Proton Countertransport and Coupled Gating in the Sarcoplasmic Reticulum Calcium Pump

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca²⁺ ions that functions via an “alternating-access” cycle mechanism. In each cycle, SERCA transports two Ca²⁺ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate.     

Hemoglobin patterns in different populations of Synbranchus marmoratus Bloch, 1795 (Pisces, Synbranchidae)

Total hemolysates of Synbranchus marmoratus Bloch, 1795 captured at four different sites in the State of S?o Paulo, Brazil, showed two different hemoglobin phenotypes when submitted to agar-starch gel electrophoresis on glass slides in basic buffer. Phenotype I was characterized by 3 hemoglobin bands. When the total hemolysate was submitted to cellulose acetate electrophoresis in basic buffer containing 6 M urea and beta-mercaptoethanol, Phenotype I showed four globins of the alpha 1, alpha 2, beta and gamma types, with 11.9 +/- 1.9 g% total hemoglobin, 45.3 +/- 3.6% globular volume, and 26.8 +/- 4.4% mean corpuscular hemoglobin concentration (MCHC). Phenotype II showed three groups of hemoglobins, with a total of up to 12 hemoglobin bands. When the total hemolysate was submitted to cellulose acetate electrophoresis in basic buffer containing 6 M urea and beta-mercaptoethanol, phenotype II showed five types of globins, denoted types alpha 1, alpha 2, gamma 1, gamma 2 and beta, having electrophoretic positions different from those of Phenotype I globins, with 18.1 +/- 3.3% total hemoglobin, 47.9 +/- 6.4% globular volume, and 37.8 +/- 4.4% MCHC. The distribution of the specimens having the two hemoglobin phenotypes is associated with the different geomorphological provinces of the State of S?o Paulo, suggesting the existence of at least two populational groups of Synbranchus marmoratus.     

In vivo immunological antitumor effect of OK-432-stimulated dendritic cell transfer after radiofrequency ablation

Radiofrequency ablation therapy (RFA) is a radical treatment for liver cancers and induces tumor antigen-specific immune responses. In the present study, we examined the antitumor effects of focal OK-432-stimulated dendritic cell (DC) transfer combined with RFA and analyzed the functional mechanisms involved using a murine model. C57BL/6 mice were injected subcutaneously with colon cancer cells (MC38) in their bilateral flanks. After the establishment of tumors, the subcutaneous tumor on one flank was treated using RFA, and then OK-432-stimulated DCs were injected locally. The antitumor effect of the treatment was evaluated by measuring the size of the tumor on the opposite flank, and the immunological responses were assessed using tumor-infiltrating lymphocytes, splenocytes and draining lymph nodes. Tumor growth was strongly inhibited in mice that exhibited efficient DC migration after RFA and OK-432-stimulated DC transfer, as compared to mice treated with RFA alone or treatment involving immature DC transfer. We also demonstrated that the antitumor effect of this treatment depended on both CD8-positive and CD4-positive cells. On the basis of our findings, we believe that combination therapy for metastatic liver cancer consisting of OK-432-stimulated DCs in combination with RFA can proceed to clinical trials, and it is anticipated to be markedly superior to RFA single therapy.     

Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction

A key step decisively affecting the catalytic efficiency of copper amine oxidase is stereospecific abstraction of substrate alpha-proton by a conserved Asp residue. We analyzed this step by pre-steady-state kinetics using a bacterial enzyme and stereospecifically deuterium-labeled substrates, 2-phenylethylamine and tyramine. A small and temperature-dependent kinetic isotope effect (KIE) was observed with 2-phenylethylamine, whereas a large and temperature-independent KIE was observed with tyramine in the alpha-proton abstraction step, showing that this step is driven by quantum mechanical hydrogen tunneling rather than the classical transition-state mechanism. Furthermore, an Arrhenius-type preexponential factor ratio approaching a transition-state value was obtained in the reaction of a mutant enzyme lacking the critical Asp. These results provide strong evidence for enzyme-enhanced hydrogen tunneling. X-ray crystallographic structures of the reaction intermediates revealed a small difference in the binding mode of distal parts of substrates, which would modulate hydrogen tunneling proceeding through either active or passive dynamics.     

Examination of drug resistance activity of human TAP-like (ABCB9) expressed in yeast

A half-type ABC transporter, human TAP-like (hTAPL) tagged with histidine cluster, was expressed in budding yeast protease-deficient strain BJ5457, and the effect of expression for resistance to peptide compounds including antibiotics and proteinase inhibitor was examined. Among these compounds, the yeast expressing hTAPL exhibits high sensitivity to valinomycin, a monovalent cation ionophore. A mutation in Walker A motif, which lost ATP-binding activity of hTAPL, eliminated the enhanced sensitivity to valinomycin. These findings suggest that the transport activity of hTAPL is important for conferring high valinomycin-sensitive phenotype to yeast.     

Dynamic redistribution of vasa homolog and exclusion of somatic cell determinants during germ cell specification in Ciona intestinalis

Ascidian embryos sequester a specific cytoplasm, called the postplasm, at the posterior pole, where many maternal RNAs and proteins accumulate. Although the postplasm is thought to act as the germ plasm, it is also highly enriched in several factors essential for somatic cell development, and how the postplasm components regulate both germ and somatic cell differentiation remains elusive. Using a vasa homolog, CiVH, and other postplasmic components as markers, we found that the postplasm-containing blastomeres, the B7.6 cells, undergo an asymmetric cell division during gastrulation to produce two distinct daughter cells: B8.11 and B8.12. Most of the postplasmic components segregate only into the B8.11 cells, which never coalesce into the gonad. By contrast, the maternal CiVH RNA and protein are specifically distributed into the B8.12 cells, which divide further and are incorporated into the gonad in juveniles. In the B8.12 cells, CiVH production is upregulated from the maternal RNA source, resulting in the formation of perinuclear CiVH granules, which may be the nuage, a hallmark of germ cells in many animal species. We propose that the redistribution of specific maternal molecules into the B8.12 cells is essential for germ-cell specification in ascidians.     

Water Extract of Ashwagandha Leaves Has Anticancer Activity: Identification of an Active Component and Its Mechanism of Action

Background

Cancer is a leading cause of death accounting for 15-20% of global mortality. Although advancements in diagnostic and therapeutic technologies have improved cancer survival statistics, 75% of the world population live in underdeveloped regions and have poor access to the advanced medical remedies. Natural therapies hence become an alternative choice of treatment. Ashwagandha, a tropical herb used in Indian Ayurvedic medicine, has a long history of its health promoting and therapeutic effects. In the present study, we have investigated an anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX).

Methodology/Principal Findings

Anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX) was detected by in vitro and in vivo assays. Bioactivity-based size fractionation and NMR analysis were performed to identify the active anticancer component(s). Mechanism of anticancer activity in the extract and its purified component was investigated by biochemical assays. We report that the ASH-WEX is cytotoxic to cancer cells selectively, and causes tumor suppression in vivo. Its active anticancer component was identified as triethylene glycol (TEG). Molecular analysis revealed activation of tumor suppressor proteins p53 and pRB by ASH-WEX and TEG in cancer cells. In contrast to the hypophosphorylation of pRB, decrease in cyclin B1 and increase in cyclin D1 in ASH-WEX and TEG-treated cancer cells (undergoing growth arrest), normal cells showed increase in pRB phosphorylation and cyclin B1, and decrease in cyclin D1 (signifying their cell cycle progression). We also found that the MMP-3 and MMP-9 that regulate metastasis were down regulated in ASH-WEX and TEG-treated cancer cells; normal cells remained unaffected.

Conclusion

We provide the first molecular evidence that the ASH-WEX and TEG have selective cancer cell growth arrest activity and hence may offer natural and economic resources for anticancer medicine.     

Detection of preclinically latent hyperperfusion due to stroke-like episodes by arterial spin-labeling perfusion MRI in MELAS patients

In stroke-like episodes (SEs) of patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), the detection of preclinically latent lesions is a challenge. We report regional cerebral hyperperfusion observed on arterial spin labeling (ASL) perfusion magnetic resonance imaging (MRI) in the preclinical phase more than 3 months before the clinical onset of SEs in 3 MELAS patients. These hyperperfused areas were not detected by conventional MRI in the preclinical phase and developed into acute lesions at the clinical onset of SEs, suggesting that ASL imaging has the potential for predicting the emergence of SEs.     

No Correlation between PNPLA3 rs738409 Genotype and Fatty Liver and Hepatic Cirrhosis in Japanese Patients with HCV

Background

Hepatitis C virus (HCV) infection is associated with the development of cirrhosis and hepatocellular carcinoma and is also related to fatty change of the liver. Variation in patatin-like phospholipase domain-containing 3 (PNPLA3) gene is associated with disease progression in nonalcoholic fatty liver disease (NAFLD). Recent reports have suggested that PNPLA3, IL28B and TLR4-associated single nucleotide polymorphisms (SNPs) may have an impact on hepatic steatosis or fibrosis in patients with chronic HCV infection.

Methods and Findings

Four SNPs (PNPLA3 rs738409, TLR4 rs4986790, TLR4 rs4986791, IL28B rs8099917) were identified in Japanese patients infected with HCV. We examined the association between the distribution of these SNP alleles and fatty change of the liver or existence of hepatic cirrhosis diagnosed by ultrasonography, one of the widely accessible and easy-to-use methods. PNPLA3 rs738409 G-allele and IL28B rs 8099917 minor allele were found in 70.0% and 31.1%, respectively. These two TLR4 SNPs were uniform in Japanese. Fatty change of the liver developed independent of the abscence of hepatic cirrhosis on sonographic findings and younger age. Hepatic cirrhosis was associated with a higher aspartate aminotransferase/platelet ratio index (APRI), no fatty change of the liver, higher BMI and higher AFP levels. No association between PNPLA3 rs738409/IL28B rs8099917 genotypes and hepatic steatosis or liver fibrosis was observed.

Conclusions

According to ultrasound examinations, no association between PNPLA3 rs738409 genotype and fatty change of the liver or hepatic cirrhosis was found in Japanese patients infected with HCV. Together, our results suggested that the mechanism of hepatic steatosis underlying HCV infection might differ from that of NAFLD and should be explored.