SEA URCHINS (BULU BABI)

By. Muhammad Fahri*
* Mahasiswa Perikanan

Bulu babi atau urchin adalah binatang kecil, berbentuk bulat, bertulang belakang, yang merupakan bagian dari kelas Echinoidea. Bulu babi ditemukan seluruh samudra di dunia. Kulit atau "Test", mebentuk putaran dan bertulang belakang, secara khas dari 3 sampai 10 cm berhadapan. Warna umum hitam dan paduan dari hijau olive,, zaitun, coklat, ungu, dan merah. Pergerakkan pelan, Makanan kebanyakan dari ganggang. Berang-Berang Laut, Ikan belut Serigala, dan pemangsa lain merupakan predator bulu babi. Bulu babi juga dipanen oleh manusia dan rusa kecil sebagai makan yang lezat.

Bulu babi adalah anggota phylum Echinodermata, meliputi bintang laut, ketimun laut, bintang rapuh, dan crinoids. Seperti echinoderms lain bulu babi mempunyai bentuk lima simetri (disebut pentamerisme) dan pergerakkan denga pertolongan ratusan tiny kecil, transparan, melekat " kaki tabung ". Pentamerous simetri tidak jelas nyata pada peristiwa kebetulan tetapi mudah dilihat di kulit bulu babi kering atau test.
Bersama dengan ketimun laut (Holothuroidea), menyusun subphylum Echinozoa, yang digambarkan terutama mempunyai bentuk globoid tanpa lengan atau memproyeksikan sinar. Ketimun Laut dan echinoids irregular mempunyai perubahan bentuk berbeda. Walaupun banyak ketimun laut mempunyai lengan bercabang disekeliling pembukaan mulutnya, ini berasal dari modifikasi kaki tabung dan tidak sama dengan lengan dari crinoids, bintang laut dan bintang rapuh.

Anatomi Dan Fisiologi

Pada mulanya, bulu babi sering terlihat sessile (diam), yaitu. tidak mampu untuk yang bergerak. Kadang-kadang tanda kehidupan yang paling menyolok adalah tulang belakang, yang dipasang pada dasar ke sendi peluru dan dapat menunjuk ke segala arah.

Kebanyakan bulu babi, sentuhan cahaya menimbulkan gerakan dan reaksi yang dapat terlihat dari tulang belakang, yang memusat ke arah titik yang disentuh. Bulu babi tidak punya mata yang dapat melihat, kaki, atau alat bergerak, tetapi dapat bergerak dengan bebas di atas permukaan atas pertolongan pelekatan kaki tabungnya, bekerja bersama dengan tulang belakang.

Diatas permukaan mulut bulu babi yaitu lokasi pertengahan mulut dapat menyusun lima gigi kalsium karbonat yang dipersatukan atau jaws, dengan struktur seperti lidah di dalam. Keseluruhan organ pengunyahan dikenal sebagai Lentera Aristotle's, nama yang berasal dari deskripsi akurat Aristotle's dalam Story of Animals nya:

… bulu babi mempunyai apa yang disebut kepala dan mulut menurun, and ditempatkan untuk isu residu atas di atas. Bulu babi juga mempunyai, lima gigi berongga di dalam, dan pada pertengahan gigi ini terdapat unsur gemuk yang melayani lidah. Kemudian kerongkongan, dan kemudian perut, dibagi menjadi lima bagian, dan terisi dengan kotoran ekskresi, semua lima bagian dipersatukan pada lubang anal, di mana kulit dilubangi untuk suatu saluran pembuangan... Pada kenyataannya anggota mulut bulu babi berlanjut dari suatu akhir ke lain, tetapi pada penampilan luar tidak demikian, tetapi kelihatan seperti suatu lentera tanduk dengan kaca tanduk dihilangkan. (Tr. D'Arcy Thompson)

Bulu babi membangun spicules, dari kristal yang tajam "tulang" itu yang mendasari endoskeleton pada fase larval. Spicule secara penuh dibentuk terdiri atas kristal tunggal dengan marfologi tidak biasa. tidak punya segi dan di dalam 48 jam dari asumsi pembuahan menjadi bentuk yang terlihat sangat mirip dengan Logo Mercedes-Benz.

Tulang belakang, dalam beberapa jenis adalah panjang dan tajam, berfungsi untuk melindungi bulu babi dari pemangsa. Tulang belakang dapat menimbulkan luka menyakitkan pada manusia jika terinjak diatasnya, tetapi tidak berbahaya serius, dan tidak dijelaskan bahwa tulang belakang sangat beracun (tidak sama dengan pedicellariae antara tulang belakang, yang beracun).

Bulu babi secara khas mempunyai tulang belakang yang panjangnya antara 1 sampai 3 cm, tebal 1 sampai 2 mm, dan tidak tajam. Diadema Antillarum, umum dikenal dengan Caribbean, mempunyai tulang belakang tipis, berpotensi berbahaya yang dapat mencapai panjang 10 sampai 30 cm. Beberapa tulang belakang bulu babi adalah beracun.

Ekologi

Makanan Bulu babi sebagian besar adalah ganggang, tetapi dapat juga hidup dengan cakupan luas dari hewan tak bertulang punggung seperti kupang, spons / bunga-karang, bintang rapuh dan crinoids. (Baumiller, Tomasz K. 2008) Bulu babi adalah salah satu dari makanan favorit berang-berang laut dan juga sumber nutrisi utama untuk ikan belut serigala. Jika tidak dikendalikan, bulu babi akan merusak lingkungan yang diciptakan oleh ahli biologi yang disebutsea urchin barren, tanpa macroalgae dan asosiasi fauna. Di mana berang-berang laut direintrodusikan di British Columbia, kesehatan ecosystem pantai meningkatkan secara dramatis

Bulu babi adalah salah satu dari model organisme tradisional dalam pengembangan biologi. Penggunaan bulu babi didalam konteks ini memulai dari era 1800an, ketika pengembangan embrio dari bulu babi mudah diamati dengan mikroskop. Bulu babi merupakan jenis pertama di mana sel sperma terbukti berperan penting didalam reproduksi oleh pembuahan sel telur.

Dengan peruntunan (sequencing) genome terbaru dari bulu babi, homology telah ditemukan antara bulu babi dan gen sistem kekebalan pada hewan bertulang belakang. Kode Bulu babi sedikitnya 222 gen Toll-like receptor (TLR) dan lebih dari 200 gen berhubungan dengan Nod-Like-Receptor (NLR) yang ditemukan pada keluarga vertebrates (Rast, JP et al. 2006). Ini membuat bulu babi menjadi organisme model berharga untuk para ahli immunologis untuk mempelajari evolusi dari imunitas bawaan.

Anatomi dewasa: Bulu babi dewasa mempunyai lima sisi simetri radial. Kulit keras, plat berkapur, disebut test. Bulu babi mempunyai badan berbentuk bulat dan tulang belakang yang panjang menyebar dari badan. Tulang belakang digunakan untuk perlindungan, untuk bergerak, dan untuk mengapung mengerat ganggang untuk makanan. Di antara tulang belakang terdapat lima dipasang baris dari tabung kecil kaki dengan pengisap yang membantu penggerakan, menangkap makanan, dan menempel pada dasar laut. Pedicellarines kecil struktur untuk menyengat yang digunakan untuk pertahanan dan untuk memperoleh makanan. Seperti semua echinoderms, bulu babi tidak mempunyai otak. Mulut seperti kuku dan terletak pada bagian bawah mempunyai 5 plat seperti gigi yang menunjuk ke arah dalam disebut Lentera Aristotle's. Anus dan pori-pori genital bulu babi berada di atas.

Diet: Bulu babi makan tumbuhan dan binatang, mencakup tumbuhan laut yang besar, bahan hasil pembusukan, ganggang, ikan mati, bunga-karang, kupang, dan teriti).

Pemangsa : Bulu babi dimangsa oleh ketam, bintang laut, keong, berang-berang laut, beberapa burung-burung, pemakan ikan (teramsuk ikan belut serigala), dan manusia.

Reproduksi: Pembuahan eksternal. Bulu Babi betina melepaskan beberapa juta telor kecil yang dibungkus cairan gel pada waktu yang sama. Telor atau sperma dilepaskan melalui lima gonopores. Perkembanga, larva kecil (disebut pluteus, mempunyai bi-lateral simetri) berenang di laut dan sperti komponen zooplankton. Setelah beberapa bulan bulu babi muda terbentuk. Waktu dari pembuahan sampai usia dewasa reproduktif adalah dari 2sampai 5 tahun.

Klasifikasi: Kerajaan Animalia (Binatang), Phylum Echinodermata (echinoderms), Kelas Echinoidea (Bulu Babi).

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Penyakit Tumor Dan Kanker

by. Muhammad Fahri*

Tumor atau kanker merupakan suatu penyakit sel yang ditandai dengan hilangnya fungsi kontrol sel terhadap regulasi daur sel maupun fungsi homeostatis sel pada organisme multiseluler. Dengan kegagalan tersebut, sel tidak dapat berproliferasi secara normal. Akibatnya, sel akan berproliferasi terus-menerus sehingga menimbulkan pertumbuhan jaringan yang abnormal (tidak terkendali).



Perkembangan tumor berupa invasi merupakan langkah awal untuk metastasis kanker yang meliputi motilitas sel, perlekatan permukaan dan aktivitas ekstraselular protease. Untuk invasi sel kanker memerlukan peningkatan migrasi, berbagai perubahan sitofisiologik yang meliputi hilangnya perlekatan sel-sel bersama dengan meningkatnya perlekatan sel-matrik, dan meningkatnya ekspresi dan aktivasi ekstraseluler protease untuk mendegradasi ekstraseluler matrik dan mengijinkan sel berinvasi dan metastasis. (Chen, P. N et al., 2006).

Sel-sel pada hati akan memperbanyak diri untuk menggantikan sel-sel yang rusak karena luka atau karena sudah tua. Seperti proses pembentukan sel lain di dalam tubuh, proses ini juga dikontrol oleh gen-gen tertentu dalam sel. Kanker hati berasal dari satu sel yang mengalami perubahan mekanisme kontrol dalam sel yang mengakibatkan pembelahan sel yang tidak terkontrol. Sel abnormal tersebut akan membentuk jutaan kopi, yang disebut klon. Mereka tidak dapat melakukan fungsi normal sel hati dan terus menerus memperbanyak diri. Sel-sel tidak normal ini akan membentuk tumor (Anonim, 2004).

Karakteristik Sel Kanker

Sel tumor ganas bersifat abnormal dalam berbagai cara di samping proliferasi yang berlebihan. Sel itu mungkin memiliki jumlah kromosom yang tidak biasa. Metabolismenya mungkin dikacaukan, dan sel itu berhenti berfungsi dengan cara konstruktif. Akibat perubahan abnormal pada permukaan sel, sel itu juga kehilangan pelekatan dengan sel di sebelahnya dan dengan matriks ekstraseluler, dan dapat menyebar ke jaringan di dekatnya. Sel kanker dapat juga berpisah dari tumor asli, dan memasuki pembuluh darah dan pembuluh limfa pada system peredaran tubuh, menyerang bagian tubuh lainnya dimana sel itu berproliferasi untuk membentuk tumor baru. Penyebaran sel kanker di luar dari tempat asalnya disebut metatesis. Jika tumor bermetatesis, pengobatan mungkin melibatkan radiasi energi tinggi dan kemoterapi dengan obat beracun yang sangat berbahaya bagi sel yang sedang aktif membelah.

Menurut Hanahan dan Weinberg (2000), sel tumor atau kanker memiliki karakteristik sebagai berikut :

Sel kanker mampu mencukupi kebutuhan sinyal pertumbuhannya sendiri. Sinyal pertumbuhan diperlukan agar sel dapat terus membelah. Berbeda dari sel normal, sel kanker dapat tetap dan terus tumbuh.

Tidak sensitif terhadap sinyal antipertumbuhan. Sel kanker tidak merespon adanya sinyal yang dapat menghentikan terjadinya pertumbuhan dan pembelahan sel. Dengan demikian, sel kanker dapat terus membelah.

Sel kanker mampu menghindar dari mekanisme apoptosis. Apoptosis merupakan program bunuh diri sel ketika sel tersebut mengalami kerusakan, baik struktural maupun fungsional, yang tidak dapat ditolerir lagi. Namun sel kanker dapat menghindar dari kematian dengan mengeblok jalur terjadinya apoptosis di dalam sel.

Sel kanker memiliki potensi tak terbatas untuk mengadakan replikasi.

Sel kanker mampu menginduksi angiogenesis untuk mencukupi kebutuhannya akan oksigen dan nutrisi. Pada tahap perkembangan tumor yang hiperproliferatif, sel-sel tumor akan mengekspresikan protein proangiogenik sehingga akan terbentuk cabang baru pada pembuluh darah yang menuju sel kanker yang kemudian akan mensuplai kebutuhan nutrisi dan oksigen dari sel kanker.

Sel kanker mampu menginvasi jaringan di sekitarnya dan membentuk anak sebar .

Sedangkan menurut Pecorino (2005) terdapat enam (6) karakter sel kanker (The six hallmark of cancer) adalah sebagai berikut ini :

Growth signal autonomy :
-Sel normal memerlukan sinyal eksternal untuk pertumbuhan dan pembelahannya
-Sel kanker mampu memproduksi growth factors dan growth factor receptors sendiri.
-Dalam proliferasinya sel kanker tidak tergantung pada sinyal pertumbuhan normal.
-Mutasi yang dimilikinya memungkinkan sel kanker untuk memperpendek Growth Factor pathways .

Evasion Growth inhibitory signals :
-Sel normal merespon sinyal penghambatan pertumbuhan untuk mencapai homeostasis. Jadi ada waktu tertentu bagi sel normal untuk proliferasi dan istirahat.
-Sel kanker tidak mengenal dan tidak merespon sinyal penghambatan pertumbuhan.
-Keadaan ini banyak disebabkan adanya mutasi pada beberapa gen (proto-onkogen) pada sel kanker.

Evasion of Apoptosis Signals :
-Sel normal akan dikurangi jumlahnya dengan mekanisme apoptosis, bila ada kerusakan DNA yang tidak bisa lagi direparasi.
-Sel kanker tidak peka terhadap sinyal apoptosis (padahal sel kanker membawa acumulative DNA error yang sifatnya irreversible)
-Kegagalan sel kanker dalam merespon sinyal apoptosis lebih disebabkan karena mutasinya gen-gen regulator apoptosis dan gen-gen sinyal apoptosis.

Unlimited replicative potential :
-Sel normal mengenal dan mampu menghentikan pembelahan selnya bila sudah mencapai jumlah tertentu dan mencapai pendewasaan. Pengitungan jumlah sel ini ditentukan oleh pemendekan telomere pada kromosom yang akan berlangsung setiap ada replikasi DNA.
-Sel kanker memiliki mekanisme tertentu untuk tetap menjaga telomere tetap panjang, hingga memungkinkan untuk tetap membelah diri.
-Kecacatan dalam regulasi pemendekan telomere inilah yang memungkinkan sel kanker memiliki unlimited replicative potential.

Angiogenesis (formation of blood vessels) :
-Sel normal memiliki ketergantungan terhadap pembuluh darah untuk mendapatkan suplay oksigen dan nutrient yang diperlukan untuk hidup. Namun, arsitektur pembuluh darah sel normal lebih seherhana atau konstan sampai dengan sel itu dewasa.
-Sel kanker mampu menginduksi angiogenesis, yaitu pertumbuhan pembuluh darah baru di sekitar jaringan kanker. Pembentukan pembuluh darah baru ini diperlukan untuk survival sel kanker dan ekspansi ke bagian lain dari tubuh (metastase).
-Kecacatan pada pengaturan keseimbangan induser angiogenik dan inhibitornya dapat mengaktifkan angiogenic switch.

Invasion and metastasis :
-Normal sel memiki kepatuhan untuk tidak berpindah ke lokasi lain di dalam tubuh.
-Perpindahan sel kanker dari lokasi primernya ke lokasi sekunder atau tertiernya merupakan faktor utama adanya kematian yang disebabkan karena kanker.
-Mutasi memungkinkan peningkatan aktivitas ensim-ensim yang terlibat invasi sel kanker (MMPs).
-Mutasi juga memungkinkan berkurangnya atau hilangnya adesi antar sel oleh molekul-molekul adisi sel, meningkatnya attachment, degragasi dan migrasi.

Menurut Vinay Kumar, et, al (2005), pada percobaan skala in vitro, perbedaan karakter antara sel normal dan kanker adalah sebagai berikut :

Sel normal akan tumbuh sebagai selapis sel (monolayer) jika dibiakan dalam petridish, semua ini karena adanya kepekaan terhadap contact inhibition dengan sel-sel tetangganya (bila sel normal sudah menyentuh sel tetangganya maka pertumbuhannya akan berhenti).

Transformed cells (sel kanker) memiliki fenotipe sebagai berikut :

-Tumbuh terus tanpa mengenal contact inhibitory signals, tumbuh menumpuk ke atas bukan sebgai monolayer.
-Dapat tumbuh dalam kondisi minim serum (serum / FBS (fetal bovine serum) berisi banyak Growth factor, sel kanker mampu memenuhi kebutuhan Growth factors sendiri.
-Morfologi sel kanker lebih membulat dengan inti yang relatif lebih besar (karena aktif membelah).

Beberapa obat antikanker yang telah dikembangkan saat ini antara lain berupa obat yang merangsang diferensiasi sel sehingga akan terjadi perubahan sifat dari sel kanker yang ganas menjadi sel jinak, obat yang dapat meningkatkan efektivitas radiasi dan obat yang mengubah respon imun sel kanker dengan sel sehat. Selain itu, telah banyak obat-obatan yang dikembangkan berdasarkan aktivitas molekuler dari sel kanker. Namun, obat-obatan tersebut mengalami permasalahan dalam hal resistensi dan toleransi obat serta selektivitas obat itu sendiri disamping dari berbagai efek samping yang dapat ditimbulkan .

NB. Dari banyak sumber.

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(Brine Shrimp Lethality Test)

by Muhammad Fahri.

Uji Toksisitas (Brine Shrimp Lethality Test)

Senyawa yang diduga memiliki aktifitas anti kanker, harus di ujikan terlebih dahulu pada hewan percobaan. Metode Brine Shrimp Lethality Test (BST) dengan menggunakan larva udang Artemia salina Leach sebagai hewan uji merupakan salah satu metode yang banyak digunakan untuk pencarian senyawa antikanker baru yang berasal dari tanaman. Hasil uji toksisitas dengan metode ini telah terbukti memiliki korelasi dengan daya sitotoksis senyawa anti kanker. Selain itu, metode ini juga mudah dikerjakan, murah, cepat dan cukup akurat (Meyer, 1982). Lebih dari itu uji larva udang ini juga digunakan untuk praskrining terhadap senyawa-senyawa yang diduga berkhasiat sebagai antitumor. Dengan kata lain, uji ini mempunyai korelasi yang positif dengan potensinya sebagai antikanker (Anderson, 1991).


Artemia salina Leach merupakan komponen dari invertebrata dari fauna pada ekosistem perairan laut. Udang renik ini mempunyai peranan yang penting dalam aliran energi dan rantai makanan. Spesies invertebrata ini umumnya digunakan sebagai organisme sentinel sejati berdasarkan pada penyebaran, fasilitas sampling, dan luasnya karakteristik ekologi dan sensifitasnya terhadap bahan kimia (Calleja M.C, Persoone G, 1992).
Pengujian Lethalitas telah digunakan dengan sukses untuk isolasi biomonitor dari cytotoxic (Siqueira, M. J et. al., 1998), antimalaria (Perez, H, et.al., 1997), insektisida (Oberlies, N. H.,et.al., 1998), dan antifeedent (Labbe, C., et.al., 1993) campuran dari ektrak tumbuhan. Hasil dari skrening dari air, hydroalcoholic dan ekstrak alkohol dari beberapa tumbuhan obat penting yang digunakan dalam pengobatan tradisional untuk lethalitas merujuk pada larva Artemia salina yang diperkenalkan.
Suatu konsentrasi mematikan (Lethal Concentration) adalah analisa secara statistik yang menggunakan uji Whole Effluent Toxicity (WET) untuk menaksir lethalitas sampel effluen. Test akut digunakan di Wisconsin untuk menaksir kondisi "akhir dari pipa" (yaitu, effluent yang tidak dilemahkan, sebagai adanya dibebaskan pada lingkungan).

Konsentrasi effluen dimana 50% dari organisme mati selama test (LC50) digunakan sebagai pemenuhan titik akhir (endpoint) untuk Test Whole Effluent Toxicity (WET) akut. Dalam rangka mengkalkulasi LC50, salah satu dari konsentrasi test harus menyebabkan > 50% kematian. LC50, yang lebih rendah berarti semakin beracun effluent tersebut. Sebagai contoh, LC50 > 100% berarti kekuatan penuh effluent tersebut tidak membunuh lebih dari separuh organisme. LC50 sama dengan 50% berarti separuh effluent mempunyai kekuatan membunuh 50% dari organisme tersebut.

Menurut Meyer dkk. (1982) tingkat toksisitas dari ekstrak tanaman dapat ditentukan dengan melihat harga LC50-nya. Apabila harga LC50 lebih kecil dari 1000 μg/ml dikatakan toksik, sebaliknya apabila harga LC50 lebih besar dari 1000 μg/ml dikatakan tidak toksik. Tingkat toksisitas tersebut akan memberi makna terhadap potensi aktivitasnya sebagai antitumor. Semakin kecil harga LC50 semakin toksik suatu senyawa.

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Alga Coklat Sargassum duplicatum

by. Muhammad Fahri*


Alga Sargassum merupakan salah satu marga Sargassum termasuk dalam kelas Phaeophyceae. Ada 150 jenis Marga Sargassum yang dijumpai di daerah perairan tropis, subtropis dan daerah bermusim dingin (Nizamuddin, 1970). Habitat alga Sargassum tumbuh diperairan pada kedalaman 0,5–10 m, ada arus dan ombak. Pertumbuhan alga ini sebagai makro alga bentik melekat pada substrat dasar perairan. Di daerah tubir tumbuh membentuk rumpun besar, panjang thalli utama mencapai 0,5-3 m dengan untaian cabang thalli terdapat kantong udara (bladder), selalu muncul di permukaan air.

Di Indonesia diperkirakan terdapat lebih dari 15 jenis alga Sargassum dan yang telah dikenal mencapai 12 jenis. Sedangkan di perairan Indo-Pasifik tercatat 58 jenis (Bosse, 1928). Alga Sargassum tumbuh sepanjang tahun, alga ini bersifat “perenial” atau setiap musim barat maupun timur dapat dijumpai di berbagai perairan.

Sargassum secara ekologis ikut andil dalam pembentukan ekosistem terumbu karang dan merupakan tempat asuhan bagi biota kecil, termasuk untuk perlindungan benih ikan dan benur udang serta sarang melekatnya telur cumi-cumi. Jenis Sargassum yang telah dipasarkan di daerah Jawa Barat dari jenis Sargassum polycystum, Sargassum binderi dan Sargassum duplicatum. Marga Sargassum mengandung bahan alginat dan iodin, bermanfaat sebagai bahan industri makanan, farmasi, kosmetik dan tekstil.

Morfologi dan Penyebaran S. duplicatum

Di dunia Sargassum spp. ada sekitar 400 spesies, sedangkan di Indonesia dikenal ada 12 jenis, yaitu : Sargassum duplicatum, S. hitrix, S. echinocarpum, S. gracilinum, S. obtuspfolium, S. binderi, S. polyceystum, S. microphylum, S. crassifolium, S. aquafolium, S. vulgare, dan S. polyceratium. Hormophysa di Indonesia dijumpai satu jenis yaitu H. tricuetra dan Turbinaria spp. ada 4 jenis yaitu T. conoides, T. conoides, T. ornata, T. murrayana dan T. deccurens (DirJen Perikanan Budidaya DKP RI, 2009).

Alga coklat Sargassum spp. termasuk tumbuhan kosmopolitan, tersebar hampir diseluruh perairan Indonesia. Penyebaran Sargassum spp. di alam sangat luas terutama di daerah rataan terumbu karang di semua wilayah perairan pantai.

Lingkungan tempat tumbuh alga Sargassum terutama di daerah perairan yang jernih yang mempunyai substrat dasar batu karang, karang mati, batuan vulkanik dan benda-benda yang bersifat massif yang berada di dasar perairan. Alga Sargassum tumbuh dari daerah intertidal, subtidal sampai daerah tubir dengan ombak besar dan arus deras. Kedalaman untuk pertumbuhan dari 0,5–10 m. Marga Sargassum termasuk dalam kelas Phaeophyceae tumbuh subur pada daerah tropis, suhu perairan 27,25 – 29,30 oC dan salinitas 32–33,5 %o. Kebutuhan intensitas cahaya matahari marga Sargassum lebih tinggi dari pada marga alga merah. Boney (1965) menyatakan pertumbuhan Sargassum membutuhkan intensitas cahaya matahari berkisar 6500–7500 lux. Alga Sargassum tumbuh berumpun dengan untaian cabang-cabang. Panjang thalli utama mencapai 1–3 m dan tiap-tiap percabangan terdapat gelembung udara berbentuk bulat yang disebut “Bladder,” berguna untuk menopang cabang-cabang thalli terapung ke arah permukaan air untuk mendapatkan intensitas cahaya matahari.

Taksonomi Sargassum duplicatum

Berdasarkan hirarki taksonomi Sargassum duplicatum menurut NODC Taxonomic Code, database (version 8.0) (1996) adalah sebagai berikut :
Kingdom : Plantae -- Planta, plantes, plants, Vegetal
Division : Phaeophyta -- algues brunes, brown algae
Class : Phaeophyceae
Order : Fucales
Family : Sargassaceae
Genus : Sargassum C. Agardh
Species : Sargassum duplicatum J. Ag.
Variety : Sargassum duplicatum duplicatum J. Ag.
Sumber : NODC Taxonomic Code, database (version 8.0)
Acquired : 1996

Kandungan S. duplicatum

Rumput laut mengandung komponen penting yang dibutuhkan dalam proses fisiologis hewan dan manusia. Rumput laut kaya akan karbohidrat, protein, lipid dan mineral dan tidak menyebabkan kerusakan pada paru-paru, ginjal, perut dan usus (Katheresan, 1992). Sehingga dapat digunakan sebagai suatu sumber potensi pangan fungsional.

Secara umum, rumput laut mempunyai kandungan nutrisi cukup lengkap. Secara kimia rumput laut terdiri dari air (27,8%), protein (5,4%), karbohidrat (33,3%), lemak (8,6%) serat kasar (3%) dan abu (22,25%). Selain karbohidrat, protein, lemak dan serat, rumput laut juga mengandung enzim, asam nukleat, asam amino, vitamin (A, B, C, D, E dan K) dan makro mineral seperti nitrogen, oksigen, kalsium dan selenium serta mikro mineral seperti zat besi, magnesium dan natrium. Kandungan asam amino, vitamin dan mineral rumput laut mencapai 10 -20 kali lipat dibandingkan dengan tanaman darat (Anonim, 2009).

Rumput laut telah banyak digunakan sebagai bahan pembuatan obat-obatan dan suplemen makanan serta difortifikasi ke produk pangan untuk meningkatkan nilai jual produk tersebut. Jenis rumput laut yang banyak digunakan untuk pembuatan obat adalah alga coklat khususnya Sargasum dan Turbinaria. Pengolahan rumput laut jenis tersebut menghasilkan ekstrak berupa senyawa natrium alginat. Senyawa alginat inilah yang dimanfaatkan dalam pembuatan obat antibakteri, anti tumor, penurunan darah tinggi dan mengatasi gangguan kelenjar.

Alga coklat ini adalah anggota Pheophyta, alga khas daerah tropik, mengandung pigmen klorofil a dan c, alfa dan beta karoten, alginat dan lain-lain. Alginat diduga dapat menurunkan kadar gula darah, kolesterol dan dapat mengabsorbsi logam berat dalam tubuh, atau dapat menyembuhkan penyakit degeneratif organ-organ tubuh, misalnya hepar, ren, dan cerebellum. Alga coklat dapat tumbuh subur disebagian besar pantai perairan laut Indonesia. Sargassum, terutama Sargassum duplicatum mengandung protein 2,97%, lemak 0,26%, zat anti tumor, algin, mineral (Ca, K, Na, Cu, Zn, Mg, I, S dan P, Fenol). Alga ini juga mengandung zat anti bakteri dan anti virus. Kandungan nutrien dan potensi zat-zat yang terdapat dalam alga laut, khususnya Sargassum belum banyak diteliti (Mulyo A.U, 2009)

Senyawa Aktif Antitumor dari S. duplicatum

Menurut Atmadja produk alam makroalga yang telah teruji aktivitas antikankernya yaitu polisakarida alga antara lain : polisakarida sulfat, sodium alginat fraksi G dan fraksi M, karagenan iota, karagenan kappa, karagenan lambda dan porphyran. Mortalitas Artemia pada larutan ekstrak E. alvarezii yang terlarut pada metanol dan kloroform, membuktikan adanya metabolisme sekunder yang bersifat polar dan nonpolar. Senyawa metabolit sekunder dari alga yang bersifat polar adalah flavonoid dan alkaloid, sedangkan senyawa yang bersifat nonpolar adalah terpenoid dan steroid (Sastrohamidjojo, 1985).

Alginat memiliki campuran struktural yang dominan dari dinding sel dan intercellular matriks pada rumput laut alga coklat (Kloareg & Quatrano, 1988). Secara luas digunakan dalam makanan, farmasi dan industri kosmetik, alginat juga dominan pada pemanfaatan industri dari rumput laut coklat. Produksi alginat secara global diperkirakan sebesar 27.000 ton per tahun, mendekati nilai sebesar US$ 230 juta (Indergaard & Ø Stgaard, 1991).

Alginat adalah garam dari asam alginic, suatu co-polymer linier dari b-1, asam 4-D-mannuronic dan a-1, asam 4-L-guluronic, dengan residu diorganisir dari kelompok asam polyguluronic dan polymannuronic, seperti sequens heteropolymeric dari asam guluronic dan mannuronic. Alginat dilaporkan dapat merangsang produksi cytokines, tumour necrosis factor-a, interleukin-1 dan interleukin-6 dari monocytes manusia (Otterlei et Al., 1991; Espevik et Al., 1993), dan tidak bisa dipisahkan dari aktivitas perlawanan antitumour terhadap model tumour murine secara in-vivo (Fujihara et al., 1984; Fujihara & Nagumo, 1992). Suntikan Intraperitoneal tunggal dari derivat alginat seperti alginate-DNM (daunomycin) pada tikus B16 menekan tumours subcutaneous yang dihasilkan kecil, tetapi secara signifikan menghalangi pertumbuhan tumor (Al-Shamkhani & Duncan, 1995).

Depolymerisasi secara biologi dari alginat adalah mengkatalisasi dengan alginate lyases (EC 4.2.2.3) melalui reaksi β-elimination, membelah rantai molekular dan menciptakan asam uronic yang tak terbungkus pada akhir tanpa pengurangan yang baru (Haugen et al., 1990). Produk akhir adalah campuran dari oligosaccharides pendek, yang menjadi bioaktif dan mempunyai aktivitas antitumour dan antivirus (Boyd & Turvey, 1978; Currie, 1983).

Adanya flavonoid dalam lingkungan sel, menyebabkan gugus OH- pada flavonoid berikatan dengan protein integral membran sel. Hal ini menyebabkan terbendungnya transpor aktif Na+ - K+. Transpor aktif yang berhenti menyebabkan pemasukan ion Na+ yang tidak terkendali ke dalam sel, hal ini menyebabkan pecahnya membran sel. (Scheuer, 1994). Pecahnya membran sel inilah yang menyebabkan kematian sel.

1. Flavonoids

Flavonoid merupakan salah satu golongan fenol alam terbesar yang terdapat dalam semua tumbuhan berpembuluh. Semua flavonoid, menurut strukturnya merupakan turunan senyawa induk flavon yang mempunyai sejumlah sifat yang sama. Dalam tumbuhan, aglikon flavonoid terdapat dalam berbagai bentuk struktur. Semuanya mengandung atom karbon dalam inti dasarnya yang tersusun dalam konfigurasi C6-C3-C6, yaitu dua cincin aromatik yang dihubungkan oleh satuan tiga karbon yang dapat atau tidak dapat membentuk cincin ketiga. Semua varian flavonoid saling berkaitan karena alur biosintesis yang sama, yang memasukkan pra zat dari alur sikimat dan alur asetat-malonat. Flavonoid dalam tumbuhan umumnya terikat sebagai glikosida, baik O-glikosida maupun C-glikosida (Markham. K.R., 1988).

2. Steroid dan Triterpenoid

Triterpenoid adalah senyawa yang kerangka karbonnya berasal dari enam satuan isoprena dan secara biosintesis dirumuskan dari hidrokarbon C30 asiklin, yaitu skualena. Senyawa ini berstruktur siklin dan nisbi rumit, kebanyakan berupa alcohol, aldehida atau asam karbohidrat. Senyawa ini tidak berwarna, berbentuk kristal, sering bertitik leleh tinggi dan aktif optik pada umumnya sukar dicirikan karena tak ada kereaktifan kimianya. Uji yang banyak digunakan adalah reaksi Lieberman-Burchard yang dengan kebanyakan triterpena dan sterol memberikan warna hijau-biru. Triterpena dapat dipilih menjadi sekurang-kurangnya empat golongan senyawa: triterpena sebenarnya, steroid, saiconon dan glikosida jantung. Kedua golongan terakhir sebenarnya triterpena atau seteroid yang terdapat sebagai glikosida (J.B. Harborne, 1987).

3. Alkaloids

Tidak ada istilah alkaloid yang memuaskan, tetapi umumnya alkaloid ini mencakup senyawa bersifat basa yang mengandung satu atau lebih atom nitrogen, biasanya dalam gabungan sebagai bagian dari sistem siklik. Alkaloid sering kali beracun bagi manusia dengan bahaya yang mempunyai aktivitas fisiologi yang menonjol sehingga digunakan secara luas dalam pengobatan. Alkaloid biasanya tak berwarna, seringkali bersifat aktif optik kebanyakan berbentuk kristal pada suhu kamar. Prazat alkaloid yang paling umum adalah asam amino, meskipun sebenarnya biosintesis kebanyakan asam amino lebih rumit. Secara kimia alkaloid merupakan suatu golongan heterogen. Banyak alkaloid bersifat terpenoid dan beberapa diantaranya dari segi biosintesis merupakan terpenoid termodifikasi alkaloid lain terutama berupa senyawa atomatik dengan gugus basa sebagai rantai samping (Harborne J.B, 1987).

NB. Dari Berbagai sumber.

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Methods Cell Culture

Muhammad Fahri*
Dari berbagai sumber bacaan.

Methods
Culture

The HeLa cell line (Scherer et al., 1953) and its derivates, were cultured at 37°C with 5% C02 in Dulbecco’s Modified Eagle's Medium (DMEM) containing 10% heat inactivated fetal calf serum (FCS), 2 mM L-glutamine, 50 μg/ml penicillin and 50 μg/ml streptomycin. Cultures at ~80% confluence were routinely split 1:5 in 10 cm culture dishes as follows. The cells were washed twice in prewarmed PBS. 1 ml PBS containing 0.25% (w/v) Trypsin was added to the dishes and placed at 37°C for 5-10 minutes. After the cells were detached from the dishes, 1 ml prewarmed culture medium was added and the cells transferred to a 50 ml falcon tube. Cells were spun down at 150 g and plated in new dishes with fresh culture medium.



Insects cells were cultured at 26°C in Grace’s medium (for Sf9 cells) or TMN-FH medium (for Hi-5 cells) containing 10% heat inactivated FCS, 2 mM L- glutamine, 50 μg/ml penicillin, 50 μg/ml streptomycin, 0.1% Pluronic F-68 and 1x Yeastolate. The Sf9 cells grew well as suspension culture and Hi-5 cells as loosely adhered cultures in Petri dishes. For Sf9 cells, 50 ml suspension cultures were maintained in 250 ml glass bottle, with shaking at 110 rpm in the 26°C incubator. For Hi-5 cells, 15 cm dishes are used to infect cells. Starting culture always had
the cell density of 0.5 million cells per ml. Cells were split twice in a week and care was taken for the cell density to never exceeded 3 million cells per ml. Human cell line transfection method Roti Fect reagent from Roth was used to make all transfections. Cells were grown to 60 – 80 % confluence. 10ug of qiagen purified DNA was used for a typical 10cm dish transfection. In tube A, 500ul of optimem (serum free medium) is added to DNA. In tube B, 500ul of optimem (serum free medium) is added to 30ul of Roti Fect reagent. Now contents of both tubes are mixed gently. The DNA – Lipid complex is allowed to form by incubating the transfection reaction for 30 minutes at room temperature. While complex is forming rinse the cells with PBS
and refill the dish with fresh serum free medium. Add DNA – Lipid complex on the cells and mix gently. Incubate for 4-6 hours. Remove the transfection medium and add complete medium to cells.

Generating stable cell lines

The HeLa cells were used to generate stable cell lines with constitutive expression of recombinant protein. A new technique of sorting sub cloning

Flow cytometry

Flow cytometry, or fluorescence-activated cell scanning or sorting (FACS analysis), is the measurement of characteristics of single cells suspended in a flowing saline stream when they flow past a series of detectors. The fundamental concept is the cells flow one at a time through a region of integration where multiple biophysical properties of each cell can be measured at rates of over 1000 cells per second. These biophysical properties are then correlated with
biological and biochemical properties of interest.

Cells subjected for FACS analysis in this study were either expressing fluorescent protein EGFP or stained with propidiumiodide (PI) as described. Cells grown on 6-well plates were harvested by centrifuging at 1200 g for 3 min as described, and then suspended in 400 μl of PBS with 0.1% EDTA in Falcon tube 2054. The cells were scanned on the FACSCalibur station (Becton Dickinson Inc.) using proper laser settings according to the suggested manufacture protocols. In order to keep the resolution as accurate as possible, the acquisition flow rate was controlled around 200 events per second by choosing proper scanning speed on the FACScalibur or diluting the cells with PBS. Dead cells and cell debris were excluded by gating the cells with a FSC threshold of 200. Doublet discrimination (DMM) to distinguish between clumped and mitotic cells was set for FL2. One negative cell line was included as autofluorescence control in each FACS analysis in order to adjust the instrumental setting properly for the positive fluorescent samples. The instrumental settings were kept consistent for all batches of samples during the time course. 10,000 cells were set as the defined scanning events for every sample. Data acquisition and control of the flow cytometer was performed with the CellQuest program (BD Biosciences, Heidelberg, Germany).

FACSorting in this study was set for collecting EGFP positive or negative cell populations. Cells grown in T25 flasks were harvested and counted. The cells were diluted with PBS at a concentration of 2X 10E6 cells per ml. The cell suspension was pipetted into 5 ml Falcon round bottom tubes (Falcon tube 35-2235) through cell-strainer cap to break up any clumped cells. The cells were sorted on FACSVantage (Becton Dickinson Inc.) and the subpopulations were
collected into 15 ml Falcon tubes with 3 ml complete medium.

Transfection of Sf9 cells with bacmid for baculoviruses preparation

Two ml of Sf9 cells with a density of 0.5 million cells per ml were put in each well of a 6-well plate. The cells were let to stand for one hour at RT. In the mean time a mixture 200 μl Grace’s media (without FCS), 15 μl DAC-30 (a kind gift from the Rezka lab, MDC, Berlin) and 10 μl Bacmid DNA (10 μg) were made and incubated at RT for 30 min. After 30 min 800 μl Grace’s media (without FCS) was added to this mixture and the mix was added to Sf9 cells in the six well plates. The Sf9 cells were washed once with Grace’s media (without FCS) before adding
the transfection mix. The cells were incubated with 1 ml transfection mix at 26°C for 4 hours. The transfection mix was removed and 2 ml of fresh Grace’s complete media was added to the culture. Cells were further grown for at 26°C for 72 hours.

After 72 hours the culture supernatant was collected and one ml of this supernatant (containing baculoviruses) was added to a 60 mm dish containing 5 ml culture volume (2.5 million cells), for further amplification of the baculovirus.

Recombinant DNA techniques

Conventional DNA preparation protocols

All standard methods like plasmid preparation, cloning methods, electrophoresis on agarose gels in TBE or TAE etc were done according to methods provided by Molecular cloning book (Sambrook and Russel, 2001) or according to the manufacturer’s instructions. Modifications of any of these protocols are indicated where necessary.

Elution of DNA from agarose gel pieces

A homemade electrophoresis device (‘salt trap’) was used for elution of DNA fragments from agarose gel pieces. The apparatus was filled with 0.5 X TBE and agarose gel pieces with DNA was placed in the slots. Any air bubbles in the passages of the apparatus were carefully removed. 80 μl of salt trap solution (3 M Sodium Acetate, 0.025% w/v Bromophenol blue dye, pH 7) was loaded in the salt trap passage. The apparatus was connected to power pack and run at constant 150 Volts for 30 minutes. Then, the blue colored salt trap solution was collected in 1.5 ml tubes and equal volume of isopropanol was added to the tube. The precipitated DNA was immediately centrifuged at 4°C, 20000 g for 20 min. The pellet was washed once in 70% ethanol dried and re-dissolved in desired volume of double distilled water.

Biochemical methods of protein purification

Cell lysis and nuclear salt extraction method

HeLa cells and insect cells were both lysed using the same protocol. Cells were harvested and washed twice in ice cold PBS. Cells were then either lysed immediately or snap frozen in freezing buffer (PBS, 2 mM MgCl2 and 10% glycerol). For lysis, ice-cold lysis buffer (PBS, 2 mM MgCl2, 0.1% NP40, 10% glycerol and 1 mM PMSF) was added to the cells. All further steps were done at 4°C. Cell were uniformly mixed and incubated for 5 min. The nuclei were pelleted by spinning the lysate at 1500 g for 4 min. The nuclei pellet was washed once in lysis buffer and salt extracted in lysis buffer with 300 mM KCl for 1 hour. The soluble proteins were collected by spinning at 20000 g for 20 min. The nuclear salt extract was used for ORC immunoprecipitations and HA affinity purification experiments.

A typical experiment with Sf9 cultures, used cells from 50 ml baculovirus infected cells (pre infection cell count of 0.5 million cells per ml). The cell pellet was lysed in 8 ml lyisis buffer. Isolated nuclei were extracted in 2 ml of lysis buffer with 300 mM salt.

SDS-gel electrophoresis and Immunoblotting

Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide gels used in this work were 1 mm thick, 8 cm wide and 9.2 cm long. The gels were poured in Hoefer mini VE Basic gel frames. The electrophoresis tank and upper chamber of the gel container were filled with 1x SDS running buffer (25 mM Tris-HCl, 192 mM glycin, 0.1% SDS). Proteins were separated through 10% or 12% polyacrylamide minigels, followed by coomassie staining, silver staining or immunoblotting for protein detection. The gels were electrophoresed at 150 V for first half an hour and then at 200 V till the bromophenol dye reached the bottom. Power was supplied from a BioRad PowerPac 400 (Pharmacia).

Transfer of proteins to PVDF membranes

Before use PVDF membranes (Immobilon P, Millipore) were treated as follows: membranes were placed in 100% methanol for 1 minute, transferred into 60% methanol. The membranes were soaked in 1x transfer buffer (25 mM Tris, 192 mM Glycin) for at least 5 minutes. Polyacrylamide gels containing separated proteins were placed on PVDF membranes sandwiched between, 2 mm whatmann paper, 1mm whatmann paper presoaked in 1x transfer buffer and placed in a semi-dry blotter (Bio-Rad). The proteins were transferred at 15V for
60 minutes. Power was supplied from a BioRad Power Pac 200 (Pharmacia). The efficiency of the transfer was confirmed by staining the PVDF membrane with Poncue-S.

Immunoblotting

PVDF membranes containing bound proteins were blocked with super blotto solution (10 mM Tris pH 8.0, 150 mM NaCl, 0.1% Tween 20, 0.5% NP40, 0.5% BSA, Fraction V, 2.5% non-fat dried milk) for one hour at RT or overnight at 4°C. The membranes were incubated with primary antibodies diluted in super blotto for 1-2 hour at RT or overnight at 4°C. The membranes were washed twice in 1x TBST (100 mM Tris-Hcl, pH 8.0, 1.54 M NaCl, 1% V/V Tween 20) for 5 minutes and incubated for one hour at RT in super blotto containing a 1:5000 dilution of horseradish peroxidase (HRP) conjugated secondary antibody. The membranes
were washed three times, for 10 minutes each in 1x TBST and the bound antibodies were visualized by chemiluminescence.

Chemiluminescence

Immunoreacted proteins were detected using the SuperSignal Pico West (Pierce). Equal volumes of the Luminol/Enhancer and Stable Peroxide solutions were mixed, poured onto the immunoblotted PVDF membranes and incubated for 2 minutes at RT. Excess solution was drained away, the blot placed in clear plastic folder and exposed to Biomax MR film (Kodak). The film was developed through an AGFA Curix 60 machine (AGFA, Germany).

Immunoprecipitation (IP)

All immunoprecipitations were done using nuclear extracts prepared by the method described earlier. When using anti-serum for IPs, the nuclear extract was pre cleared with pre immune serum from the same rabbit. 10 μl of preimmune serum and 7.5 μl protein A coupled sepharose beads (Pharmacia) was added to 400 μl of nuclear extract (from 1 million cells) and incubated at 4°C on shaker for 2 hours. The beads were then spun down (1000 rpm, 4°C, 4 min) and 10 μl of
anit-serum was added to the supernatant. The anti-serum was incubated with cleared nuclear extract overnight at 4°C on shaker. Next day, 7.5 μl Protein A coupled beads were added to the tube and incubated further for 2 hours at 4°C. The IP beads were spun down (150 g, 4°C, 4 min) and washed 5 times with total of 5 ml lysis buffer (0.1% NP40, 1X PBS, 2mM MgCl2, 1mM DTT, 10% Glycerol).

When using a monoclonal antibody for immunoprecipitation, either antibody coupled to beads was purchased or covalently coupled. 2.5 μl of the antibodycoupled beads were added to 200 μl of nuclear extract (from 2-3 million cells) and incubated overnight at 4°C on shaker. The beads were washed with lysis buffer next day, 5 times with a total of 5 ml buffer. Then the beads were boiled in 1X SDS loading buffer and separated on SDS-PAGE. The proteins were transferred on PVDF membrane and detected by immunoblotting.

HA-affinity purification of recombinant protein complex

Sf9 cells were infected with baculoviruses coding for different HsOrc proteins and were harvested 60 hours post infection. Cells were lysed and nuclear extract was made following the method described before. 25 μl of 50% HA ab-agarose slurry was added to 1 ml of nuclear extract (corresponding to 1.25 X 107 infected Sf9 cells) and incubated overnight at 4°C on an overhead rotor. The antibody binding was done in siliconized 1.5 ml eppendorf tubes. The beads were washed 5 times with a total of 5 ml ice cold lysis buffer. The proteins bound to the antibody beads were eluted by cleaving off the tag on Orc, using TEV protease enzyme (Invitrogen). For TEV digestions, 1X TEV protease enzyme buffer and 1 μl of TEV protease enzyme (10 units) was added to the washed beads in a total volume of 100 μl. The beads were incubated at 16°C for 2 hours for TEV digestion. The eluate was collected by spinning the tubes and taking the
supernatant without disturbing the beads pellet. ORC complex from 12.5 million cells were eluted in 140 μl 1X TEV buffer. Care was taken not to let the antibody coupled agarose beads to dry at any step of the experiment.

TCA precipitation of protein

100% Trichloro acetic acid (TCA) solution was mixed with 1/10 volume of 10% Sodium deoxycholate (DOC) solution before use. The mixing was done at room temperature, on shaker for half an hour. 1/4 volume of TCA/DOC mixture was added to the protein solution, and was incubated on ice for 40 min after a quick vortex. The samples were centrifuged at 20000 g for 20 min at 4°C. The supernatant was poured off and 100 μl ice cold Acetone was added to the pellet. The samples were centrifuged again at 20000 g for 20 min at 4°C. The pellet was
air dried at RT for 30 min to 1 hour. The pellet was dissolved in minimal volume of SDS-sample loading buffer. The presence of trace amounts of acetone sometimes gave yellow color to the sample and it was titrated with 1 M Tris HCl pH 8.5 to get the normal blue color (color is indicative of the sample pH).

In vivo DNA binding assay

All transfections were made in HeLa cells by Roti fect transfection method. The expression vector TO4/LacZ has 2xtetO sequences surrounding the TATA box of the CMV promoter (i.e. a “repression reporter”). Tet repressors (TR6) or Tettransactivators in the cell switch the expression of ß-galactosidase OFF and ON in absence and presence of Dox, respectively. In contrast, the pTRE/LacZ plasmid has a Tet responsive promoter (TRE, containing multimerized tet operators in front of minimal CMV promoter i.e. an “activation reporter”). Here, the Tet transactivator switches the expression of ß-galactosidase ON and OFF in absence and presence of Dox, respectively. Tet repressor should not induce expression of ß- galactosidase. After 3 days incubation, cells were harvested and ß-galactosidase activity is measure by Galacto-Light Kit (Applied biosystems) as per manufacturer’s protocol. As an internal control 10ng firefly luciferase expressing plasmid is co-transfected with all expression vectors. Relative intensity of luciferase is used to normalize the ß-galactosidase activity.

Electrophorectic mobility shift Assay

EMSAs were performed as described previously (Baron et al., 1997). Breifly, HEK 293 cells were grown in 10 cm dishes to 50-60% confluency and transfected via the Lipofectamine procedure with 10 ug of plasmid DNA encoding the various Orc fusions. 72 hours post-transfection total cell extracts were prepared as described before. Aliquots of the extracts (10 ul) were mixed with 10 ul of binding buffer (20 mM MgCl2, 20 mM Tris, pH 7.5, 10% glycerol, 2 mg/ml herring sperm
DNA and 1 mg/ml bovine serum albumin, 2X protease inhibitor cocktail, 2mM ATP) and 2fmol 32P-end labeled tetO DNA (34 bp synthesized oligos). After 25 min, the reaction mixture was loaded onto a 5% polyacrylamide/0.13% bisacrylamide gel containing 5% glycerol. Electrophoresis was carried out in 0.5x TBE (45 mM Tris base, 45 mM boric acid and 1 mM EDTA) at 10 V/cm.

Short term plasmid Replication assay

Plasmids are transfected into HEK 293 cells with Roti Fect transfection as described before. Cells are harvested 72 hours post transfection. Plasmid is extracted by method of Hirt (Hirt) as follows. Cells were washed twice with PBS and lysed with 1% SDS in 1X TE (Tris 8.0, EDTA 10mM) on the plate itself. 5M NaCl is added to the final concentration of 1M. Lysate is incubated in 4°C overnight and centrifuged at 15000 xg for 30 min at 4°C. Supernatant is incubated with 200ug/ml of RNAase for 1 hour at 37°C. Subsequently, 100ug/ml of Proteinase K is added at 37°C for 1 hour. After phenol:choloroform extraction DNA is ethanol precipitated in suspended in TE buffer or autoclaved water. To analyze the replication, extracted plasmids are linearized and digested with DpnI together a unique cutter for 3 hour to overnight. The DpnI resistant bands are analyzed by southern hybridization using appropriate radiolabeled DNA probes. Efficiency of DpnI digestion is tested by spiking 1ug of bacterial plasmid from DH5α or GM47 (Dam - strain) into Hirt extracts from untransfected cells.

Southern Hybridization

Digested DNA is resolved on 1% agarose gel. After analyzing the gel under UV, it’s incubated with denaturation buffer for 30 minutes to get appropriate target for the radiolabeled probe. Overnight capillary transfer is used to transfer DNA to charged nylon membrane. After UV cross-linking, membrane is incubated with pre hybridization buffer at 65°C for 2 - 4 hours. In the mean time, probe is radiolabeled with [α-P32] CTP using NEblot kit. For SV40 replication
experiments, AgeI/BsrGI digested fragment of pEGFP-C1 (Clontech) was used as probe. For tetO dependent experiments, XcmI/BglII fragment cut out from scTet plasmid was used as probe. Overnight incubation with radiolabeled probe allows hybridization of probe with specific target DNA on nylon membrane.Membrane is exposed to phosphor plate after three washes and analyzed with phosphor imager.

Biotin-streptavidin coupling

For a standard coupling reaction, either 100pmol of 77bp biotinylated annealed tetO or 150pmol of 34bp biotinylated annealed tetO is used per mg of streptavidin coated paramagnetic beads. Beads are suspended in 100 ul of high salt binding buffer after two washes in the same buffer. Afterwards biotinylated DNA is incubated with the beads for 1-2 hours at 25°C with gentle shaking. The magnet is applied to collect the bound DNA. After three stringent washes with
binding buffer. DNA coupled beads are resuspended in 100 ul of same buffer and stored in 4°C

Biotin DNA- Protein interaction

10pmol of streptavidin coupled DNA is used in a standard binding reaction. Final binding reaction contains 1x binding buffer, 60mM NaCl, 1mM ATP, 2x protease inhibitor cocktail, 1mg/ml ssDNA, 2mg/ml BSA and 10 fold excess of Poly (dA). Poly (dT). Purified ORC is incubated with the binding reaction for 10 minutes at 4°C. Then tetO is added to the reaction. After 30 minutes incubation at 4°C, magnet is applied to collect the bound protein. Beads are boiled with 10ul of 1x SDS buffer after three stringent washes with 1x binding buffer. Protein is loaded on 10% SDS gel and silver stained.

Purification of recombinant ORC using Talon beads

In parallel to HA antibody purification, ORC is also purified through His-tag using talon beads (Clontech). Protein was expressed in 15 cm dishes with Hi-5 cells at a density of one million cells/ ml. Cells were harvested and washed twice with PBS. lysis buffer. Nuclear extract is made as described before. Typical volume of nuclear extracts obtained from a 15cm dish is 1ml. To this, 50 -60ul of talon beads is added. Beads have to be washed twice in the lysis buffer before adding to nuclear extracts. After incubation at 4°C for 2 h, bound protein is spun down at
700g for 2 minutes. 4-5 washes with Lysis buffer (without 300mM KCl), Histagged protein is eluated batch-wise in lysis buffer with 400mM imidazole, pH 5.0. Two rounds of elution at 4°C incubation for 30 min each, generally gives more than 80% of bound protein. Eluted protein is snap frozen and stored in -80°C.

New The Mechanisms Of Gene Regulation

Muhammad Fahri*
Mahasiswa Pasca Biotek Perikanan UB malang

New Research Into The Mechanisms Of Gene Regulation
Source: Penn State

A team led by Penn State's Ross Hardison, T. Ming Chu Professor of Biochemistry and Molecular Biology, has taken a large step toward unraveling how regulatory proteins control the production of gene products during development and growth. Working with collaborators including Drs. Mitchell Weiss and Gerd Blobel at Children's Hospital of Philadelphia, they focused specifically on the complex process of producing red blood cells (erythrocytes). These cells contain large amounts of hemoglobin, a molecule essential for transporting oxygen throughout the body. Abnormalities in hemoglobin figure in many serious diseases, such as sickle-cell disease, and abnormalities in producing blood cells can lead to leukemias. The work will be published in the December 2009 issue of the journal Genome Research.

As erythroid cells mature into red blood cells, the transcription factor, GATA-1, turns the genes responsible for making different proteins on and off. Hardison's team worked with a special strain of mouse erythroid cells that lack the gene gata-1. These cells could not mature into red blood cells unless the researchers added the protein GATA-1 experimentally. This procedure allows the investigators to monitor how the genes respond to GATA-1.

GATA-1 binds to special sites on the cell's DNA. The first step of the project was to locate the genes that are affected by GATA-1, so the researchers conducted a genome-wide search after adding GATA-1 to the cells. Using microarrays developed by newer methods of manufacturing which allow for a much higher density of probes, the team examined 19,000 mouse genes using 45,000 probe sets, many more than previous researchers had been able to study.

They found that adding GATA-1 affected 2,616 genes significantly, which was defined as showing at least a twofold change in the amount of the gene's product. Substantially more genes (1,568) were turned down or off by GATA-1 than were turned on (1,048) by GATA-1. The rest showed little or no response to the addition of GATA-1.

Could the differences in gene response and expression be explained by the spatial patterning of GATA-1 binding? The team used two independent methods of mapping where the GATA-1 binding sites lay on the DNA, and how far those binding sites were from the responsive genes. The two methods yielded strikingly similar information. In all, they found 15,360 DNA segments occupied by GATA-1.

Genes responsive to GATA-1 tend to occur in clusters on the DNA, with each cluster being separated from the next by long regions without any responsive genes.

"We think each cluster of responsive genes may represent a regulatory domain," says Hardison. "This work also highlights the importance of local regulation by transcription factors."

One of the fascinating discoveries of this research is how the genes that are enhanced by GATA-1 differ from those that are repressed. Nearly all responsive genes (more than 88%) lie within 100 kilobase pairs (kb) of a GATA-1 site and more than 60% of those that are up-regulated are less than 5 kb away from the closest GATA-1 site. In contrast, 60% of repressed genes are up to 33 kb away from the closest DNA sites occupied by GATA-1. Enhanced genes also have more nearby GATA-1 sites than repressed genes do.

The team also found evidence that the specific motif on the DNA sites to which the GATA-1 binds when it enhances genes is strongly conserved evolutionarily, while the binding motifs for repressed genes are not so strongly conserved across different species. This suggests that the selective pressure to increase gene expression is stronger than that to decrease gene expression.

Another issue is whether or not GATA-1 forms a complex with TAL1 (T-cell acute lymphocytic leukemia protein 1) at the binding sites. Almost all (at least 83%) of the activated genes have one or more complexes of GATA-1 and TAL1 nearby. A substantial fraction (38%) of the repressed genes lack or have decreased amounts of TAL1 after GATA-1 is added. Thus for many genes, the presence of TAL1 at the GATA-1 binding sites distinguishes activation from repression. However, another class of repressed genes, comprising 62% of the total, have complexes of GATA-1 and TAL1 in their vicinity.

"It is not a simple relationship," explains Hardison. "Genes that are activated almost invariably have complexes of GATA-1 and TAL1 at the binding sites, and genes lacking TAL1 at the GATA1-binding sites are almost always down-regulated. This suggests a model in which the GATA-1/TAL1 complex is involved in gene activation. However, we also see a large number of repressed genes with both proteins at the binding sites. These data indicate that there must be at least two mechanisms for repressing genes in these red blood cells, and only one of those mechanisms involves a loss of TAL1," concludes Hardison.

Another factor involved in regulating the expression of genes responsive to GATA-1 is the modification of histone H3, a protein that coils and compresses DNA inside the nucleus. Overall, Hardison's team found that histone modification is common in the large regions of the DNA that contain the GATA-1 responsive genes and the GATA-1 binding sites, but histone modification does not occur in the adjacent "dead zones" that lack genes responsive to GATA-1.

One particular histone modification, the trimethylation of amino acid lysine at position 27 of histone H3 -- or H3K27me3 -- is well-known to be associated with repression of some genes. By analyzing the location and amount of H3K27me3 around GATA1-responsive genes, the team gained new insights into how this modification influences the expression of genes. As expected, genes enhanced by GATA-1 have very little H3K27me3. However, the two classes of repressed genes differ in the level of H3K27me3. Like enhanced genes, the repressed genes that retain TAL1 after the addition of GATA-1 have low levels of H3K27me3. Those genes that lose some or all of their TAL1 after adding GATA-1 have relatively high levels of H3K27me3.

One class of down-regulated genes, perhaps the one with low TAL1 and increased H3K27me3, may be directly repressed by GATA-1. Possibly GATA-1 recruits other proteins that help reduce the level of expression. This repressed state appears to be indicated by the presence of H3K27me3.

In addition, the team hypothesizes that there is another, indirect mechanism for repressing genes. They suggest that the capacity for a cell to transcribe genes and manufacture their products is limited. In effect, if the cell has a series of transcription factories -- each of which has particular favored "customers" (genes) -- then placing a big order for one set of customers (the genes promoted by GATA-1) will indirectly cause the gene products for other customers (those not being promoted by GATA-1) to be diminished. This scenario implies that all the GATA-1 responsive genes are served by a limited number of local transcription factories with limited capacities.

"Though we developed this hypothesis about gene regulation by studying erythrocytes," comments Hardison, "it could obviously apply to the more general function of many different types of gene regulation. Since almost every disease is related in some way to gene expression, this could provide a powerful new model for thinking about many different diseases and their treatment.

"For example," Hardison continues, "sickle-cell disease is a devastating illness affecting more than 70,000 Americans1. It is caused by mutations in hemoglobin genes. All adults still produce some amount of fetal hemoglobin, and that amount differs among individuals. Some sickle-cell patients produce relatively high levels of it, and these patients have much milder symptoms. If we could learn how to repress the genes that produce the defective hemoglobin and promote those that produce the fetal hemoglobin, we may be able to develop an important new therapy to combat this disease."

Other collaborators in the research are graduate students and research associates at Penn State (Yong Cheng, Weishing Wu, Swathi Ashok Kumar, David C. King, Kuan-Bei Chen, Ying Zhang, Daniela Drautz, Belinda Giardine), faculty at Penn State (Stephan A. Schuster, professor of biochemistry and molecular biology; Webb Miller, professor of biology and computer science and engineering; Francesca Chiaromonte, associate professor of statistics and health evaluation sciences; and Yu Zhang, assistant professor of statistics), and students and post-doctoral fellows at Children's Hospital of Philadelphia (Duonan Yu, Wulan Deng, Tamara Tripic).

This research was supported by funding from the National Institutes of Health, the Gordon and Betty Moore Foundation, and the Leukemia and Lymphoma Society.

http://www.cdc.gov/Features/SickleCell/ accessed Nov. 8, 2009
http://news.biocompare.com/News/NewsStory/300296/NewsStory.html

CONTACTS
Ross Hardison: (+1)814-863-0113, rch8@psu.edu
Barbara Kennedy (PIO): 814-863-4682, science@psu.edu

Researchers Identify Role of Gene in Tumor Development, Growth and Progression
Source: Virginia Commonwealth University

Virginia Commonwealth University Massey Cancer Center and VCU Institute of Molecular Medicine researchers have identified a gene that may play a pivotal role in two processes that are essential for tumor development, growth and progression to metastasis. Scientists hope the finding could lead to an effective therapy to target and inhibit the expression of this gene resulting in inhibition of cancer growth.

According to Paul B. Fisher, M.Ph., Ph.D., professor and chair of the Department of Human and Molecular Genetics, director of the VCU Institute of Molecular Medicine in the VCU School of Medicine, and program leader of Cancer Molecular Genetics at the Massey Cancer Center, the team has shown that astrocyte elevated gene-1, AEG-1, a cancer promoting gene, is involved in both oncogenic transformation, which is the conversion of a normal cell to a cancer cell, and angiogenesis, which is the formation of new blood cells. Oncogenic transformation and angiogenesis are critical for tumor development, growth and progression to metastasis.

In the study published online the week of Nov. 16 in the Early Edition of the journal Proceedings of the National Academy of Sciences, researchers employing a series of molecular studies reported that the elevated expression of AEG-1 is involved with turning normal cells into cancer cells.

According to Fisher, when AEG-1 was expressed in normal immortal rat embryo fibroblast cells it converted these cells into transformed cells that induced rapidly growing aggressive cancers when injected into animals. AEG-1 expressing cells displayed enhanced expression of genes regulating blood vessel formation, thereby contributing to tumorigenicity. The team has further defined the pathways in target cells that are activated by AEG-1 and mediate its oncogenic and angiogenic inducing properties.

“Our goal is to understand the functions of a novel gene AEG-1 that plays an essential role in tumor progression, with potential to develop effective therapeutic approaches for multiple cancers through targeted inhibition of this novel molecule or its downstream regulated processes,” said Fisher, who is the first incumbent of the Thelma Newmeyer Corman Endowed Chair in Cancer Research with the VCU Massey Cancer Center.

“We believe it will pave the way for ameliorating the sufferings of scores of cancer patients by uncovering new and effective avenues for treatment,” he said.

To expand the work on AEG-1, the VCU Department of Human and Molecular Genetics, Institute of Molecular Medicine and Massey Cancer Center recently received a National Cancer Institute grant totaling $1.6 million to study the AEG-1 gene in the context of malignant brain tumors such as glioblastoma multiforme, or GBM. According to Fisher, who is the primary investigator for the study, the work will extend the understanding of this gene and how it may serve as an oncogenic, or transforming gene.

“Cancer development and progression are multi-factor and multi-step processes that occur in a temporal manner. As mentioned above AEG-1 clearly has multiple roles in various steps of tumor progression, including tumor cell growth, insensitivity to growth-inhibitory signals, including chemotherapeutic agents, invasion, angiogenesis and metastasis,” explained Fisher.
“In addition, AEG-1 has been known to have oncogenic roles in various cancers including glioma (CNS tumor), neuroblastoma, liver cancer, breast cancer, prostate cancer, lung cancer, and esophageal squamous cell carcinoma. These important correlations make this gene an intriguing molecule to study with potential to serve as a direct target for cancer therapy,” he said.

The gene was discovered in 2002 in Fisher’s laboratory while he was at the Columbia University College of Physicians and Surgeons in New York.

Fisher worked with a team that included VCU School of Medicine researchers Zao-zhong Su, Ph.D., associate professor in the VCU Department of Human and Molecular Genetics; Devanand Sarkar, M.B.B.S., Ph.D., assistant professor and Harrison Endowed Scholar in Cancer Research at the VCU Massey Cancer Center, the VCU Institute of Molecular Medicine and the Department of Human and Molecular Genetics; Hyun Yong Jeon, M.S., research assistant with the VCU Department of Human and Molecular Genetics; Luni Emdad, M.D., Ph.D., with the Mount Sinai School of Medicine in New York; and Habib Boukerche, Ph.D., a senior scientist with the University Lyon 1 in France.

EDITOR’S NOTE: A copy of the study is available for reporters by email request from pnasnews@nas.edu.

About VCU and the VCU Medical Center:

Virginia Commonwealth University is a major, urban public research university with national and international rankings in sponsored research. Located on two downtown campuses in Richmond, VCU enrolls more than 32,000 students in 205 certificate and degree programs in the arts, sciences and humanities. Sixty-five of the programs are unique in Virginia, many of them crossing the disciplines of VCU’s 15 schools and one college. MCV Hospitals and the health sciences schools of Virginia Commonwealth University compose the VCU Medical Center, one of the nation’s leading academic medical centers. For more, see www.vcu.edu.

About the VCU Massey Cancer Center:

The VCU Massey CancerCenter is one of 63 National Cancer Institute-designated institutions that leads and shapes America’s cancer research efforts. Working with all kinds of cancers, the Center conducts basic, translational and clinical cancer research, provides state-of-the-art treatments and promotes cancer prevention and education. Since 1974, Massey has served as an internationally recognized center of excellence. It offers more clinical trials than any other institution in Virginia, serving patients in Richmond and in four satellite locations. Treating all kinds of cancers, its 1,000 researchers, clinicians and staff members are dedicated to improving the quality of human life by developing and delivering effective means to prevent, control and, ultimately, to cure cancer. Visit Massey online at www.massey.vcu.edu or call 1-877-4-MASSEY


Paradoxical Protein Might Prevent Cancer
Source: Karolinska Institutet

One difficulty with fighting cancer cells is that they are similar in many respects to the body's stem cells. By focusing on the differences, researchers at Karolinska Institutet have found a new way of tackling colon cancer. The study is presented in the prestigious journal Cell

Molecular signal pathways that stimulate the division of stem cells are generally the same as those active in tumour growth. This limits the possibility of treating cancer as the drugs that kill cancer cells also often adversely affect the body's healthy cells, particularly stem cells. A new study from Karolinska Institutet, conducted in collaboration with an international team of scientists led by Professor Jonas Frisén, is now focusing on an exception that can make it possible to treat a form of colon cancer.

The results concern a group of signal proteins called EphB receptors. These proteins stimulate the division of stem cells in the intestine and can contribute to the formation of adenoma (polyps), which are known to carry a risk of cancer. Paradoxically, these same proteins also prevent the adenoma from growing unchecked and becoming cancerous.

The new results show that EphB controls two separate signal pathways, one of which stimulates cell division and the other that curbs the cells' ability to become cancerous. Using this knowledge, the scientists have identified a drug substance called imatinib, which can inhibit the first signal pathway without affecting the other, protective, pathway.
"Imatinib or a similar substance could possibly be used for preventing the development of cancer in people who are in the risk zone for colon cancer instead of intestinal resection," says Maria Genander, one of the researchers involved in the study.

Imatinib has so far proved to inhibit cell division in intestinal tumour cells in vitro and in mice. The substance is a component of the drug Glivec, which is used, amongst other things, in the treatment of certain forms of leukaemia. Whether it can also be used against adenoma and colon cancer in humans remains to be seen. The company that manufactures the drug did not fund the study.

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