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Biomedical subjects

K J Schray

Publications and source records attributed to K J Schray.

At least 19 recordsLinked to original sources
PubMed1995

Retroviral-like sequences specifically expressed in the rat ovary detect genetic differences between normal and transformed rat ovarian surface epithelial cells.

We have identified a repetitive DNA element in the rat genome that we demonstrate to be suitable to detect molecular genetic differences between normal and malignantly transformed rat ovarian surface epithelial cells by genome scanning. With fluorescence in situ hybridization, we show that these elements are widely distributed in the rat genome, and that a member of this family is present within a homogeneously staining chromosomal region of tumorigenic rat ovarian surface epithelial cells. The homogeneously staining chromosomal region infers the presence of an amplified DNA sequence in the tumor cells, and we provide molecular evidence for an amplicon in this DNA by genome scanning using a probe related to these elements. Sequence analysis revealed that these elements have the structural features of retroviral DNA. We show that they are transcriptionally active in the rat ovary, but not in a wide range of other normal rat tissues. In situ hybridization to ovarian tissue sections revealed that the elements are expressed in granulosa and theca interna cells and in the surface epithelial cells adjacent to preovulatory follicles. Based on this property, we refer to these retroviral-like elements as ovary-specific transcribed sequences. Partial sequence analysis of multiple members of this family and the range in transcript sizes strongly suggest that multiple members of this retrovirus-like family are transcriptionally active. We speculate that binding by one or a combination of ovary-specific transcription factors to regulatory sequences within the genomic ovary-specific transcribed sequences units contributes to the tissue specificity of their expression.

Animals↗
PubMed1992

An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide.

An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1988

Interactions of porphyrins and transfer RNA.

The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(II), manganese(III) and zinc(II) complexes with brewer's yeast type V phenylalaninyl tRNA were evaluated by UV-visible spectroscopy, circular dichroism and melting temperature studies over a range of magnesium ion concentrations and ionic strengths. Scatchard analysis of absorption spectra of the porphyrins in the presence of tRNA showed the free base, copper and zinc porphyrins to have binding constants of 7.3 X 10(7), 1.7 X 10(6) and 2.3 X 10(8), respectively; the manganese(III) complex did not demonstrate changes in its electronic spectra that enable the calculation of a binding constant. The results of the spectroscopic studies indicate a mode of binding for the free base, copper(II) and zinc(II) complexes that is neither intercalative nor simply outside electrostatic. The magnitude of the binding constants and the UV-visible results support intercalation, but the analyses of the thermal denaturation studies and the circular dichroism evaluations suggest that the porphyrins are associating at a single site in a fold of the tertiary structure of the tRNA close to several crucial hydrogen bonds, perhaps in the vicinity of the P10 loop. That the manganese(III) complex does not bind in this site points to constraints on the axial thickness of a molecule that may be accommodated in this locus.

Cations, Divalent↗
PubMed1987

Radiosensitizer conjugation to the carcinoma 19-9 monoclonal antibody.

Misonidazole was covalently conjugated (3-68 mol drug/mol antibody) to 19-9 monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen as a method for targeting radiosensitizing agents. This attachment was accomplished by the mixed anhydride method using the hemisuccinate derivative of misonidazole. Evaluation of conjugates in vitro shows a loss of antibody binding activity with increasing loading levels; however, significant binding activity is retained even at relatively high sensitizer/antibody ratios. This observation was consistent in three binding assays: a competitive radioimmunoassay; an enzyme immunoassay; and an affinity column assay. From these studies, it was concluded that the optimal loading factor for misonidazole-antibody conjugates, when it is used for immunochemotherapy lies between 8 and 15. In vitro release studies indicated that conjugates are hydrolytically stable (t1/2 = 4 days) under physiological conditions.

Antibodies, Monoclonal↗
PubMed1987

Further studies on the catalytic mechanism of human liver alpha-L-fucosidase.

Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) with [1-3H]conduritol C trans-epoxide revealed that there are four active sites per tetrameric enzyme complex. Solvent isotope effect experiments give evidence for a proton transfer at the rate-limiting step in catalysis. Transglycosylase activity was observed using methanol as an alternative glycone acceptor to produce methyl alpha-L-fucoside, suggesting that alpha-L-fucose is formed when water is the acceptor. Initial burst kinetics experiments suggest that a glycosyl-enzyme intermediate is formed, although the magnitude of the burst is not stoichiometric with the number of active sites. These data, along with previous results, suggest a general acid-general base catalytic mechanism involving double inversion of stereochemistry at C-1 of fucose, as well as the formation of either a covalent glycosyl-enzyme intermediate or a tight ion pair between a charged active-site residue and a hypothetical fucosyl oxocarbonium ion intermediate.

Affinity Labels↗
PubMed1987

Macromolecular attachment as a metabolic stabilizer for a labile radiosensitizer.

4-Nitro-5-sulfonylimidazoles represent a class of hypoxic cell radiation sensitizers whose in vitro activity greatly exceeds that of the current clinical standard, misonidazole. However, in vivo studies with these 4,5-disubstituted imidazoles have shown that a rapid reaction with circulating thiols decomposes the agent and compromises its clinical utility. Drug-macromolecule conjugates prepared in this study from poly-L-glutamate, succinylated poly-L-lysine, dextran, and a succinylated polylysine-antibody, all demonstrated protection of the drug from glutathione displacement. 1-Methyl-4-nitro-5-imidazolyl 4-aminophenyl sulfone conjugated to a succinylated poly-L-lysine-antibody was greater than 7 times less reactive than the unbound drug. Polymer transport may offer a useful tumor-delivery mechanism for these highly reactive radiation sensitizers.

Antibodies, Monoclonal↗
PubMed1986

Active-site-directed inactivation of human liver alpha-L-fucosidase by conduritol C trans-epoxide.

Conduritol C trans-epoxide was found to inactivate human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51), exhibiting an apparent dissociation constant of 43 mM. The cis-isomer of the inactivator had no apparent effect on the enzyme's activity. The pH profile for the inactivation yielded two apparent pK values of approx. 3.7 and 6.1 alpha-L-Fucose (a competitive inhibitor) was effective in protecting the enzyme from inactivation. These results are consistent with a requirement for two amino acid side chains at the active site involved in the reaction of the enzyme with conduritol C trans-epoxide.

Binding Sites↗
PubMed1986

A spectrophotometric assay for nanogram quantities of biotin and avidin.

Parameters and conditions of an enzyme based assay for biotin and avidin are presented. Biotinylated glucose-6-phosphate dehydrogenase when complexed with avidin becomes inactivated. Thus it was possible to construct a competitive assay system for biotin. The assay is sensitive between 100-500 ng/ml and could detect as little as 10 ng in 0.1 ml with a between run error of 2.4%. It requires a 60 min incubation at 21 degrees C and 5 min to assay. The avidin assay, based on the degree of inactivation of biotinylated-glucose-6-phosphate dehydrogenase in relation to the concentration of avidin, could detect as little as 0.25 ng in 0.1 ml or 2.5 ng/ml with an assay time of 10 min with a between run error of 3.9%. Both assays are rapid with significant improvements over other non-isotopic methods in sensitivity and comparable to radioisotopic methods in sensitivity with the added advantage of ease of method.

Avidin↗
PubMed1985

Improved biotinylation of glucose-6-phosphate dehydrogenase using active-site-blocking agents.

Characteristics of the biotinylation of glucose-6-phosphate dehydrogenase are presented. The enzyme is inactivated in the presence of N-hydroxysuccinimido biotin but can be protected by an appropriate concentration of NADPH used as an active-site blocker. A Ki of 1.6 +/- 1.0 microM calculated for NADPH for this protection shows it to be an active-site phenomena. Enzyme inactivation is irreversible with consistent kinetic results requiring the presence of 10 mM EDTA. An improved methodology was developed for biotinylation allowing 100% protection of the enzyme with loading factors up to 30.8 mol of biotin per enzyme.

Binding Sites↗
PubMed1985

Studies on the catalytic residues at the active site of human liver alpha-L-fucosidase.

Kinetic studies and chemical modifications were performed on purified human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) in an attempt to identify the catalytic residues at the active site. Plots of log Vmax vs. pH (computer-fitted to a theoretical model) displayed two apparent pK values, of approx. 3.8 and 7.3. The temperature dependence of these pK values yielded heats of ionization of 3 and 0 kcal/mol from Van't Hoff plots for the lower and higher pK values, respectively. Reaction of alpha-L-fucosidase with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sodium p-(hydroxymercuri)benzoate resulted in complete inactivation of the enzyme. Other nonspecific inactivators had little or no effect on enzyme activity. These results suggest two carboxyl groups whose ionization state is important to activity, a non-active-site cysteine residue important to activity, and at least one active-site carboxyl group.

Binding Sites↗
PubMed1985

Protein quantitation of as low as 10-ng/ml concentrations by competitive binding to polystyrene latexes.

A protein quantitation method which offers protein detection as low as 10 ng protein/ml and accurate quantitation as low as 30-100 ng protein/ml, depending on the protein, has been designed. The assay, which is relatively quick and simple to perform, utilizes the strong, nonspecific adsorption of proteins onto polystyrene latexes. A competition is created between a marker enzyme and the analyte protein for a limited amount of latex surface area. Due to inactivation of the enzyme upon binding to a hydrophobic latex surface, measurement of enzyme activity allows determination of the bound/free enzyme ratio and thus the competing protein concentration. Considerations of sensitivity and simplicity are suggested to make this assay superior to others presently available.

Alkaline Phosphatase↗
PubMed1981

Comparative inactivation and inhibition of the anomerase and isomerase activities of phosphoglucose isomerase.

Several metabolic compounds have been found to be competitive inhibitors of the anomerase activity of phosphoglucose isomerase (EC 5.3.1.9).Ki values for erythrose 4-phosphate, 6-phosphogluconate, and fructose 1,6-bisphosphate for the anomerase reaction are 0.32 muM, 21 muM, and 84 muM respectively at 0 degree and pH 8.2. A significant difference between the fructose 1,6-bisphosphate inhibition constants for both activities was found (Ki(isomerase) = 800 muM and Ki(anomerase) = 140 muM). Also the Km values for both activities were found to be significantly different (Km(isomerase) = 140 muM and Km(anomerase) = 3.6 muM). Attempts to independently alter the anomerase to isomerase activity ratio through protein modification yielded mixed results. While several modifying reagents destroyed the catalytic activities at identical rates, inactivation by iodoacetamide or pyridoxal 5' phosphate sensitized photo-oxidation displayed differential initial effects on the two activities with the anomerase activity being the less affected. These data support the theory that an imidazole residue is catalytically important for isomerization, but less so for anomerization.

Animals↗
PubMed1981

Coated tube enzyme immunoassay: factors affecting sensitivity and effects of reversible protein binding to polystyrene.

Coated tube enzyme immunoassay using alkaline phosphatase conjugated to rabbit (anti-human IgG) antiserum was studied to determine conditions of maximum sensitivity. The competitive binding assay utilized showed a large increase in sensitivity with immobilized antigen levels below the levels giving rise to the maximum in the coating-antigen dilution series. The effects of reversible antigen binding to the solid phase were investigated by comparison of untreated polystyrene tubes, polystyrene tubes treated with glutaraldehyde and glass tubes activated with an aminosilane. The use of glutaraldehyde treated tubes reduced, and the use of activated glass tubes prevented the time dependent release of immobilized antigen seen with the untreated polystyrene tubes. By comparison of these solid phases, it is shown that reversible antigen immobilized in a competitive binding assay gives rise to poorer conjugate binding (three-fold), and poorer sensitivity (six-fold). A noncompetitive response was found to occur at high free antibody levels and low competing antigen concentrations. This binding behavior is moderated by the minimization of the reversible antigen immobilization.

Animals↗