Abdul Rauf Shakoori

Phenotypic transcription factors epigenetically mediate cell growth control

Ribosomal RNA (rRNA) genes are down-regulated during osteogenesis, myogenesis, and adipogenesis, necessitating a mechanistic understanding of interrelationships between growth control and phenotype commitment. Here, we show that cell fate-determining factors [MyoD, myogenin (Mgn), Runx2, C/EBPβ] occupy rDNA loci and suppress rRNA expression during lineage progression, concomitant with decreased rRNA expression and reciprocal loss of occupancy by c-Myc, a proliferation-specific activator of rRNA transcription. We find interaction of phenotypic factors with the polymerase I activator upstream binding factor UBF-1 at interphase nucleoli, and this interaction is epigenetically retained on mitotic chromosomes at nucleolar organizing regions. Ectopic expression and RNA interference establish that MyoD, Mgn, Runx2, and C/EBPβ each functionally suppress rRNA genes and global protein synthesis. We conclude that epigenetic control of ribosomal biogenesis by lineage-specific differentiation factors is a general developmental mechanism for coordinate control of cell growth and phenotype.

Transcriptional corepressor TLE1 functions with Runx2 in epigenetic repression of ribosomal RNA genes.

Epigenetic control of ribosomal RNA (rRNA) gene transcription by cell type-specific regulators, such as the osteogenic transcription factor Runx2, conveys cellular memory of growth and differentiation to progeny cells during mitosis. Here, we examined whether coregulatory proteins contribute to epigenetic functions that are mitotically transmitted by Runx2 in osteoblastic cells. We show that the transcriptional corepressor Transducin Like Enhancer-1 (TLE1) associates with rRNA genes during mitosis and interphase through interaction with Runx2. Mechanistically, depletion of TLE1 relieves Runx2-mediated repression of rRNA genes transcription and selectively increases histone modifications linked to active transcription. Biologically, loss of TLE-dependent rRNA gene repression coincides with increased global protein synthesis and enhanced cell proliferation. Our findings reinforce the epigenetic marking target genes by phenotypic transcription factors in mitosis and demonstrate a requirement for retention of coregulatory factors to sustain physiological control of gene expression during proliferation of lineage committed cells.

Oct-2 DNA binding transcription factor: functional consequences of phosphorylation and glycosylation

Phosphorylation and O-GlcNAc modification often induce conformational changes and allow the protein to specifically interact with other proteins. Interplay of phosphorylation and O-GlcNAc modification at the same conserved site may result in the protein undergoing functional switches. We describe that at conserved Ser/Thr residues of human Oct-2, alternative phosphorylation and O-GlcNAc modification (Yin Yang sites) can be predicted by the YinOYang1.2 method. We propose here that alternative phosphorylation and O-GlcNAc modification at Ser191 in the N-terminal region, Ser271 and 274 in the linker region of two POU sub-domains and Thr301 and Ser323 in the POUh subdomain are involved in the differential binding behavior of Oct-2 to the octamer DNA motif. This implies that phosphorylation or O-GlcNAc modification of the same amino acid may result in a different binding capacity of the modified protein. In the C-terminal domain, Ser371, 389 and 394 are additional Yin Yang sites that could be involved in the modulation of Oct-2 binding properties.

Immediate‐early gene regulation by interplay between different post‐translational modifications on human histone H3

In mammalian cells, induction of immediate‐early (IE) gene transcription occurs concomitantly with histone H3 phosphorylation on Ser 10 and is catalyzed by mitogen‐activated protein kinases (MAPKs). Histone H3 is an evolutionarily conserved protein located in the core of the nucleosome, along with histones H2A, H2B, and H4. The N‐terminal tails of histones protrude outside the chromatin structure and are accessible to various enzymes for post‐translational modifications (PTM). Phosphorylation, O‐GlcNAc modification, and their interplay often induce functional changes, but it is very difficult to monitor dynamic structural and functional changes in vivo. To get started in this complex task, computer‐assisted studies are useful to predict the range in which those dynamic structural and functional changes may occur. As an illustration, we propose blocking of phosphorylation by O‐GlcNAc modification on Ser 10, which may result in gene silencing in the presence of methylated Lys 9. Thus, alternate phosphorylation and O‐GlcNAc modification on Ser 10 in the histone H3 protein may provide an on/off switch to regulate expression of IE genes. J. Cell. Biochem. 103: 835–851, 2008.

Prevalence of hepatitis B and C in internally displaced persons of war against terrorism in Swat, Pakistan

BACKGROUND Hepatitis B and C are the most common blood-borne liver infections worldwide. According to the recent estimates, 270-300 million people worldwide are infected with hepatitis C virus (HCV) and more than 2 billion people have been infected with the hepatitis B virus (HBV). Transmission of these viruses is carried out by exposure to infectious blood or body fluids containing blood. METHODS Five-hundred and ninety (290 males and 300 females, 5-65 years) internally displaced persons (IDPs) from Swat area of northern Pakistan were screened for hepatitis B surface antigen (HBsAg) and anti-HCV antibodies using immune-chromatography kits. The subjects, positive for virus-related antibody, were further confirmed for viral RNA (for HCV) and DNA (for HBV) in the blood by polymerase chain reaction (PCR) amplification. The virus of PCR-confirmed HCV-individuals was further genotyped and the prevalence of HCV infection was determined with respect to age, sex, history of exposure to blood or surgical operation and different types of liver diseases. RESULTS The HCV infection was found to be the predominant liver infection in the population which was 91% of the positive cases, as against 9% of HBV. Among the HCV-positive subjects (68% females, 32% males) 56% were asymptomatic. No co-incidence of HBV and HCV was found in any subject. Genotype 3a was the dominant strain of HCV followed by 2a > 3b > 1b > 1a, 2b. CONCLUSION The viral hepatitis among the apparently healthy population of a relatively natural and pollution free environment refers to an alarming condition about liver infections, particularly of HCV, in Pakistan.

Growth and survival of protozoa isolated from a tannery effluent

Industrial effluent from a tannery was used for the growth of algae in a medium containing various inorganic salts. Growth of algal cells became visible after 7 d. Two species of protozoa were observed to proliferate in the algal culture containing no organic supplement in the medium. The culture was kept bacteria-free by the use of antibiotics and was perpetuated for at least 150 d with no decline in the protozoan population. Efficient growth of protozoa in a culture of algae elucidated new modes of nutrition in protozoa. Cr(VI) was added to the medium to check the resistance of algae and protozoa against this heavy metal. Protozoa showed different degrees of resistance. The results indicate the importance of algae and protozoa in the process of bioremediation.

SP-D polymorphisms and the risk of COPD.

Introduction: There are limited data linking serum levels of surfactant protein D, its genetic polymorphisms to the risk of Chronic Obstructive Pulmonary Disease (COPD). Objectives: We sought to investigate these relationships using a case control study design. Methods: Post bronchodilator values of FEV1/FVC <0.7 were used to diagnose COPD patients (n = 115). Controls were healthy subjects with normal spirometry (n = 106) Single nucleotide polymorphisms (rs721917, rs2243639, rs3088308) were genotyped using polymerase chain reaction (PCR) and restriction analysis. Serum SP-D levels were measured using a specific immunoassay. Results: Allele ‘A’ at rs3088308 (p < 0.00, B = −0.41) and ‘C’ allele at rs721917 (p = 0.03; B = −0.30) were associated with reduced serum SP-D levels. Genotype ‘T/T’ at rs721917 was significantly associated with risk of COPD (p = 0.01). Patients with repeat exacerbations had significantly higher serum SP-D even after adjusting for genetic factors. Conclusions: We report for the first time that rs3088308 is an important factor influencing systemic SP-D levels and confirm the previous association of rs721917 to the risk of COPD and serum SP-D levels.

Ethanol Extract of Cissus quadrangularis Enhances Osteoblast Differentiation and Mineralization of Murine Pre-Osteoblastic MC3T3-E1 Cells.

Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti‐osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre‐osteoblast cell line, MC3T3‐E1. Ethanolic extract of CQ (CQ‐E) was found to affect growth kinetics of MC3T3‐E1 cells in a dosage‐dependent manner. High concentrations of CQ‐E (more than 10 μg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non‐toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ‐E. CQ‐E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose‐dependent effect of CQ‐E with lower concentrations exhibiting anabolic and osteogenic properties. J. Cell. Physiol. 232: 540–547, 2017.

Molecular Characterization of a Copper Metallothionein Gene from a Ciliate Tetrahymena farahensis

A new copper metallothionein (TfCuMT) gene has been identified from a locally isolated ciliate Tetrahymena farahensis. It contains 327 nucleotides encoding a peptide chain of 108 amino acids and belongs to class MTT2 and subfamily 7b. Amplification from both gDNA and mRNA confirmed the intronless nature of this gene. Like most of the metallohtioneins, cysteine residues contribute nearly 30% content with the specific CKC motifs. Structural repeats present in peptide sequence of TfCuMT indicate internal duplication of gene at some stage of gene evolution. The predicted model of copper metallothionein protein showed that copper ions are mainly chelated by thiol sulfur of cysteine residues and are embedded in the folds of polypeptide chain. For in vivo expression of TfCuMT in Escherichia coli host cells the classical stop codons, which coded for glutamine in the ciliate were mutated to CAA and CAG through site directed mutagenesis. The mutated gene showed higher expression in pET28a expression vector compared with pET21a. Optimum expression was obtained after 6–8 h of 0.1 mM IPTG induction. Stability of His tagged TfCuMT in 5% SDS was low, with half‐life of about 104 min. Presence of 1.0 μM copper increased the expression level by 1.65‐fold. Presence of 100 μM Cysteine in culture medium caused 2.4‐fold increase in expression level. His tagged TfCuMT was purified through affinity chromatography using NTN‐His binding resin in the presence of 0.1 M imidazole and NaCl. The modeled structure of the TfCuMT showed a cleft for Cu binding with correct orientation of Cys residues in the motif CKC. J. Cell. Biochem. 117: 1843–1854, 2016.

Anti-proliferative and genotoxic effects of arsenic and lead on human cells in vitro

Rapid industrialization and anthropogenic activities are main cause of environmental pollution, leading to an alarming situation. Both arsenic and lead are very dangerous to human health as exposure to these metal results in several abnormalities. In the present study, toxic effects of arsenic and lead were tested on HeLa cells. Cells were exposed to different concentrations of arsenic and lead and anti-proliferative response was tested using neutral red uptake assay. Comet assay was conducted to test the effect of arsenic and lead on DNA. Change in morphology and death of cells was observed after exposure to both metals. Comets were observed in the case of treated samples giving an indication of DNA damage. Ten different comet parameters were investigated for control and heavy metals treated samples. Present study clearly demonstrates that both arsenic and lead have anti-proliferative effect on HeLa cells and they also cause DNA damage. Overall, effects of arsenic exposure were more severe as compared to lead.