Decreased plasma levels of nitric oxide metabolites, angiotensin II, and aldosterone in spontaneously hypertensive rats exposed to 5 mT static magnetic field

Previously, we found that whole body exposure to static magnetic fields (SMF) at 10 mT (B(max)) and 25 mT (B(max)) for 2-9 weeks suppressed and delayed blood pressure (BP) elevation in young, stroke resistant, spontaneously hypertensive rats (SHR). In this study, we investigated the interrelated antipressor effects of lower field strengths and nitric oxide (NO) metabolites (NO(x) = NO(2)(-) + NO(3)(-)) in SHR. Seven-week-old male rats were exposed to two different ranges of SMF intensity, 0.3-1.0 mT or 1.5-5.0 mT, for 12 weeks. Three experimental groups of 20 animals each were examined: (1) no exposure with intraperitoneal (ip) saline injection (sham-exposed control); (2) 1 mT SMF exposure with ip saline injection (1 mT); (3) 5 mT SMF exposure with ip saline injection (5 mT). Arterial BP, heart rate (HR), skin blood flow (SBF), plasma NO metabolites (NO(x)), and plasma catecholamine levels were monitored. SMF at 5 mT, but not 1 mT, significantly suppressed and retarded the early stage development of hypertension for several weeks, compared with the age matched, unexposed (sham exposed) control. Exposure to 5 mT resulted in reduced plasma NO(x) concentrations together with lower levels of angiotensin II and aldosterone in SHR. These results suggest that SMF may suppress and delay BP elevation via the NO pathways and hormonal regulatory systems.     

v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.     

Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.     

Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben)

The bacterial diversity occurring in traditional Moroccan soft white cheese, produced in eight different regions in Morocco, was studied. A total of 164 lactic acid bacteria were isolated, purified and identified by whole-cell protein fingerprinting and rep-PCR genomic fingerprinting. The majority of the strains belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Enterococcus. Sixteen species were identified: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, Lactococcus garvieae, Lactococcus raffinolactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc citreum, Eterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus saccharominimus and Streptococcus sp.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

The 1.49 A resolution crystal structure of PsbQ from photosystem II of Spinacia oleracea reveals a PPII structure in the N-terminal region

We report the high-resolution structure of the spinach PsbQ protein, one of the main extrinsic proteins of higher plant photosystem II (PSII). The crystal structure shows that there are two well-defined regions in PsbQ, the C-terminal region (residues 46-149) folded as a four helix up-down bundle and the N-terminal region (residues 1-45) that is loosely packed. This structure provides, for the first time, insights into the crucial N-terminal region. First, two parallel beta-strands cross spatially, joining the beginning and the end of the N-terminal region of PsbQ. Secondly, the residues Pro9-Pro10-Pro11-Pro12 form a left-handed helix (or a polyproline type II (PPII) structure), which is stabilized by hydrogen bonds between the Pro peptide carbonyl groups and solvent water molecules. Thirdly, residues 14-33 are not visible in the electron density map, suggesting that this loop might be very flexible and presumably extended when PsbQ is free in solution. On the basis of the essential role of the N-terminal region of PsbQ in binding to PSII, we propose that both the PPII structure and the missing loop are key secondary structure elements in the recognition of specific protein-protein interactions between PsbQ and other oxygen-evolving complex extrinsic and/or intrinsic proteins of PSII. In addition, the PsbQ crystal coordinates two zinc ions, one of them is proposed to have a physiological role in higher plants, on the basis of the full conservation of the ligand protein residues in the sequence subfamily.     

Assembly of TbetaRI:TbetaRII:TGFbeta ternary complex in vitro with receptor extracellular domains is cooperative and isoform-dependent

Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.     

A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant-negative activity

Thyrotropin-releasing hormone is inactivated in the extracellular space by a membrane-bound peptidase, pyroglutamyl aminopeptidase II (PPII), a member of the M1 family of zinc metallopeptidases. The functional significance of multiple PPII RNA species expression is unknown. We detected, in rat tissues, a RNA species derived from an alternative processing at the exon 14-intron 14 boundary. The alternatively processed RNA encoded a shorter version of PPII (PPII*), lacking part of the C-terminal domain. PPII* was expressed in COS-7 (or C6 glioma) cells but it did not exhibit any PPII activity. Co-transfection of PPII and increasing amounts of PPII* expression vectors resulted in a dose-dependent reduction in PPII activity and the formation of covalent PPII-PPII* heterodimers. PPII* is therefore a powerful dominant-negative isoform of PPII, and heterodimerization may be its mechanism of action. Natural expression of shortened versions of M1 aminopeptidases may constitute a new mode of regulation of their activity.     

TGF-beta control of cell proliferation

This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.