Differential Effects of Cystathionine-γ-lyase-Dependent Vasodilatory H(2)S in Periadventitial Vasoregulation of Rat and Mouse Aortas

Background

Hydrogen sulfide (H₂S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H₂S are not fully understood. We tested the hypotheses that (1) H₂S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K_v) K⁺ channels and (2) that H₂S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway.

Methodology/Principal Findings

Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H₂S donors. KCNQ-type K_v channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H₂S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition.

Conclusions/Signficance

Our results demonstrate potent vasorelaxant effects of H₂S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H₂S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H₂S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H₂S/KCNQ pathways to influence states of vascular dysfunction may be possible.     

Electro-optical measurements on aqueous suspension of purple membrane from Halobacterium halobium

The permanent dipole moment, polarizability, and the retinal angle of Halobacterium halobium purple membranes were determined at different pH values. All of the parameters have a maximum between pH 5 and 6. There is a reversal in the direction of the permanent dipole moment near pH 5. The value of permanent dipole moment was determined to be 60 D/protein at pH 6.6, and the value obtained for polarizability was 3 X 10(-28) Fm2/membrane fragment. The retinal angle of all-trans retinal was 0.8 degrees smaller than that of the 13-cis conformation.     

Synthesis of hepcidin derivatives in order to develop standards for immune adsorption method

MeCN, acetonitrile; ECL, enhanced chemiluminescence; EDT, 1,2‐ethanedithiole; HEPC12‐A, rabbit anti‐human hepcidin IgG, affinity purified; HEPC13‐A, rabbit anti‐mouse/human hepcidin IgG, affinity purified; HEPC61‐P, human hepcidin‐25 control/blocking synthetic peptide; HRP, horseradish peroxidase; IL‐6, interleukin‐6; KLH, keyhole limpet hemocyanin; LEAP, liver‐expressed antimicrobial peptide; NEM, N‐ethylmaleimide; NMP, N‐methyl‐pirrolidone; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; SELDI‐TOF‐MS, surface‐enhanced laser desorption ionization–time‐of‐flight mass spectrometry; TMB, tetramethylbenzidin; TNF‐α, tumor necrosis factor‐α Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A five-year study of autotrophic winter picoplankton in Lake Balaton,Hungary

Picoeukaryotes dominate the phytoplankton of Lake Balaton—the largest shallow lake in Central Europe—in the winter period. We examined the annual dynamics of picoplankton abundance and composition in the lake in order to establish if the picoeukaryotes merely survive the harsher winter conditions or they are able to grow in the ice-covered lake when the entire phytoplankton is limited by low light and temperature. Lake Balaton has an annual temperature range of 1–29°C, and it is usually frozen between December and February for 30–60 days. In the spring-autumn period phycocyanin and phycoerythrin rich Cyanobacteria are the dominant picoplankters, and picoeukaryotes are negligible. Our five-year study shows the presence of three types of picophytoplankton assemblages in Lake Balaton: (1) Phycoerythrin-rich Cyanobacteria—the dominant summer picoplankters in the mesotrophic lake area; (2) Phycocyanin-rich Cyanobacteria—the most abundant summer picoplankters in the eutrophic lake area and; (3) Picoeukaryotes—the dominant winter picoplankters in the whole lake. The observed winter abundance of picoeukaryotes was high (up to 3 × 10⁵ cells ml⁻¹), their highest biomass (520 μg l⁻¹) exceeded the maximum summer biomass of picocyanobacteria (500 μg l⁻¹). Our results indicate that the winter predominance of picoeukaryotes is a regular phenomenon in Lake Balaton, irrespective of the absence or presence of the ice cover. Picoeukaryotes are able to grow at as low as 1–2°C water temperature, while the total phytoplankton biomass show the lowest annual values in the winter period. In agreement with earlier findings, the contribution of picocyanobacteria to the total phytoplankton biomass in Lake Balaton is inversely related to the total phytoplankton biomass, whereas no such relationship was observable in the case of picoeukaryotes.     

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.     

Potentiation of liposome-induced complement activation by surface-bound albumin

Large anionic multilamellar liposomes containing 71% membrane cholesterol (MLV) caused complement (C) activation in human serum in vitro, as reflected in significant rises in S protein-bound terminal complex (SC5b-9) and C3a-desarg levels. Increasing the albumin content in serum by 1-4 g/100 ml led to 50-100% further increase in MLV-induced C activation, while higher amounts of exogenous human serum albumin (HSA) gradually lost the capability to potentiate liposomal C activation. HSA alone had no influence on SC5b-9 formation at any level below 12%. Complement activation by liposomes and the potentiating effect of supplemental HSA were greatly reduced or eliminated in the absence of C1q or in the presence of 10 mM EGTA/2.5 mM Mg²⁺, pointing to the involvement of the classical pathway. Potentiation of C activation by supplemental HSA was not unique to MLV-induced activation, as deposition of HSA on the membrane of ‘Centricon’ ultrafiltration units also potentiated the C-activating effect of the polycarbonate membrane. Fatty acid (FA) or non-monomeric protein contamination in HSA were unlikely to be playing a role in the described effects, as 96% pure, FA-rich (Buminate) and 99% pure, FA-free HSA had identical effects on liposomal C activation. While highlighting a new modulatory mechanism on liposomal C activation, the above data raise the possibility that deposition of extravasated HSA at sites of tissue injury may serve a hitherto unrecognized proinflammatory function.     

An iron-containing superoxide dismutase from Anacystis nidulans.

Superoxide dismutase (SOD) was isolated and purified from Anacystis nidulans to near electrophoretic homogeneity. The enzyme has a molecular weight of 37,500, as determined by gel filtration and SDS-gel electrophoresis. The enzyme molecule consists of two subunits of identical molecular weight. Proton-induced X-ray elemental analysis (PIXE) showed that the SOD of A. nidulans is an iron-containing enzyme; the Fe:enzyme mol ratio was found to be 1. The EPR spectra indicated that the active center contains high-spin ferric ion. Based on quantitative EPR data, we conclude that eseentially all iron ions were detected in the EPR experiments and were present in the Fe3+ active center. Effective g'-values were calculated from computer-simulated spectra and analysis of the g'-value anisotropy of the +/-3/2 Kramers doublet made the calculation of crystal field parameters possible. The symmetry of the Fe3+ ion in the SOD molecule was found to be close to rhombic (E/D=0.240).     

Comparative analysis of the complete mitochondrial genomes of Aspergillus niger mtDNA type 1a and Aspergillus tubingensis mtDNA type 2b.

To understand the differences in the organization of mitochondrial genomes of the very closely related Aspergillus niger and Aspergillus tubingensis species, we determined the complete genome sequence of the 1a mtDNA type of A. niger and 2b mtDNA type of A. tubingensis and now we provide a comparative analysis of the two mtDNAs. We found that (1) the organization (gene content and order) of the two genomes is almost identical and (2) the size difference between them is principally attributed to the different intron content of their cox1, atp9 and ndh4L genes.     

Polyethylene-glycol induced fusion of bacterial protoplasts

Summary Direct selection for recombinants by supplemented minimal media from polyethylene-glycol (PEG)-induced fusion of protoplasts of polyauxotrophic strains of B. megaterium revealed striking physiological influences on the yield of recombinants. Cytoplasmic state of the protoplasts to be fused, rather than genetic events, determined the number of colonies obtained on the selection media. It is suggested that the physiological effects primarily influenced the ability of the fused protoplasts to revert to bacillary form.