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Biomedical subjects

R E Hurst

Publications and source records attributed to R E Hurst.

At least 55 records · Page 3Linked to original sources
PubMed1988

The identification of a heparin-binding protein on the surface of bovine sperm.

We report the identification of a sperm surface protein which binds tightly to heparin. The protein was isolated by affinity chromatography on heparin agarose, and its affinity for heparin was confirmed by electrophoresis in the presence of heparin under non-denaturing conditions. The protein consists of a single polypeptide chain with a molecular weight of 45,000, as determined by electrophoresis under denaturing conditions. The protein may bind glycosaminoglycans in vivo and play a part in initiating the capacitation/acrosome reaction.

Animals↗
PubMed1988

Equilibrium binding of Hoechst 33258 and Hoechst 33342 fluorochromes with rat colorectal cells.

We examined the biophysical characteristics of the interaction of Hoechst 33258 and 33342 dyes with normal rat colorectal cells as functions of fixation and solution composition. Classical dye-binding techniques were used to investigate the stoichiometry and binding constants with whole cells, and quantitative fluorescence image analysis was used to specifically study nuclear dye binding in intact cells. In aqueous solution, H-33258 dye bound cooperatively with intact cells, with a binding constant of between 3-4 x 10(5). In ethanolic solution, binding appeared less cooperative, although Scatchard analysis could not be used. The binding constant was slightly lower (2 x 10(5)), but the total number of cell binding sites was decreased by a factor of 5, reflecting a great decrease in cytoplasmic sites. QFIA studies identified conditions optimal for DNA quantitation under which the fluorescence signal was independent of dye or cell concentration. The proportionality between absolute nuclear fluorescence intensity and DNA content was established, and the upper limit of DNA content of normal colorectal cells was also determined.

Animals↗
PubMed1987

DNA cytometry and cytology by quantitative fluorescence image analysis in symptomatic bladder cancer patients.

A semi-automated quantitative fluorescence image analysis (QFIA) technique was developed with the Leitz TAS-Plus to detect bladder cancer using hyperploidy in urinary cells. Absolute nuclear fluorescence intensity (ANFI) (emission at 540 nm with excitation at 436 nm) of individual acridine-orange-stained cells was quantitated using (1) QFIA and (2) simple filter microspectrofluorophotometry (SFM). Both methods employed an internal phosphor particle standard which, when once calibrated against the DNA content of normal cells, obviates the necessity of routinely calibrating against normal cells in each sample. Results of SFM and QFIA were compared with routine Papanicolaou (Pap) cytopathology, using histopathology as the diagnostic standard in 272 samples from 67 symptomatic patients. The sensitivities for detecting low-grade transitional-cell carcinoma were 86% for SFM, 76% for QFIA, and 33% for Pap cytology. QFIA and SFM were significantly more sensitive at detecting bladder cancer than was Pap (0.01 greater than p greater than 0.001). Comparison of sensitivity obtained with bladder washings and urine samples showed that noninvasively obtained urines can be used. ANFI also detected recurrent and precancerous bladder lesions and kidney, ureter, and prostate lesions. This approach may prove generally useful in quantifying biochemical and immunological probes and should be broadly applicable as a research tool for studying the relationship of biochemical markers in the pathogenesis of disease and as a test for cancer control.

Carcinoma, Transitional Cell↗
PubMed1987

Functional and structural characteristics of the glycosaminoglycans of the bladder luminal surface.

The glycosaminoglycan layer of bladder has been proposed to play a crucial role in protecting the bladder from harmful substances in urine. Rats were partially cystectomized to determine whether bladder glycosaminoglycans are routinely eluted from the bladder surface in detectable quantities. Cystectomy produced no detectable qualitative or quantitative changes in excreted GAG thereby showing that most urinary glycosaminoglycan originates in the kidney and not from the bladder. Damaging the glycosaminoglycan layer by a dilute acid wash, however, leads to a consistent decrease in the output of urinary GAG which recovers to normal at the same rate as the layer regenerates. This suggests that the newly exposed sites tightly bind urinary GAG. We suggest that such binding may be a component of the normal physiological defense mechanism of the bladder. The bladder glycosaminoglycan layer was isolated, dilute acid being used to elute ionically-bound material and brief trypsinization to elute intercalated proteoglycans from the luminal surface. The GAG from the luminal surface, which was present at a density of one chain per 50 nm.2 of bladder surface, was quite different in composition from that isolated from the whole bladder.

Animals↗
PubMed1985

Heparin binding is necessary, but not sufficient, for fibronectin aggregation. A fluorescence polarization study.

Analysis of parameters governing heparin binding to fibronectin indicates that heparin binding is a necessary, but insufficient, condition for fibronectin cryoprecipitation. Heparin binding to fibronectin is a rapid, readily reversible event which can occur under several conditions which prohibit fibronectin cryoprecipitation. While cryoprecipitation of fibronectin is abolished at temperatures in excess of 10 degrees C, appreciable heparin binding to fibronectin does occur even at 40 degrees C. While increasing ionic strength and pH inhibit both heparin binding and cryoprecipitation of fibronectin, heparin binding can still occur at high ionic strengths and pH values which completely abolish cryoprecipitation. Scatchard analysis of fluorescent polarization data reveals a biphasic heparin binding curve with high and low affinity Kd values of 3.5 X 10(-8) and 10(-6) M, respectively. In contrast to heparin binding, fibronectin aggregation is a cooperative phenomenon. Fibronectin cryoprecipitation is greatly reduced at temperatures above 10 degrees C, at pH values above pH 10, and at ionic strengths above 0.3 M. Thus, heparin binding and protein aggregation are separate events which occur during fibronectin cryoprecipitation. Results obtained here via fluorescence polarization in conjunction with other physical measurements suggest that a decrease in flexibility of the fibronectin molecule is associated with the protein aggregation step of cryoprecipitation. The role of heparin in the mechanism of fibronectin cryoprecipitation is discussed.

Animals↗
PubMed1984

Structural analysis of heparin by methylation and g.l.c.-m.s.: preliminary results.

Heparin is a complex mixture of polysaccharides differing in biological activity and structure, and attempts to relate this activity to structure have suffered, owing to a lack of sufficiently sensitive and specific analytical methods. Application of methylation analysis to determination of the structure of heparin is described. Carboxyl-reduced heparin was converted into its pyridinium salt, this was dissolved in Me2SO, and free OH and NH groups were methylated with dimethylsulfinyl anion. Sulfate groups were removed by solvolysis, and after dialysis, the polymer was acetylated and depolymerized by acetolysis. The resulting monosaccharides were converted into alditol acetates, which were separated by capillary, gas-liquid chromatography, and identified by both electron impact and chemical ionization mass spectrometry. Seventeen different monosaccharides were identified in the hydrolyzate. All of the expected internal hexosaminyl and glycosyluronic residues were identified. Although several sugars were identified as nonreducing termini, only a hexosamine 6-sulfate was identified as a reducing-terminus sugar. The results indicate that methylation analysis of heparins and other complex, sulfated glycosaminoglycans is feasible.

Chemical Phenomena↗
PubMed1983

Structure-activity relationships of heparin. Independence of heparin charge density and antithrombin-binding domains in thrombin inhibition by antithrombin and heparin cofactor II.

To better understand how heparin structure affects its activity the relationships between the functional domains for inhibitor binding and charge density were investigated to determine how these domains affect heparin-mediated thrombin inhibition by two different heparin-dependent protease inhibitors, antithrombin (AT) and heparin cofactor II (HC II). A series of heparins, fractionated systematically by charge density, was further fractionated on antithrombin agarose to isolate more homogeneous subfractions that were either inactive or highly active with respect to thrombin inhibition by AT. With AT, the activities of the AT-active subfractions increased sharply with heparin charge density, while those with little or no affinity for AT were virtually inactive. In contrast, with HC II inhibitor, the activities of the heparins depended only upon their charge densities and were independent of AT affinity. At any given charge density, the heparin before fractionation by AT affinity and the fractions that were highly active and inactive with AT were all equally active with HC II. The two inhibitors also differed in their reactivity with heparan sulfate and dermatan sulfate. A charge-density effect with the subfractions having similar high affinity for AT demonstrates that charge density represents a heparin functional domain that is independent of the AT-binding domain. The behavior of the AT-inactive heparins, being fully active with HC II, demonstrates the functional domain necessary for AT binding is not needed to produce HC II activity.

Animals↗
PubMed1983

Glycosaminoglycans in human neutrophils and leukemic myeloblasts: ultrastructural, cytochemical, immunologic, and biochemical characterization.

Chondroitin sulfate is known to be present in normal and leukemic myeloid cells; however, its definitive subcellular location and association with other glycosaminoglycans (GAGs) has not been demonstrated. We have studied the type and distribution of GAGs in neutrophil granule subpopulations of normal and leukemic myeloid cells using ultrastructural, cytochemical, immunologic, and biochemical methods. At the ultrastructural level, high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) stained sulfated glycoconjugates selectively in immature primary granules of normal promyelocytes and Auer rods and immature granules of leukemic myeloblasts. Staining was weak or absent in mature primary granules, whereas tertiary granules stained moderately. Primary granule staining with HID-TCH-SP was greatly diminished by prior treatment of the specimens with chondroitinase ABC and/or nitrous acid, indicating the presence of chondroitin sulfate and N-sulfated glycosaminoglycan. Immunostaining of myeloid cells with a rabbit antichondroitin 4-sulfate and ferritin-conjugated goat anti-rabbit IgG sequence resulted in staining of most primary granules. Biochemical analysis of GAGs from leukemic myeloblasts containing primary granules and Auer rods, but lacking secondary and tertiary granules, revealed 8 x 10(-17) mole of uronic acid/cell and electrophoretic and sulfaminohexose analysis showed 60%-70% chondroitin sulfate AC of heterogeneous molecular weight, 20%-30% of a GAG that most closely resembled heparan sulfate, and 10% dermatan sulfate. The lack of significant HID-TCH-SP staining of sulfate iin sites other than Auer rods and primary granules in leukemic myeloblasts indicates that these granules contain the chondroitin, dermatan, and heparan sulfate isolated from the same specimen. Similar GAGs are present in primary granules of normal cells as evidenced by their cytochemical and immunostaining properties. Thus, these studies demonstrate a heterogeneous population of GAGs not previously identified and localize these substances to the primary granule of leukemic and normal cells.

Acid Phosphatase↗
PubMed1982

Platelet factor four and protamine sulfate neutralization of heparin fractionated according to anionic charge density.

The role of heparin structure in neutralization by the neutralizing substances (NS) platelet factor 4 (PF4) and protamine sulfate (PS) was investigated using a thrombin clotting assay and a series of more homogeneous heparin fractions varying systematically in charge density (Z). For a given heparin, plotting inverse clotting times measured without NS, and in the presence of PF4 or PS, vs heparin concentration yielded approximately parallel straight lines displaced horizontally according to the amount of NS. Potencies of heparin fractions in the absence of NS, or in the presence of PF4 of PS, depended almost identically upon Z2. Small but significant quantitative differences in potency among equivalent fractions from different heparins showed both PF4 and PS had a slight preference for the least active subfraction of decolorized heparins, but for the most active subfraction of undecolorized heparins. Neutralization of heparin by PF4 and PS probably proceeds by similar mechanisms, but the details of structure outside the antithrombin III-binding oligosaccharide of heparin may enter in differently.

Animals↗
PubMed1982

Heterogeneity in the composition of commercial heparins: comparison of anticoagulant activities and biochemical compositions of anionic density-fractionated heparins.

In this study the nature of compositional heterogeneity in commercial heparins was investigated. Five hog-mucosal (HM) and one beef-lung (BL) heparin were subjected to partition fraction in a two-phase system of 1-butanol containing hexadecylpyridinium chloride/aqueous NaCl, a system which fractionates heterogeneous heparins larger than 10,000 daltons according to anionic density. The heparins differed markedly in purity as determined by both the galactosamine content of the unfractionated preparations and the amount of uronate extracted at lower NaCl concentrations in the fractionation scheme. All fractions afforded linear plots of the logarithm of clotting time vs heparins concentration in the APTT assay with human plasma, thereby permitting measurement of concentration--independent specific activities. Equivalent fractions from different HM heparins had similar compositions, and with the exception of an unbleached sample, had equivalent specific APTT anticoagulant activities. These latter were linearly related to the square of anionic density. In contrast, the BL heparin behaved quite differently. Its most abundant fractions were extracted at higher NaCl concentrations than was the case for HM heparins, little systematic variation in anionic density was observed, and its fractions had much lower specific activities than equivalent HM fractions.

Animals↗
PubMed1981

Heparan sulfate of AH-130 ascites hepatoma cells: a cell-surface glycosaminoglycan not displaced by heparin.

This study reports on the ultrastructural location and biophysical properties of cell-associated glycosaminoglycans of AH-130 cells, an azo dye-induced ascites hepatoma. Earlier studies have shown that a low-sulfated heparan sulfate, which comprises 93% of their total glycosaminoglycan (GAG) content, is associated with these cells. High-iron diamine, an ultrastructural stain for sulfated glycoconjugates, stained the hepatoma cell surfaces heavily. With the exception of occasional light staining in a few cytoplasmic granules, intracellular organelles did not stain with this method. The lack of an extensive pool of intracellular GAG was confirmed by quantitative fluorescence microscopy of cells vitally stained with acridine orange. The nature of the binding of the cell-surface heparan sulfate was explored by competitive binding studies with exogenous heparin. When cells were incubated with exogenous heparin, release of heparan sulfate into the medium was not detected, although heparin was bound. We conclude that low-sulfated heparan sulfate is an integral component of the AH-130 hepatoma cell surface and is bound at a site different than heparin.

Acridine Orange↗
PubMed1981

Urinary glycosaminoglycan excretion as a biochemical marker in patients with bladder carcinoma.

Urinary glycosaminoglycan excretion was examined in 25 individuals with bladder cancer in comparison to glycosaminoglycan excretion by eight normal individuals. Urinary glycosaminoglycan was isolated by gel filtration and quantified as macromolecular uronate concentration. Electrophoresis in calcium acetate and densitometry of Alcian blue-stained electrophoretograms were used to separate and quantify the relative amounts of individual glycosaminoglycans. Elevated excretion of macromolecular uronate was noted in 53% of the cancer cases. The highest levels were found among individuals with metastatic disease. Three electrophoretic bands were always detected in the control and cancer groups: chondroitin sulfate, heparan sulfate (both confirmed by chemical and enzymatic degradation), and a third band (Band 1) of unknown composition. A fourth band, corresponding to dermatan sulfate, was seen in some high-grade metastatic tumors. Band 1 excretion was elevated in a significant fraction of all patients. Seven of 12 metastatic cases but only two of 13 localized cases showed increased heparan sulfate excretion. Diagnostic limits were drawn from the observed distributions of normals, and with these limits 92% of the cancer cases, including 12 of 12 metastatic cases, could be identified. The results strongly suggest noninvasive urinary glycosaminoglycan analysis may well provide a new biochemical approach for detecting and monitoring the pathogeneses of bladder cancer.

Chondroitin Sulfates↗
PubMed1980

Glycosaminoglycan excretion in osteogenesis imperfecta.

This study was carried out in order to determine whether osteogenesis imperfecta affected individuals showed a different pattern of glycosaminoglycan excretion in urine than do normals. Quantitative excretion was compared by three different methods, and the qualitative composition of the excreted glycosaminoglycan was compared by electrophoresis. No difference was noted in the amount of glycosaminoglycan excreted by normal or affected individuals as measured by macromolecular uronic acid, Alcian Blue bound by urinary constituents or amount of glycosaminoglycan actually isolated by partition. However, the affected individuals showed a statistically significant increase in Alcian Blue binding over macromolecular uronic acid not seen in the normal group, which may possibly indicate the excretion of a substance capable of binding Alcian Blue, but which is not glycosaminoglycan, by osteogenesis imperfecta affected individuals. No qualitative differences in glycosaminoglycan excretion between the two groups were detectable by electrophoresis.

Adolescent↗
PubMed1980

Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells.

The thermodynamic binding parameters for intracellular heparin-acridine orange (AO) complexes were determined for Furth murine mastocytoma cells and were found to agree with 1) results from binding studies on heparin-AO complexes in solution, and 2) with biochemical analyses of the cells. The cells exhibited cooperative binding with a binding constant of 1.18 x 10(6) M-1. The cooperative binding constant of heparin-AO in 1 mM buffer was found to be 1.13 x 10(6) M-1. The addition of 1 mM NaCl to heparin-AO system in vitro detectably decreased the cooperative binding constant. Low ionic strength is the only condition in solution under which the cell and solution binding constants are equal. The cells have an average of 1.2 x 10(-14) mol of AO binding sites per cell. Using the biochemically measured heparin content per cell and the amount of AO bound by heparin in solution, 8 x 10(15) mol of sites/cell can be attributed to heparin. The remaining cellular binding sites (4 x 10(-15) mol of sites per cell) are essentially all accounted for by AO binding to DNA, the amount of which is calculated from its previously determined thermodynamic binding parameters. A theoretical isotherm, calculated from the binding parameters of both heparin-AO in solution and DNA-AO complexes in situ, agreed closely with the isotherm experimentally determined for the Furth mastocytoma cells. Ligand-binding analysis yields a binding constant, which may aid in identification of cellular bipolymers, and the number of ligand binding sites per cell. The latter is a measure of the amount of a given intracellular biopolymer present.

Acridine Orange↗