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Physiological Relevance (physiological + relevance)
Selected AbstractsRegulatory Mechanisms and Physiological Relevance of a Voltage-Gated H+ Channel in Murine Osteoclasts: Phorbol Myristate Acetate Induces Cell Acidosis and the Channel Activation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2003Hiroyuki Mori Abstract The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. Introduction: H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings. Methods: To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). Results and Conclusions: Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (,pH), indicating that the H+ channel changed its activity by sensing ,pH. The reversal potential (Vrev) was a valuable tool for the real-time monitoring of ,pH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 ,M) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of Vrev, suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K+ -rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In ,80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the ,pH changes, implying that both ,pH-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward Vrev. These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C. [source] Serine/threonine kinase PKR: A sentinel kinase that discriminates a signaling pathway mediated by TLR4 from those mediated by TLR3 and TLR9AMERICAN JOURNAL OF HEMATOLOGY, Issue 7 2007Yusuke Asakura Abstract Cells of the innate immune system discriminate between "noninfectious self" and "infectious nonself" via pattern recognition receptors known as Toll-like receptors (TLRs). Though TLRs and the related interleukin 1 receptors share considerable homology in their cytoplasmic domains and adaptor molecules, signaling cascades may substantially differ from one another depending on the adaptor proteins recruited. Here we show that ectopic overexpression of catalytically inactive dominant-negative PKR expression system suppressed NF- , B activation mediated by TLR3, TLR9, TNF receptor 1 and 2 (TNF-R 1/2), but not by TLR4. Physiological relevance of the observations described here are discussed. Am. J. Hematol 2007. © 2006 Wiley-Liss, Inc. [source] Lectin-based electrophoretic analysis of the expression of the 35,kDa inter-,-trypsin inhibitor heavy chain H4 fragment in sera of patients with five different malignanciesELECTROPHORESIS, Issue 12 2008Emida Mohamed Abstract A 35,kDa glycoprotein whose abundance was previously demonstrated to be enhanced in sera of patients with endometrial adenocarcinoma (n,=,12), was isolated from pooled sera of three of the cancer patients using champedak galactose-binding lectin affinity chromatography in the present study. Subjecting it to 2-DE and MS/MS, the glycoprotein was identified as the O -glycosylated fragment of inter-,-trypsin inhibitor heavy chain H4 (ITIH4). When compared to control sera (n,=,17), expression of the 35,kDa ITIH4 cleavage fragment was demonstrated to be significantly enhanced in sera of patients with breast carcinoma (n,=,10), epithelial ovarian carcinoma (n,=,10), and germ cell ovarian carcinoma (n,=,10) but not in patients with nasopharyngeal carcinoma (n,=,13) and osteosarcoma (n,=,7). The lectin-based electrophoretic bioanalytical method adopted in the present study may be used to assess the physiological relevance of ITIH4 fragmentation and its correlation with different malignancies, their stages and progression. [source] Proteomic analysis of cellular responses to low concentration N -methyl- N,-nitro- N -nitrosoguanidine in human amnion FL cellsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2004Jinghua Jin Abstract We have shown previously that exposure to a low concentration of N -methyl- N,-nitro- N -nitrosoguanidine (MNNG) induces comprehensive changes in the protein expression profile of human amnion FL cells, including the induction, suppression, upregulation, and downregulation of various proteins. In addition, by proteomic analysis combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry, some of the induced and suppressed proteins were identified. In this study, we identified an additional 18 proteins among those that were either up- or downregulated by MNNG treatment. The proteins identified were a heterogeneous group that included several zinc finger proteins, proteins involved in signal transduction, cytoskeletal proteins, cell-cycle regulation proteins, and proteins with unknown functions. The involvement of these proteins in the cellular responses to alkylating agents has not been reported before and their physiological relevance is not clear. Therefore, our findings may help better understand the global cellular stress responses to chemical carcinogens, and may lead to new studies on the functions of these MNNG-responsive proteins. Furthermore, some of these proteins may serve as biomarkers for detecting exposure of human populations to environmental carcinogens. Environ. Mol. Mutagen. 43:93,99, 2004. © 2004 Wiley-Liss, Inc. [source] Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2008Akiko Nagasaka Abstract Osteopontin (OPN), originally discovered in bone as an extracellular matrix protein, was identified in many cell types in the immune system, presumably being involved in many aspects of pathogenesis of inflammatory and immune diseases. Mast cells are also involved in such pathological aspects by secreting multiple mediators. However, it has not been determined whether mast cells produce OPN and whether it affects their function. To test this, we used murine fetal skin-derived cultured mast cells (FSMC) and bone marrow-derived cultured mast cells. We found that OPN was spontaneously produced by FSMC and inducible by ionomycin and Fc,RI aggregation in bone marrow-derived cultured mast cells. In the presence of mast cell growth factors, FSMC were similarly generated from both OPN-deficient (OPN,/,) and -sufficient (OPN+/+) mice without significant differences in yield, purity, granularity, and viability. Using OPN,/, FSMC, we found that recombinant OPN augmented IgE-mediated degranulation and induced FSMC chemotaxis. Both effects were mediated by OPN receptors (i.e. CD44 and integrin,,v). IgE-mediated passive cutaneous anaphylaxis was significantly reduced in OPN,/, mice compared with OPN+/+ mice, indicating physiological relevance of OPN. These results indicate that OPN is a mast cell mediator, enhances mast cell responses to antigen, and thus may influence mast cell-related pathological conditions. See accompanying commentary at http://dx.doi.org/10.1002/eji200738131 [source] Endocannabinoids mediate muscarine-induced synaptic depression at the vertebrate neuromuscular junctionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Zachary Newman Abstract Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using the Ceratomandibularis muscle from the lizard Anolis carolinensis we asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB1 cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M3 muscarinic acetylcholine (ACh) receptors. N -(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB1 antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB1 receptor agonist, mimics M3 activation and occludes the effect of muscarine. As for its mechanism of action, ACPA reduces the action-potential-evoked calcium transient in the nerve terminal and this decrease is more than sufficient to account for the observed inhibition of neurotransmitter release. Similar to muscarine, the inhibition of synaptic transmission by ACPA requires nitric oxide, acting via the synthesis of cGMP and the activation of cGMP-dependent protein kinase. 2-Arachidonoylglycerol (2-AG) is responsible for the majority of the effects of eCB as inhibitors of phospholipase C and diacylglycerol lipase, two enzymes responsible for synthesis of 2-AG, significantly limit muscarine-induced inhibition of neurotransmitter release. Lastly, the injection of (5Z,8Z,11Z,14Z)- N -(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (an inhibitor of eCB transport) into the muscle prevents muscarine, but not ACPA, from inhibiting ACh release. These results collectively lead to a model of the vertebrate neuromuscular junction whereby 2-AG mediates the muscarine-induced inhibition of ACh release. To demonstrate the physiological relevance of this model we show that the CB1 antagonist N -(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide prevents synaptic inhibition induced by 20 min of 1-Hz stimulation. [source] Gene Transfer Strategies for the PhysiologistEXPERIMENTAL PHYSIOLOGY, Issue 6 2000Liang-Fong Wong Foreign genes can be introduced into whole animals using methods of germline transgenesis and somatic gene delivery. While germline transgenesis can generate useful animal models for genetic studies, it can be costly, time-consuming and requires the use of a large number of animals. An alternative means of gene transfer is to deliver genes to somatic cells using non-viral and viral technologies. Non-viral methods such as naked DNA injection, electroporation and liposome/cation lipid-mediated gene transfer are relatively inefficient. In contrast, viruses are effective vehicles that carry foreign genes into a cell rapidly and efficiently. Here we illustrate the usefulness of adenoviral vectors to express a potent and specific inhibitor of cAMP-dependent protein kinase (PKA) to study the role of cyclic 3,,5,-cyclic AMP (cAMP) in the osmotic regulation of the vasopressin gene in a transgenic rat model. The ability to modify endogenous systems within specific cells in a whole animal model allows gene effects to be studied with physiological relevance. The combination of molecular biology and integrative physiology is a powerful application that can aid in the elucidation of how gene function can translate into complex systems in an organism [source] Transcription elongation factor S-II maintains transcriptional fidelity and confers oxidative stress resistanceGENES TO CELLS, Issue 10 2003Hiroshi Koyama Background:, During transcription elongation, RNA polymerase II is arrested on the template when incorrect ribonucleotides are incorporated into the nascent transcripts. Transcription factor S-II enhances the excision of these mis-incorporated nucleotides by RNA polymerase II and stimulates transcription elongation in vitro. This mechanism is considered to be transcriptional proof-reading, but its physiological relevance remains unknown. Results:, We report that S-II contributes to the maintenance of transcriptional fidelity in vivo. We employed a genetic reporter assay utilizing a mutated lacZ gene from which active ,-galactosidase protein is expressed when mRNA proof-reading is compromised. In S-II-disrupted mutant yeasts, ,-galactosidase activity was ninefold higher than that in wild-type. The S-II mutant exhibited sensitivity to oxidants, which was suppressed by introduction of the S-II gene. The mutant S-II proteins, which are unable to stimulate transcription by RNA polymerase II in vitro, did not suppress the sensitivity of the mutants to oxidative stress or maintain transcriptional fidelity. Conclusion:, These results suggest that S-II confers oxidative stress resistance by providing an mRNA proof-reading mechanism during transcription elongation. [source] Estradiol levels in prepubertal boys and girls , analytical challengesINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2004Katrine Bay Summary Increasing evidence points at an important function of low concentrations of estradiol (E2) in prepubertal boys and girls. E2 serum levels in prepubertal children are, however, often immeasurable in conventional E2 assays. This strongly hampers further investigation of the physiological relevance of E2 in children. In addition, there is an increasing concern of the potential effect of exposure to endocrine disrupters with estrogenic or antiandrogenic activity on pubertal development. A requirement of assessing the instance for this concern, adds further to the demands for applicable methodologies for the evaluation of the sensitivity of the organism to low E2 concentrations. Traditionally, E2 is measured by use of the radioimmunoassay (RIA). As an ultrasensitive alternative to the RIA, a recombinant cell bioassay has been developed. In this review, methodological aspects for these methods of analysis are examined and their applicability for evaluation of low E2 serum concentrations in children is estimated. Furthermore, available data on E2 levels in prepubertal boys and girls are evaluated and discussed, taking into consideration the limitations of the methods of analysis. In conclusion, there is a pronounced demand for new and improved methods of analysis for accurate and sensitive evaluation of low concentrations of E2. [source] Inorganic phosphate as a signaling molecule in osteoblast differentiation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2003George R. Beck Jr. Abstract The spatial and temporal coordination of the many events required for osteogenic cells to create a mineralized matrix are only partially understood. The complexity of this process, and the nature of the final product, demand that these cells have mechanisms to carefully monitor events in the extracellular environment and have the ability to respond through cellular and molecular changes. The generation of inorganic phosphate during the process of differentiation may be one such signal. In addition to the requirement of inorganic phosphate as a component of hydroxyapatite mineral, Ca10(PO4)6(OH)2, a number of studies have also suggested it is required in the events preceding mineralization. However, contrasting results, physiological relevance, and the lack of a clear mechanism(s) have created some debate as to the significance of elevated phosphate in the differentiation process. More recently, a number of studies have begun to shed light on possible cellular and molecular consequences of elevated intracellular inorganic phosphate. These results suggest a model in which the generation of inorganic phosphate during osteoblast differentiation may in and of itself represent a signal capable of facilitating the temporal coordination of expression and regulation of multiple factors necessary for mineralization. The regulation of protein function and gene expression by elevated inorganic phosphate during osteoblast differentiation may represent a mechanism by which mineralizing cells monitor and respond to the changing extracellular environment. J. Cell. Biochem. 90: 234,243, 2003. Published 2003 Wiley-Liss, Inc. [source] Ontogeny of Plurihormonal Cells in the Anterior Pituitary of the Mouse, as Studied by Means of Hormone mRNA Detection in Single CellsJOURNAL OF NEUROENDOCRINOLOGY, Issue 8 2002E. Seuntjens Abstract The expression of mRNA of growth hormone (GH), prolactin (PRL), pro-opiomelanocortin (POMC) and the common glycoprotein hormone ,-subunit (,GSU) was studied by means of single cell reverse transcriptase-polymerase chain reaction in male mouse pituitary cells at key time points of fetal and postnatal development: embryonic day 16 (E16); postnatal day 1 (P1) and young-adult age (P38). At E16, the hormone mRNAs examined were detectable, although only in 44% of total cells. Most of the hormone-positive cells expressed only one of the tested hormone mRNAs (monohormonal) but 14% of them contained more than one hormone mRNA (plurihormonal cells). Combinations of GH mRNA with PRL mRNA, of ,GSU mRNA with GH and/or PRL mRNA and of POMC mRNA with GH and/or PRL mRNA or ,GSU mRNA were found. As expected, the proportion of hormone-positive cells rose as the mouse aged. The proportions of plurihormonal cells followed a developmental pattern independent of that of monohormonal cells and characteristic for each hormone mRNA examined. Cells coexpressing POMC mRNA with GH or PRL mRNA significantly rose in proportion between E16 and P1, while the proportion of cells coexpressing GH and PRL mRNA markedly increased between P1 and P38. The occurrence of cells displaying combined expression of ,GSU mRNA with GH and/or PRL mRNA did not significantly change during development. Remarkably, the population of cells expressing PRL mRNA only, was larger at E16 than at P1 and expanded again thereafter. In conclusion, the normal mouse pituitary develops a cell population that is capable of expressing multiple hormone mRNAs, thereby combining typical phenotypes of different cell lineages. These plurihormonal cells are already present during embryonic life. This population is of potential physiological relevance because development-related factors appear to determine which hormone mRNAs are preferentially coexpressed. Coexpression of multiple hormone mRNAs may represent a mechanism to respond to temporally increased endocrine demands. The data also suggest that the control of combined hormone expression is different from that of single hormone expression, raising questions about the current view on pituitary cell lineage specifications. [source] Neuronal coupling via connexin36 contributes to spontaneous synaptic currents of striatal medium-sized spiny neuronsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2008Damian M. Cummings Abstract Gap junctions provide a means for electrotonic coupling between neurons, allowing for the generation of synchronous activity, an important contributor to learning and memory. Connexin36 (Cx36) is largely neuron specific and provides a target for genetic manipulation to determine the physiological relevance of neuronal coupling. Within the striatum, Cx36 is more specifically localized to the interneuronal population, which provides the main inhibitory input to the principal projection medium-sized spiny neurons. In the present study, we examined the impact of genetic ablation of Cx36 on striatal spontaneous synaptic activity. Patch-clamp recordings were performed from medium-sized spiny neurons, the primary target of interneurons. In Cx36 knockout mice, the frequencies of both excitatory and inhibitory spontaneous postsynaptic currents were reduced. We also showed that activation of dopamine receptors differentially modulated the frequency of GABAergic currents in Cx36 knockout mice compared with their wild-type littermates, suggesting that dopamine plays a role in altering the coupling of interneurons. Taken together, the present findings demonstrate that electrical coupling of neuronal populations is important for the maintenance of normal chemical synaptic interactions within the striatum. © 2008 Wiley-Liss, Inc. [source] Biological, pharmaceutical, and analytical considerations with respect to the transport media used in the absorption screening system, Caco-2JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2003Françoise M. Ingels Abstract During the evaluation and selection of drug candidates, the Caco-2 cell culture system is commonly used for the determination of intestinal transport characteristics and to anticipate permeability limited drug absorption. Although classic HBSS-like buffered salt solutions are commonly used to perform Caco-2 transport experiments, different shortcomings (e.g., adsorption and low solubility) have been associated with the use of plain aqueous buffers. As transport experiments performed with unoptimized conditions may compromize the value of the Caco-2 model as a permeation screening tool, many efforts have been made to optimize the experimental conditions of Caco-2 transport assays. In this minireview, the hurdles associated with the use of saline aqueous buffers in Caco-2 transport experiments are summarized and the different options, which have been proposed to overcome these issues, are reviewed and discussed. Biologically, pharmaceutically, as well as analytically relevant media affecting the outcome of the transport experiments are described. Unfortunately, up to now, no systematic studies comparing the different experimental conditions have been performed, jeopardizing the possibility to define a (single) optimal solution to overcome the different issues associated with the use of saline aqueous buffers. Based on the reported options it can be proposed to use DMSO (,1%) in standard screening procedures for the ranking of compounds based on their apical to basolateral transport. If compounds are not soluble in DMSO 1%, dimethylacetamide (3%) or N -1-methyl-pyrrolidone (2.5%) are good alternatives. However, these options do not imitate the in vivo situation. If one wants to take into account the physiological relevance of the media, the use of a biologically relevant apical medium (e.g., FASSIF) in combination with an analytically friendly, sink condition creating basolateral solvent (e.g., containing a micelle forming agent) can be suggested. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1545,1558, 2003 [source] Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic miceJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2003M. Eren Summary., Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal ,2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-,1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-,1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal ,2.9 kb promoter. [source] The liver stage of Plasmodium berghei inhibits host cell apoptosisMOLECULAR MICROBIOLOGY, Issue 3 2005Claudia Van De Sand Summary Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite,host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei -infected cells and compared their level of expression and their distribution to that of non-infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non-infected HepG2 cells, whereas P. berghei -infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)-,/d -galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non-treated control mice as well as in TNF-,-treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death. [source] Spectrum separation resolves partial-volume effect of MRSI as demonstrated on brain tumor scansNMR IN BIOMEDICINE, Issue 10 2008Yuzhuo Su Abstract Magnetic resonance spectroscopic imaging (MRSI) is currently used clinically in conjunction with anatomical MRI to assess the presence and extent of brain tumors and to evaluate treatment response. Unfortunately, the clinical utility of MRSI is limited by significant variability of in vivo spectra. Spectral profiles show increased variability because of partial coverage of large voxel volumes, infiltration of normal brain tissue by tumors, innate tumor heterogeneity, and measurement noise. We address these problems directly by quantifying the abundance (i.e. volume fraction) within a voxel for each tissue type instead of the conventional estimation of metabolite concentrations from spectral resonance peaks. This ,spectrum separation' method uses the non-negative matrix factorization algorithm, which simultaneously decomposes the observed spectra of multiple voxels into abundance distributions and constituent spectra. The accuracy of the estimated abundances is validated on phantom data. The presented results on 20 clinical cases of brain tumor show reduced cross-subject variability. This is reflected in improved discrimination between high-grade and low-grade gliomas, which demonstrates the physiological relevance of the extracted spectra. These results show that the proposed spectral analysis method can improve the effectiveness of MRSI as a diagnostic tool. Copyright © 2008 John Wiley & Sons, Ltd. [source] Permeant anions contribute to voltage dependence of ClC-2 chloride channel by interacting with the protopore gateTHE JOURNAL OF PHYSIOLOGY, Issue 14 2010Jorge E. Sánchez-Rodríguez It has been shown that the voltage (Vm) dependence of ClC Cl, channels is conferred by interaction of the protopore gate with H+ ions. However, in this paper we present evidence which indicates that permeant Cl, ions contribute to Vm -dependent gating of the broadly distributed ClC-2 Cl, channel. The apparent open probability (PA) of ClC-2 was enhanced either by changing the [Cl,]i from 10 to 200 mm or by keeping the [Cl,]i low (10 mm) and then raising [Cl,]o from 10 to 140 mm. Additionally, these changes in [Cl,] slowed down channel closing at positive Vm suggesting that high [Cl,] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the PA(Vm) curves were nearly identical with Cl, or Br, but shifted to negative voltages in the presence of SCN, ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN,/Cl, mixture in agreement with the idea that pore occupancy by different permeant anions modifies the Vm dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl, ions dwell in the central binding site. In contrast, when Cl, ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl, -dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl,]o enhances channel opening when the [Cl,]i is in the physiological range. [source] Synapses on NG2-expressing progenitors in the brain: multiple functions?THE JOURNAL OF PHYSIOLOGY, Issue 16 2008Vittorio Gallo Progenitor cells expressing the proteoglycan NG2 represent approximately 5% of the total cells in the adult brain, and are found both in grey and white matter regions where they give rise to oligodendrocytes. The finding that these cells receive synaptic contacts from excitatory and inhibitory neurons has not only raised major interest in the possible roles of these synapses, but also stimulated further research on the developmental and cellular functions of NG2-expressing (NG2+) progenitors themselves in the context of neural circuit physiology. Here we review recent findings on the functional properties of the synapses on NG2+ cells in grey and white matter regions of the brain. In this review article we make an attempt to integrate current knowledge on the cellular and developmental properties of NG2+ progenitors with the functional attributes of their synapses, in order to understand the physiological relevance of neuron,NG2+ progenitor signal transmission. We propose that, although NG2+ progenitors receive synaptic contact in all brain regions where they are found, their synapses might have different developmental and functional roles, probably reflecting the distinct functions of NG2+ progenitors in the brain. [source] Transport of Benzo[,]pyrene in the Dually Perfused Human Placenta Perfusion Model: Effect of Albumin in the Perfusion MediumBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2009Line Mathiesen Foetal exposure to this substance is highly relevant but is difficult to estimate. The human placenta is unique compared to other species; since it is available without major ethical obstacles, we have used the human placenta perfusion model to study transport from mother to foetus. Placentas were donated after births at Rigshospitalet in Copenhagen from pregnant mothers who signed an informed consent. BaP is lipophilic and studies using cell culture medium in 6-hr placenta perfusions showed minimal transport through the placenta. To increase the solubility of BaP in perfusion medium and to increase physiological relevance, perfusions were also performed with albumin added to the perfusion medium [2 and 30 mg/ml bovine serum albumin (BSA) and 30 mg/ml human serum albumin (HSA)]. The addition of albumin resulted in increased transfer of BaP from maternal to foetal reservoirs. The transfer was even higher in the presence of an HSA formulation containing acetyltryptophanate and caprylate, resulting in a foetal,maternal concentration (FM) ratio of 0.71 ± 0.10 after 3 hr and 0.78 ± 0.11 after 6 hr, whereas the FM ratio in perfusions without albumin was only 0.05 ± 0.03 after 6 hr of perfusion. Less BaP accumulated in placental tissue in perfusions with added albumin. This shows that transplacental transport of the pro-carcinogenic substance BaP occurs, and emphasizes the importance of adding physiological concentrations of albumin when studying the transport of lipophilic substances. [source] Effect of intermediate-purity factor VIII (FVIII) concentrate on lymphocyte proliferation and apoptosis: transforming growth factor-, is a significant immunomodulatory component of FVIIIBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2001G. Hodge Factor concentrates have been shown to have a variety of immunomodulatory effects in vitro. The presence of plasma-derived factor VIII (pdFVIII) has been shown to diminish lymphocyte proliferative response to mitogens. Recently, we have shown the presence of transforming growth factor-, (TGF-,) as an immunomodulatory component present in plasma-derived FVIII concentrate. However, the addition of neutralizing antibody to TGF-, did not abrogate the inhibitory effect of pdFVIII on monocyte cytokine production, suggesting the presence of other, as yet undetermined, immunomodulatory agent/s in pdFVIII. To further characterize the immunomodulatory effects of pdFVIII, the in vitro effect of pdFVIII concentrate on proliferation and apoptosis of mitogen-stimulated T cells was studied using whole blood and purified T cells. The presence of pdFVIII increased the apoptosis of phytohaemagglutinn (PHA) -stimulated CD4 and CD8 T-cell subsets as determined by Annexin V binding and DNA fragmentation. T-cell subsets showed a pdFVIII dose-dependent inhibition of entry into S-phase and G1 arrest. Addition of neutralizing anti-TGF-, reduced some of these changes. To determine the physiological relevance of these findings, blood samples from five patients receiving FVIII prophylaxis were similarly studied ex vivo and showed significantly increased apoptosis of T-cell subsets as determined by Annexin V staining. TGF-, has been reported to be a potent inhibitor of T-cell proliferation, arresting the cell cycle in G1 phase and causing apoptosis. Together, these findings suggest that TGF-, is a significant immunomodulatory component of pdFVIII concentrates. [source] Do studies in caveolin-knockouts teach us about physiology and pharmacology or instead, the ways mice compensate for ,lost proteins'?BRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2007P A Insel A wide array of phenotypic changes have been reported in mice with knockout of expression of caveolin-1. Neidhold et al. (2007) describe results in this issue that continue this trend by showing that saphenous arteries from adult caveolin-1 knockout mice lack caveolae, lose ,1 -adrenoceptor-promoted relaxation, gain ,3 -adrenoceptor-promoted relaxation but show no change in vasomotor response to ,2 -adrenoceptor activation. Neither the physiological importance for wild-type animals nor the mechanistic basis for these changes is clear. Although the caveolin-1 knockout and wild-type mice express similar levels of the receptor mRNAs, the protein expression of the receptors is not specified and represents, in our view, an important limitation of the study. We also question the physiological relevance of the findings and ask: Do studies in total body/lifespan caveolin-knockout mice further understanding of physiology and pharmacology or do they primarily characterize secondary consequences? We propose that alternative approaches that decrease caveolin expression in a temporally and spatially discrete manner are more likely to facilitate definitive conclusions regarding caveolin-1 and its role in regulation of , -adrenoceptors and other pharmacological targets. British Journal of Pharmacology (2007) 150, 251,254. doi:10.1038/sj.bjp.0706981 [source] Proceedings of the Australian Physiological and Pharmacological Society Symposium: New Frontiers in Muscle Research Gene transfer: manipulating and monitoring function in cells and tissuesCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2001Rekha G Panchal SUMMARY 1. The ectopic expression of genes has proven to be an extremely valuable tool for biologists. The most widely used systems involve electrically or chemically mediated transfer of genes to immortalized cell lines and, at the other end of the spectrum, transgenic animal models. As would be expected, there are compromises to be made when using either of these broad approaches. Immortalized cell lines have limited ,physiological relevance' and transgenic approaches are costly and out of the reach of many laboratories. There is also significant time required for the de novo generation of a transgenic animal. 2. As a viable alternative to these approaches, we describe the use of recombinant adenovirus and Sindbis virus to deliver genes to cells and tissues. 3. We exemplify this approach with studies from our laboratories: (i) an investigation of Ca2+ handling deficits in cardiac myocytes of hypertrophied hearts using infection with recombinant adenovirus encoding either green fluorescent protein (GFP) or the sarcoplasmic/endoplasmic reticulum calcium-ATPase (Serca2a); (ii) a study of the mechanism of macrophage/microglial migration by infection of embryonic phagocytes with a GFP-encoding virus and coculture with brain slices to then track the movement of labelled cells; and (iii) we are also exploiting the natural tropism of the Sindbis virus to label neurons in hippocampal brain slices in culture to resolve high-resolution structure and to map neuronal connectivity. 4. Further development of these approaches should open new avenues of investigation for the study of physiology in a range of cells and tissues. [source] | |||||||||||||||||||||||||