NHE blockade inhibits chemokine production and NF-kappaB activation in immunostimulated endothelial cells

Na⁺/H⁺exchanger (NHE) activation has been documented to contribute toendothelial cell injury caused by inflammatory states. However, therole of NHEs in regulation of the endothelial cell inflammatoryresponse has not been investigated. The present study tested thehypothesis that NHEs contribute to endothelial cell inflammationinduced by endotoxin or interleukin (IL)-1. NHE inhibition usingamiloride, 5-(N-ethyl-N-isopropyl)-amiloride, and5-(N-methyl-N-isobutyl)amiloride as well as thenon-amiloride NHE inhibitors cimetidine, clonidine, and harmalinesuppressed endotoxin-induced IL-8 and monocyte chemoattractant protein(MCP)-1 production by human umbilical endothelial vein cells (HUVECs). The suppressive effect of amiloride on endotoxin-induced IL-8 production was associated with a decreased accumulation of IL-8 mRNA.NHE inhibitors suppressed both inhibitory (I)B degradation andnuclear factor (NF)-B DNA binding, suggesting that a decrease inactivation of the IB-NF-B system contributed to the suppression of HUVEC inflammatory response by NHE blockade. NHE inhibition decreased also the IL-1-induced HUVEC inflammatory response, becauseamiloride suppressed IL-1-induced E-selectin expression on HUVECs.These results demonstrate that maximal activation of the HUVECinflammatory response requires a functional NHE.

     

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.     

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions: IMPLICATIONS FOR BINDING SPECIFICITY*

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K_D values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m⁻¹ s⁻¹ and FGF-9, 130,000 m⁻¹ s⁻¹). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.     

The Role of Catechol-O-Methyl Transferase Val(108/158)Met Polymorphism (rs4680) in the Effect of Green Tea on Resting Energy Expenditure and Fat Oxidation: A Pilot Study

Introduction

Green tea(GT) is able to increase energy expenditure(EE) and fat oxidation(FATox) via inhibition of catechol-O-methyl transferase(COMT) by catechins. However, this does not always appear unanimously because of large inter-individual variability. This may be explained by different alleles of the functional COMT Val108/158Met polymorphism that are associated with COMT enzyme activity; high-activity enzyme, COMT^H(Val/Val genotype), and low-activity COMT^L(Met/Met genotype).

Methods

Fourteen Caucasian subjects (BMI: 22.2±2.3 kg/m², age: 21.4±2.2 years) of whom 7 with the COMT^H-genotype and 7 with the COMT^L-genotype were included in a randomized, cross-over study in which EE and substrate oxidation were measured with a ventilated-hood system after decaffeinated GT and placebo(PL) consumption.

Results

At baseline, EE, RQ, FATox and carbohydrate oxidation(CHOox) did not differ between groups. Significant interactions were observed between COMT genotypes and treatment for RQ, FATox and CHOox (p<0.05). After GT vs. PL, EE(GT: 62.2 vs. PL: 35.4 kJ.3.5 hrs; p<0.01), RQ(GT: 0.80 vs. PL: 0.83; p<0.01), FATox(GT: 18.3 vs. PL: 15.3 g/d; p<0.001) and CHOox(GT: 18.5 vs. PL: 24.3 g/d; p<0.001) were significantly different for subjects carrying the COMT^H genotype, but not for subjects carrying the COMT^L genotype (EE, GT: 60.3 vs. PL: 51.7 kJ.3.5 hrs; NS), (RQ, GT: 0.81 vs. PL: 0.81; NS), (FATox, GT: 17.3 vs. PL: 17.0 g/d; NS), (CHOox, GT: 22.1 vs. PL: 21.4 g/d; NS).

Conclusion

Subjects carrying the COMT^H genotype increased energy expenditure and fat-oxidation upon ingestion of green tea catechins vs, placebo, whereas COMT^L genotype carriers reacted similarly to GT and PL ingestion. The differences in responses were due to the different responses on PL ingestion, but similar responses to GT ingestion, pointing to different mechanisms. The different alleles of the functional COMT Val108/158Met polymorphism appear to play a role in the inter-individual variability for EE and FATox after GT treatment.

Trial Registration

Nederlands Trial register NTR1918     

Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture

Neurotransmission requires anterograde axonal transport of dense core vesicles (DCVs) containing neuropeptides and active zone components from the soma to nerve terminals. However, it is puzzling how one-way traffic could uniformly supply sequential release sites called en passant boutons. Here, Drosophila neuropeptide-containing DCVs are tracked in vivo for minutes with a new method called simultaneous photobleaching and imaging (SPAIM). Surprisingly, anterograde DCVs typically bypass proximal boutons to accumulate initially in the most distal bouton. Then, excess distal DCVs undergo dynactin-dependent retrograde transport back through proximal boutons into the axon. Just before re-entering the soma, DCVs again reverse for another round of anterograde axonal transport. While circulating over long distances, both anterograde and retrograde DCVs are captured sporadically in en passant boutons. Therefore, vesicle circulation, which includes long-range retrograde transport and inefficient bidirectional capture, overcomes the limitations of one-way anterograde transport to uniformly supply release sites with DCVs.     

Xenobiotic metabolizing enzyme gene polymorphisms predict response to lung volume reduction surgery

Background

In the National Emphysema Treatment Trial (NETT), marked variability in response to lung volume reduction surgery (LVRS) was observed. We sought to identify genetic differences which may explain some of this variability.

Methods

In 203 subjects from the NETT Genetics Ancillary Study, four outcome measures were used to define response to LVRS at six months: modified BODE index, post-bronchodilator FEV₁, maximum work achieved on a cardiopulmonary exercise test, and University of California, San Diego shortness of breath questionnaire. Sixty-four single nucleotide polymorphisms (SNPs) were genotyped in five genes previously shown to be associated with chronic obstructive pulmonary disease susceptibility, exercise capacity, or emphysema distribution.

Results

A SNP upstream from glutathione S-transferase pi (GSTP1; p = 0.003) and a coding SNP in microsomal epoxide hydrolase (EPHX1; p = 0.02) were each associated with change in BODE score. These effects appeared to be strongest in patients in the non-upper lobe predominant, low exercise subgroup. A promoter SNP in EPHX1 was associated with change in BODE score (p = 0.008), with the strongest effects in patients with upper lobe predominant emphysema and low exercise capacity. One additional SNP in GSTP1 and three additional SNPs in EPHX1 were associated (p < 0.05) with additional LVRS outcomes. None of these SNP effects were seen in 166 patients randomized to medical therapy.

Conclusion

Genetic variants in GSTP1 and EPHX1, two genes encoding xenobiotic metabolizing enzymes, were predictive of response to LVRS. These polymorphisms may identify patients most likely to benefit from LVRS.     

Contribution of the FtsQ transmembrane segment to localization to the cell division site

The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.     

The Natural Antimicrobial Carvacrol Inhibits Quorum Sensing in Chromobacterium violaceum and Reduces Bacterial Biofilm Formation at Sub-Lethal Concentrations

The formation of biofilm by bacteria confers resistance to biocides and presents problems in medical and veterinary clinical settings. Here we report the effect of carvacrol, one of the major antimicrobial components of oregano oil, on the formation of biofilms and its activity on existing biofilms. Assays were carried out in polystyrene microplates to observe (a) the effect of 0–0.8 mM carvacrol on the formation of biofilms by selected bacterial pathogens over 24 h and (b) the effect of 0–8 mM carvacrol on the stability of pre-formed biofilms. Carvacrol was able to inhibit the formation of biofilms of Chromobacterium violaceum ATCC 12472, Salmonella enterica subsp. Typhimurium DT104, and Staphylococcus aureus 0074, while it showed no effect on formation of Pseudomonas aeruginosa (field isolate) biofilms. This inhibitory effect of carvacrol was observed at sub-lethal concentrations (<0.5 mM) where no effect was seen on total bacterial numbers, indicating that carvacrol''s bactericidal effect was not causing the observed inhibition of biofilm formation. In contrast, carvacrol had (up to 8 mM) very little or no activity against existing biofilms of the bacteria described, showing that formation of the biofilm also confers protection against this compound. Since quorum sensing is an essential part of biofilm formation, the effect of carvacrol on quorum sensing of C. violaceum was also studied. Sub-MIC concentrations of carvacrol reduced expression of cviI (a gene coding for the N-acyl-L-homoserine lactone synthase), production of violacein (pigmentation) and chitinase activity (both regulated by quorum sensing) at concentrations coinciding with carvacrol''s inhibiting effect on biofilm formation. These results indicate that carvacrol''s activity in inhibition of biofilm formation may be related to the disruption of quorum sensing.     

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.     

Tuberculosis Treatment in HIV Infected Ugandans with CD4 Counts >350 Cells/mm3 Reduces Immune Activation with No Effect on HIV Load or CD4 Count

Background

Both HIV and TB cause a state of heightened immune activation. Immune activation in HIV is associated with progression to AIDS. Prior studies, focusing on persons with advanced HIV, have shown no decline in markers of cellular activation in response to TB therapy alone.

Methodology

This prospective cohort study, composed of participants within a larger phase 3 open-label randomized controlled clinical trial, measured the impact of TB treatment on immune activation in persons with non-advanced HIV infection (CD4>350 cells/mm³) and pulmonary TB. HIV load, CD4 count, and markers of immune activation (CD38 and HLA-DR on CD4 and CD8 T cells) were measured prior to starting, during, and for 6 months after completion of standard 6 month anti-tuberculosis (TB) therapy in 38 HIV infected Ugandans with smear and culture confirmed pulmonary TB.

Results

Expression of CD38, and co-expression of CD38 and HLA-DR, on CD8 cells declined significantly within 3 months of starting standard TB therapy in the absence of anti-retroviral therapy, and remained suppressed for 6 months after completion of therapy. In contrast, HIV load and CD4 count remained unchanged throughout the study period.

Conclusion

TB therapy leads to measurable decreases in immune activation in persons with HIV/TB co-infection and CD4 counts >350 cells/mm³.