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Biomedical subjects

D A Blake

Publications and source records attributed to D A Blake.

At least 19 recordsLinked to original sources
PubMed1992

Heparin- and sulfatide-binding peptides from the type I repeats of human thrombospondin promote melanoma cell adhesion.

Peptides from the three type I repeats of human endothelial cell thrombospondin, containing the consensus sequence-Trp-Ser-Xaa-Trp-, bind to sulfated glycoconjugates including heparin and sulfatide. The peptides are potent inhibitors for the binding of thrombospondin, laminin, or apolipoprotein E to these ligands. The thrombospondin peptides that inhibit heparin binding, but not adjacent peptides from the thrombospondin sequence containing the previously identified adhesive motif Val-Thr-Cys-Gly, promote melanoma cell adhesion when immobilized on plastic. Melanoma cell adhesion to the immobilized peptides is inhibited by soluble recombinant heparin-binding fragment of thrombospondin. The peptides also inhibit heparin-dependent binding of thrombospondin or laminin to human melanoma cells. The active peptides lack any previously identified heparin-binding consensus sequences and most do not contain any basic amino acids. Studies with homologous peptides showed that the tryptophan residues are required for binding. Adjacent basic residues in the second type I repeat enhance binding to heparin but not to sulfatide. Thus the type I peptides of thrombospondin define a distinct class of heparin-binding peptides.

Amino Acid Sequence↗
PubMed1990

High-pressure, anion-exchange chromatography of proteoglycans.

Although high-performance liquid chromatography has been used extensively to characterize the glycosaminoglycan chains of proteoglycans, very few researchers have reported the use of this technology for the separation of intact proteoglycan species. The high molarity denaturing buffers required for proteoglycan disaggregation and separation are often not compatible with the low back-pressure limitations imposed by many of the HPLC systems designed for the separation of biological macromolecules. In this study, heparan sulfate and dermatan sulfate proteoglycans, obtained by the metabolic labeling of cultured corneal endothelial cells, were rapidly and completely separated in less than an hour in a high-pressure liquid chromatography system. The separation, which used a Dionex BioLC system equipped with a Pharmacia Superloop and a ProPac PA1 column, also effected a greater than 10-fold concentration of the proteoglycans during the separation procedure. All buffers were 8 M in urea, and the back-pressures generated during the separation were well below the limit of the system. The pooled fractions from the ion-exchange column were subsequently analyzed for glycosaminoglycan composition and molecular size. The system was able to resolve dermatan sulfate-substituted species from heparan sulfate-substituted species in a single chromatographic step. The proteoglycan nature of the recovered products was established by Sepharose CL-4B chromatography and gel electrophoresis.

Animals↗
PubMed1990

Thrombospondin: biosynthesis, distribution, and changes associated with wound repair in corneal endothelium.

Thrombospondin is a cell adhesion molecule which interacts via specific domains with a wide array of extracellular matrix components, including fibrinogen, fibrin, fibronectin, collagen, and heparan sulfate proteoglycan. Although this protein has been localized in several human tissues, its presence in corneal tissues had not been previously established. In the present study, we have demonstrated that cultured bovine corneal endothelial cells synthesize thrombospondin and incorporate it into their extracellular matrix. We have also shown immunofluorescently the presence and distribution of thrombospondin in these cultured cells and in the noninjured and injured corneal endothelium in situ. Ultrastructural immunoperoxidase cytochemistry revealed that thrombospondin could be displaced from the cell surface by heparin, but not by keratan sulfate. Confluent cultures of corneal endothelium synthesize and secrete the three cell adhesion proteins laminin, thrombospondin, and fibronectin in the ratios 1:8.2:51.8. Only the laminin B chains were detected in immunoprecipitates. Immunofluorescent studies of these cultured cells, using a polyclonal antiserum raised against purified thrombospondin, revealed a low level of fluorescence associated with the cell layer but a punctate fluorescent pattern at the level of the extracellular matrix. Noninjured corneal endothelium in situ also demonstrated a low level of fluorescence throughout the cell layer. However, this dramatically changed after a circular freeze injury to the tissue. By 24 h after wounding, cells surrounding the injury zone displayed a prominent fluorescence that was still observed at 48 h post-injury. In addition to its increased intracellular fluorescence, thrombospondin was also localized as migration tracks, oriented in the direction of cellular migration into the wound site. Thus, in corneal endothelium, thrombospondin appears to play a major role in injury-induced cell migration in situ along a natural basement membrane.

Animals↗
PubMed1989

Biosynthesis of galactogen: identification of a beta-(1----6)-D-galactosyltransferase in Helix pomatia albumen glands.

A beta-(1----6)-D-galactosyltransferase has been purified over 2000-fold by affinity chromatography on UDP-p-aminophenyl-Sepharose. The enzyme, from a pellet fraction (8000 x g) of Helix pomatia albumen gland, catalyzes transfer of D-galactose from UDP-galactose to a (1----6) linkage on acceptor H. pomatia galactogen. Three other polymers served as acceptors: beef lung galactan, Lymnaea stagnalis galactogen and arabinogalactan from larch wood. To determine the linkage specificity of the enzyme, it was incubated with UDP-D-galactose and acceptor galactogen that had been tritiated previously by treatment with galactose oxidase and [3H]KBH4. The [3H]galactogen reaction product was recovered, methylated, hydrolyzed and acetylated; tritiated derivatives were identified by mass spectroscopy of effluent fractions separated by gas chromatography. This analysis revealed that (1----6)-linked galactosyl groups had been added to the enzyme-treated acceptor galactogen. Also identified was a hydrolytic enzyme that removed terminal alpha 1,2-linked L-galactosyl residues from H. pomatia galactogen.

Animals↗
PubMed1989

A colorimetric assay for the measurement of D-glucose consumption by cultured cells.

A colorimetric method is described for measuring glucose consumption by tissue culture cells. This procedure, which utilizes the coupled activities of glucose oxidase and horseradish peroxidase, is insensitive to the spectral interferences caused by the phenol red and sera present in most tissue culture media. The spectral properties (absorbance maxima and apparent absorption coefficients) and stability of a large number of chromogenic horseradish peroxidase substrates were surveyed for their ability to perform in an assay for glucose in the presence of phenol red and sera components. One of these chromophores, the product of an oxidative couple between 4-aminoantipyrine and N-ethyl-N-sulfopropyl-m-toluidine, was subsequently used to develop a fixed time assay for glucose in media samples. The assay required only 10 microliters of media in a 1-ml assay volume; reproducibility studies showed variabilities of less than 5% in the assay of a single sample, and values obtained in glucose analyses correlated well with those obtained using commercially available test kits. The assay was used to study the rate of glucose consumption in two different cell types, bovine corneal endothelial cells and human diploid fibroblasts.

Animals↗
PubMed1989

The Michaelis constants of a nonchromogenic substrate may be determined using a chromogenic substrate.

A general method is presented for the determination of the KM and Vmax for a nonchromogenic substrate in an experimental system where a chromogenic and a nonchromogenic substrate compete for the active site of a single enzyme. Entire progress curves of absorbance versus time for the transformation of the chromogenic substrate must be obtained in the absence and presence of the nonchromgenic substrate. Two quantities may then be extracted from the entire kinetic curves: the value of a delta Area, the area bounded by the kinetic traces of absorbance versus time in the presence and absence of the nonchromogenic substrate; and the value of delta t(5%), the time required to transform 5% of the chromogenic substrate in the presence of the nonchromogenic substrate minus the corresponding time required in its absence. The values of KM and Vmax for the nonchromogenic substrate may be obtained from the dependencies of delta Area and delta t(5%) upon the concentration of the nonchromogenic substrate. The ability of this procedure to yield the correct values of KM and Vmax was demonstrated using beta-lactamase, beta-galactosidase, alkaline phosphatase, and 19 chromogenic/nonchromogenic substrate pairs. This method is equally valid in the absence or presence of competitive product inhibition and should be applicable to any enzyme-catalyzed, strongly exergonic reaction.

Alkaline Phosphatase↗
PubMed1986

Purification and characterization of a UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase from Ehrlich ascites tumor cells.

A UDP-Gal:N-acetyllactosaminide alpha (1,3)-galactosyltransferase from Ehrlich ascites tumor cells has been purified over 200,000-fold to apparent electrophoretic homogeneity. The purified enzyme transfers D-galactosyl groups from UDP-Gal to beta-D-Gal-(1,4)-D-GlcNAc in alpha-linkage. The apparent Km values for donor and acceptor substrates are 12.6 microM and 1.15 mM, respectively. The trisaccharides beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)- or (1,6)-D-Man exhibit a Km 5-fold lower than that of N-acetyllactosamine, and an even more pronounced effect is observed with the biantennary pentasaccharide beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)-[beta-D-Gal(1, 4)-beta-D-GlcNAc-(1,6)]-D-Man (Km 0.10 mM). The transferase shows a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with an apparent subunit molecular weight of 80,000, exhibits a pH optimum at 6.2, and requires Mn2+ ions and detergent for enzymatic activity. Specificity studies using immobilized oligosaccharides show that the minimum acceptor structure for the alpha-galactosyltransferase is N-acetyllactosamine. The narrow specificity of the alpha-galactosyltransferase is indicated by the fact that lactose, beta-D-Gal(1,3)-D-GlcNAc, and beta-D-Gal(1,4)-[alpha-L-Fuc(1,3)]-D-GlcNAc are very poor acceptors. The enzyme differs from the blood-group B-specified galactosyltransferase in that the sequence alpha-L-Fuc(1,2)-beta-D-Gal(1,4)-D-GlcNAc is not an acceptor. Oligosaccharides, glycoproteins, glycolipids, and glycosaminoglycans containing the terminal nonreducing N-acetyllactosamine unit all serve as acceptors for the enzyme.

Animals↗
PubMed1985

Lack of maternal metabolic, endocrine, and environmental influences in the etiology of cleft lip with or without cleft palate.

We studied metabolic, endocrine, and environmental factors in 59 women who had delivered a child with cleft lip with or without cleft palate (CL +/- CP) and compared these values with those of 56 mothers of unaffected children. There was no significant difference between the two groups with respect to race, age, weight, height, education, parity, menstrual history, medical illnesses, or the use of contraceptives, tobacco, alcohol, or caffeine. All patients had a normal XX karyotype confirmed by the fluorescent banding technique. The two groups demonstrated no significant difference in test results of serum chemistries, glucose tolerance, serum or erythrocyte folate, vitamin A, carotene, corticoids, prolactin T4, free T4, urine 17-ketosteroids, 17-hydroxysteroids, total estrogens, or pregnanediol. Urinalyses revealed no group differences in the presence of barbiturates, amphetamines, salicylates, or benzodiazepines. The percentage of immunologic studies reflecting susceptibility to toxoplasmosis, rubella, cytomegalic inclusion disease, and herpes was not different between the two groups. The only statistically significant metabolic differences between the two groups were serum alkaline phosphatase, creatinine, creatinine clearance, and creatinine clearance/m2. Phenytoin pharmacokinetics and urinary metabolic patterns were compared in a subgroup of ten mothers of affected children and ten mothers from the control group. No significant differences were observed. However, a brief course of phenytoin treatment induced a greater inhibition of the folate tolerance test in controls than in mothers of children with clefts.

Adult↗
PubMed1984

Synthesis of N-acetyllactosamine containing a D-[6-3H]galactopyranosyl group.

A simple and convenient method for the introduction of radiolabel onto C-6' of N-acetyllactosamine is described. 1-N-Benzyl-3-O-beta-D-galactopyranosyl-D-arabinosylamine (1) was synthesized from 3-O-beta-D-galactopyranosyl-D-arabinose as described by Lee and Lee. Compound 1 was oxidized with D-galactose oxidase, and the product reduced with KB3H4 to introduce the label at C-6'. After dilution with unlabeled material, the N-benzyl-3-O-beta-D-[6-3H]galactopyranosyl-D-arabinosylamine was converted into 2-(benzylamino)-2-deoxy-4-O-D-[6-3H]galactopyranosyl-D-glucononitrile , which was subjected to simultaneous hydrogenolysis of the benzylamino and nitrile groups. N-Acetylation of the amino group as described by Alais and Veyrières afforded the crystalline title compound in 63% yield.

Amino Sugars↗
PubMed1984

Substrate specificity of D-galactose oxidase. Evidence for the oxidation of internally linked galactosyl residues of Helix pomatia galactogen.

Linkage analysis of the carbohydrate portion of glycoproteins and glycolipids is widespread. Sequential treatment with D-galactose oxidase and tritiated borohydride is a standard method for incorporation of radioactive marker into what has been assumed to be exclusively terminal residues of D-galactose or N-acetyl-D-galactosamine. The data presented here establishes the ability of D-galactose oxidase to act upon a specific subterminal D-galactosyl residue, [----2)-D-Gal(1----], as well as upon terminal nonreducing galactosyl residues. Helix pomatia galactogen, a high molecular weight galactose homopolymer, was sequentially treated with D-galactose oxidase and tritiated borohydride. The 3H-galactogen was recovered and analyzed to determine which galactosyl units carried radioactive label. After complete methylation and then acid hydrolysis of 3H-galactogen, its partially methylated galactosyl components were reduced and acetylated for identification by gas chromatography and mass spectroscopy. Radioactivity was located by collection of effluent fractions during gas chromatography. The only subterminal residue to be labeled was the 2-linked D-galactose, although another with a free oxidizable 6-carbon was present, 3-linked D-galactose, [----3)-D-Gal(1----]. Linkage analysis of internal radiolabeled galactosyl residues could be used to detect changes in saccharide structure during cellular events.

Animals↗
PubMed1984

Zymogen activation: a new system for homogeneous ligand-binding assay.

In this ligand-binding assay procedure, sensitivity is enhanced by successive generation of enzyme active sites via two zymogens from the blood-coagulation cascade: Factor X and prothrombin. A protease fraction from Russell's viper venom (RVV) acts upon Factor X, initiating a two-step cascade that culminates in generation of thrombin, the activity of which is monitored with a chromogenic substrate. In the model presented here, the analyte of interest, biotin, is covalently coupled to Factor X. In the presence of avidin, a biotin-binding protein, RVV cannot initiate cascade activity; however, the inhibition can be competitively overcome by addition of free biotin to the reaction mixture. In a complete system, the dose-response curve is linear from 20 to 100 nmol of biotin per liter. Such an assay offers improved sensitivity over many radioisotope-independent immunoassay methods, and may be applicable to a wide variety of analytes.

Avidin↗
PubMed1983

Thromboxane production in the pregnant rat: differential recovery by platelets and uterus following aspirin administration.

Radioimmunologic data provide evidence that the pregnant rat uterus produces thromboxane B2 (TXB2). To provide further evidence that this radioimmunologic compound is TXB2, an extract of media incubated with uteri from 21-day pregnant rats was passed through a silicic acid column and each 1-ml eluate was tested for its ability to displace tritiated TXB2 from antibody. One peak was found and it corresponded to that of authentic TXB2 eluted through an identical column. Rechromatographing the peak on a thin-layer plate, the radioimmunologic peak again corresponded with the TXB2 standard. Since blood platelets are a major source of thromboxane, their presence in the vasculature of tissues makes them a possible contaminating factor. Following aspirin (300 mg) intubation into rats on either gestational Day 18, 19 or 20, in vitro production of the TXB2 by isolated uteri and washed platelets was determined and compared to the same tissues from untreated rats. When aspirin was administered 1 day prior to autopsy, TXB2 production by uterine tissue was 32% of the control uterus. Platelet TXB2 production was 25% of control platelets. When aspirin was administered 2 days prior to autopsy, uterine TXB2 production increased to 60% of the control, while platelet TXB2 was 43% of the control. When aspirin was administered 3 days prior to autopsy, uterine TXB2 production was higher than that of control, while platelet TXB2 production was 54% of the control. The more rapid recovery of TXB2 by uterine tissue compared to platelets suggest that the TXB2 produced by uterine tissue is not due solely to platelet contamination.

Animals↗
PubMed1983

Hysterosalpingography in cynomolgus monkeys.

This paper describes recent success in performing hysterosalpingography in cynomolgus monkeys. The animals were anesthesized, and a needle was manipulated through the cervical canal into the endometrial cavity for injection of contrast material. Difficulties in threading the needle through the canal were due to several blind pouches formed by folds in the cervical mucosa. The uterine lumen and both tubes could be visualized in over 75% of the cases. The study demonstrates that the hysterosalpingography technique can be performed satisfactorily in cynomolgus monkeys.

Animals↗
PubMed1982

Pharmacokinetic studies on quinacrine following intrauterine administration to cynomolgus monkeys.

Recent efforts have been made to develop a chemical oviductal occluding agent. Intrauterine quinacrine has been used in certain areas of the world with moderate success in effected tubal closure. This report presents the pharmacokinetics of a quinacrine solution (30 mg) as administered to cynomolgus monkeys via the intrauterine route, compared with intravascular injection. The data show rapid transfer of the drug from the uterine to the vascular compartment and uptake by almost all tissues examined. Although plasma concentrations disappear within 24 hours, levels can be detected in most tissues for at least 1 week following intrauterine injection. After 28 days, however, tissue levels of the drug are absent or near the limit of detection.

Animals↗
PubMed1982

Thromboxane B2, 6-keto-prostaglandin F1 alpha, and prostaglandin F2 alpha production by contracting pregnant rate uteri in vitro.

While prostaglandin production by uterine tissue has been shown to be involved in the contractile mechanism of this tissue, less attention has focused upon the involvement of other prostanoids. We have simultaneously measured in vitro isometric contractility of pregnant rat uteri with the release of prostaglandin F2 alpha (PGF), 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) and thromboxane B2 (TXB2) into the bathing medium under various conditions. Frequency of uterine contractions and integrated contractile force (ICF) increased from 15 days of gestation and peaked at the time of parturition. Activity was generally greatest during the first 15 min of incubation except during parturition and on Day 1 postpartum when the uterine segment remained active for 1 h experimental period. Indomethacin (INDO) significantly reduced contractile activity regardless of gestational stage. PGF, TXB2, and 6-k-PGF1 alpha increased with gestational age, peaking at the time of parturition. Production was greatest during the first 15 min of incubation and INDO inhibited production of each prostanoid regardless of gestational stage. Imidazole (100 micrograms/ml) inhibited TXB2 production without affecting PGF or 6-k-PGF1 alpha levels. Frequency of contraction and ICF were not affected by imidazole treatment despite TXB2 reduction. These data demonstrate that the in vitro uterus from pregnant rats is capable of producing prostanoids other than prostaglandins and their production generally parallels uterine contractile activity. Thus, the possibility that these prostanoids are involved in physiologic changes during parturition warrants further investigation.

6-Ketoprostaglandin F1 alpha↗