Significance of anti-HBc screening of blood donors & its association with occult hepatitis B virus infection: Implications for blood transfusion.

Background & objective: Expansions of blood donor screening and improved laboratory detection of viral markers have remarkably reduced the risk for infection with transfusion-transmitted viruses. This study was aimed to evaluate the presence of anti-HBc and to determine the presence or absence of HBV DNA in the serum samples from HBsAg negative, anti-HBc positive blood donors in a tertiary care hospital blood bank from Delhi. Methods: A total of 2175 HBsAg negative, first time volunteer blood donors were included in the study from blood bank, Lok Nayak Hospital, New Delhi. The blood specimens from all these subjects were evaluated for anti-HBV-core antigen (anti-HBc) serology, anti-HBV-surface antigen (anti-HBs) titres and HBeAg. The presence of HBV DNA was evaluated by testing, through polymerase chain reaction (PCR) techniques. Results: Of the 2175 HBsAg negative voluntary blood donors, 413 (19.8%) were tested to be positive for anti-HBc alone. Of these, 153 (group-I) were anti-HBs negative whereas group-II comprises a total of 260 anti-HBs positive cases i.e. 89 out of 413 had anti-HBs titres of 10-99 IU/l and the remaining 171 had anti-HBs titres of 100-500 IU/l. HBV DNA was detected in 7.5 per cent anti-HBc positive samples irrespective of anti-HBs status. Interpretation & conclusions: Our results showed that 18.9 per cent of our donor population was anti-HBc reactive, and hence inclusion of anti-HBc testing will lead to a high discard rate. The presence of HBV DNA in fairly high percentage of anti-HBc positive samples highlighted the need for a stringent and better screening system to prevent occult HBV infection.

Effect of sodium nitroprusside on H+-ATPase activity and ATP concentration in Candida albicans.

ATP hydrolysis by plasma membrane H+-ATPase from Candida albicans has been investigated in presence of nitric oxide and various nutrients (sugars and amino acids). Sodium nitroprusside (SNP) was used as nitric oxide donor. It was found that ATP concentration decreased in SNP treated cells which was more in presence of sugars like glucose, xylose and 2-deoxy-D-glucose and amino acids as compared to their respective controls. The activity of H+-ATPase from plasma membrane decreased by 70 % in SNP treated cells. Both in vivo and in vitro treatments of SNP showed almost similar effects of decrease in ATPase activity. Effect of SNP was more pronounced in presence of nutrients. Interestingly, it was observed that vanadate did not show any independent effect in presence of nitric oxide. Several workers have reported similar type of results with other P-type ATPases. For the first time, it was observed in the present study that in presence of nitric oxide, H+-ATPase activity decreased like other P-type ATPases. Our study indicated that NO had a significant effect on ATP synthesis and activity of H+- ATPase. In the presence of NO, the ATP concentration was decreased indicating it affected mitochondrial electron transport chain. It may be concluded that NO, not only affects (inhibit) mitochondrial electron transport chain but also interferes with H+- ATPase of plasma membrane by changing its conformation resulting in decreased activity.

Effect of nitric oxide on H+ -efflux in presence of various nutrients in Candida albicans.

In the present study tentative link has been established between H+ -efflux and effect of NO in presence of various nutrients (glucose, 2-deoxy-D-glucose, xylose, proline, glutamic acid and lysine) in C. albicans using sodium nitroprusside (SNP) as a potent source of NO. It was observed that there was a decreasing trend in pH with time, in control, while SNP treated cells showed an initial decline in pH for 10-15 min, followed by an increase in pH up to 30 min. In presence of glucose there was an enhancement in H+ -efflux by 9-fold whereas proline, glutamic acid and lysine showed enhancement by 3, 6 and 1.5-fold respectively. Similar trends in increase in pH after 15 min in SNP treated cells of Candida was observed in presence of all nutrients used. It was demonstrated for the first time that H+ -ATPase of C. albicans was affected by NO.

Effect of phosphocreatine on H+ extrusion, pHi and dimorphism in Candida albicans.

Candida albicans is an opportunistic pathogen. Its proliferation in human hosts is believed to be controlled by immunologic mechanisms. The plasma membrane of the fungus possesses an H(+)-ATPase (PM-ATPase) which actively extrudes protons to generate an electrochemical gradient which is used in co-transport of nutrients. This ATPase is associated with the growth, dimorphism and pathogenicity of the fungus. The physiological concentration of phosphocreatine (PCr) is 20-35 mM in skeletal muscles. H(+)-extrusion in Candida cells was strongly inhibited by PCr; 44% at 20 mM and 69% at 40 mM. H(+)-extrusion was stimulated 6.2-fold in the presence of 10 mM glucose. This glucose stimulated extrusion was inhibited significantly by PCr; 36% at 20 mM and 53% at 40 mM. The intracellular pH pattern of cells destined to differentiate was greatly altered in the presence of PCr. Evagination time for control cells was between 90-120 min. PCr, delayed dimorphism, reduced the population of cells differentiating to hyphae and also reduced the length of hyphae after each time interval. Only 60% differentiation was observed with 10 mM PCr and 40% for higher PCr concentration even after 210 min. Direct interaction of PM-ATPase and PCr has been demonstrated by difference spectrum measurement employing stopped flow spectrophotometer. It can be concluded that PCr may be playing a significant role in checking growth and pathogenesis of C. albicans.

A kinetic scheme for the early phase of ATP hydrolysis by actomyosin ATPase and its bioenergetic implications.

By following the absorption pattern of the dye orthocresol red in stopped flow spectrophotometer we have studied the H+ liberation in the early phase of ATP hydrolysis by myosin and actomyosin ATPases at different molar ratios of ATP:ATPase. In the case of myosin alone, we observe alkalination up to a molar ratio of 10:1 and net acidification above 30:1. In the case of actomyosin, hydrolysis results in acidification for all ratios. Estimation of Pi generated in the early phase, employing a coupled enzyme system, gives Pi early burst magnitude of 6.2/head of myosin. Interpreting alkalination as release of HPO2(4-) unaccompanied by H+ in the case of myosin for molar ratios less than 10:1 together with the results of Pi estimation we deduce that between 6 to 10, H+ ions are withheld by myosin in the early burst phase. Polymerized actin was found to induce concomitant release of H+ during the early phase of ATP hydrolysis. A kinetic scheme is proposed for actomyosin ATPase which encompasses the pre-steady state as well. Bioenergetic significance of these protons held by the myosin heads for the process of muscular contraction is discussed.