Ahyar Ahmad

WD repeats of the p48 subunit of chicken chromatin assembly factor-1 required for in vitro interaction with chicken histone deacetylase-2.

Chromatin assembly factor-1 (CAF-1) is essential for chromatin assembly in eukaryotes, and comprises three subunits of 150 kDa (p150), 60 kDa (p60), and 48 kDa (p48). We cloned and sequenced cDNA encoding the small subunit of the chicken CAF-1, chCAF-1p48. It consists of 425 amino acid residues including a putative initiation Met, possesses seven WD repeat motifs, and contains only one amino acid change relative to the human and mouse CAF-1p48s. The immunoprecipitation experiment followed by Western blotting revealed that chCAF-1p48 interacts with chicken histone deacetylases (chHDAC-1 and -2) in vivo. The glutathione S-transferase pulldown affinity assay revealed the in vitro interaction of chCAF-1p48 with chHDAC-1, -2, and -3. We showed that the p48 subunit tightly binds to two regions of chHDAC-2, located between amino acid residues 82–180 and 245–314, respectively. We also established that two N-terminal, two C-terminal, or one N-terminal and one C-terminal WD repeat motif of chCAF-1p48 are required for this interaction, using deletion mutants of the respective regions. These results suggest that chCAF-1p48 is involved in many aspects of DNA-utilizing processes, through alterations in the chromatin structure based on both the acetylation and deacetylation of core histones.

RbAp48 is essential for viability of vertebrate cells and plays a role in chromosome stability.

RbAp46/48, histone chaperone, is a family of evolutionarily conserved WD40 repeat-containing proteins, which are involved in various chromatin-metabolizing processes, but their in vivo functional relevance is yet unclear. In order to examine the biological role of pRbAp48 in chicken DT40 cells, we generated a tetracycline-inducible system for conditional RbAp48-knockout cells. Depletion of RbAp48 led to delayed S phase progression associated with slow DNA synthesis and nascent nucleosome formation, followed by accumulation in G2/M phase, finally leading to cell death. Prior to cell death, these cells exhibited aberrant mitosis such as highly condensed and abnormal chromosome alignment on the metaphase plate, leading to chromosome missegregation. Depletion of RbAp48 also caused dissociation of heterochromatin protein 1 (HP1) from pericentromeric heterochromatin. Furthermore, depletion of RbAp48 from cells led to elevated levels of acetylation and slightly decreased levels of methylation, specifically at Lys-9 residue of histone H3. These results suggest that RbAp48 plays an important role in chromosome stability for proper organization of heterochromatin structure through the regulation of epigenetic mark.

Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study

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 the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT83, have been examined as TB vaccine candidate. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of this 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 E. coli BL21 strain and produces 48 kDa protein as well as GST-MPT83 fusion protein.

TOXICITY AND FREE RADICAL SCAVENGING ACTIVITY OF RHIZOPHORA MUCRONATA LAMK. BARK EXTRACTS FROM KENDARI BAY OF SOUTHEAST SULAWESI PROVINCE INDONESIA

The aim of this study was to evaluate the toxicity and free radical scavenging activity of Rhizophora mucronata Lamk. bark extracts from the mangrove forest of Kendari Bay, Southeast Sulawesi Province Indonesia. The extracts were obtained by maceration of R. mucronata Lamk. bark with n-hexane, ethyl acetate, and methanol, respectively. Toxicity test was conducted by using Brine Shrimp Lethal Test (BSLT) method with Artemia salina Leach as the animal test, while free radical scavenging activity test was carried out by using DPPH (1,1-diphenyl 2-picryl-hydrazyl) free radical. The test results showed n-hexane, ethyl acetate and methanol extracts had LC50 values of 136.40 μg/mL, 82.43 μg/mL and 109.38 μg/mL, respectively. The IC50 values for the free radical scavenging activity of the n-hexane, ethyl acetate, and methanol extracts were 122.19 μg/mL, 37.84 μg/mL, 29.32μg/mL, respectively. Result of phytochemical test showed n-hexane extract was dominated by terpenoid and steroid group compounds, while flavonoids, alkaloids, and tannins were predominantly found in both ethyl acetate and methanol extracts. Extracts of R. mucronata bark, especially ethyl acetate extracts had toxic properties for A. salina larvae, therefore it has potential to be developed as an anti-cancer drug. Metahanol extract, on the other hand, indicated strong anti-oxidant activity, then it is potential to develop it as cosmetics agent.