MOLECULAR CLONING, EXPRESSION AND FUNCTIONAL INTERACTION OF p48 SUBUNIT OF CHICKEN CHROMATIN ASSEMBLY FACTOR 1 WITH HISTONE DEACETYLASE 2 AND HISTONE ACETYLTRANSFERASE 1

We cloned and sequenced cDNA encoding p48 subunit of the chicken CAF-1, chCAF-1p48, and histone\ud acetyltransferase-1, chHAT-1 from chicken DT40 cell lines. We showed that the p48 subunit of CAF-1 tightly binds to\ud two regions of chicken histone deacetylase 2, chHDAC-2, located between amino acid residues 82-180 and 245-\ud 314, respectively. We also established that two N-terminal, two C-terminal, or one N-terminal and one C-terminal\ud WD repeat motif of chCAF-1p48 are required for this interaction. The GST pulldown assay, involving truncated and\ud missense mutants of chCAF-1p48, revealed not only that a region containing the seventh WD dipeptide motif of\ud chCAF-1p48, comprising amino acids 376-405, binds to chHAT-1 in vitro, but also that mutation of the motif has no\ud influence on the in vitro interaction. We also established that the region, which is located between amino acids 380-\ud 408 of chHAT-1 and contains a leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. Mutation\ud on each of four Leu residues in the leucine zipper motif of chHAT-1 causes the disappearance of the interaction with\ud chCAF-1p48. These results should be useful information for understanding the participation of chCAF-1p48 protein\ud as histones chaperone in DNA-utilizing processes, such as replication, recombination, repair and gene expression in\ud DT40 chicken B cell

EXPRESSION AND FUNCTIONAL INTERACTION OF CHICKEN RbAp46 POLYPEPTIDE WITH HISTONES, HISTONE DEACETYLASE-1, AND HISTONE ACETYLTRANSFERASE-1

In this study, we cloned and sequenced cDNA encoding the chicken p46 polypeptide, RbAp46. The cDNA\ud encoding a protein consists of 424 amino acids is a member of the WD protein family, with seven WD repeat motifs,\ud and exhibits 90.3% identity to RbAp48, and 94.3% identity to the human RbAp46. The RbAp46 fusion protein were\ud synthesized by in vitro translation system and in Escherichia coli under induction by 50 ??M IPTG and single step\ud purified with glutathione-Agarose beads, showed that GST-tagged protein of approximately 72 kDa. The in vitro\ud experiment established that RbAp46 interacts with chicken histones, chHDAC-1, and chHAT-1. The in vitro\ud immunoprecipitation experiment, involving truncated mutants of RbAp46, revealed not only that two regions\ud comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions\ud comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. Furthermore, the GST pulldown\ud affinity assay, involving truncated mutants of RbAp46, revealed that a region comprising amino acids 359-404 binds\ud to chHAT-1 in vitro. Taken together, these results indicate not only that RbAp46 should participate differentially in a\ud number of DNA-utilizing processes through interactions of its distinct regions with histones and chHAT-1, but also\ud that the proper propeller structure of RbAp46 is not necessary for its interaction with chHAT-1

EFFECTOF HIRA PROTEINONTRANSCRIPTIONALREGULATIONSOF GENESIN VERTEBRATECELLS

ABSTRACT For better understanding of DNA replicating-coupled chromatin assembly and transcription regulation in eukaryotes, we studied biochemical and genetic analysis of nuclear-related proteins from chicken DT40 cell lines. The genetic analysis of some nuclear proteins, such as HIRA and CAF-1, indicated that these proteins could play overlappingroles in chromatin dynamics and is consistent with the finding that HIRA protein exhibited binding ability to histones and ASF-1, as also ASF-1 bound directly with CAF-1p60. In this study, revealed not only that the Nterminal and C-terminal halves of HIRA mediate individually transcription repressions but also that even one of the sevenWDdipeptide motifs and the LXXLL motif of HIRA are required for these mediations in vivo. Finally,we found that HIRA-mediatedrepression is sensitive to tricostatin TSAand it co-represses transcription together with HDAC-2. We believe our findings will contribute to a major break-throughin future studies on the specific, individual roles of HIRA involved in numerous DNA-utilizing processes, through the formation and/or maintenance of the chromatin structurein vertebratecells. Keywords: histone regulator, transcriptionalrepression,tricostatin,chromati

PURIFICATION AND IMMOBILIZATION OF L-ASPARAGINASE ENZYME FROM THE THERMOPHILIC BACTERIA Bacillus licheniformis STRAIN HSA3-1a

L-Asparaginase gives a great benefit in the cancer treatment, especially in acute\ud lymphoblastic leukemia. L-Asparaginase is also proven to reduce the acrylamide\ud content in the foods. The objective of this study was to perform immobilization and\ud characterization L-Asparaginase produced from Bacillus licheniformis Strain HSA3-\ud 1a. The results showed that the free form L-Asparaginase from B.\ud licheniformis HSA3-1a has optimum activity at pH 8 and 50oC, with a specific activity\ud of 616.26 IU/mg protein and stabilized at the optimum pH and temperature for 60\ud minutes. The immobilized L-Asparaginase with activated glutaraldehyde-carbon\ud carrier has optimum activity at pH 7 and 60??C with a specific activity of 499.27 IU/mg\ud protein and stabilized at the optimum pH and temperature for 60 minutes. The\ud immobilized L-Asparaginase can retain its activity by 84.79% after 2 times repeated\ud use

TOXICITY AND ANTIMICROBIAL ACTIVITY FROM EXTRACT AND OLEANAN DERIVATIVE COMPOUNDS OF THE BARK MELOCHIA UMBELLATE (HOUTT) STAPF VAR. DEGRABRATA

Oleanan derivative compound namely 3-acetyl-12-oleanen-28-oic acid, has been isolated from n-hexane fraction of the bark M. umbellate (Houtt) Stapf var. degrabrata The molecular structure was determined by IR spectroscopy, NMR 1D and 2D (1H-NMR, 13C-NMR, DEPT, COSY, HMQC and HMBC). The results of test bioactivity to hexane, chloroform, ethyl acetic, methanol extracts and compound 1 mentioned above showed that they are toxic to A. salina (brine shrimp) with LC50 361.93 to 460 ??g/mL. The most toxic one was the compound 1 with the LC50 value of 361.93 ??g/mL. At a concentration of 1000 ??g/mL, the extract of hexane, methanol and compound 1 showed very high inhibition to the growth of B. subtilis and C.albican, whereas the extract of ethyl acetate had very high inhibition effect to the growth of S. aureus and A. niger. The inhibition zone of the three extracts and compound 1 were greater than 14 mm. On the other hand, the extracts of chloroform gave weak inhibition against bacteria and fungi

BIOETHANOL PRODUCTION FROM CELLULOSE IN RED ALGAE Gracilaria verrucosa BY SEPARATED HYDROLYSIS AND FERMENTATION SYSTEM USING Trichoderma viride AND Zymomonas mobilis

In this study, renewable marine cellulose from red algae Gracilaria verrucosa was \ud utilized for the production of bioethanol. Bioethanol from the red alga cellulose was \ud produced by the enzymatic hydrolysis and fermentation methods and the conversion \ud value of the cellulose in Gracilaria verrucosa was estimated. Trichoderma viride \ud fungus and Zymomonas mobilis bacterium were used for enzymatic hydrolysis and \ud bioethanol fermentation, respectively. Enzymatic hydrolysis and fermentation \ud optimization were carried out by varying pH and period of hydrolysis or fermentation. \ud Research results indicate that the optimum condition of the enzymatic hydrolysis \ud cellulose is obtained at pH 5.5 in 6 days, whereas the optimum condition of bioethanol \ud fermentation is obtained at pH 6.0 in 7 days. The bioethanol concentration as \ud measured by Gas Chromatography, showed that one kilogram of cellulose in \ud Gracilaria verrucosa produces 23.01% bioethanol with the concentration of 29.60%

ISOLATION AND CHARACTERIZATION OF BIOACTIVE PROTEIN FROM GREEN ALGAE Halimeda macrobola AS ANTIOXIDANT AND ANTICANCER AGENT

A protein fraction isolated from green algae Halimeda macrobola taken from the sea of Selayar and Kapoposang Island inSouth Sulawesi was tested forantioxidant and anticancer properties.The protein was isolated using buffer Tris (hydroxymethyl) amino methane. Initial purification of protein uses conducted by using the fractionation method with ammonium sulphate, followed by a dialysis process. The protein concentration was determined by Lowry method. The antioxidant assay was done by using DPPH method and the anticancer activity test by Brine Shrimp Lethality Test (BSLT) methods. Anticancer activity was further confirmed by antimitotic test using urchinzygote cells. The results showed that the protein concentration of the crude extract was 0.920 mg/mL. The highest concentration of protein fractions was indicated by the fraction 40-60%,with 1.015 mg/mL. The strong antioxidant activity wasnshown in the protein fraction of 0-20% saturation with IC50 values of 0.110 mg/mL.The highest activity in the anticancer tests was shown in fractions 0-20% saturation with LC50 values of 0.29??g/mLand IC50 value of 53.80 ??g/mL. The protein fraction 0-20% saturation had a potential to be developed as an alternative antioxidant and anticancer agent

IMPLICATION OF CLAY MODIFIED ELECTRODE ON CYANIDE BIOSENSOR

A novel, inexpensive and simple amperometric biosensor based on immobilization of \ud tyrosinase enzyme onto bentonite and modified bentonite is applied for determination of \ud cyanide. The determination of cyanide was performed via its inhibiting action on the \ud tyrosinase electrode. Measurement was carried out using cathecol as substrate. The \ud enzymatically generated quinoid products were electroreduced at 0.1 V vs Ag/AgCl. An \ud extremely sensitive detection limit (2 x 10-7 M) was obtained for cyanide. Enzyme \ud immobilitation onto an modified bentonite seems to cause an increase in cyanide inhibition because of the greater surface area

CLONING AND EXPRESSION OF MPT83 GEN FROM Mycobacterium tuberculosis LOCAL STRAIN IN E. coli BL21 AS VACCINE CANDIDATE OF TUBERCULOSIS

The appearance of Mycobacterium tuberculosis strains leading to drug resistance has caused new problems in TB treatment in various parts of the world and forces WHO to declare TB as a global emergency. With the increase of TB drug resistance, it is convinced that a more effective vaccine development will stop or decrease the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT8, have been examined as TB vaccine candidate, using DNA recombinant technology. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with electron microscope and cytometry circulation. MPT83 can also stimulate humoural as well as T-cell immune response. Next, MPT83 is an M. tuberculosis surface antigen that can induct CD4 lymphocyte T in order to produce Interleukin-2 that has a central role in triggering immune against TB. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of the research is to conduct amplification of MPT83 antigen cloning, and expression of its antigen on E coli bacteria. From the result of the research, it is expected that raw material to produce TB vaccine as well as a high-quality antigen can be obtained. The band of DNA in PCR product is 660 bp, while the one in pGEMT-Easy-MPT83 recombinant plasmid is 3678 bp. This is expressed in BL21 E coli strain and produces 44 kDa protein as well as GST-MPT83 fusion protein

ISOLASI DAN KARAKTERISASI PROTEIN BIOAKTIF DARI SPONS Callyspongia.sp SEBAGAI ZAT ANTIOKSIDAN

ABSTRAK\ud \ud Spons Callyspongia sp. merupakan salah satu biota laut yang banyak tersebar diperairan pulau Barang Lompo Sulawesi Selatan. Spons ini menarik untuk diteliti bioaktivitas dari fraksi proteinnya. Protein tersebut diisolasi dengan menggunakan buffer Tris (hidroksimetil) amino metana. Ekstrak kasar difraksinasi dengan penambahan amonium sulfat pada tingkat kejenuhan 0-20%, 20-40%, 40-60%, dan 60-80%. Pemurnian protein dilakukan dengan cara dialisis menggunakan kantong selofan. Kadar protein ditentukan berdasarkan metode Lowry. Jumlah protein tertinggi terdapat pada fraksi 60-80% sebesar 290,4 mg. Pengujian aktivitas antioksidannya menggunakan metode DPPH dengan spektrofotometri UV-Vis diamati absorbansinya pada ?? = 500 nm. Aktivitas antioksidan yang kuat terdapat pada fraksi protein 0-20% kejenuhan dengan nilai IC50 sebesar 61,62??g/mL. Sedangkan aktivitas antioksidan yang sangat kuat ditunjukkan oleh fraksi protein 20-40%, 40-60%, dan 60-80% kejenuhan dengan nilai IC50 masing-masing sebesar 28,76 ??g/mL, 13,90 ??g/mL, dan 35,66 ??g/mL. Hasil dari penelitian menunjukkan masing-masing fraksi protein tersebut terdapat perbedaan aktivitas antioksidan yang mampu mencegah terjadinya proses oksidasi.\ud \ud Kata kunci: Spons Callyspongia. sp, Antioksidan, DPP