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Biomedical subjects

Norbert Weiss

Publications and source records attributed to Norbert Weiss.

At least 19 recordsLinked to original sources
PubMed2007

[Role of P/Q calcium channel in familial hemiplegic migraine].

Voltage-dependent calcium channels constitute one of the main pathways of calcium entry into neurons. They are the principal actors of synaptic transmission by controlling the release of neurotransmitters. They also contribute to numerous other cell functions, such as gene expression or synaptogenesis. These channels, by their essential cell functions, are at the origin of numerous channelopathies resulting from mutations of the genes encoding their different subunits. Familial Hemiplegic Migraine (FHM) represents one such example of these channelopathies. In this human disease, genetic studies have demonstrated the implication of the CACNA1A gene in a type 1 form of FHM. This gene encodes for the Ca(v)2.1 subunit of P/Q calcium channels and is the target of numerous mutations affecting the properties of channel activity. The question on how discrete mutations of this gene are able to alter the activity of the channel and contribute to the physiopathology of FHM remains an open question. The functional characterization of mutated channels in various heterologous expression systems, as well as in vivo in an animal model, provides a molecular scheme of the physiopathology of FHM in which neurons, astrocytes and blood circulation act in concert.

Amino Acid Sequence↗
PubMed2006

Importance of voltage-dependent inactivation in N-type calcium channel regulation by G-proteins.

Direct regulation of N-type calcium channels by G-proteins is essential to control neuronal excitability and neurotransmitter release. Binding of the G(betagamma) dimer directly onto the channel is characterized by a marked current inhibition ("ON" effect), whereas the pore opening- and time-dependent dissociation of this complex from the channel produce a characteristic set of biophysical modifications ("OFF" effects). Although G-protein dissociation is linked to channel opening, the contribution of channel inactivation to G-protein regulation has been poorly studied. Here, the role of channel inactivation was assessed by examining time-dependent G-protein de-inhibition of Ca(v)2.2 channels in the presence of various inactivation-altering beta subunit constructs. G-protein activation was produced via mu-opioid receptor activation using the DAMGO agonist. Whereas the "ON" effect of G-protein regulation is independent of the type of beta subunit, the "OFF" effects were critically affected by channel inactivation. Channel inactivation acts as a synergistic factor to channel activation for the speed of G-protein dissociation. However, fast inactivating channels also reduce the temporal window of opportunity for G-protein dissociation, resulting in a reduced extent of current recovery, whereas slow inactivating channels undergo a far more complete recovery from inhibition. Taken together, these results provide novel insights on the role of channel inactivation in N-type channel regulation by G-proteins and contribute to the understanding of the physiological consequence of channel inactivation in the modulation of synaptic activity by G-protein coupled receptors.

Animals↗
PubMed2006

Introducing an alternative biophysical method to analyze direct G protein regulation of voltage-dependent calcium channels.

Direct G protein inhibition of voltage-dependent calcium channels is currently indirectly assessed by the gain of current produced by depolarizing prepulse potentials (PP). Indeed, PPs produce a channel opening- and time-dependent dissociation of G proteins from the channel that is responsible for the increase in Ca(2+) permeation. Parameters of G protein dissociation are essential to describe the characteristic landmark modifications in channel activities that underlie G protein regulation. From the kinetics and opening-dependence of this dissociation, crucial biophysical parameters are extracted such as the extent and the rate of G protein unbinding from the channel. Unfortunately, the method used so far assumes that G protein regulated channels undergo the same inactivation kinetics than control channels. Herein, we demonstrate for the first time that G protein-bound channels undergo a much slower inactivation than control channels. We thus introduce a novel simple-to-use method that avoids the use of PPs and that is not affected by potential changes in channel inactivation kinetics conferred by G protein binding. This method extracts G protein unbinding parameters from ionic currents induced by regular depolarizing pulses by separating the ionic currents due to non-regulated channels from the ionic currents that result from a progressive departure of G proteins from regulated channels.

Animals↗
PubMed2006

Contribution of the kinetics of G protein dissociation to the characteristic modifications of N-type calcium channel activity.

Direct G protein inhibition of N-type calcium channels is recognized by characteristic biophysical modifications. In this study, we quantify and simulate the importance of G protein dissociation on the phenotype of G protein-regulated whole-cell currents. Based on the observation that the voltage-dependence of the time constant of recovery from G protein inhibition is correlated with the voltage-dependence of channel opening, we depict all G protein effects by a simple kinetic scheme. All landmark modifications in calcium currents, except inhibition, can be successfully described using three simple biophysical parameters (extent of block, extent of recovery, and time constant of recovery). Modifications of these parameters by auxiliary beta subunits are at the origin of differences in N-type channel regulation by G proteins. The simulation data illustrate that channel reluctance can occur as the result of an experimental bias linked to the variable extent of G protein dissociation when peak currents are measured at various membrane potentials. To produce alterations in channel kinetics, the two most important parameters are the extents of initial block and recovery. These data emphasize the contribution of the degree and kinetics of G protein dissociation in the modification of N-type currents.

Analgesics, Opioid↗
PubMed2006

[Voltage-dependent calcium channels at the heart of pain perception].

Voltage-dependent calcium channels represent a major pathway of calcium entry into neurons, where they participate actively to cell excitability and to the molecular processes of synaptic transmission. For that reason, they have been the direct or indirect pharmacological targets of analgesics and this long before their implication in the physiology of nociception had been demonstrated. These last years, the still more refined molecular characterization of these channels and their associated regulatory subunits and the demonstration of their implication in nociceptive processes indicates that these structures are prime pharmacological targets for the management of pain. Herein, we detail the recent breakthroughs on calcium channel structure, function and pharmacology, review the implication of calcium channels in the transmission of nociception, and evaluate their importance as targets for the treatment of pain perception. The search for specific inhibitors of voltage-dependent calcium channels appears as a prelude to the development of new promising analgesic molecules.

Afferent Pathways↗
PubMed2006

Aged garlic extract improves homocysteine-induced endothelial dysfunction in macro- and microcirculation.

Endothelial dysfunction caused by increases in vascular oxidant stress that decrease bioavailable nitric oxide (NO) plays a critical role in the vascular pathobiology of hyperhomocysteinemia. Boosting cellular glutathione levels or increasing the activity of cellular glutathione peroxidase can compensate for homocysteine's effects on endothelial function. Aged garlic extract (AGE) contains water- and oil-soluble sulfur compounds that modify the intracellular thiol and redox state, minimize intracellular oxidant stress, and stimulate NO generation in endothelial cells and animals. We performed a placebo-controlled, blinded, crossover trial to examine whether AGE reduces macro- and microvascular endothelial dysfunction during acute hyperhomocysteinemia induced by an oral methionine challenge in healthy subjects. Acute hyperhomocysteinemia leads to a significant decrease in flow-mediated vasodilation of the brachial artery as determined by vascular ultrasound, indicative of macrovascular endothelial dysfunction. In addition, acute hyperhomocysteinemia leads to a decrease in acetylcholine-stimulated skin perfusion as measured by laser-Doppler flowmetry. This indicates microvascular endothelial dysfunction, which is presumably a result of impairment of the endothelium-derived hyperpolarizing factor pathway. Pretreatment with AGE for 6 wk significantly diminished the adverse effects of acute hyperhomocysteinemia in both vascular territories. We conclude that AGE may at least partly prevent a decrease in bioavailable NO and endothelium-derived hyperpolarizing factor during acute hyperhomocysteinemia. This pilot study warrants further investigations on the effects of AGE on endothelial dysfunction in patients with other cardiovascular risk factors or established vascular disease and on the clinical outcome of patients with cardiovascular disease.

Biological Availability↗
PubMed2006

Aged garlic extract inhibits homocysteine-induced CD36 expression and foam cell formation in human macrophages.

Elevated plasma homocysteine (Hcy) levels have been recognized as an independent risk factor for atherosclerotic vascular disease. During formation of early atherosclerotic lesions, expression of CD36, a class B scavenger receptor on macrophages, is crucially involved in the uptake of oxidized low-density lipoprotein (OxLDL) and foam-cell formation. We therefore determined the effects of Hcy on CD36 expression and foam cell formation in human monocytes/macrophages (THP-1) using flow cytometry, and the effects of aged garlic extract (AGE) on this process. Incubation of THP-1 cells with Hcy (200 micromol/L) for 72 h in the presence of phorbol 12-myristate 13-acetate (PMA) (10 nmol/L) caused a 37.8+/-5.2% increase in CD36 expression compared with PMA-stimulated cells without Hcy (P<0.01). Coincubation with AGE (5 g/L) significantly suppressed CD36 expression by 61.8+/-13.9%, compared with control conditions, and by 48.6+/-9.0% compared with Hcy-incubated cells (P<0.01). THP-1 cells in the presence of PMA (10 nmol/L) were incubated with Hcy or AGE for 72 h followed by incubation with 1,1'-dioctadecyl-3,3,3'3'-tetra-methylindocyanide percholorate (DiI)-labeled OxLDL for 3 h, and fluorescence intensity was measured by flow cytometry. AGE also inhibited DiI-labeled OxLDL uptake into PMA-stimulated THP-1 cells by 85.6+/-2.8% (P<0.01), but Hcy had no effects on it. Our data indicate that AGE inhibits CD36 expression and OxLDL uptake in macrophages and suggest that the extract could modulate the formation of early atherosclerotic lesions.

Antigens, CD↗
PubMed2005

Stereospecific and redox-sensitive increase in monocyte adhesion to endothelial cells by homocysteine.

OBJECTIVE: Previous studies have shown that elevated homocysteine (Hcy) levels promote the development of atherosclerotic lesions in atherosclerosis-prone animal models. There is evidence that oxidant stress contributes to Hcy's deleterious effects on the vasculature. The accumulation and adhesion of monocytes to the vascular endothelium is a critical event in the development of atherosclerosis. We investigated the effects of Hcy on the interaction between human endothelial cells (EC) (EC line EA.hy 926 and primary human umbilical vein EC [HUVEC]) and the monocytic cell line THP-1, and the impact of vascular oxidant stress and redox-sensitive signaling pathways on these events. METHODS AND RESULTS: L-Hcy, but not D-Hcy, increases the production of reactive oxygen species inside EC, enhances nuclear factor(NF)-kappaB activation, and stimulates intercellular adhesion molecule-1 (ICAM-1) RNA transcription and cell surface expression. This leads to a time- and dose-dependent increase in monocyte adhesion to ECs. Pretreatment of ECs with superoxide scavengers (MnTBAP and Tiron) or with an inhibitor of NF-kappaB activation abolished Hcy-induced monocyte adhesion, ICAM-1 expression, and nuclear translocation of NF-kappaB. CONCLUSIONS: These findings suggest that reactive oxygen species produced under hyperhomocysteinemic conditions may induce a proinflammatory situation in the vessel wall that initiates and promotes atherosclerotic lesion development.

Atherosclerosis↗
PubMed2005

How do G proteins directly control neuronal Ca2+ channel function?

Ca2+ entry into neuronal cells is modulated by the activation of numerous G-protein-coupled receptors (GPCRs). Much effort has been invested in studying direct G-protein-mediated inhibition of voltage-dependent CaV2 Ca2+ channels. This inhibition occurs through a series of convergent modifications in the biophysical properties of the channels. An integrated view of the structural organization of the Gbetagamma-dimer binding-site pocket within the channel is emerging. In this review, we discuss how variable geometry of the Gbetagamma binding pocket can yield distinct sets of channel inhibition. In addition, we propose specific mechanisms for the regulation of the channel by G proteins that take into account the regulatory input of each Gbetagamma binding element.

Amino Acid Sequence↗
PubMed2005

Cellulomonas bogoriensis sp. nov., an alkaliphilic cellulomonad.

An alkaliphilic, slightly halotolerant, chemo-organotrophic, Gram-positive, rod-shaped bacterium, strain 69B4(T), was isolated from the sediment of the littoral zone of Lake Bogoria, Kenya. Phylogenetically, it is a member of the genus Cellulomonas, showing less than 97.5 % sequence similarity to the type strains of other Cellulomonas species. The highest level of similarity, albeit moderate, was found with respect to Cellulomonas cellasea DSM 20118(T). Chemotaxonomic properties confirm the 16S rRNA gene-based generic affiliation, i.e. a DNA G+C content of 71.5 mol%, anteiso-C(15:0) and C(16:0) as the major fatty acids, MK-9(H(4)) as the major isoprenoid quinone, a peptidoglycan containing L-ornithine as the diamino acid and D-aspartic acid in the interpeptide bridge and phosphatidylglycerol as the only identified main polar lipid. The strain is aerobic to facultatively anaerobic, being capable of growth under strictly anaerobic conditions. Optimal growth occurs between pH values 9.0 and 10.0. On the basis of its distinct phylogenetic position and metabolic properties, strain 69B4(T) represents a novel species of the genus Cellulomonas, for which the name Cellulomonas bogoriensis sp. nov. is proposed. The type strain is 69B4(T) (=DSM 16987(T)=CIP 108683(T)).

Bacterial Typing Techniques↗
PubMed2004

Cavbeta-subunit displacement is a key step to induce the reluctant state of P/Q calcium channels by direct G protein regulation.

P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary beta(3)-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.

Animals↗
PubMed2004

L-Homocysteine and L-homocystine stereospecifically induce endothelial nitric oxide synthase-dependent lipid peroxidation in endothelial cells.

Atherothrombotic cardiovascular disease associated with hyperhomocysteinemia has been proposed to result, at least in part, from increased vascular oxidative stress. Here we characterize one mechanism by which homocyteine may induce a vascular cell type-specific oxidative stress. Our results show that L-homocysteine at micromolar levels stereospecifically increases lipid peroxidation in cultured endothelial cells, but not in vascular smooth muscle cells or when medium is incubated in the absence of cells. Consistent with these observations, homocysteine also increases the formation of intracellular reactive oxygen species. The pro-oxidant effect of homocysteine can be fully replicated by an equivalent concentration of homocystine (i.e., an oxidized form of homocysteine), but not with cysteine or glutathione. Homocyst(e)ine-dependent lipid peroxidation is independent of H(2)O(2) and alterations in glutathione peroxidase activity, but dependent on superoxide. Mechanistically, the pro-oxidant effect of homocysteine appears to involve endothelial nitric oxide synthase (eNOS), as it is blocked by the eNOS inhibitor L-N(G)-nitroarginine methyl ester. Thus, homocyst(e)ine actively promotes oxidative stress in endothelial cells via an eNOS-dependent mechanism.

Animals↗
PubMed2004

Effect of 5-lipoxygenase on the development of pulmonary hypertension in rats.

5-Lipoxygenase (5-LO) and its downstream leukotriene products have been implicated in the development of pulmonary hypertension. In this study, we examined the effects of 5-LO overexpression in rat lungs on pulmonary hypertension using a recombinant adenovirus expressing 5-LO (Ad5-LO). Transthoracic echocardiography and right heart catheterization data showed that 5-LO overexpression in the lung did not cause pulmonary hypertension in normal rats; however, it markedly accelerated the progression of pulmonary hypertension in rats treated with monocrotaline (MCT). An increase in pulmonary artery pressure occurred earlier in the rats treated with MCT + Ad5-LO (7-10 days) compared with those treated with control vector, MCT + adenovirus expressing green fluorescent protein (AdGFP), or MCT alone (15-18 days). The weight ratio of the right ventricle to left ventricle plus septum was higher in the MCT + Ad5-LO group than that of the MCT + AdGFP or MCT group (0.45 +/- 0.08 vs. 0.35 +/- 0.03 or 0.33 +/- 0.06). Lung tissue histological sections from MCT + Ad5-LO rats exhibited more severe inflammatory cell infiltration and pulmonary vascular muscularization than those from MCT + AdGFP- or MCT-treated rats. Administration of 5-LO inhibitors, zileuton or MK-886, to either MCT- or MCT + Ad5-LO-treated rats prevented the development of pulmonary hypertension. These data suggest that 5-LO plays a critical role in the progression of pulmonary hypertension in rats and that the detrimental effect of 5-LO is manifest only in the setting of pulmonary vascular endothelial cell dysfunction.

Animals↗
PubMed2004

Gulosibacter molinativorax gen. nov., sp. nov., a molinate-degrading bacterium, and classification of 'Brevibacterium helvolum' DSM 20419 as Pseudoclavibacter helvolus gen. nov., sp. nov.

A Gram-positive, molinate-degrading bacterium, strain ON4(T) (=DSM 13485(T)=LMG 21909(T)), was isolated from a mixed bacterial culture able to mineralize the herbicide molinate. The strain was strictly aerobic, oxidase- and catalase-positive and non-acid-fast, with a growth temperature of 10-41 degrees C. It contained the major menaquinone MK-9 and a cell-wall peptidoglycan based on D-ornithine. 16S rDNA sequence analysis revealed that the strain formed a distinct line of descent in the family Microbacteriaceae, showing the highest 16S rDNA similarity ( approximately 95 %) to members of the genus Curtobacterium and 'Brevibacterium helvolum' DSM 20419 (=ATCC 13715). The latter was reported to have the cell-wall peptidoglycan type B2gamma and the major menaquinone MK-9, which are typical of Clavibacter, but it is clearly separated from this genus at the phylogenetic level. Based on low values of 16S rDNA sequence similarity to previously described genera and their distinctive phenotypic characteristics, it is proposed that strains ON4(T) and 'B. helvolum' DSM 20419 be classified as two novel genera and species, with the respective names Gulosibacter molinativorax gen. nov., sp. nov. and Pseudoclavibater helvolus gen. nov., sp. nov.

Actinomycetales↗
PubMed2004

Repositioning of charged I-II loop amino acid residues within the electric field by beta subunit as a novel working hypothesis for the control of fast P/Q calcium channel inactivation.

We have investigated the contribution of the Ca(v)beta subunits to the process of inactivation dependent of the I-II loop of Ca(v)alpha(2.1). Two amino acid residues located in the alpha1 interaction domain (AID) of the I-II loop of Ca(v)alpha(2.1) (Arg(387) and Glu(388)) have been directly implicated in voltage-dependent inactivation of this channel. Various point mutations of these residues disrupt the interaction between the I-II loop and the III-IV loop, and thereby modify the inactivation properties of the channel by accelerating its kinetics and shifting the steady-state inactivation curve towards hyperpolarized potentials. A similar disruption is produced by Ca(v)beta(4) subunit association with the I-II loop. Moreover, in the presence of Ca(v)beta(4) subunit, introducing negatively charged residues at positions 387 or 388 slows inactivation kinetics down, whereas introducing positive charges has the opposite effect. The shift of the steady-state inactivation curve is also amino acid charge-dependent. In contrast, mutation of Arg(387) or Glu(388) does not alter the differential regulation of the different Ca(v)beta isoforms on inactivation. These results suggest that the expression of Ca(v)beta(4) alters the contribution of charged residues at positions 387 and 388 to inactivation. We discuss these results with regard to the actual hypotheses on the mechanisms of calcium channel inactivation. We introduce the working concept that Ca(v)beta-subunits produce a conformational repositioning of charged AID residues within the electric field.

Alanine↗
PubMed2003

Inhibition of voltage-gated calcium channels by sequestration of beta subunits.

The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.

Animals↗
PubMed2003

Jeotgalicoccus halotolerans gen. nov., sp. nov. and Jeotgalicoccus psychrophilus sp. nov., isolated from the traditional Korean fermented seafood jeotgal.

Two Gram-positive, non-motile, non-spore-forming, halotolerant and moderately halophilic cocci (strains YKJ-101T and YKJ-115T) were isolated from the traditional Korean fermented seafood jeotgal, and were investigated using a polyphasic taxonomic approach. Phylogenetic analysis of 16S rDNA sequences showed that strains YKJ-101T and YKJ-115T are most closely related to the cluster comprising two Salinicoccus species. The peptidoglycan type of the strains is A3alpha, based on L-Lys-Gly(3-4)-L-Ala(Gly), and the predominant menaquinone is MK-7. Strains YKJ-101T and YKJ-115T have cellular fatty acid profiles containing major amounts of saturated, unsaturated and branched fatty acids; the major fatty acids are anteiso-C15 : 0 and iso-C15 : 0. The cellular polar lipids are phosphatidylglycerol, diphosphatidylglycerol and unidentified phospholipids. Strains YKJ-101T and YKJ-115T have identical DNA G + C contents of 42 mol%. The 16S rDNA similarity between strains YKJ-101T and YKJ-115T is 98% and the mean level of DNA-DNA relatedness between the two strains is 13.4%. On the basis of phenotypic and phylogenetic data and genomic distinctiveness, it is proposed that strains YKJ-101T and YKJ-115T should be placed in a new genus, Jeotgalicoccus gen. nov., as two distinct new species, for which the names Jeotgalicoccus halotolerans sp. nov. and Jeotgalicoccus psychrophilus sp. nov. are proposed. The type strains are YKJ-101T (=KCCM 41448T =JCM 11198T) and YKJ-115T (=KCCM 41449T =JCM 11199T), respectively.

Fatty Acids↗