow intra and inter-specific in different Brazilian regions and biomes?”
10. The risk of dispersing the transgene with the wild flora and fauna shall not be disregarded, since the herbicide-tolerant gene may grant a selective advantage to the host organism in agrosystems managed with Herbicide Tolerant Technology.
11. The lack of studies in the report of the application for commercial release does not enable assessing the direct toxicity of glyphosate penetrating plant tissues on non-target organisms that feed on such plants.
12. Genetically modified cotton is different from the non-genetically modified cotton.
13. The proceedings fail to show experimental results proving that genetically modified cotton has a yield higher than conventional cotton, as suggested in the initial part.
14. It is indispensable that the applicant conduct experiments in successive cultivars to assess the consumption evolution of the pesticide that is part of the Roundup Ready Cotton Event MON 1445 technology, since the experience with Roundup Ready Soybeans became known only after its commercial release.
15. Commercial release of herbicide-resistant transgenic plants accelerate the appearance of plants that are resistant to such pesticides.
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