Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p)

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.     

Genome-wide copy number analysis uncovers a new HSCR gene: NRG3

Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50 × 10(-5)), particularly for those encompassing genes (p = 5.00 × 10(-6)). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64 × 10(-3)). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36 × 10(-5)) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR-associated syndromes showed that while syndromic-HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50 × 10(-5)), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00 × 10(-6)) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.     

Discovery of nor-seco himbacine analogs as thrombin receptor antagonists

Discovery of a novel nor-seco himbacine analog as potent thrombin receptor (PAR-1) antagonist is described. Despite low plasma level, these new analogs showed excellent ex vivo efficacy in the monkey platelet aggregation assay. A potent hydroxy metabolite generated in vivo was identified as the agent responsible for the ex vivo efficacy. Following this discovery, the metabolite series was optimized to obtain analogs that showed very good ex vivo efficacy along with excellent pharmacokinetic profile in c. monkey.     

Association of cortactin and fascin-1 expression in gastric adenocarcinoma: correlation with clinicopathological parameters.

Cortactin and fascin-1 are important factors in tumor progression. We tested the hypothesis that cortactin and fascin-1 expression correlates with clinicopathological parameters of gastric adenocarcinoma. Immunohistochemical analysis of cortactin and fascin-1 was done using tissue microarrays of 100 surgical specimens, including 20 well-differentiated, 20 moderately differentiated, and 60 poorly differentiated gastric adenocarcinomas. Among the 20 well-differentiated gastric adenocarcinomas, 15 cases (75%) showed negative or weak staining (1+); 5 cases (25%) had moderate (2+) or strong (3+) cortactin expression. Among the 60 poorly differentiated gastric adenocarcinomas, more than three-quarters of the cases (76.7%) had moderate or strong cortactin expression; 14 cases (23.3%) had weak staining. Of 20 well-differentiated gastric adenocarcinoma cases, 14 (70%) showed negative or weak staining of fascin-1, whereas nearly one-third (30%) had moderate or strong expression. Among the 60 poorly differentiated gastric adenocarcinomas, 32 (53.3%) exhibited moderate or strong fascin-1 expression; fewer than half of the cases showed negative or weak staining. Higher intensity of cortactin and fascin-1 staining correlated directly with more-advanced cancer stages (TNM) and inversely with survival rates. Our findings suggest the possibility that pharmacological inhibitors of cortactin and fascin-1 activity may slow down tumor progression and prolong survival time in patients with gastric adenocarcinomas.     

Helicobacter pylori (H. pylori) molecular signature in conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma

Conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal marginal zone B-cell lymphoma that is characterized by an exaggerated clonal expansion of B cells, which implicate a pathological proliferative response to antigen(s) including bacteria. Helicobacter pylori (H. pylori) infection is recognized as one of the causative agents of gastric MALT lymphoma; however, it has not been reported in extra gastric MALT lymphoma. We studied 5 patients (4 adults and 1 child) with salmon-colored conjunctival lesions. One patient also had a history of abnormal bone marrow biopsy a year earlier with lymphoid aggregates involving 5% of the overall bone marrow. The conjunctival lesions of the 5 patients were biopsied. Histopathological diagnoses were consistent with conjunctival MALT lymphoma. Lymphoma and normal conjunctival cells were microdissected using laser capture microscopy or manual techniques. DNA was extracted and subjected to PCR amplification using H. pylori gene-specific primers from the urease B and vac/m2 gene. Cells from chronic conjunctivitis (normal lymphocytes), conjunctival human T-cell lymphotropic virus type-1/adult T-cell leukemia/lymphoma (HTLV-1/ATL), and orbital B-cell lymphoma were also microdissected, processed and analyzed. PCR amplification and Southern blot hybridization demonstrated H. pylori DNA in the conjunctival MALT lymphoma cells of 4/5 cases. The negative case was the one with a history of abnormal bone marrow. In contrast, H. pylori gene was not detected in normal conjunctival cells from the cases of MALT lymphoma or the lymphocytes, ATL and orbital B-lymphoma cells from the controls. These data suggest that H. pylori may play a role in conjunctival MALT lymphoma.     

OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L

OPA1, a dynamin-related guanosine triphosphatase mutated in dominant optic atrophy, is required for the fusion of mitochondria. Proteolytic cleavage by the mitochondrial processing peptidase generates long isoforms from eight messenger RNA (mRNA) splice forms, whereas further cleavages at protease sites S1 and S2 generate short forms. Using OPA1-null cells, we developed a cellular system to study how individual OPA1 splice forms function in mitochondrial fusion. Only mRNA splice forms that generate a long isoform in addition to one or more short isoforms support substantial mitochondrial fusion activity. On their own, long and short OPA1 isoforms have little activity, but, when coexpressed, they functionally complement each other. Loss of mitochondrial membrane potential destabilizes the long isoforms and enhances the cleavage of OPA1 at S1 but not S2. Cleavage at S2 is regulated by the i-AAA protease Yme1L. Our results suggest that mammalian cells have multiple pathways to control mitochondrial fusion through regulation of the spectrum of OPA1 isoforms.     

Anti-retroviral treatment outcomes among older adults in Zomba district, Malawi

Background

There are approximately 3 million people aged 50 and older in sub-Saharan Africa who are HIV-positive. Despite this, little is known about the characteristics of older adults who are on treatment and their treatment outcomes.

Methods

A retrospective cohort analysis was performed using routinely collected data with Malawi Ministry of Health monitoring tools from facilities providing antiretroviral therapy services in Zomba district. Patients aged 25 years and older initiated on treatment from July 2005 to June 2010 were included. Differences in survival, by age group, were determined using Kaplan–Meier survival plots and Cox proportional hazards regression models.

Results

There were 10,888 patients aged 25 and older. Patients aged 50 and older (N = 1419) were more likely to be male (P<0.0001) and located in rural areas (P = 0.003) than those aged 25–49. Crude survival estimates among those aged 50–59 were not statistically different from those aged 25–49 (P = 0.925). However, survival among those aged 60 and older (N = 345) was worse (P = 0.019) than among those 25–59. In the proportional hazards model, after controlling for sex and stage at initiation, survival in those aged 50–59 did not differ significantly from those aged 25–49 (hazard ratio 1.00 (95% CI: 0.79 to 1.27; P = 0.998) but the hazard ratio was 1.46 (95% CI: 1.03 to 2.06; P = 0.032) for those aged 60 and older compared to those aged 25–49.

Conclusions

Treatment outcomes of those aged 50–59 are similar to those aged 25–49. A better understanding of how older adults present for and respond to treatment is critical to improving HIV services.     

Enhanced resistance of restraint-stressed mice to sepsis

Sepsis remains a major health concern across the world. The effects of stress on host resistance to sepsis are still not very clear. To explore the effects of chronic stress on sepsis(') we examined the impact of restraint stress on the resistance of mice to sepsis. Interestingly, it was found that restraint stress enhanced the antisepsis resistance of mice and the concentrations of the proinflammatory cytokines IL-1, IL-6, IL-12, and TNF-alpha in the blood of stressed mice were dramatically reduced post Escherichia coli infection or LPS treatment as compared with that of controls (p < 0.05). In addition, the mRNA expressions of glucocorticoid-induced leucine zipper (GILZ) were up-regulated in the spleen and peritoneal macrophages of mice receiving restraint stress or dexamethasone treatment. These results demonstrate that restraint stress enhances the resistance of mice to sepsis, supporting corticotherapy for sepsis and proposing restraint-stressed mouse as an animal model to elucidate mechanisms of stress-associated, antisepsis resistance.     

Evaluating the potential of the extract of Perilla sp. as a natural insecticide for Bemisia tabaci (Hemiptera: Aleyrodidae) on sweet peppers

The sweetpotato whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), is a major pest on greenhouse crops including sweet pepper (Capsicum annuum L.), which is one of the leading greenhouse crops in South Korea. Synthetic insecticides, especially the neonicotinoids, have been used to conventionally control this pest. There have been continuous efforts to develop plant‐derived compounds as insecticides, deterrents, and repellents to reduce spraying synthetic insecticides. To develop new plant‐extract insecticides, we investigated the insecticidal effects of Perilla sp. (Perilla frutescens var. crispa) extract on B. tabaci in laboratory conditions. The Perilla sp. extract induced 90 % mortality within one hour, but phytotoxicity symptoms on sweet pepper leaves were also observed. We monitored the population change and spatial distribution of adult B. tabaci in an experimental sweet pepper greenhouse using yellow sticky traps, and analyzed distribution patterns by spatial analysis with distance indices (SADIE). Based on monitoring data and SADIE analysis, we concluded that B. tabaci aggregated near the greenhouse entrances, and it showed aggregation and association pattern as time passed. Therefore, we recommend spraying Perilla sp. extract near the entrances or wild host before the pest population penetrates. It will be one of the alternative pest management strategies to reduce B. tabaci population with fewer negative effects from chemical insecticide. Further study is required to reduce the phytotoxicity symptoms from Perilla sp. extract spray and insecticidal effect should be evaluated under field conditions.