Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.
Mol Biol Cell 1992 Apr
PMID:Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. 149 62

Point mutation in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaden their substrate spectrum towards all beta-lactams except cephamicins and imipenem. We describe here hybridization techniques for the detection of point mutations by non-radioactive oligonucleotide probes with plasmid DNA carrying bla T genes immobilized in polystyrene microwells. After hybridization in discriminating conditions with corresponding biotinylated oligonucleotide probes, the hybrids were detected by using a streptavidin-alkaline phosphatase conjugate and a fluorogenic substrate, 4-methylumbelliferyl-phosphate. The adsorption of DNA to microwells used in the present work was found to be independent of Mg2+ and Na+ concentrations. By this method, less than 3 fmols of target DNA were sufficient for the detection of point mutation.
Mol Cell Probes 1992 Feb
PMID:Detection of point mutation in bla T genes of Enterobacteriaceae by biotinylated oligonucleotide probes using microwell hybridization and enzymofluorometric method. 154 33

Using homopolymeric units of either phenylalanine or tryptophan to replace the natural core segment of the Escherichia coli alkaline phosphatase signal peptide, the hydrophobicity requirements for protein export and processing were further explored. The mutant signal peptide containing polyphenylalanine functioned at least as efficiently as the wild-type, while the signal incorporating polytryptophan was dysfunctional. The transport properties of these mutants confirm our work with sequences rich in aliphatic residues; namely that a high mean hydrophobicity per residue is critical for complete and rapid precursor processing and for translocation of the protein. The efficient transport properties of the polyphenylalanine-containing signal peptide demonstrate that neither the bulky, aromatic nature of phenylalanine nor the unusually high hydrophobicity of this mutant peptide adversely alters function. This study also suggests that the low occurrence of phenylalanine in natural signal sequences is not of functional consequence but probably reflects the low number of DNA codons for this residue. The polytryptophan-containing precursor was membrane inserted but not translocated. This type of transport defect suggests that this is a weakly hydrophobic signal peptide, consistent with hydropathy scales, which indicate that tryptophan is comparable to alanine. This application of polymeric sequences provides a function-based assay for the evaluation of amino acid hydrophobicity.
J Mol Biol 1992 Mar 05
PMID:Signal sequences containing multiple aromatic residues. 154 10

Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on SDS-PAGE gels. Most of the receptor protein is present in the higher molecular mass form. Labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Upon alkaline phosphatase treatment a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform was seen, indicating that the observed 110 to 112 kDa upshift reflects androgen receptor phosphorylation. Furthermore, it is shown that both isoforms can bind hormone and undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Androgen receptor heterogeneity in LNCaP cells is caused by a hormone independent phosphorylation step. 156 42

Escherichia coli lac permease is a polytopic integral membrane protein with six translocated (periplasmic) domains. Individual N-terminal cytoplasmic regions and membrane-spanning segments adjacent to each of the periplasmic domains acted as export signals for an attached sensor protein (alkaline phosphatase). However, the export activity of one of the spanning segments was considerably lower than that of the others, and was limited by the presence of a positively charged residue (Arg302). These observations are compatible with models of membrane protein insertion in which hydrophilic domains are translocated independently. However, the results suggest that efficient translocation may sometimes require interaction between individual spanning segments.
J Mol Biol 1992 Apr 05
PMID:Membrane protein spanning segments as export signals. 156 45

The nuclear restructuring that occurs between insemination and full pronuclear formation in pig eggs is accompanied by posttranslational changes to specific egg proteins. Sperm penetration begins in vitro at 3 hr postinsemination (hpi). By 5 hr, decondensing sperm heads and anaphase II plates are observed in 50% of eggs, and, by 8 hpi, both male and female pronuclei have formed. Three consistent changes to the pattern of newly synthesised proteins are triggered in this period; they affect the 46K, 25K, and 22K polypeptides. Changes are also triggered in the 180-200K polypeptides and in the 14K polypeptides, but these are highly variable. The same changes in the prefertilization pattern were observed when prelabelled eggs were used and new protein synthesis was suppressed. The first and most abrupt change involves the apparent catabolic elimination of a group of 46K unphosphorylated polypeptides (pl 7.3-6.4), whose synthesis was greatest before germinal vesicle breakdown but declined slowly in the final phase of maturation, then declined precipitously after activation. Ageing (beyond maturation) also leads to the disappearance of these polypeptides. The progressive disappearance of a set of 25K polypeptides and the concomitant appearance of a dominant 22K polypeptide is the most characteristic fertilization-induced modification to porcine egg proteins. These modifications begin within 1 hr of sperm penetration or activation, are specific to the pig, and involve heavily phosphorylated polypeptides (25K, pl 6.7-6.0) whose synthesis is begun in the early metaphase I stage. Dual ([35S] and [32P]) labelling, protein blocking experiments, and use of alkaline phosphatase suggest that dephosphorylation selectively affects these 25K polypeptides and is mainly or wholly responsible for converting them (completely within 6 hr) to a single, new (22K, pl 7.6) species that is positively charged. The 25K/22K polypeptide modification has a close temporal relationship with the formation of the male and female pronuclei.
Mol Reprod Dev 1992 Apr
PMID:Protein and nuclear changes in pig eggs at fertilization. 157 Nov 62

The immortalized rat calvarial bone cell line RCT-1 responds to treatment with retinoic acid (RA) by increased expression of osteoblast phenotype-related features, including the induction of liver/bone/kidney alkaline phosphatase (ALP) activity. ALP mRNA could not be demonstrated in unstimulated cells, but was first detected in cells treated for 6 h with 1 microM RA. Cycloheximide failed to block the RA induction of ALP mRNA, indicating that de novo protein synthesis was not a requirement for the RA effect and that the ALP gene may be a direct target for RA action. This was confirmed by nuclear run-on assays, which demonstrated a 2.5-fold increase in the abundance of ALP transcripts after 6 h of RA treatment. To determine whether the RA responsiveness was mediated by a specific segment of the ALP promoter, RCT-1 cells were transfected with a series of plasmids containing deletions of the 5'-flanking sequence of the human ALP gene fused to the chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured in cells cultured in the presence of RA or vehicle. All but the smallest construct, which contained 44 basepairs up-stream of the initiation of transcription, were found to mediate a 2- to 3-fold increase in the expression of CAT activity in response to RA. Furthermore, when the region -108 to -45 of the human ALP gene was inserted into the expression vector pBLcat2, in a position immediately up-stream of the herpes simplex virus thymidine kinase promoter, the construct was found to mediate a 2-fold enhancement of CAT activity in response to RA. In gel retardation assays, a major band was present corresponding to the formation of a complex between the 32P-labeled probe containing the -108 to -45 sequence and proteins present in nuclear extracts of RCT-1 cells stimulated for 3 h with RA. These data suggest that the sequence of 64 basepairs (-108 to -45) 5' to the transcription start site is involved in the RA inducibility of the human ALP gene.
Mol Endocrinol 1992 Apr
PMID:Retinoic acid stimulates transcriptional activity from the alkaline phosphatase promoter in the immortalized rat calvarial cell line, RCT-1. 158 26

The effect of acute dopamine (DA) antagonist treatment on neuronal proneurotensin (NT) mRNA was investigated in the rat striatum using a technique of non-radioactive in situ hybridisation. Adult Wistar rats were given a single intraperitoneal injection of either raclopride (D2 antagonist), SCH 23390 (D1 antagonist) or its inactive isomer SCH 23388 and left to survive for 3 h. Their brains were rapidly removed and striatal sections processed for in situ hybridisation using an alkaline phosphatase (AP) labelled oligonucleotide specific for NT mRNA. Blockade of the DA D2 receptors by a single injection of raclopride resulted in an increase in the number of NT mRNA containing cells in the dorsal lateral rim of the striatum adjacent to the corpus callosum. In contrast, no such increase was observed following blockade of the DA D1 receptors with SCH 23390. These findings demonstrate that NT mRNA expression is differentially regulated in the adult rat striatum by selective D1 and D2 antagonists.
Brain Res Mol Brain Res 1991 Mar
PMID:Differential effects of acute dopaminergic D1 and D2 receptor antagonists on proneurotensin mRNA expression in rat striatum. 164 36

We describe some properties on an Mr 30,000 thermolabile and trypsin-sensitive protein that activates phospholipase A2 (PLA2) and which was isolated from nervous tissue of the marine mollusk, Aplysia californica. A similar protein is present in rat cerebral cortex. This protein was partially purified from crude homogenates of nervous tissue by ion exchange chromatography on DEAE-Sephadex followed by size-exclusion high performance liquid chromatography (HPLC). It is loosely associated with membrane fractions, and is extracted by 0.05% Tween 20. Although similar in size to several previously described PLA2-stimulating proteins from non-neural mammalian cells and tissues, it differs from them in some aspects of biological activity. The protein promotes the release of eicosanoids from the membranes of intact Aplysia neurons prelabeled with [3H]arachidonic acid and appears to be an in vitro substrate for protein kinase C (PKC). PLA2-stimulating activity is greatly enhanced after exposing isolated ganglia to phorbol dibutyrate (PDBu) and is reduced by treatment with immobilized E. coli alkaline phosphatase. These observations suggest that phosphorylation of this stimulatory protein by PKC regulates PLA2 in neurons.
Brain Res Mol Brain Res 1991 Mar
PMID:A phospholipase A2-stimulating protein regulated by protein kinase C in Aplysia neurons. 164 37

We have recently demonstrated that a 200-kDa antigen that serves as a target of antibodies acting in synergy with praziquantel is linked to the surface membrane of Schistosoma mansoni by a glycosylphosphatidylinositol (GPI) anchor. In the present study we have examined the potential role of this GPI anchor in the therapeutic action of praziquantel by monitoring the release of surface antigens from living adult schistosomes cultured in the presence or absence of praziquantel and exogenous phospholipases. Phosphatidylinositol-specific phospholipase C (PIPLC) selectively released the 200-kDa antigen from the surface of adult schistosomes, as determined by immunoprecipitation experiments; none of the other GPI-anchored proteins, including alkaline phosphatase and a 22-kDa protein, were released by this enzyme. Anti-cross-reacting determinant antiserum (anti-CRD), which recognizes an epitope on GPI-anchored proteins only after the anchor has been removed by PIPLC, specifically precipitated the 200-kDa antigen, confirming the cleavage of its anchor. When the worms were exposed to both praziquantel and PIPLC, the amount of 200-kDa cleaved from the worms was increased five-fold. The selective release of this antigen was also detected by indirect immunofluorescent labeling of praziquantel-exposed adult worms cultured in the presence of phospholipases. Taken together these observations suggest that modulation of the phospholipase-mediated release of GPI-anchored antigens by praziquantel may contribute to the therapeutic action of the drug.
Mol Biochem Parasitol 1991 May
PMID:Selective release of a glycosylphosphatidylinositol-anchored antigen from the surface of Schistosoma mansoni. 164 1


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