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Query: UNIPROT:P06889 (Mol)
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It is currently thought that nuclear pore complexes (NPCs) primarily govern nucleocytoplasmic interactions via selective recognition and active transport of macromolecules. However, in various nuclear preparations, patch-clamp and fluorescence, luminiscence and ion microscopy support classical microelectrode measurements indicating that monoatomic ion flow across the nuclear envelope (NE) is strictly regulated. Gating of large conductance nuclear envelope ion channels (NICs) somewhat resembles that of gap junctional channels. In other respects, NICs are distinct in that they require cytosolic factors, are blocked by wheat germ agglutinin and are blocked and/or modified by antibodies to epitopes of NPC glycoproteins. Therefore, NIC activity, recorded as electrical current/conductance is likely to be intrinsic to NPCs. This observation suggests a potential use for the patch-clamp technique in establishing the mechanisms underlying nuclear pore gating in response to cytosolic and nucleosolic factors such as transcription and growth factors, oncogene and proto-oncogene products and receptors for retinoids, steroids and thyroid hormone. NIC activity may also be useful in evaluating the mechanisms of nuclear import of foreign nucleic acid material such as that contained in virons and viroids. Finally, in consideration to the electrophysiological data accumulated so far, the study of nuclear pore ion channel activity may help our understanding of other important issues such as cell suicide, programmed cell death or apoptosis.
Mol Membr Biol
PMID:Nuclear pore complex ion channels (review). 753 9

An Aedes albopictus cell line previously shown to be deficient in thymidine kinase activity was transfected with a thymidine kinase gene (tk) from Herpes simplex virus. Survival of the transfected lines in a 'TK+ selective medium' indicated that the viral gene was actively expressed at a level sufficient to rescue the TK-deficient phenotype of the parent line. Unlike the parental cells, TK+ transformants (TK6:hsv cells) were sensitive to 5-bromodeoxyuridine, and contained DNA corresponding to the constructs introduced by transfection. This TK selection system will facilitate recovery of other cotransfected, non-selectable mosquito genes in cultured cells. Transformed cells were treated with several antiviral drugs to define conditions for a 'suicide selection' system, in which cells expressing the viral thymidine kinase enzyme (TK) under the control of an inducible promoter would be selectively destroyed, whereas cells expressing the endogenous mosquito enzyme would remain relatively unaffected. The anti-herpetic drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) showed greater cytotoxicity against transformed cells expressing the viral enzyme, and less toxicity to wild-type mosquito cells. This cell culture system provides a model for initial evaluation of suicide selection systems that may ultimately be adapted to the mosquito using sex- or tissue-specific promoters to drive expression of heterologous genes.
Insect Mol Biol 1995 May
PMID:Evaluation of a viral thymidine kinase gene for suicide selection in transfected mosquito cells. 755 Nov 94

Dividing eukaryotic cells expressing the herpes simplex virus type 1 thymidine kinase (TK) gene are sensitive to the cytotoxic effect of nucleoside analogs such as acyclovir or ganciclovir (GCV). Transgenic mice with cell-targeted expression of this conditional toxin have been used to create animals with temporally controlled cell-specific ablation. In these animal models, which allow the study of the physiological importance of a cell type, males are sterile. In this study, we showed that this phenomenon is due to testis-specific high-level expression of short TK transcripts initiated mainly upstream of the second internal ATG of the TK gene. This expression is DNA methylation independent. To obtain a suicide gene that does not cause male infertility, we generated and analyzed the properties of a truncated TK (delta TK) lacking the sequences upstream of the second ATG. We showed that when expressed at sufficient levels, the functional properties of delta TK are similar to those of TK in terms of thymidine or GCV phosphorylation. This translated into a similar GCV-dependent toxicity for delta TK- or TK-expressing cells, both in vitro and in transgenic mice. However, delta TK behaved differently from TK in two ways. First, it did not cause sterility in delta TK transgenic males. Second, low-level delta TK RNA expression did not confer sensitivity to GCV. The uses of delta TK in cell-specific ablation in transgenic mice and in gene therapy are discussed.
Mol Cell Biol 1995 Oct
PMID:A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice. 756 81

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.
J Steroid Biochem Mol Biol 1995 Sep
PMID:Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs. 757 11

The first required step in ecdysteroid (molting hormone) biosynthesis, dietary cholesterol (C) conversion to 7-dehydrocholesterol (7dC) via 7,8-dehydrogenation, is mediated by a microsomal cytochrome-P450 monooxygenase specific to the larval prothoracic gland. A subsequent series of unknown "black-box" oxidations of 7dC result in the unusual ring geometry (cis-A/B) and functionality (6-keto-7-ene-14-alpha-ol) of the ecdysteroids and has been thought to involve the initial formation of alpha-5,6-epoxy-7-dehydrocholesterol (alpha epo7dC). Pharmacological studies indicated that conversion of C to 7dC in prothoracic gland homogenates was strongly and equally inhibited by the isomeric cholesterol substrate analogues alpha- and beta-5,6-epoxycholesterol (alpha- and beta epoC) and alpha- and beta-5,6-iminocholesterol (alpha- and beta iminoC). With respect to the conversion of C to ecdysteroids by disrupted glands, however, the two alpha-isomeric substrates were 10-fold more inhibitory than were their beta-analogues. Indeed, alpha amino C was as active as the non-specific pyrimidyl cytochrome-P450 monooxygenase inhibitor fenarimol that shows moderate toxicity in many insect species. All four cholesterol analogues competitively inhibited cholesterol 7,8-dehydrogenation, but only alpha epoC and possibly alpha iminoC were desaturated to delta 7-products. Although the KmS (and KiS) for all the substrates were similar (1.7-6.0 x 10(-5) M), the Vmax for alpha epoC dehydrogenation was eight-fold higher than that of C, making it a superior substrate for following this reaction in ecdysteroidogenic tissues rich in endogenous C. The 7,8-dehydrogenation of alpha epoC and alpha iminoC by prothoracic glands would produce the potentially reactive intermediates, alpha epo7dC and alpha imino7dC, respectively. They, in turn, could then undergo facile, acid-catalyzed ring-opening to the allylic-stabilized carbo-cation electrophiles. These very reactive, transient species, if formed in the active site of the monooxygenase, would then alkylate either the heme group or the apoprotein of the cytochrome or both, leading to the irreversible inhibition of the enzyme. The present data show that alpha epoC and probably alpha iminoC are mechanism-based suicide inhibitors of the enzyme catalyzing cholesterol 7,8-dehydrogenation and may be the prototypes of a new class of selective insect control agents.
Insect Biochem Mol Biol 1995 Jun
PMID:Stereospecific, mechanism-based, suicide inhibition of a cytochrome P450 involved in ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta. 762

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as theta-toxin or perfringolysin O, and in the chromosomal plc gene, which encodes the alpha-toxin or phospholipase C. The resultant mutants failed to produce detectable theta-toxin or alpha-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the plc mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of alpha-toxin in gas gangrene or clostridial myonecrosis.
Mol Microbiol 1995 Jan
PMID:Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene. 774 41

A cDNA clone (cNPK15) was isolated from tobacco cells in suspension culture, which encodes a predicted protein kinase of 422 amino acids. The predicted NPK15 protein consists of a hydrophobic region near the amino-terminus, a linker domain and the catalytic domain of a protein-serine/threonine kinase in the carboxyl-half. NPK15 was not found to be closely related to any reported protein, but its putative catalytic domain shares some structural similarity with those of receptor-like protein kinases of plants, such as ZmPK1 from Zea mays and TMK1 from Arabidopsis, even though no receptor-like domain is found in NPK15. Recombinant NPK15 expressed in Escherichia coli as a fusion protein was found capable of autophosphorylation and of phosphorylation of the histone H1 protein on both serine and threonine residues. Upon overexpression of cNPK15 under control of the promoter of cauliflower mosaic virus 35S RNA in tobacco cells, into which it had been introduced by Agrobacterium-mediated transformation, the NPK15 gene acted as a "suicide" gene and blocked proliferation of the host cells. By contrast, such a suicide effect was not observed with the gene for a kinase-negative mutant protein in which the nucleotide sequence for the ATP-binding site had been mutated or with a mutant derivative encoding a protein in which the hydrophobic region had been deleted. Thus, the protein kinase activity of NPK15 and the hydrophobic region of the protein are responsible for the suicide effect. The NPK15 protein kinase seems to be associated with specific cellular functions. Southern blot analysis with cNPK15 as the probe detected several fragments in restriction digests of genomic DNAs from both tobacco and other members of the Solanaceae. This results suggests that NPK15-related genes constitute a small gene family in the genomes of Solanaceae.
Mol Gen Genet 1994 Oct 17
PMID:NPK15, a tobacco protein-serine/threonine kinase with a single hydrophobic region near the amino-terminus. 784 51

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide' vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18 kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18 kDa gene and expression in M. smegmatis was obtained.
Mol Microbiol 1993 Dec
PMID:Construction and use of integrative vectors to express foreign genes in mycobacteria. 793 74

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.
Mol Cell Biol 1994 Nov
PMID:Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination. 793 64

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were subcloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or alpha Gal(1-4)beta Gal-coated latex beads. CBA mice (N = 100) were challenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.
Mol Microbiol 1993 Oct
PMID:Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of alpha Gal(1-4) beta Gal binding in virulence of a wild-type strain. 796 11


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