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1.
Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle 总被引:16,自引:3,他引:13 下载免费PDF全文
Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle. 相似文献
2.
Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability 总被引:14,自引:0,他引:14
We have analyzed nucleic acid and amino acid sequence alignments of a
variety of voltage-sensitive ion channels, using several methods for
phylogenetic tree reconstruction. Ancient duplications within this family
gave rise to three distantly related groups, one consisting of the Na+ and
Ca++ channels, another the K+ channels, and a third including the cyclic
nucleotide-binding channels. A series of gene duplications produced at
least seven mammalian homologues of the Drosophila Shaker K+ channel;
clones of only three of these genes are available from all three mammalian
species examined (mouse, rat, and human), pointing to specific genes that
have yet to be recovered in one or another of these species. The
Shaw-related K+ channels and the Na+ channel family have also undergone
considerable expansion in mammals, relative to flies. These expansions
presumably reflect the needs of the high degree of physiological and
neuronal complexity of mammals. Analysis of the separate domains of the
four-domain channels (Ca++ and Na+) supports their having evolved by two
sequential gene duplications and implies the historical existence of a
functional two-domain channel.
相似文献
3.
Prashant G. Bhat Ajay M. V. Kumar Balaji Naik Srinath Satyanarayana Deepak KG Sreenivas A. Nair Suryakanth MD Einar Heldal Donald A. Enarson Anthony J. Reid 《PloS one》2013,8(12)
Background
Severe acute malnutrition (SAM) is the most serious form of malnutrition affecting children under-five and is associated with many infectious diseases including Tuberculosis (TB). In India, nutritional rehabilitation centres (NRCs) have been recently established for the management of SAM including TB. The National TB Programme (NTP) in India has introduced a revised algorithm for diagnosing paediatric TB. We aimed to examine whether NRCs adhered to these guidelines in diagnosing TB among SAM children.Methods
A cross-sectional study involving review of records of all SAM children identified by health workers during 2012 in six tehsils (sub-districts) with NRCs (population: 1.8 million) of Karnataka, India.Results
Of 1927 identified SAM children, 1632 (85%) reached NRCs. Of them, 1173 (72%) were evaluated for TB and 19(2%) were diagnosed as TB. Of 1173, diagnostic algorithm was followed in 460 (37%). Among remaining 763 not evaluated as per algorithm, tuberculin skin test alone was conducted in 307 (41%), chest radiography alone in 99 (13%) and no investigations in 337 (45%). The yield of TB was higher among children evaluated as per algorithm (4%) as compared to those who were not (0.3%) (OR: 15.3 [95%CI: 3.5-66.3]). Several operational challenges including non-availability of a full-time paediatrician, non-functioning X-ray machine due to frequent power cuts, use of tuberculin with suboptimal strength and difficulties in adhering to a complex diagnostic algorithm were observed.Conclusion
This study showed that TB screening in NRCs was sub-optimal in Karnataka. Some children did not reach the NRC, while many of those who did were either not or sub-optimally evaluated for TB. This study pointed to a number of operational issues that need to be addressed if this collaborative strategy is to identify more TB cases amongst malnourished children in India. 相似文献4.
Noureddine Zebda Yufeng Tian Xinyong Tian Grzegorz Gawlak Katherine Higginbotham Albert B. Reynolds Anna A. Birukova Konstantin G. Birukov 《The Journal of biological chemistry》2013,288(25):18290-18299
p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820–843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation. 相似文献
5.
Birukov KG Csortos C Marzilli L Dudek S Ma SF Bresnick AR Verin AD Cotter RJ Garcia JG 《The Journal of biological chemistry》2001,276(11):8567-8573
The Ca(2+)/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436-505) that contains potentially important consensus sites for phosphorylation by p60(Src) kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction of V(max), and a 2-fold increase in K(0.5 CaM) when compared with the SM MLCK isoform, whereas K(m) was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60(Src) in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was observed over a broad range of submaximal [Ca(2+)] levels with comparable EC(50) [Ca(2+)] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60(Src) are Tyr(464) and Tyr(471) within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60(Src)-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of nonmuscle MLCK and vascular cell function. 相似文献
6.
The ability of inflammatory cytokine TGF-beta1 to alter endothelial cell phenotype suggests its role in the regulation of vascular endothelial cell permeability. We demonstrate that depletion of TGF-beta1 receptor ALK5 and regulatory protein Smad4, but not ALK1 receptor attenuates TGF-beta1-induced permeability increase and significantly inhibits TGF-beta1-induced EC contraction manifested by actin stress fiber formation and increased MLC and MYPT1 phosphorylation. Consistent with these results, EC treatment with SB 431542, an inhibitor of ALK5 but not ALK1 receptor, significantly attenuates TGF-beta1-induced permeability. Thus, our data demonstrate for the first time direct link between TGF-beta1-mediated activation of ALK5/Smad and EC barrier dysfunction. 相似文献
7.
Dudek SM Jacobson JR Chiang ET Birukov KG Wang P Zhan X Garcia JG 《The Journal of biological chemistry》2004,279(23):24692-24700
We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P. 相似文献
8.
Birukova AA Adyshev D Gorshkov B Bokoch GM Birukov KG Verin AD 《American journal of physiology. Lung cellular and molecular physiology》2006,290(3):L540-L548
Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation. 相似文献
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10.
Anna A. Birukova Patrick A. Singleton Grzegorz Gawlak Xinyong Tian Tamara Mirzapoiazova Bolot Mambetsariev Oleksii Dubrovskyi Olga V. Oskolkova Valery N. Bochkov Konstantin G. Birukov 《Molecular biology of the cell》2014,25(13):2006-2016
Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell–cell junctions. Defects in the endothelial barrier become an initiating factor in several pathologies, including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis, and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We previously reported that oxidized phospholipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [OxPAPC]) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examines the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane–localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane–localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEMs) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1, Src and Fyn tyrosine kinases, and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyperpermeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from interleukin-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity. 相似文献