Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1–61) of p53 via an extended binding interface, with dissociation constant of 12.7 (± 0.7) μM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1–40) and TAD2 (residues 41–61) subdomains of p53. HmtSSB enhances intrinsic 3′-5′ exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) present at 3′-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.     

Inhibition of NF-kappaB sensitizes A431 cells to epidermal growth factor-induced apoptosis,whereas its activation by ectopic expression of RelA confers resistance

Epidermal growth factor (EGF) is a well known mitogen, but it paradoxically induces apoptosis in cells that overexpress its receptor. We demonstrate for the first time that the EGF-induced apoptosis is accelerated if NF-kappaB is inactivated. To inactivate NF-kappaB, human epidermoid carcinoma cells (A431) that overexpress EGF receptor were stably transfected with an IkappaB-alpha double mutant construct. Under the NF-kappaB-inactivated condition, A431 cells were more sensitive to EGF with decreased cell viability and increased externalization of phosphatidylserine on the cell surface, DNA fragmentation, and activation of caspases (3 and 8 but not 9), typical features of apoptosis. These results were further supported by the potentiation of the growth inhibitory effects of EGF by chemical inhibitors of NF-kappaB (curcumin and sodium salicylate) and the protective role of RelA evidenced by the resistance of A431-RelA cells (stably transfected with RelA) to EGF-induced apoptosis. EGF treatment or ectopic expression of RelA in A431 cells induced DNA binding activity of NF-kappaB (p50 and RelA) and the expression of c-IAP1, a downstream target of NF-kappaB. A431-RelA cells exhibited spontaneous phosphorylation of Akt (a downstream target of phosphatidylinositol 3-kinase and regulator of NF-kappaB) and EGF treatment stimulated it further. Blocking this basal Akt phosphorylation with LY294002, an inhibitor of phosphatidylinositol 3-kinase, did not affect their viability but blocking of EGF-induced phosphorylation of Akt sensitized the otherwise resistant A431-RelA cells to EGF-mediated growth inhibition. Our results favor an anti-apoptotic role for NF-kappaB in the regulation of EGF-induced apoptosis.     

Cashew nut shell oil as a potential feedstock for biodiesel production: An overview

Biodiesel outperforms diesel in emissions and engine performance. They burn efficiently in diesel engines and are eco-friendly. Since cashew nut shell liquid (CNSO) is waste, commercial biodiesel production from it should be profitable. CNSO is cheap and can reduce cashew processing factory waste. From cashew kernels, CNSL is extracted using various mechanical, thermal, and solvent extraction techniques. This article examines current research into using cashew nutshell liquid biodiesel (CNSLBD) in diesel engines. The work also discusses Indian biodiesel demand, availability, export information, life cycle cost analysis, cost economics of per hectare yield, Indian government initiative of CNSO. This review also evaluates the viability of this fuel as an alternative energy source. CNSLBD is a prospective alternative fuel that has the potential to benefit both the cashew nut industry and the energy industry. In addition to this, the study examines the procedures for extracting CNSO. According to the findings of the study, CNSO is a prospective alternative fuel that has the potential to benefit both the cashew nut industry and the energy industry.     

Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

 Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28–30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76±0.78 mg·g⁻¹ wet tissue in normal unexposed rats; 15.82±2.30 mg·g⁻¹ wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure. Glutamine synthetase activity in muscle was significantly higher in the 14-day exposed group (4.32 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹) in comparison to normal (1.53 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹); this parameter had decreased by 40% following 21 days of exposure. These results suggest that since no dramatic changes in the levels of protein were observed in the muscle and liver, there is an alteration in glutaminase and glutamine synthetase activity in order to maintain nitrogen metabolism in the initial phase of hypoxic exposure. Received: 30 March 1998 / Revised: 18 November 1998 / Accepted: 25 November 1998     

High-Risk Cervical Human Papillomavirus Infections among Human Immunodeficiency Virus-Positive Women in the Bahamas

Background

High-risk (HR) HPV genotypes other than 16 and 18 have been detected in a significant proportion of immunocompromised females. We aim to evaluate the frequency of HR HPV genotypes in a population of HIV-positive Caribbean women.

Methods

One hundred sixty-seven consecutive, non-pregnant, HIV-positive females ≥18 years were recruited in this study. Each participant received a vaginal examination, PAP smear, and completed a questionnaire. DNA was extracted for HPV testing in 86 patients.

Results

Mean age was 39.1 years for women positive for HR HPV and 43.1 years for women negative for HR HPV (P value  = 0.040). 78% (130/167) of the women had HR HPV infections; the prevalence of abnormal cervical cytology was 38% among women who were HR HPV-positive compared to women who were HR HPV-negative (22%). Fifty-one percent of the 86 women with available genotype carried infections with HPV 16 and/or HPV 18; genotypes of unknown risk were also frequently observed. Women who had a CD4+ count of ≤200 had 7 times increased odds of carrying HR HPV infection in comparison to women with CD4+>200.

Conclusions

HR HPV infections in HIV infected females may consist of more than just HPV 16 and 18, but also HPV 52 and 58. Further studies are needed to determine whether HPV 52 and 58 play a significant role in the development of cervical cytological abnormalities in HIV+ women.     

Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.