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)

Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.
J Steroid Biochem Mol Biol 1992 Dec
PMID:Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement. 147 60

An acidic glycoconjugate could be extracted from a delipidated residue fraction of [3H]galactose, [3H]mannose or [32P]orthophosphate metabolically labeled Entamoeba histolytica with water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactively labeled glycoconjugate comprised 50-55% of the total [3H]galactose label incorporated into macromolecules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the radiolabeled glycoconjugate showed two diffuse smears centering around 110 kDa and 45 kDa. Similar profiles were observed for both [3H]galactose- and [32P]orthophosphate-labeled glycoconjugate. No such bands were visible in [35S]methionine-labeled material. The hydrophobic nature of this glycoconjugate was inferred from its chromatographic behavior on phenyl-Sepharose. The molecule was rendered hydrophilic after digestion with phosphatidylinositol-specific phospholipase C. It was also sensitive to deamination by nitrous acid. Mild acid hydrolysis led to its fragmentation into smaller molecules as revealed by Sepharose 4B chromatography. Paper chromatographic analysis of the depolymerized [3H]galactose- and [3H]mannose-labeled fragments revealed that each was sensitive to alkaline phosphatase. The major dephosphorylated fragment migrated as an apparent galactose and mannose containing disaccharide which migrated identically to the Gal beta 1-4Man disaccharide derived from the lipophosphoglycan of Leishmania donovani. The above data support the existence of a major acidic glycoconjugate in E. histolytica bearing striking structural similarities to the lipophosphoglycan of Leishmania.
Mol Biochem Parasitol 1992 Nov
PMID:Identification and partial characterization of a lipophosphoglycan from a pathogenic strain of Entamoeba histolytica. 147 94

We have constructed a series of interspecific somatic cell hybrids between the human osteoblast-like osteosarcoma, TE85, and a mouse fibrosarcoma, La-t-. In these whole-cell hybrids, we observed a 10-fold reduction of human liver/bone/kidney (L/B/K) alkaline phosphatase steady-state mRNA and alkaline phosphatase protein activity. The phenomenon of loss of tissue-specific gene expression has been termed extinction. Subclones of these hybrids were isolated, which reexpressed the alkaline phosphatase gene product. These late-passage hybrids had a reduced number of mouse fibroblast chromosomes when compared to earlier passages. This suggests that a trans-acting negative regulatory element, encoded in the fibroblast genome, regulates expression of L/B/K alkaline phosphatase. This is the first evidence that extinction plays a role in the regulation of osteoblast gene expression.
Somat Cell Mol Genet 1992 Sep
PMID:Extinction of liver/bone/kidney alkaline phosphatase in osteosarcoma hybrid cells. 147 9

A non-isotopic microtitre plate-based assay method was devised for detection of products of the polymerase chain reaction. This assay involves affinity immobilization of the biotinylated amplification products in microtitre plate wells and their fluorescence detection by their hybridization with an oligonucleotide probe linked to alkaline phosphatase. An advantage of this procedure is that the immobilization and hybridization are carried out simultaneously in the wells, thus shortening the assay time. The assay method was applied to the detection of the tdh gene of Vibrio parahaemolyticus. Seven copies of the target chromosome could be detected in about 45 min after 35 cycles of amplification.
Mol Cell Probes 1992 Dec
PMID:Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification: application to detection of the tdh gene of Vibrio parahaemolyticus. 148 Jan 88

A cerium-based incubation medium, developed for the light microscopical demonstration of alkaline phosphatase activity, was tried out for the electron microscopical demonstration of this enzyme in kidney and heart muscle of the rat. The medium is very stable and the pH is in the optimum range of the enzyme. The medium consists of 14 mM CeCl3, 11 mM Na-citrate, 4 mM MgCl2, 10 mM p-nitrophenyl phosphate, 0.18 M glycine/NaOH buffer, pH 9.3. Other concentrations of cerium and citrate were tried out as well but 14 mM CeCl3, and 11 mM Na-citrate gave the best results with a small amount of non-specific reaction product in the nucleus that can be largely avoided by postincubation rinsing in cerium-containing buffer. In the kidney reaction product was only present along the microvilli of the proximal tubular epithelial cells. In the glomerulus no reaction product could be found whereas light microscopical cryotome sections contained activity in the glomerulus. Replacement of glutaraldehyde by formaldehyde fixatives resulted in reaction product in glomerular and tubular basement membranes, on podocyte plasma membranes and in tubular basal infoldings. In glutaraldehyde-fixed heart muscle, reaction product was present in the basement membranes and on lateral plasma membranes of endothelial cells of blood capillaries.
Cell Mol Biol (Noisy-le-grand)
PMID:Electron microscopical demonstration of alkaline phosphatase activity with the cerium-based method in citrate-containing medium at pH 9.3 and the influence of glutaraldehyde fixation. 148 7

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.
Mol Biol Cell 1992 Dec
PMID:Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. 2279 87

The uropathogenic Gram-negative bacterium Proteus mirabilis exhibits a form of multicellular behaviour termed swarming, which involves cyclical differentiation of typical vegetative cells into filamentous, multinucleate, hyperflagellate swarm cells capable of rapid and co-ordinated population migration across surfaces. We observed that differentiation into swarm cells was accompanied by substantial increases in the activities of intracellular urease and extracellular haemolysin and metalloprotease, which are believed to be central to the pathogenicity of P. mirabilis. In addition, the ability of P. mirabilis to invade human urothelial cells in vitro was primarily a characteristic of differentiated swarm cells, not vegetative cells. These virulence factor activities fell back as the cells underwent cyclical reversion to the vegetative form (consolidation), in parallel with the diagnostic modulation of flagellin levels on the cell surface. Control cellular alkaline phosphatase activities did not increase during differentiation or consolidation. Non-flagellated, nonmotile transposon insertion mutants were unable to invade urothelial cells and they generated only low-level activities of haemolysin, urease and protease (0-10% of wild type). Motile mutants unable to differentiate into swarm cells were comparably reduced in their haemolytic, ureolytic and invasive phenotypes and generated threefold less protease activity. Mutants that were able to form swarm cells but exhibited various aberrant patterns of swarming migration produced wild-type activities of haemolysin, urease and protease, but their ability to enter urothelial cells was three- to 10-fold lower.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Microbiol 1992 Jun
PMID:Co-ordinate expression of virulence genes during swarm-cell differentiation and population migration of Proteus mirabilis. 149 87

A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active HlyD-LacZ fusion proteins were only generated when lacZ was fused to hlyD within the first 180 bp (60 amino acids). HlyD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD). Active insertions of phoA into the middle region of hlyD were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1992 Jul
PMID:A topological model for the haemolysin translocator protein HlyD. 149 79

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


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