Geldanamycin disrupts platelet-membrane structure, leading to membrane permeabilization and inhibition of platelet aggregation.
(73/2081)
Geldanamycin (GA), a benzoquinoid ansamycin antibiotic, has been used as a tyrosine kinase inhibitor and an anti-tumour agent and is known to bind to heat-shock protein 90. In the present study on human platelets we have found that GA inhibited platelet aggregation induced by ADP, thrombin and the thrombin-receptor-activating peptide and caused platelet plasma-membrane damage, detected by leakage of adenine nucleotides as well as serotonin. Scanning electron microscopy (SEM) revealed that platelet exposure to GA led to the formation of holes or fenestrations in the platelet plasma membrane, confirming GA's ability to initiate membrane damage. In addition, GA itself caused both the dephosphorylation and phosphorylation of proteins in resting platelets and prevented agonist-induced phosphorylation of pleckstrin, the 20-kDa myosin light chain and other proteins. Another ansamycin, herbimycin A, also inhibited platelet aggregation, but caused minimal membrane permeabilization, as detected by (3)H release from platelets labelled previously with [(3)H]adenine, and much less membrane damage, revealed by SEM. Overall, GA is able to disrupt membrane structure and inhibit platelet aggregation, an ability which may be linked to alterations in the activity of protein kinases and phosphatases. (+info)
Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.
(74/2081)
Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model. (+info)
Pressure-induced upregulation of preproendothelin-1 and endothelin B receptor expression in rabbit jugular vein in situ : implications for vein graft failure?
(75/2081)
Upregulation of endothelin-1 (ET-1) synthesis in venous bypass grafts in response to arterial levels of blood pressure may play a major role in graft failure. To investigate this hypothesis, isolated segments of the rabbit jugular vein were perfused at physiological (0 to 5 mm Hg) and nonphysiological (20 mm Hg) levels of intraluminal pressure. As judged by reverse transcription-polymerase chain reaction analysis (mRNA level), neither endothelin-converting enzyme nor endothelin A receptor expression appeared to be pressure sensitive. In contrast, there was a profound and time-dependent increase in endothelial prepro-ET-1 mRNA and intravascular ET-1 abundance (by ELISA) as well as in smooth muscle endothelin B receptor mRNA and functional protein (by superfusion bioassay) on raising the perfusion pressure from 5 to 20 mm Hg, but not from 0 to 5 mm Hg, for up to 12 hours. Video microscopy analysis revealed that the segments were distended by 75% at 5 mm Hg and near maximally at 20 mm Hg compared with the resting diameter at 0 to 1 mm Hg. Treatment of the segments with actinomycin D (1 micromol/L), the specific protein kinase C inhibitor, Ro 31-8220 (0.1 micromol/L), or the c-Src family-specific tyrosine kinase inhibitor, herbimycin A (0.1 micromol/L), demonstrated that the pressure-induced expression of these gene products occurs at the level of transcription and requires activation of protein kinase C, but not c-Src. In venous bypass grafts such deformation-induced changes in gene expression may contribute not only to acute graft failure through ET-1-induced vasospasm but also to endothelin A receptor- and/or endothelin B receptor-mediated smooth muscle cell hyperplasia and graft occlusion. (+info)
Degradation of heterotrimeric Galpha(o) subunits via the proteosome pathway is induced by the hsp90-specific compound geldanamycin.
(76/2081)
One mechanism utilized by cells to maintain signaling pathways is to regulate the levels of specific signal transduction proteins. The compound geldanamycin (GA) specifically interacts with heat shock protein 90 (hsp90) complexes and has been widely utilized to study the role of hsp90 in modulating the function of signaling proteins. In this study, we used GA to demonstrate that levels of heterotrimeric Galpha subunits can be regulated through interactions with hsp90. In a dose-dependent manner, GA significantly reduced the steady state levels of endogenous Galpha(o) expression in two cell lines (PC12 and GH3) and had a similar effect on Galpha(o) transiently expressed in COS cells. Galpha(o) synthesis and degradation was studied in PC12 cells and in transiently transfected COS cells. (35)S labeling followed by immunoprecipitation demonstrated no effect of GA on the rate of Galpha(o) synthesis, but GA accelerated degradation of Galpha(o) in both PC12 cells and COS cells. The use of inhibitors, including lactacystin (a proteosome-specific inhibitor), suggests that Galpha(o) is predominantly degraded through the proteosome pathway. In vitro translated (35)S-labeled Galpha(o) could be detected in hsp90 immunoprecipitates, and this interaction did not require N-terminal myristoylation. Taken together, these results suggest that heterotrimeric Galpha(o) subunits are protected from degradation by interaction with hsp90 and that the interaction of Galpha subunits with heat shock proteins may be a general mechanism for regulating Galpha levels in the cell. (+info)
Inhibition of I kappaB-alpha phosphorylation at serine and tyrosine acts independently on sensitization to DNA damaging agents in human glioma cells.
(77/2081)
Molecular mechanisms and/or intrinsic factors controlling cellular radiosensitivity are not fully understood in mammalian cells. The recent studies have suggested that nuclear factor kappaB (NF-kappaB) is one of such factors. The activation and regulation of NF-kappaB are tightly controlled by IkappaB-alpha, a cellular inhibitory protein of NF-kappaB. Most importantly, phosphorylation regulates activity of the inhibitor IkappaB-alpha, which sequesters NF-kappaB in the cytosol. Two different pathways for the phosphorylation of IkappaB-alpha are demonstrated, such as serine (at residues 32 and 36) and tyrosine (at residue 42) phosphorylations. To assess a role of the transcription factor, NF-kappaB, on cellular sensitivity to DNA damaging agents, we constructed three different types of expression plasmids, i.e. S-IkappaB (mutations at residues 32 and 36), Y-IkappaB (mutation at residue 42) and SY-IkappaB (mutations at residues 32, 36 and 42). The cell clones expressing S-IkappaB and Y-IkappaB proteins became sensitive to X-rays as compared with the parental and vector-transfected cells. The cell clones expressing SY-IkappaB were further radiosensitive. By the treatment with herbimycin A, an inhibitor of phosphorylation, the X-ray sensitivity of cells expressing SY-IkappaB did not change, while that of the cells expressing S-IkappaB and Y-IkappaB and the parental cells was enhanced. Change in the sensitivity to adriamycin and UV in those clones was very similar to that in the X-ray sensitivity. The inhibition of IkappaB-alpha phosphorylation at serine and tyrosine acts independently on the sensitization to X-rays, adriamycin and UV. These findings suggest that the transcriptional activation induced by NF-kappaB may play a role in the DNA damage repair. The present study proposes a possibility that the inactivation of NF-kappaB by inhibition of both serine and tyrosine phosphorylations may be useful for the treatment of cancer in radio- and chemotherapies. (+info)
Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells.
(78/2081)
Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs. (+info)
Tyrosine kinase inhibitors reduce bcl-2 expression and induce apoptosis in androgen-dependent cells.
(79/2081)
The signal transduction pathway showing how androgen withdrawal induces apoptosis in androgen-dependent cells has not been clearly understood. In these studies, we focused on the behavior of tyrosine kinases in androgen-dependent cells and investigated its correlation with apoptosis and bcl-2 expression. We used SC2G, an androgen-dependent mouse mammary carcinoma cell line, which had been cloned from Shionogi Carcinoma 115 (SC115). When SC2G cells were cultured with herbimycin A (HMA), a potent tyrosine kinase inhibitor, the number of viable cells decreased significantly after 24 h. Terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling and flow cytometric analysis of annexin V staining showed that HMA induced apoptosis of SC2G cells. The level of bcl-2 mRNA in SC2G cells was suppressed by HMA in a dose-dependent manner on RT-PCR. Preincubation with caspase inhibitors protected HMA-induced apoptosis of SC2G cells. When a human bcl-2 gene was transfected in SC2G cells and overexpressed, SC2G cells seemed to acquire tolerance for HMA. These data indicate that HMA-sensitive tyrosine kinase(s) can regulate apoptosis and inhibit bcl-2 expression in SC2G mouse androgen-dependent cells. Tyrosine kinase(s) seemed to be a member of signal transduction between androgen receptor activation and bcl-2 expression. (+info)
Protein tyrosine kinase regulates the number of renal secretory K channels.
(80/2081)
The apical small conductance (SK) channel plays a key role in K secretion in the cortical collecting duct (CCD). A high-K intake stimulates renal K secretion and involves a significant increase in the number of SK channels in the apical membrane of the CCD. We used the patch-clamp technique to examine the role of protein tyrosine kinase (PTK) in regulating the activity of SK channels in the CCD. The application of 100 microM genistein stimulated SK channels in 11 of 12 patches in CCDs from rats on a K-deficient diet, and the mean increase in NP(o), a product of channel number (N) and open probability (P(o)), was 2.5. In contrast, inhibition of PTK had no effect in tubules from animals on a high-K diet in all 10 experiments. Western blot analysis further shows that the level of cSrc, a nonreceptor type of PTK, is 261% higher in the kidneys from rats on a K-deficient diet than those on a high-K diet. However, the effect of cSrc was not the result of direct inhibition of channel itself, because addition of exogenous cSrc had no effect on SK channels in inside-out patches. In cell-attached patches, application of herbimycin A increased channel activity in 14 of 16 patches, and the mean increase in NP(o) was 2.4 in tubules from rats on a K-deficient diet. In contrast, herbimycin A had no effect on channel activity in any of 15 tubules from rats on a high-K diet. Furthermore, herbimycin A pretreatment increased NP(o) per patch from the control value (0.4) to 2.25 in CCDs from rats on a K-deficient diet, whereas herbimycin failed to increase channel activity (NP(o): control, 3.10; herbimycin A, 3.25) in the CCDs from animals on a high-K diet. We conclude that PTK is involved in regulating the number of apical SK channels in the kidney. (+info)