Induction of Apoptosis in G1/S Blocked HeLa Cells by R-Roscovitine: A Preliminary Study

HeLa cells are human cervical cancer cells with HR HPV-18 genes integrated in the genome. The functions of tumor suppressor proteins p53 and pRB are abrogated and cell cycle regulation becomes nonfunctional. The aim of the present study was to investigate whether the CDK inhibitor R-Roscovitine would allow the G₁/S blocked HeLa cells to enter into mitosis prematurely and induce apoptosis. HeLa cells blocked in G₁/S border were treated with different concentrations of Roscovitine for 4 and 18 h respectively. Induction of apoptosis was studied by FACS and DNA fragmentation. Presence of γH2AX in the treated cells was studied by confocal microscopy. Expression levels of CASP3, CDKN1A i.e. p21 (Cip1/Waf1) and Bcl2 were studied by semi-quantitative RT-PCR to analyze the role played by these proteins in Roscovitine induced apoptosis in G₁/S blocked HeLa cells. Results indicate that the Roscovitine allowed the thymidine blocked HeLa cells to enter into mitosis prematurely. Presence of γH2AX loci in treated cells indicates DNA damage in prematurely mitotic cells. Analysis of DNA fragmentation and chromatin condensation confirmed apoptosis as the possible mechanism of Roscovitine induced cell death. Our results also reveal that Roscovitine induced apoptosis is associated with the overexpression of CASP3, p21 (cip1/waf1) and Bcl2.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

PprA Contributes to Deinococcus radiodurans Resistance to Nalidixic Acid,Genome Maintenance after DNA Damage and Interacts with Deinococcal Topoisomerases

PprA is known to contribute to Deinococcus radiodurans'' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA''s role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans''s capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB.     

Effect of organic anions on the crystallization of the Ca2(+)-ATPase of muscle sarcoplasmic reticulum

The effect of varying the solute species on the crystallization of the Ca2(+)-ATPase from rabbit muscle reticulum (SR) is reported. We have found that substitution of KCl with salts of organic acids in the crystallization protocol reported by Pikula et al. has a profound effect on the size of two-dimensional crystalline arrays. Crystalline arrays of up to 3 microns diameter have been obtained by incubating purified calcium ATPase in standard crystallization medium but with 0.8 M sodium propionate substituted for KCl. These two-dimensional (2-D) arrays display a reduced tendency to stack in addition to having larger planar dimensions. Increasing the KCl concentration does not have the same effect on stacking or crystal growth that sodium propionate has. The production of 2-D sheets has some dependence on the hydrocarbon chain length of the salt because crystals formed in propionate were larger and less stacked than those formed in acetate or formate. There seems to be no dependence on cation. These observations suggest that in addition to reducing the forces that lead to stacking of the sheets, propionate may facilitate incorporation of the detergent-solubilized protein into the 2-D sheet.     

Inhibition of superoxide dismutase by nitroprusside and electron spin resonance observations on the formation of a superoxide-mediated nitroprusside nitroxyl free radical

Nitroprusside appears to inhibit the known types of superoxide dismutases irrespective of their metal prosthetic group and regardless of the source from which the enzymes were isolated. Thus the copper-zinc enzyme from bovine erythrocyte or Neurospora crassa behaved identically as did the manganese enzymes from Escherichia coli or red alga and the iron enzyme from E. coli and a blue-green alga. The inhibition was dose dependent with a Ki = 2.5 X 10(-5) for nitroprusside. Nitroprusside does not bind to the copper moiety of copper-zinc enzyme and seems to compete with O2- for superoxide dismutase. These inhibitions by nitroprusside, which were elicited not only in purified enzymes but also in crude soluble extracts of biological samples, were rapidly reversible. Nitroprusside was found to react with O2- to form a paramagnetic species with three absorption lines of equal width with a separation AN = 15.0 G and a g value of 2.028. The spin adduct appears to be a nitroxide radical and was stable for several minutes.     

Penetration of soil aggregates of finite size

Summary Maximum penetrometer pressure was measured on artificial soil aggregates of finite size (2–29 mm) using blunt probes (total cone angle 60°) driven at 3 mm min⁻¹. Maximum penetrometer pressure increased asymptotically with increase in dimensionless aggregate radius,b/a, wherea andb are the probe and aggregate radii, respectively. A theory was developed for penetration of blunt probes into soil aggregates of finite size. The theory assumed that plastic failure occurs out to a radius,R, and that beyond this only elastic straining occurs. This theory can be applied to estimate the radial and tangential stresses adjacent to a blunt probe. The estimated radial and tangential stresses increased with increase in dimensionless aggregate radius,b/a. The radius of the plastic front,R, around the probe is predicted to increase with increased aggregate size. The results also demonstrate the effect of soil shear cohesion and internal friction angle onR. The results are discussed with reference to root penetration.