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81.
The effects of alterations in extracellular calcium concentration on prostaglandin (PGE) and thromboxane (TXB2) syntheses were studied in isolated epithelial cells from the urinary bladder of the toad, Bufo marinus. In epithelial cells prepared using collagenase, basal iPGE synthesis was greater than iTXB2 synthesis. Increasing extracellular calcium from zero to 1 mm increased iPGE synthesis and decreased iTXB2 synthesis equivalently such that total conversion of endogenous arachidonate to these two metabolites was unaltered. Vasopressin stimulated iPGE and iTXB2 syntheses when the incubation buffer contained 1 mm calcium but had no effect in the presence of 0.4 μm calcium. In contrast, using an EDTA isolation method, basal iPGE and iTXB2 syntheses were equal in the presence of zero calcium. Increasing extracellular calcium concentration to 1 mm caused a greater enhancement in iTXB2 synthesis compared to iPGE. Increasing extracellular calcium to 2 mm was associated with a decline in iPGE and iTXB2 syntheses back to the levels observed with no calcium added to the medium. The effect of increasing the calcium concentration was greater in phosphate than in bicarbonate buffer. In a Tris buffer the effect of altered calcium was almost completely abrogated. These studies demonstrate that the choice of buffer and alterations in extracellular calcium concentration differentially alter basal arachidonic acid metabolism to prostaglandins and thromboxane in isolated toad urinary bladder cells. The results suggest that there may exist several endogenous pools of arachidonic acid which are differentially influenced by calcium. Furthermore, the pool sensitive to vasopressin has an absolute requirement for calcium. 相似文献
82.
Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line 总被引:4,自引:0,他引:4
R Binder C C MacDonald J B Burch C B Lazier D L Williams 《Molecular endocrinology (Baltimore, Md.)》1990,4(2):201-208
A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
83.
The major and minor chicken vitellogenin genes are each adjacent to partially deleted pseudogene copies of the other. 总被引:4,自引:2,他引:2 下载免费PDF全文
The major chicken vitellogenin gene (VTGII) has previously been cloned and sequenced. We now report the isolation of genomic clones that encompass a minor chicken vitellogenin gene (VTGIII) which is also expressed in the liver in response to estradiol. Our analysis reveals that a pseudogene for VTGII (psi VTGII) lies 1,426 base pairs upstream of this VTGIII gene. A reevaluation of published sequence data reveals that the converse is also true, namely, that a pseudogene for VTGIII (psi VTGIII) lies 1,345 base pairs downstream of the VTGII gene. Our results show that a 335-base-pair deletion has removed the psi VTGIII promoter and cap site but left residual estrogen response element in a region where nuclease-hypersensitive sites have been reported to be induced in response to estradiol. 相似文献
84.
Evidence that chicken CR1 elements represent a novel family of retroposons. 总被引:6,自引:1,他引:5 下载免费PDF全文
We report the first precise delineation of a chicken CR1 element and show that it is flanked by a 6-base-pair target site duplication that occurred when this repetitive element transposed. The 3' end of this CR1 element is defined by an 8-base-pair imperfect direct repeat, and we infer that this sequence represents the 3' end of all intact CR1 elements. In contrast, the 5' ends are not unique, and we argue that this variation existed at the time each element transposed. We also provide evidence that CR1 elements transposed into preferred target sites. CR1 elements therefore appear to represent a novel class of passive retroposons. 相似文献
85.
Identification and sequence analysis of the 5' end of the major chicken vitellogenin gene. 总被引:26,自引:9,他引:17 下载免费PDF全文
J B Burch 《Nucleic acids research》1984,12(2):1117-1135
We have precisely determined the positions of the first three exons for the major chicken vitellogenin gene (VTG II) by a combination of S1 nuclease protection, primer extension and DNA sequencing experiments. In addition, we have determined the nucleotide sequences of the 5' flanking nuclease hypersensitive sites that we have previously shown are induced during the estrogen mediated activation of the VTG II gene in liver (1). One of these sites is found to be nearly identical to the enhancer core sequence of SV40. A computer assisted analysis of the DNA sequences upstream from the VTG II gene has revealed four short (7 to 9 base pair) sequence elements that are present in similar positions flanking the other major estrogen inducible gene for liver, very low density apolipoprotein II (apoVLDL II). For VTG II, these sequences are located between two of the induced nuclease hypersensitive sites that are liver specific. Sequences homologous to one element, located approximately 100 base pairs upstream from the mRNA cap sites of the VTG II and apoVLDL II genes, are also observed for three estrogen inducible genes that are expressed in the oviduct, although for each of these genes the sequence falls further upstream, between -220 and -200. We suggest that these conserved sequences may be important in mediating the tissue specific responses of these genes to estrogen. 相似文献
86.
87.
NPC 15437 interacts with the C1 domain of protein kinase C. An analysis using mutant PKC constructs 总被引:1,自引:0,他引:1
We recently demonstrated that 2,6,diamino-N-[( 1-(oxotridecyl)-2-piperidinyl]methyl)-hexanamide (NPC 15437) is a selective inhibitor of PKC interacting at the regulatory domain of the enzyme. To further investigate the interaction of NPC 15437 with PKC we expressed a series of cDNAs encoding mutant PKC molecules in COS7 cells. NPC 15437 had no effect on the protein kinase activity of mutants lacking the N-terminal region of the C1 domain. Further, NPC 15437 was a competitive inhibitor of the activation of PKC alpha by phorbol ester and attenuated the binding of phorbol ester to the enzyme in intact cells. The present study demonstrates that mutant enzyme constructs can be used to localize the site of interaction of NPC 15437 with PKC to residues 12-42, which encodes the pseudosubstrate binding domain and part of the first cysteine-rich repeat sequence. 相似文献
88.
D-Phe7-substituted peptide bradykinin antagonists are not substrates for kininase II 总被引:1,自引:0,他引:1
The bradykinin receptor antagonists [D-Phe7]bradykinin, D-Arg[Hyp3,D-Phe7]bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]bradykinin were tested for their ability to serve as substrates for kininase II (angiotensin converting enzyme) purified from rabbit lung. By HPLC, the peptides were not measurably degraded over 30 minutes. Under identical conditions, bradykinin was completely degraded to bradykinin (1-7). When hippuryl-His-Leu was used as a substrate for kininase II, the D-Phe7-substituted bradykinins acted as weak noncompetitive inhibitors. While the peptides were poor substrates for kininase II, they were short-lived when injected intravenously. D-Arg[Hyp3,D-Phe7]bradykinin was completely degraded to small fragments in less than 2 minutes. In diluted serum in vitro, a single product was observed with elution consistent with loss of arginine, suggestive of metabolism by kininase I. 相似文献
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90.