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Biomedical subjects

B M Shapiro

Publications and source records attributed to B M Shapiro.

At least 91 records · Page 5Linked to original sources
PubMed1977

Binding of concanavalin A to the surface of sea urchin eggs and its alteration upon fertilization.

The binding of concanavalin A (Con A) to Strongylocentrotus purpuratus eggs has been investigated using 125I-concanavalin A (125I-Con A). The lectin binds specifically to the egg surface and does not produce agglutination of the eggs. High 125I-Con A concentrations are necessary to saturate all of the surface binding sites. Scatchard plots of the binding data are biphasic and may be interpreted as showing two main classes of sites. Unfertilized eggs have 4 X 10(8) high affinity sites/egg (Ka=8 X 10(-7) M) and 4.5 X 10(8) istes of lower affinity (Kb=4 X 10(-6) M). The sites may be assigned to different layers of the egg surface by studying the effects of removal of egg coats on the binding reaction. Removal of the jelly coat by washing eggs at pH 4.5 has no effect on binding. However, disruption of the vitelline layer with dithiothreitol leads to a decrease in the number of high affinity sites. After fertilization, the high affinity sites are found upon the fertilization membrane produced from the vitelline layer. Low affinity sites predominate in the plasma membrane, and are not affected by treatments which alter the vitellinelayer. The number of low affinity sites double upon fertilization, with the insertion of new membrane into the egg surface, as a result of cortical granule exocytosis. The doubling of sites is not due to hyaline material released from the cortical vesicles at fertilization, and thus these sites appear to reside upon the new membrane that is inserted from the cortical vesicles. If eggs are activated with ammonia, bypassing the cortical reaction, no change in the binding of ConA occurs. Con A inhibits fertilization at concentrations higher than 0.1 mg/ml, where less than 50% of the high affinity (viteline layer) binding sites are occupied, and there is little binding to the low affinity (plasma membrane) sites. Thus, the interaction of sperm with vitelline layer components may be an obligatory step in the fertilization process. A fraction of the 125I-Con A binding sites is cleaved from the egg surface upon fertilization or after activation by the calcium ionophore A23187. This release of Con A binding sites occurs during the limited proteolysis of surface components that accompanies the cortical reaction, and does not occur with ammonia activation of the egg, where the cortical reaction does not occur. Thus, the changes in Con A binding at fertilization are caused by the massive cortical granule exocytosis that occurs within minutes of sperm penetration, and not by activation of the egg per se.

Animals↗
PubMed1977

Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks.

One feature of fertilization is the alteration of the vitelline layer, by components released from the egg, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. The following evidence indicates that crosslinking and hardening are caused by the production of diand trityrosyl residues, by oxidation of protein-bound tyrosyl residues in the presence of a peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit many peroxidases. A peroxidase, here called the ovoperoxidase, is released from eggs at fertilization. This enzyme is inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN(3), phenylhydrazine, K(4)Fe(CN)(6), sodium sulfite, and glycine ethyl ester. In addition, tyramine and N-acetyltyrosine both inhibit hardening, but O-methyltyrosine does not. Dityrosyl and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues have been identified by cellulose phosphate column chromatography, thin-layer chromatography, and amino acid analysis. The amino acid data have been used to estimate that there is one dityrosine crosslink per 55,000 daltons of protein. We suggest that, by catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks the entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg.

Amino Acids↗
PubMed1977

Molecular mechanisms of fertilization.

The fertilization process serves the species as a mechanism for increasing its genetic diversity and ultimately its evolutionary persistence. For the biologist, it provides many fundamental problems for study. Several different types of membrane fusion reactions occur, each with a specific role in the sequence of fertilization. The sperm faces problems in transport and interaction with the egg; the egg modifies its surface and its internal metabolism in response to fusion with the sperm; and all these processes contain a wealth of interesting biochemical and biophysical problems. Many systems under study are easily manipulated, and, as a result, different portions of the fertilization sequence can be studied in isolation. Despite the enormous progress and insights made on this problem over the last century, our understanding of fertilization is still tentative, and the above discussion should be viewed as a progress report. We are just beginning to obtain some of the clues to unravel the mechanism of the complex machinery that Aristotle predicted was in the egg.

Acrosin↗
PubMed1976

The NADH dehydrogenase of the respiratory chain of Escherichia coli. I. Properties of the membrane-bound enzyme, its solubilization, and purification to near homogeneity.

The NADH dehydrogenase of the Escherichia coli respiratory chain has been identified by the following properties: (a) its location in membrane vesicles; (b) its inhibition by AMP in a fashion similar to that of the NADH oxidase; (c) its specificity for NADH, but not NADPH, with the same Km for NADH as that of the NADH oxidase; (d) its sensitivity when membrane-bound to inhibition by dicoumarol, rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide, which are also inhibitors for the NADH oxidase. The NADH-dehydrogenase of the cytosol fraction (assayed as NADH-dichlorphenolindophenol reductase activity) differs substantially from the membrane-bound activity both in substrate specificity and in the inhibitors of the reaction. The respiratory chain NADH dehydrogenase was extracted from isolated membrane vesicle preparations by solubilization in Triton X-100, and was purified in buffers containing that detergent. The purification employed chromatography on DEAE-cellulose, precipitation by 30% ethanol, and chromatography on hydroxyalapatite and DEAE-agarose. The most highly purified preparations of the enzyme were homogeneous in migration on polyacrylamide gels containing Triton X-100, at pH 9.5, where one band accounted for all of the protein and activity. Electrophoresis on polyacrylamide gels containing sodium dodecul sulfate showed 1 band of molecular weight 38,000, which accounted for over 75% of the protein on the gel. Because of requirements for either Triton X-100 or phospholipid for activity of the purified enzyme, it is difficult to estimate the level of purification achieved over isolated membrane vesicles. However, we estimate that the enzyme was purified some 30-fold over membrane vesicles, or some 300-fold over whole cells.

Adenosine Monophosphate↗
PubMed1976

The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.

2,6-Dichloroindophenol↗
PubMed1976

Changes in the topography of the sea urchin egg after fertilization.

Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.

Animals↗
PubMed1976

Use of polymyxin B, levallorphan, and tetracaine to isolate novel envelope mutants of Escherichia coli.

Mutants of Escherichia coli were isolated by their resistance to the bacteriocidal effects of the membrane-active drugs polymyxin B, levallorphan, and tetracaine. The mutants were examined for additional changes in cellular physiology evoked by the lesions; many polymyxin-resistant strains had a concomitant increased sensitivity to anionic detergents, and several strains of each type had concomitant alterations in generation time and morphology. Mutants of each class (polymyxin resistant, tetracaine resistant, and levallorphan resistant) were transduced into recipient strains. The levallorphan resistance site (lev) was located at approximately 9 min on the E. coli chromosome. Polymyxin (pmx) and tetracaine (tec) resistance loci were also transduced. The lev and tec strains had a slight prolongation of generation time, in contrast with their isogenic wild-type strains. The tec transductant produced long filaments in the absence of tetracaine and had an altered colonial morphology, it reverted at high frequency, with the morphological abnormalities reverting along with the tetracaine resistance. The pmx transductant had an increased sensitivity to levallorphan and to anionic detergents. In contrast, both lev and tec mutants were more resistant to acriflavine than was the wild type or the pmx transductant. The pmx, lev, and tec loci differed in sensitivity to mitomycin C; the lev strain was more resistant, the tec strain was more sensitive, and the pmx strain was much more sensitive than the wild type. There was no difference in sensitivity to several other dyes and detergents, colicins, or T bacteriophage between the transductant and isogenic wild-type strains. Thus, lev, tec, and pmx loci confer more subtle alterations in the permeability barrier than do lipopolysaccharide-deficient mutants previously studied.

Acriflavine↗
PubMed1975

Dental management of the patient with biliary atresia.

Biliary atresia is a disease of unknown origin characterized by partial or total absence of the biliary tract. While this condition is rare, the medical and surgical management makes the ramifications for dental treatment increasingly complex. This article reviews the disease and its complications and documents dental treatment of two patients.

Bile Ducts↗
PubMed1972

Isolation and some properties of cell envelope altered mutants of Escherichia coli.

Mutants of Escherichia coli which have a defect in their permeability barrier were selected. The technique used was to employ a strain of E. coli having a deletion in the gene for lactose permease and to select for mutants which can grow on lactose at 40 C. Twenty such mutants were isolated and six of these were found to be more sensitive to actinomycin D, sodium deoxycholate, and sodium dodecyl sulfate than was the parental strain. They were also more sensitive to the antibiotics vancomycin and bacitracin, which inhibit peptidoglycan biosynthesis. These mutants were no more sensitive to several different colicins or phages than was the wild-type strain. One of the mutants selected by this technique has an abnormal morphology when grown on certain carbon sources in minimal medium, and this mutant is more extensively studied in the accompanying paper.

Bacitracin↗