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B M Shapiro

Publications and source records attributed to B M Shapiro.

At least 73 records · Page 4Linked to original sources
PubMed1982

Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin, Strongylocentrotus purpuratus.

The internal pH (pHi) of sperm of the sea urchin, Strongylocentrotus purpuratus, was estimated by measuring the accumulation of the weak bases [14C]methylamine, [14C]diethylamine, or 9-aminoacridine under conditions where cellular respiration was activated or inhibited. When 9-aminoacridine fluorescence measurements are corrected for binding to intracellular components, the pHi estimates agree quantitatively with those obtained from [14C]amine distributions. The pHi of sperm decreased when the intracellular [K+] was elevated above the physiological value of 10 mM, or when the external pH was substantially decreased below the physiological 8.0, or when Na+ was absent from the seawater. At the decreased pHi values, sperm respiration and motility were inhibited; conversely, both respiration and motility increased when the pHi was elevated. Increased respiration occurred whether the pHi was increased by altering the external pH, [K+] or [Na+], or by the addition of NH4Cl to the medium. In all cases, the activation of respiration and motility were linked, suggesting a unitary control mechanism, some possibilities for which are presented.

Animals↗
PubMed1982

Assembly of the fertilization membrane of the sea urchin: isolation of a divalent cation-dependent intermediate and its crosslinking in vitro.

To analyze the mechanism of assembly of the fertilization membrane of the sea urchin Strongylocentrotus purpuratus, we inhibited the ovoperoxidase that catalyzes dityrosine formation to isolate an uncrosslinked, soft fertilization membrane (SFM). The SFM intermediates were stabilized by divalent cation-dependent interactions: in the absence of divalent cations, the SFM became amorphous and less refractile and released proteins into the surrounding medium. We term the remaining structures "wraiths." The rate of this disaggregation was increased in solutions of low ionic strength, but 5-10 mM divalent cations (Ca2+, Mg2+, Mn2+ or Ba2+) prevented disaggregation. Wraiths could be reassembled into structures that resembled SFM by readdition of divalent cations. The SFM contained active ovoperoxidase and could be hardened in vitro by washing away the ovoperoxidase inhibitor and adding H2O2. After hardening, certain proteins of over 100 kd were excluded from SDS-polyacrylamide gels, suggesting that these proteins contain the substrates for crosslinking. We propose that the SFM is a divalent cation-dependent intermediate on the pathway of fertilization membrane assembly containing tyrosyl residues that are appropriately juxtaposed for crosslinking.

Calcium↗
PubMed1981

Membrane potential depolarization and increased intracellular pH accompany the acrosome reaction of sea urchin sperm.

The intracellular pH and membrane potential in sperm of the sea urchin Strongylocentrotus purpuratus were investigated by using fluorescent and radiolabeled probes. The weak bases [14C]methylamine, [14C]diethylamine, and 9-aminoacridine were concentrated within sperm 5-fold or greater. The weak acid [14C]dimethyloxazolidine-2,4-dione (DMO) was excluded from sperm. These data suggested that the apparent intracellular pH is acidic with respect to seawater (pH 8.0). Induction of the acrosome reaction caused efflux of the amines and uptake of DMO, consistent with an increase in apparent intracellular pH of 0.1-0.2 pH unit. The presence of an internally negative membrane potential was indicated by estimating the distribution of [3H]tetraphenylphosphonium (Ph4P+) and [14C]SCN-. From SCN- exclusion we estimated a value of about -30 mV for the nonmitochondrial membrane potential, whereas from Ph4P+ accumulation an apparent potential of -90 to -150 mV was demonstrated. The membrane potentials obtained with Ph4P+ and SCN- were dependent upon the external K+ concentration, with increasing K+ leading to depolarization. Induction of the acrosome reaction led to efflux of Ph4P+ and uptake of SCN- for an approximate depolarization of about 30 mV, primarily due to the collapse of the plasma membrane potential.

Acrosome↗
PubMed1979

Metabolic similarities between fertilization and phagocytosis. Conservation of a peroxidatic mechanism.

At the time of fertilization, sea urchin eggs release a peroxidase which, together with H2O2 generated by a respiratory burst, is responsible for hardening of the fertilization membrane. We demonstrate here that the ovoperoxidase of unfertilized eggs is located in cortical granules and, after fertilization, is concentrated in the fertilization membrane. Fertilization of sea urchin eggs or their parthenogenetic activation with the ionophor A23187 also results in (a) the conversion of iodide to a trichloroacetic acid-precipitable form (iodination), (b) the deiodination of eggs exogenously labeled with myeloperoxidase and H2O2, (c) the degradation of thyroxine as measured by the recovery of the released radioiodine at the origin and in the inorganic iodide spot on paper chromatography, and (d) the conversion of estradiol to an alcohol-precipitable form (estrogen binding). The iodination reaction and the binding of estradio occurs predominantly in the fertilization membrane where the ovoperoxidase is concentrated. From the estimation of the kinetics of incorporation of iodine, we determine that the peroxidative system is active for 30 min after fertilization, long after hardening of the fertilization membrane is complete. Most of the bound iodine is lost during the hatching process. Iodination of albumin is catalyzed by the material released from the egg during fertilization, when combined with H2O2 and iodide. Iodination, thyroxine degradation, and estradiol binding are inhibited by azide, cyanide, aminotriazole, methimazole, ascorbic acid and ergothioneine, all of which can inhibit peroxidase-catalyzed reactions. These responses of the sea urchin egg to fertilization are strikingly similar to the changes induced in polymorphonuclear leukocytes by phagocytosis and, in both instances, a peroxidative mechanism may be involved.

Animals↗
PubMed1979

After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos.

Sea urchin and mouse sperm that are labeled on their surfaces with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TMRTC) or 125I-diiodofluorescein isothiocyanate (125IFC) remain viable and can fertilize eggs. When sea urchin eggs were fertilized with 125IFC-labeled sperm, the radioactivity from the sperm was quantitatively transferred to the egg (at a ratio of one sperm equivalent per egg) and persisted in the embryo as it developed to the pluteus larval state (5 days at 12 degrees C). The radioactivity was acid-precipitable and was associated with the particulate fraction of embryo homogenates. In addition, FITC-labeled sea urchin sperm were used to fertilize eggs, and the labeled components were followed by fluorescence microscopy. In the embryo, labeled sperm components were present as a discrete patch that was partitioned unequally during early cleavages. In experiments using mouse sperm labeled with TMRTC, the labeled sperm components were also transferred to the embryo as a discrete patch that was again distributed unequally after cleavage. This physiological cell fusion system therefore has distinctive characteristics: there is limited lateral mobility of surface components, which have a low turnover rate unlike that see in other systems. In this paper, we discussed the possible morphogenetic role of this unusual behavior.

Animals↗
PubMed1979

Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components.

The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti-IFC antibodies, to isolate such proteins from other cellular constituents.

Animals↗
PubMed1979

Lipid and lipopolysaccharide composition of Escherichia coli surface-altered mutants selected for resistance to levallorphan, tetracaine, and polymyxin.

Certain mutants of Escherichia coli with an altered permeability barrier have an essentially normal lipopolysaccharide, fatty acid, and phospholipid content, with a slight increase in the membrane protein:lipid ratio. The phospholipid metabolism of the lev and tec strains shows an abnormal response to growth in the selective agents levallorphan and tetracaine, respectively.

Cell Membrane↗
PubMed1979

Membrane events of fertilization in the sea urchin.

Four important events of fertilization in the sea urchin are: 1) the acrosome reaction of the sperm, 2) sperm-egg fusion, 3) the cortical reaction of the egg, and 4) the formation of the fertilization coat. The acrosome reaction is triggered by contact of the sperm with the jelly coat, a complex extracellular matrix surrounding the egg. This causes rapid fluxes of ions, fusion of the acrosome membrane with the plasma membrane, and extension of the acrosomal filament. The acrosome membrane inserted into thesperm plasma membrane covers the acrosomal filament and contacts the plasma membrane of the egg to initiate sperm-egg fusion. One consequence of sperm-egg fusion is insertion of the sperm plasma membrane into the egg plasma membrane, producing a mosaic patch. The sperm components inserted persist in development and can be identified by quantitative methods after gastrulation. Another consequence of sperm-egg fusion is the cortical reaction in which thousands of vesicles fuse with the egg surface, thereby adding their membranes to the egg plasma membrane and releasing their contents upon the egg surface. This results in an approximate doubling of the amount of membrane on the egg surface in a few seconds and produces a mosaic topography. The excess surface membrane is accommodated by elongation of egg microvilli. The cortical reaction causes a detachment of the egg glycocalyx or vitelline membrane, and this layer is elevated from the egg surface. Contents of the cortical granules combine with and alter the vitelline membrane by a hardening reaction to produce the fertilization coat. Hardening involves a peroxidase-mediated tyrosine crosslinking, requiring a burst of oxygen consumption by the egg to generate hydrogen peroxide and resulting in chemiluminescence. These events are followed by activation of metabolic processes in the egg and changes which protect the egg against polyspermy.

Acrosome↗
PubMed1978

Hydrogen peroxide production, chemiluminescence, and the respiratory burst of fertilization: interrelated events in early sea urchin development.

After fertilization of the sea urchin, Strongyl-ocentrotus purpuratus, a crosslinked fertilization membrane is formed; the crosslinks (dityrosine residues) are synthesized in a reaction catalyzed by an ovoperoxidase that is released from the cortical granules during fertilization. The substrate for ovoperoxidase activity, hydrogen peroxide, is generated by the egg coincident with the "respiratory burst" that follows parthenogenetic activation by the divalent ionophore A23187 or fertilization. This burst of oxygen consumption may be almost quantitatively accounted for by hydrogen peroxide evolution, as measured by the peroxidase-catalyzed quenching of scopoletin fluorescence. Neither the burst of oxygen consumption nor hydrogen peroxide production occurs when the inhibitor of cortical granule discharge, procaine, is present at fertilization. Fertilization or parthenogenetic activation with A23187 also is associated with a burst of light emission. This chemiluminescence is inhibited in vivo by inhibitors of the ovoperoxidase, such as 3-amino-1,2,4-triazole, phenylhydrazine, sulfite, or azide. A crude ovoperoxidase preparation catalyzes hydrogen peroxide-dependent chemiluminescence that is similarly inhibited. Thus, the bursts of oxygen uptake, peroxide production, and chemiluminescence appear to be several manifestations of the peroxidative system released at fertilization. This system may additionally be responsible for spermicidal activity and thus may act as a component of the block to polyspermy.

Animals↗
PubMed1977

Specific phospholipid requirement for activity of the purified respiratory chain NADH dehydrogenase of Escherichia coli.

The highly purified respiratory chain NADH dehydrogenase (EC 1.6.99.3) of Escherichia coli is inactive in the absence of detergent or phospholipid. Triton X-100 is the detergent that gives optimal activity, but the Triton X-100-activated enzyme is stimulated an additional 2-fold by E. coli phospholipids. Phosphatidylglycerol and diphosphatidylglycerol are the most effective lipid activators. The activated complex prepared with diphosphatidylglycerol is stable, whereas that with phosphatidylglycerol loses activity rapidly. Maximum activation by phospholipids occurs after preincubation at 0 degrees C and at pH 7. Triton X-100 is required at low concentrations for lipid activation, but high concentrations interfere with the activation. When the enzyme is optimally activated by phospholipids, it may be additionally activated 2-fold by spermidine, but not by magnesium. In contrast, the Triton X-100-activated form of the enzyme is stimulated by several divalent cations, without specificity. Thus, the most stable, active form of the purified NADH dehydrogenase is generated in the presence of diphosphatidylglycerol and spermidine.

Cations, Divalent↗