Promoter hypermethylation of tumour suppressor genes (p14/ARF and p16/INK4a): case–control study in North Indian population

The epigenetic modifications have been reported to be key factors in breast carcinogenesis. In the current study, it has been tried to determine the methylation status of two tumour suppressor genes p14/ARF and p16/INK4a in 150 breast cancer patients as well as 150 controls by using MSP-PCR. There was, highly significant difference in methylation of p14/ARF and p16/INK4a (P = 0.000) between patients and controls. Methylation of both the genes together significantly increased the risk of breast cancer by 12.31 folds. The present study concludes that hypermethylation of p14/ARF and p16/INK4a promoters demonstrate significant association with the risk of breast cancer, hence indicating these as important tumour suppressor genes involved in the pathogenesis of breast cancer in North Indian population (i.e. Punjab, Haryana, Uttar Pradesh, Himachal Pradesh as well as Union Territory of Chandigarh).     

The Blepharis persica seed hydroalcoholic extract synergistically enhances the apoptotic effect of doxorubicin in human colon cancer and gastric cancer cells

The goal of this survey is to evaluate the anti-proliferative effects of the hydroalcholic extract of Blepharis persica seeds and its synergic effect on doxorubicin (DOX) in human colon cancer (HT-29) and gastric cancer cell (AGS) lines. 70% Ethanol was used for extraction of B. persica seed. Aluminum–chloride colorimetric and Folin–Ciocalteu reagent methods were used to measure total flavonoid and total phenolic contents of the extract respectively. Gas chromatography-mass spectrometry (GC-MS) analysis of the B. persica extract was performed on GC-MS equipment after silylation. HT-29, AGS, and human fibroblast (SKM) cell lines were treated by different concentration of the B. persica extract, (DOX) and the combination of extraction and DOX. The cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay while the apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. The changes in expression levels of BAX and BCL-2 were determined using Real-Time RT-qPCR. GC-MS analysis of the hydroalcoholic extract from B. persica seeds revealed 24 major components. The MTT assay revealed the cytotoxicity against three cell lines and also it was shown that 125 ng/mL of DOX and 0.625 mg/mL of B. persica extract had synergistic behavior against HT29 cell line. These results showed B. persica extract induced apoptosis in AGS and HT29 cells and its extract caused dose-dependent increase in up-regulation of BAX level (p?<?0.05) and down-regulation of BCL₂ (p?<?0.05). B. persica showed the synergistic effect in combination with DOX on HT29 cell line. These findings demonstrated a basis for further studies on the characterization and mechanistic evaluation of the bioactive compounds of B. persica extract which had antiproliferative effects on cancer cell lines.

     

Caveolin-Na/K-ATPase interactions: Role of transmembrane topology in non-genomic steroid signal transduction

Progesterone and its polar metabolite(s) trigger the meiotic divisions in the amphibian oocyte through a non-genomic signaling system at the plasma membrane. Published site-directed mutagenesis studies of ouabain binding and progesterone-ouabain competition studies indicate that progesterone binds to a 23 amino acid extracellular loop of the plasma membrane α-subunit of Na/K-ATPase. Integral membrane proteins such as caveolins are reported to form Na/K-ATPase-peptide complexes essential for signal transduction. We have characterized the progesterone-induced Na/K-ATPase-caveolin (CAV-1)-steroid 5α-reductase interactions initiating the meiotic divisions. Peptide sequence analysis algorithms indicate that CAV-1 contains two plasma membrane spanning helices, separated by as few as 1-2 amino acid residues at the cell surface. The CAV-1 scaffolding domain, reported to interact with CAV-1 binding (CB) motifs in signaling proteins, overlaps transmembrane (TM) helix 1. The α-subunit of Na/K-ATPase (10 TM helices) contains double CB motifs within TM-1 and TM-10. Steroid 5α-reductase (6 TM helices), an initial step in polar steroid formation, contains CB motifs overlapping TM-1 and TM-6. Computer analysis predicts that interaction between antipathic strands may bring CB motifs and scaffolding domains into close proximity, initiating allostearic changes. Progesterone binding to the α-subunit may thus facilitate CB motif:CAV-1 interaction, which in turn induces helix-helix interaction and generates both a signaling cascade and formation of polar steroids.     

Mechanism of the control of (Na+ + K+)-ATPase by long-chain acyl coenzyme A

Long-chain fatty acid esters of CoA activate (Na+ + K+)-ATPase (the sodium pump) when ATP is suboptimal. To explore the nature of the interactions of these CoA derivatives with the pump, reversible effects of palmitoyl-CoA on the purified membrane-bound kidney enzyme were studied under conditions where interference from the irreversible membrane-damaging effect of the compound was ruled out. With 50 microM ATP, while saturating palmitoyl-CoA increased (Na+ + K+)-ATPase activity, it caused partial inhibition of Na+-ATPase activity without affecting the steady-state level of the phosphoenzyme. Palmitoyl-CoA did not change the K0.5 of ATP for Na+-ATPase, but it altered the complex Na+ activation curve to suggest the antagonism of the low-affinity, but not the high-affinity, Na+ sites. At a low ATP concentration (0.5 microM), K+ inhibited Na+-ATPase as expected. In the presence of palmitoyl-CoA and 0.5 microM ATP, however, K+ became an activator, as it is at high ATP concentrations. The activating effect of palmitoyl-CoA on (Na+ + K+)-ATPase activity was reduced with increasing pH (6.5-8.5), but its inhibitory effect on Na+-ATPase was not altered in this pH range. The data show two distinct actions of palmitoyl-CoA: 1) blockade of the extracellular "allosteric" Na+ sites whose exact role in the control of the pump is yet to be determined, and 2) activation of the pump through increased rate of K+ deocclusion. Since in their latter action the fatty acid esters of CoA are far more effective than ATP at a low-affinity regulatory site, we suggest that these CoA derivatives may be the physiological ligands of this regulatory site of the pump.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

(Na+ + K+)-ATPase: On the number of the ATP sites of the functional unit

Questions concerning the number of the ATP sites of the functional unit of (Na⁺ + K⁺)-ATPase (i.e., the sodium pump) have been at the center of the controversies on the mechanisms of the catalytic and transport functions of the enzyme. When the available data pertaining to the number of these sites are examined without any assumptions regarding the reaction mechanism, it is evident that although some relevant observations may be explained either by a single site or by multiple ATP sites, the remaining data dictate the existence of multiple sites on the functional unit. Also, while from much of the data it is clear that the multiple sites of the unit enzyme represent the interacting catalytic sites of an oligomer, it is not possible to rule out the existence of a distinct regulatory site for ATP in addition to the interacting catalytic sites. Regardless of the ultimate fate of the regulatory site, any realistic approach to the resolution of the kinetic mechanism of the sodium pump should include the consideration of the established site-site interactions of the oligomer.     

Biocontrol of Rhizoctonia solani damping-off of sugar beet with native Streptomyces strains under field conditions

A 3-year study was conducted to evaluate the effectiveness of two disease-suppressive Streptomyces spp. to control sugar beet Rhizoctonia solani damping off under field conditions. Streptomyces seed treatments reduced seedling damping off in naturally (2005) and artificially (2006 and 2007) infested soils. All biocontrol agents provided better efficacy than Vitavax to control seedling damping-off. There were no significant differences among Streptomyces isolates. Isolate C increased plant stand by 19.5, 50.5 and 53.75% in 2005, 2006 and 2007, respectively. Evaluation of final harvest revealed that the root yield of the biocontrol agents increased compared to untreated control in these years.