E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

EBV latent antigen EBNA3C is indispensible for in vitro B-cell immortalization resulting in continuously proliferating lymphoblastoid cell lines (LCLs). EBNA3C was previously shown to target pRb for ubiquitin-proteasome mediated degradation, which facilitates G1 to S transition controlled by the major transcriptional activator E2F1. E2F1 also plays a pivotal role in regulating DNA damage induced apoptosis through both p53-dependent and -independent pathways. In this study, we demonstrate that in response to DNA damage LCLs knocked down for EBNA3C undergo a drastic induction of apoptosis, as a possible consequence of both p53- and E2F1-mediated activities. Importantly, EBNA3C was previously shown to suppress p53-induced apoptosis. Now, we also show that EBNA3C efficiently blocks E2F1-mediated apoptosis, as well as its anti-proliferative effects in a p53-independent manner, in response to DNA damage. The N- and C-terminal domains of EBNA3C form a stable pRb independent complex with the N-terminal DNA-binding region of E2F1 responsible for inducing apoptosis. Mechanistically, we show that EBNA3C represses E2F1 transcriptional activity via blocking its DNA-binding activity at the responsive promoters of p73 and Apaf-1 apoptosis induced genes, and also facilitates E2F1 degradation in an ubiquitin-proteasome dependent fashion. Moreover, in response to DNA damage, E2F1 knockdown LCLs exhibited a significant reduction in apoptosis with higher cell-viability. In the presence of normal mitogenic stimuli the growth rate of LCLs knockdown for E2F1 was markedly impaired; indicating that E2F1 plays a dual role in EBV positive cells and that active engagement of the EBNA3C-E2F1 complex is crucial for inhibition of DNA damage induced E2F1-mediated apoptosis. This study offers novel insights into our current understanding of EBV biology and enhances the potential for development of effective therapies against EBV associated B-cell lymphomas

Effect of antibiotics on paraoxonase1: ã-lactamase-like activity of paraoxonase1

Paraoxonase1 (PON1), a serum enzyme associated exclusively with High Density Lipoprotein (HDL) is responsible for most of HDLs cardioprotective properties. PON1 activity is a measure of HDL functionality and thus accurate measurement of PON1 activity becomes clinically important. However, factors in the serum like Ca2+, triglycerides, inflammatory cytokines, drugs and environmental toxins might interfere with PON1 measurement. In our current investigation, we have tested the effects of commonly used antibiotics- Tetracycline, Spectinomycin, Gentamicin and Penicillin G on PON1 activity. Tetracycline reduced PON1 activity by 90 at concentration as low as 0.25 mg/ml while the others increased PON1 activity at the same concentration. Upon increasing the concentration of these antibiotics, PON1 activity decreased but not below the control. Because Penicillin G contains a â-lactam ring that resembles lactone ring structure and because PON1 has lactonase activity, we used Penicillin G as a substrate for PON1 and found that Penicillin G was cleaved by PON1. The cleaved product could be separated by TLC and was subjected to 1H-NMR analysis. Penicillin G inhibited the growth of Bacillus subtilis. But upon treatment with PON1, its antibiotic activity was lost. These results suggest that PON1 may have a â-lactamase-like activity in addition to lactonase activity

Purification of human serum paraoxonase: a simple and rapid method

Paraoxonase/arylesterases (EC.3.1.8.2) is an enzyme found tightly associated with high density lipoprotein particle in serum. Because of its unique enzyme activity, antioxidant property and its role as an anti atherosclerotic molecule, various methods are used for its purification from human serum. Methods involved in its purification are elaborate and complicated. Also the yield and final activity are highly variable. Here, we report a 2 step method of purification involving affinity chromatography on cibacron blue sepharose followed by gel filtration on sephadex G50.The final preparation was 27.7 fold purified compared with the serum and gave a single band in SDS-PAGE by silver staining

First report of Fusarium oxysporum causing Fusarium wilt on Thuja orientalis in India

Fusarium oxysporum was recorded for the first time on Thuja orientalis trees in the Mysore University campus during 2006--07. Thuja trees are important because of their antifungal and antibacterial properties. The fungus was isolated from the wilted plant parts and subsequent reinoculation of the same to healthy plants confirmed the pathogen as the causal organism

Ebf1 heterozygosity results in increased DNA damage in pro-B cells and their synergistic transformation by Pax5 haploinsufficiency.

Ebf1 is a transcription factor with documented dose dependent functions in normal and malignant B-lymphocyte development. To understand more about the roles of Ebf1 in malignant transformation, we investigated the impact of reduced functional Ebf1 dosage on mouse B-cell progenitors. Gene expression analysis suggested that Ebf1 was involved in the regulation of genes important for DNA repair as well as cell survival. Investigation of the DNA damage in steady state as well as after induction of DNA damage by UV light, confirmed that pro-B cells lacking one functional allele of Ebf1 display signs of increased DNA damage. This correlated to reduced expression of DNA repair genes including Rad51 and chromatin immunoprecipitation data suggested that Rad51 is a direct target for Ebf1. Although reduced dosage of Ebf1 did not significantly increase tumor formation in mice, a dramatic increase in the frequency of pro-B cell leukemia was observed in mice with combined heterozygous mutations in the Ebf1 and Pax5 genes revealing a synergistic effect of combined dose reduction of these proteins. Our data suggest that Ebf1 controls DNA repair in a dose dependent manner providing a possible explanation to the frequent involvement of EBF1 gene loss in human leukemia

Abstracts of National Conference on Biological, Biochemical, Biomedical, Bioenergy, and Environmental Biotechnology

This book contains the abstracts of the papers presented at the National Conference on Biological, Biochemical, Biomedical, Bioenergy, and Environmental Biotechnology (NCB4EBT-2021) Organized by the Department of Biotechnology, National Institute of Technology Warangal, India held on 29–30 January 2021. This conference is the first of its kind organized by NIT-W which covered an array of interesting topics in biotechnology. This makes it a bit special as it brings together researchers from different disciplines of biotechnology, which in turn will also open new research and cooperation fields for them. Conference Title: National Conference on Biological, Biochemical, Biomedical, Bioenergy, and Environmental BiotechnologyConference Acronym: NCB4EBT-2021Conference Date: 29–30 January 2021Conference Location: Online (Virtual Mode)Conference Organizer: Department of Biotechnology, National Institute of Technology Warangal, Indi