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991.
In the cholecystokinin (CCK)hyperstimulation model of acute pancreatitis, two early intracellularevents, activation of trypsinogen and activation of nuclear factor-B(NF-B), are thought to be important in the development of thedisease. In this study, the relationship between these two events wasinvestigated. NF-B activity was monitored by using a DNA bindingassay and mob-1 chemokine gene expression. Intracellulartrypsin activity was measured by using a fluorogenic substrate.Protease inhibitors including FUT-175, Pefabloc, and E-64d preventedCCK stimulation of intracellular trypsinogen and NF-B activation.Likewise, the NF-B inhibitors pyrrolidine dithiocarbamate andN-acetyl-L-cysteine inhibited CCK stimulation ofNF-B and intracellular trypsinogen activation. These resultssuggested a possible codependency of these two events. However, CCKstimulated NF-B activation in Chinese hamster ovary-CCKAcells, which do not express trypsinogen, indicating that trypsin is notnecessary for CCK activation of NF-B. Furthermore,adenovirus-mediated expression in acinar cells of active p65 subunitsto stimulate NF-B, or of inhibitory B- molecules to inhibitNF-B, did not affect either basal or CCK-mediated trypsinogenactivation. Thus trypsinogen and NF-B activation are independentevents stimulated by CCK. 相似文献
992.
用差异显示PCR法筛选与血管外膜细胞表型转化相关的基因 总被引:8,自引:2,他引:6
为筛选血管外膜成纤维细胞(adventitial fibroblast,AF)与肌成纤维细胞(myofibroblast,MF)间表型转化有关的基因,实验建立了大鼠胸主动脉AF和MF两种细胞模型,用差异显示聚合酶链反应(DD-PCR)技术获得表达差异片段,对差异片段进行克隆和测序分析,并用定量PCR和Northern blot对差别显示结果进行验证。用反义核酸转染技术观察骨桥蛋白(osteopontin,OPN)对AF迁移的影响。结果表明,两种表型细胞存在明显的基因表达差异,其中一个在MF下调的差异片段与GenBank中NADH脱氢酶亚单位5(NADH dehydrogenase subunit 5,Nd5)基因高度同源。另一个在MF上调的差异片段与OPN基因同源。上述差异表达结果被定量PCR及Northern blot证实。此外还有4个表达序列标志(expressed sequence-tag,EST)在GenBank中未查到同源序列。反义OPN寡脱氧核甘酸可抑制AF的迁移活动。结果提示,AF转化为MF可能与ND5基因下调、OPN上调及其它未知基因的表达改变有关。应用反义技术适度抑制OPN表达在防治血管重塑中具有重要作用。 相似文献
993.
Ji J Salapatek AM Diamant NE 《American journal of physiology. Gastrointestinal and liver physiology》2000,279(5):G951-G960
The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential. 相似文献
994.
Thirteen basidiospore-derived isolates of Pleurotus ostreatus f6 strain differing in the level of ligninolytic enzyme production and other characteristics (mycelium extension rate, colony morphology) from the parental strain were cultivated on natural substrates. Under these conditions ligninolytic enzyme activity, loss of organic mass, polycyclic aromatic hydrocarbons (PAHs) degradation and colonization of sterile and nonsterile soil were studied. The activity of ligninolytic enzymes was substantially higher in straw than in liquid culture, although the differences between the isolates were less pronounced on this substrate. Some of the isolates showed a very good ability to decompose the lignocellulosic substrate (straw) and a relatively high loss of organic mass was found after 50 days of cultivation in these strains. The original strain f6 and isolates B13 and B26 successfully degraded all seven tested PAH compounds present in experimental soil samples, but the higher or lower ligninolytic enzyme production of isolates tested had no substantial effect on the extent of the degradation. In our screening, six basidiospore-derivedisolates growing well in nonsterile soil were found, whichcould be suitable for the prospective biotechnological exploitation. 相似文献
995.
Greene AL Lalli MJ Ji Y Babu GJ Grupp I Sussman M Periasamy M 《The Journal of biological chemistry》2000,275(32):24722-24727
The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart. 相似文献
996.
997.
Ji H Zhai Q Zhu J Yan M Sun L Liu X Zheng Z 《Biochemical and biophysical research communications》2000,270(1):267-271
To find a possible signal interacting with the Jak3 N-terminal, we screened the human peripheral blood cDNA library through both a two-hybrid system and a tyrosine-phosphorylation-modified two-hybrid system using the N-terminal of Jak3 as bait. Results showed that one new homologue of myosin heavy chain, designated MAJN (molecule associated with Jak3 N-terminal), could bind to Jak3 in a tyrosine-phosphorylation-independent manner. The interaction between Jak3 and MAJN was further confirmed by immunoprecipitation in BAF-B03 beta cells. To investigate the function of MAJN, we have constructed the BAF-B03 beta/MAJN cell line that stably expresses MAJN and found that overexpression of MAJN can partially inhibit the apoptosis induced by interleukin-2 deprival. Further studies are needed to elucidate how MAJN executes its function to antagonize BAF-B03beta cell death in the absence of IL-2. 相似文献
998.
For human (HIV) and simian (SIV) immunodeficiency viruses, the gp41 envelope protein undergoes a receptor-activated conformational change from a labile native structure to an energetically more stable fusogenic conformation, which then mediates viral-cell membrane fusion. The core structure of fusion-active gp41 is a six-helix bundle in which three antiparallel carboxyl-terminal helices are packed against an amino-terminal trimeric coiled coil. Here we show that a recombinant model of the SIV gp41 core, designated N36(L6)C34, forms an alpha-helical trimer that exhibits a cooperative two-state folding-unfolding transition. We investigate the importance of buried polar interactions in determining the overall fold of the gp41 core. We have replaced each of four polar amino acids at the heptad a and d positions of the coiled coil in N36(L6)C34 with a representative hydrophobic amino acid, isoleucine. The Q565I, T582I, and T586I variants form six-helix bundle structures that are significantly more stable than that of the wild-type peptide, whereas the Q575I variant misfolds into an insoluble aggregate under physiological conditions. Thus, the buried polar residues within the amino-terminal heptad repeat are important determinants of the structural specificity and stability of the gp41 core. We suggest that these conserved buried polar interactions play a role in governing the conformational state of the gp41 molecule. 相似文献
999.
Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release 总被引:17,自引:0,他引:17
Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release. 相似文献
1000.