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Mutations in the human homolog of the Vhlh gene [encoding the von-Hippel Lindau (VHL) protein] lead to tumor development. In mice, depletion of Vhlh in pancreatic ß-cells causes perturbed glucose homeostasis, but the role of this gene in other pancreatic cells is poorly understood. To investigate the function of VHL/HIF pathway in pancreatic cells, we inactivated Vhlh in the pancreatic epithelium as well as in the endocrine and exocrine lineages. Our results show that embryonic depletion of Vhlh within the pancreatic epithelium causes postnatal lethality due to severe hypoglycemia. The hypoglycemia is recapitulated in mice with endocrine-specific removal of Vhlh, while animals with loss of Vhlh predominantly in the exocrine compartment survive to adulthood with no overt defects in glucose metabolism. Mice with hypoglycemia display diminished insulin release in response to elevated glucose. Significantly, the glucagon response is impaired both in vivo (circulating glucagon levels) as well as in an in vitro secretion assay in isolated islets. Hypoxia also impairs glucagon secretion in a glucagon-expressing cell line in culture. Our results reveal a novel role for the hypoxia/HIF pathway in islet hormone secretion and maintenance of the fine balance that allows for the establishment of normoglycemia. 相似文献
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J Puri J H Pierce T Hoffman 《Prostaglandins, leukotrienes, and essential fatty acids》1992,45(1):43-48
The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E2 (PGE2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE2 production. Addition of ST638, a tyrosine kinase inhibitor, to cells transfected with c-fms in the absence of CSF-1 inhibited PGE2 production within 10-60 min. Its addition to the same cells in the presence of CSF-1 induced an opposite effect, but required longer treatment (24 h). In either cell type, AA release was not affected by this agent. These data indicate that CSF-1 may regulate cyclooxygenase activity. The different effect of CSF-1 receptor on PGE2 production in the presence or absence of CSF-1 and the opposite effect of a tyrosine kinase inhibitor on PGE2 suggest that both the receptor alone or the receptor-ligand complex may transduce an active, but different, signal through tyrosine phosphorylation. CSF-1 receptor and CSF-1 may exert separate, but related, effects on phospholipase A2 and cyclooxygenase activity which, in concert, or along with other tyrosine kinases, regulate prostaglandin production. 相似文献
5.
Inactivation of yeast hexokinase by o-phthalaldehyde: evidence for the presence of a cysteine and a lysine at or near the active site 总被引:1,自引:0,他引:1
Yeast hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a homodimer, was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics over a wide range of the inhibitor concentration. The second-order-rate constant for the inactivation of hexokinase was estimated to be 45 M-1.s-1. Hexokinase was protected more by sugar substrates than by nucleoside triphosphates during inactivation by o-phthalaldehyde. Absorption spectrum (lambda max 338 nm), and fluorescence excitation (lambda max 363 nm) and emission (lambda max 403 nm) spectra of the hexokinase-o-phthalaldehyde adduct were consistent with the formation of an isoindole derivative. These results also suggest that sulfhydryl and epsilon-amino functions of the cysteine and lysine residues, respectively, participating in the isoindole formation are about 3 A apart in the native enzyme. About 2 mol of the isoindole per mol of hexokinase dimer were formed following complete loss of the phosphotransferase activity. Chemical modification of hexokinase by iodoacetamide in the presence of mannose resulted in the modification of six sulfhydryl groups per mol of hexokinase with retention of the phosphotransferase activity. Subsequent reaction of the iodoacetamide modified hexokinase with o-phthalaldehyde resulted in complete loss of the phosphotransferase activity with concomitant modification of the remaining two sulfhydryl groups of hexokinase. Chemical modification of hexokinase by iodoacetamide in the absence of mannose resulted in complete inactivation of the enzyme. The iodoacetamide inactivated hexokinase failed to react with o-phthalaldehyde as evidenced by the absence of a fluorescence emission maximum characteristic of the isoindole derivative. The holoenzyme failed to react with [5'-(p-fluorosulfonyl)benzoyl]adenosine. The dissociated hexokinase could be inactivated by [5'-(p-fluorosulfonyl)benzoyl]adenosine; the degree of inactivation paralleled the extent of reaction between o-phthalaldehyde and the nucleotide-analog modified enzyme. Thus, it is concluded that two cysteines and lysines at or near the active site of the hexokinase were involved in reaction with o-phthalaldehyde following complete loss of the phosphotransferase activity. An important finding of this investigation is that the lysines, involved in isoindole formation, located at or near the active site are probably buried.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
6.
Peptide mapping analysis of the avian progesterone receptor 总被引:2,自引:0,他引:2
Progesterone receptor from the chicken oviduct has been shown to exist as two 8 S forms (I and II). Form I contains a protein of Mr = 75,000 and form II contains a protein of Mr = 110,000. In addition to these hormone-binding proteins, both receptor forms contain a protein with Mr = 90,000 that does not bind steroid. To investigate the possibility that these proteins are structurally related, they were isolated by preparative sodium dodecyl sulfate gel electrophoresis and subjected to peptide mapping analyses after digestion with Staphylococcus aureus V-8 protease, papain, or alpha-chymotrypsin. Receptor proteins labeled with [32P]orthophosphate in tissue minces were also subjected to peptide mapping analysis. The electrophoretic patterns of peptide fragments of the 90-kDa protein from receptor forms I and II were identical but were different from the peptide patterns obtained from the 75- and 110-kDa proteins which generated similar peptide patterns, indicating that these are structurally related. However, some differences were evident, indicating that these latter two proteins are not identical substrates for proteases. A one-dimensional comparison of the phosphopeptide patterns from the 75- and 110-kDa proteins also showed them to be similar, but not identical. Two-dimensional maps of phosphopeptides generated from the 75- and 110-kDa protein after complete tryptic digestion revealed multiple sites of phosphorylation which were identical except for one phosphopeptide that was unique to the 110-kDa protein. These results show the two progesterone-binding proteins to be very similar in structure, but to differ considerably from the 90-kDa protein. 相似文献
7.
Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
A new pretreatment technique has been developed in which the lignocellulosic material is subjected to the action of steam and high-pressure carbon dioxide before being explosively discharged through a defibrating nozzle of novel design. Operating at 200 degrees C and gas pressures in the range of 3.45-13.8 MPa, exploded products with maximum in vitro cellulase digestibilities of 81, 78, and 75% were obtained from wheat straw, bagasse, and Eucalyptus regnans woodchips, respectively. The treatment times required to obtain substrates of maximum digestibility were 5 min for wheat straw and bagasse and 15 min for E. regnans. Analysis of the exploded products indicated that the pretreatment had substantially removed and solubilized the hemicellulose fraction of the feed materials, giving an autohydrolysis liquor rich in xylose and a fibrous residue primarily composed of alpha-cellulose and lignin. The fibrous residue was readily amenable to cellulase hydrolysis, with saccharification being completed within 48 h. The theoretical energy demand for gas compression in the pretreatment process has been calculated at 11 k Wh/ton raw material when the digester is operated at 3.45 MPa and a packing density of 250 kg raw material/m(3) digester volume. 相似文献
9.
Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment. 总被引:10,自引:3,他引:7
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Marked differences were observed between the H2 and H3 strains of influenza virus in their sensitivity to pretreatment at low pH. Whereas viral fusion and hemolysis mediated by influenza virus X:31 (H3 subtype) were inactivated by pretreatment of the virus at low pH, influenza virus A/Japan/305/57 (H2 subtype) retained those activities even after a 15-min incubation at pH 5.0 and 37 degrees C. Fusion with erythrocytes was measured by using the octadecylrhodamine-dequenching assay with both intact virions and CV-1 monkey kidney cells expressing hemagglutinin (HA) on the plasma membrane. To study the nature of the differences between the two strains, we examined the effects of low-pH treatment on the conformational change of HA by its susceptibility to protease digestion, exposure of the fusion peptide, and electron microscopy of unstained, frozen, hydrated virus. We found that the respective HA molecules from the two strains assumed different conformational states after exposure to low pH. The relationship between the conformation of HA and its fusogenic activity is discussed in the context of these experiments. 相似文献
10.
Summary Immunotherapy with interleukin 2 (IL-2) alone or in combination with lymphokine-activated killer cells can mediate tumor regression in mice and in man. Further dose escalation of IL-2 along with lymphokine-activated killer cells has been prevented by the development of a vascular leak syndrome produced by IL-2. Because we have found that interferon (IFN-) or tumor necrosis factor (TNF-) has synergistic antitumor effects when administered together with IL-2, we have tested the vascular leakage induced by these lymphokine combinations. We used a murine model to quantify vascular leakage by measuring the extravasation of 125I-albumin from the intravascular space as well as the wet and dry lung weights after treatment with different cytokines. Cytokines (or Hanks balanced salt solution) were administered to C57BL/6 mice and 4 h after the last injection the vascular leak was quantified. IFN- alone did not cause extravasation of radiolabel or increase in wet lung weights, though when given in combination with IL-2, significantly greater extravasation (P<0.01) as well as increase in lung water weights (P<0.05) was observed compared to the response in mice treated with IL-2 alone. IFN- in combination with IL-2 induced significant vascular leakage earlier than the response induced by IL-2 alone. For example treatment with IFN- and IL-2 induced accumulation of 14674±605 cpm in the lungs at day 1 while IL-2 alone induced 12340±251 cpm. The degree of vascular leakage was highly related to the dose of IFN- administered along with IL-2 and increased vascular leak syndrome was evident even at low doses (5000 units) of IFN-. Immunosuppression of mice by pretreatment irradiation (500 rad) markedly decreased the development of vascular leak syndrome induced by IL-2 and IFN-. Interestingly IFN- and TNF- did not induce vascular leakage in the lungs when given alone, and did not add or synergize with IL-2 in causing the syndrome. Thus the administration of IFN- in combination with IL-2 produces a dose-limiting vascular leakage that is more severe than that caused by IL-2 alone, and may be mediated, directly or indirectly by host radiosensitive cells.
Abbreviations used: LAK, lymphokine-activated killer; IFN, interferon; TNF, tumor necrosis factor; IL-2, interleukin-2 相似文献