Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata.

The possibility that the surface of the egg of the sea urchin Arbacia punctulata contains a species-specific receptor for sperm has been investigated. The extent of fertilization of eggs of A. punctulata, which is proportional to the number of sperm, is unaffected by the presence of either eggs or membranes prepared from eggs of Strongylocentrotus purpuratus. In marked contrast, membranes prepared from eggs of A. punctulata quantitatively inhibit fertilization of A. punctulata eggs by A. punctulata sperm. Several lines of evidence indicate that this inhibition is due to the presence of a membrane-associated glycoprotein that binds to the sperm, thus preventing them from interacting with receptor on the surface of the eggs. First, eggs treated with trypsin are incapable of being fertilized, although they can be activated with the Ca2+ ionophore A23187. Moreover, membranes prepared from eggs pretreated with trypsin do not inhibit fertilization of eggs. Second, receptor isolated in soluble form from surface membranes binds to sperm and thus prevents them from fertilizing eggs; the inhibition by soluble receptor is species-specific. Third, the soluble receptor binds to concanavalin A-Sepharose. Fourth, eggs are incapable of being fertilized if they are pretreated with concanavalin A. The specificity of inhibition, and the affect of trypsin and concanavalin A on intact eggs, suggest that the receptor is a species-specific macromolecule located on the surface of the eggs. The sensitivity of the receptor to trypsin, and its ability to bind to concanavalin A, indicate that it is a glycoprotein.

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs.

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.

Purification and partial amino acid sequence analysis of human erythrocyte acetylcholinesterase.

A single step immunoaffinity purification procedure for human erythrocyte acetylcholinesterase is described which permitted the isolation of milligram quantities of enzyme from 10 U of erythrocytes, with 113,000-fold purification and a yield of about 22%. In SDS-PAGE analysis, the enzyme corresponds to a disulfide linked dimer of 140 kDa which is converted to a 70 kDa monomer upon disulfide reduction. The tryptic peptides generated from purified enzyme were separated by reverse-phase HPLC. Five of these peptides were analysed to determine the amino acid sequences. The obtained sequences showed no homology to the already known amino acid sequences for human serum and brain butyrylcholinesterase and Torpedo californica acetylcholinesterase.

Identification of mammalian sperm surface antigens: II. Characterization of an acrosomal cap protein and a tail protein using monoclonal anti-mouse sperm antibodies.

Monoclonal anti-mouse sperm antibodies have been produced by fusing mouse myeloma cells with spleen cells from rats immunized with epididymal sperm of C3H mice, Immunoprecipitation and immunoperoxidase techniques showed that one such monoclonal antibody, AMS IV-33, recognized a 200 000 dalton protein localized on the acrosomal cap of the sperm cell. Two other monoclonal antibodies AMS IV-54 and -76, reacted with a 68 000 dalton component on the surface of the sperm tail. Both antigenic targets were species specific and were present in about equal amounts on sperm from several different strains of mice. The tail protein was sperm specific, whereas the antibody reacting with the acrosomal cap protein also appeared to react somewhat with antigens present in other mouse tissues.

Epidemiologic predictors of hepatitis C virus infection in pregnant women.

OBJECTIVE: To identify sensitive epidemiologic predictors of a positive hepatitis C virus antibody test in asymptomatic persons, and to compare the cost of testing only persons with an epidemiologic predictor to that of universal screening. METHODS: Seventeen hundred consecutive pregnant women were tested by enzyme-linked immunosorbent assay for antibody to hepatitis C virus. Seventy-five subjects tested positive and were compared with 257 pregnant women who tested negative. Cohort and control patients were interviewed and their medical records were reviewed to identify those with chosen predictors of a positive hepatitis C virus antibody test. RESULTS: Seventy-four of 75 cohort patients and 108 of 257 controls had one or more predictors of a positive antibody test. Cohort patients were significantly more likely (P < .001) to have the following: human immunodeficiency virus infection, a sex partner with a risk factor for hepatitis, age greater than 30 years, and a history of drug use, blood transfusion, sexually transmitted disease, hepatitis, or incarceration. The sensitivity and specificity of a single predictor in identifying a person with a positive test were 99 and 58%, respectively. The cost of finding a single individual with a positive antibody test by universal screening was $674, compared to $303 by selectively screening persons with one or more predictors of a positive antibody test. CONCLUSIONS: Most individuals with positive hepatitis C virus antibody tests can be identified on the basis of epidemiologic predictors, reducing the cost of testing by 55%. These patients may receive appropriate medical therapy, and their children may be evaluated for possible infection by vertical transmission of hepatitis C virus.

Identification of mammalian sperm surface antigens. I. Production of monoclonal anti-mouse sperm antibodies.

Surface antigens of mammalian sperm were studied by use of monoclonal antibodies (MAs). Six hybridoma cell lines were obtained by fusion of mouse myeloma cells with spleen cells from rats immunized with unwashed, epididymal sperm from C3H mice. Quantitative assessment of antibody binding, using a solid phase, antibody-protein A assay, indicated that four MAs bound to integral, sperm surface antigens; two others bound to nonintegral sperm antigens or epididymal fluid components. Immunofluorescence studies showed specific binding of individual MAs to localized regions: acrosome, midpiece, and midpiece and tail. All of these MAs inhibited sperm-egg binding, and those to the midpiece and/or tail immobilized sperm cells. The monoclonal antibodies provide probes for immunochemical characterization of sperm antigens and for elucidation of the role of the antigens in sperm.

Response of monkeys to porcine zona pellucida as detected by a solid-phase radioimmunoassay.

The immunological response of cynomolgus monkeys (Macaca fascicularis) to immunization was evaluated utilizing collagenase-isolated pig zona pellucida. Six weeks after initial immunization a high serum titer of antibody was exhibited. Serum antibody titers demonstrated a noticeable decline 5 months after booster injections were discontinued. The assay method used is rapid and is capable of detecting antibody in serum dilutions of 1:78,000, as compared to 1:125,000 with the indirect fluorescence assay.

Specific adhesion of rat hepatocytes to beta-galactosides linked to polyacrylamide gels.

Rat hepatocytes, isolated by a collagenase perfusion technique, specifically bind to polyacrylamide gel containing covalently immobilized 6-aminohexyl beta-D-galactopyranosyl groups. Less than 5% of these cells bind to polyacrylamide or to gels with the following covalently linked ligands: 6-aminohexanol, or the 6-aminohexyl D-pyranosides of alpha-mannose, beta-glucose, beta-2-acetamido-2-deoxyglucose, beta-cellobiose, beta-maltose, or beta-melibiose. Cell binding to beta-D-galactoside gels occurs after a lag period at 37 degrees and 65 to 100% (depending on the cell preparation) of the cells adhere. The duration of the lag period is inversely related to the beta-D-galactoside content of the gel but preincubation of the cells at 37 degrees reduces the lag period. Cell-gel binding is a threshold phenomenon. Adhesion of cells to gels does not occur when the glycoside concentration is less than about 900 nmol per cm2 x 0.25 mm thick gel piece. Above this critical concentration, cell-gel binding occurs and becomes maximal when the concentration is increased by only 20%. If these in vitro results apply to cellular interactions in vivo, they suggest that slight changes in the levels of cell surface or extracellular matrix carbohydrates may profoundly influence the behavior of neighboring cells.

Studies on the intercellular adhesion of rat and chicken hepatocytes. Tissue-specific adhesion in mixtures of hepatocytes and heart myocytes.

We previously reported that chicken and rat hepatocytes isolated from young adult animals displayed adhesive specificity in that they adhered preferentially to the homologous cell type. However, since we had used cells from two widely divergent species, it was not clear whether the cells were capable of distinguishing their own cell type from cells of other tissues of the same animal. The present experiments were aimed at determining whether, given the choice of adhering to cells obtained from another tissue from the same animal, cells still preferentially adhered to their own cell type, i.e. whether they showed tissue-specific adhesion. An improved collagenase perfusion procedure was developed for preparing single, viable heart myocytes. Cell adhesion experiments were then performed with hepatocytes and myocytes obtained from a single rat or chicken. Marked tissue-specific adhesion was observed under all conditions tested, which included varying the ratio of each cell type (hepatocytes or myocytes), stationary or gyratory conditions, the presence or absence of serum and certain metal ions, etc. The demonstration of tissue-specific adhesion among hepatocytes and myocytes isolated from the same animal is consistent with the idea that the two cell types contain different cell surface components required for cell-cell recognition. Furthermore, that the hepatocytes (and myocytes) can show tissue-specific adhesion validates the use of cells from adult animals for biochemical studies on intercellular adhesion.

Studies on the intercellular adhesion of rat and chicken hepatocytes. Conditions for stimulation by liver plasma membranes.

A new assay was developed for measuring the stimulation of intercellular adhesion of rat hepatocytes by rat liver plasma membranes. Aggregates formed in the presence of the membranes are separated from single cells by filtration, and the number of cells in the aggregates is determined by their lactate dehydrogenase content. The formation of aggregates was specifically stimulated by rat liver plasma membranes and the rate of aggregate formation was proportional to the quantity of added membranes. The effects of divalent cations on the initial rates of rat and chicken hepatocytes adhesion were also examined. Optimal rates of adhesion of rat hepatocytes were obtained in the presence of physiological levels of Mg2+, and adhesion was inhibited by Ca2+, whereas optimal rates of chicken hepatocyte adhesion were obtained in the presence of physiological levels of Ca2+. Plasma membrane stimulation of hepatocyte adhesion also showed the same divalent ion requirements with the respective cell types. The stimulatory activity in the rat plasma membranes determined by the filter assay was found to be sensitive both to trypsin digestion and to mild periodate oxidation. The activity was also completely resistant to vigorous reductive alkylation. These results taken together suggest that the rat plasma membrane stimulatory activity is associated with a glycoprotein(s).

Incomplete intertrochanteric fractures: imaging features and clinical management.

PURPOSE: To present the imaging findings and treatment options for incomplete intertrochanteric fractures. MATERIALS AND METHODS: Among 31 patients with the magnetic resonance (MR) imaging diagnosis of incomplete intertrochanteric fracture, 30 also underwent radiography. MR and radiographic findings were compared. Note was made of fracture length and extent as depicted on the coronal and axial MR images, treatment (surgical vs conservative), and follow-up. RESULTS: Correlation between radiographic and MR findings was poor. Incomplete intertrochanteric fracture was the prospective radiographic diagnosis in only one case. Fracture in 18 patients was treated surgically and in 13 was managed conservatively. In both groups, the average age of the patients and length of the fractures and the percentage of separate fractures involving the greater trochanter and crossing the midline of the femur in the axial plane were the same. Fractures crossed the midline in the coronal plane in 50% of the surgical group but in only 23% of the nonsurgical group. Average time from injury to ambulation was 2 days less in the surgical group, but no difference in functional status was found subjectively between the two groups at clinical follow-up. CONCLUSION: Incomplete intertrochanteric fractures are a previously unrecognized subset of intertrochanteric fractures that are diagnosed unequivocally only with MR imaging.

Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.