Biomedical subjects
Publications and source records attributed to P Talbot.
A competitive radioimmunoassay has been developed for the differentiation of poliovirus subtypes using highly purified (35S)-methionine labelled virus and specific adsorbed sera to virulent and Sabin vaccine strains. The technique enables the antigenic relatedness of poliovirus strains or isolates to be examined.
A solid phase radioimmunoassay has been developed for the determination of poliovirus D-antigen over the range 1-100 D-units/ml. A dilution series of a D-antigen standard or of the samples to be assayed is incubated in the wells of a flexible vinyl Microtiter plate coated with D-antigen specific antibody. The poliovirus D-antigen bound to the antibody coated wells is quantitated by addition of the same antibody radioactively labelled with n-succinimidyl (2, 3-3H) propionate; the D-antigen content of the samples is determined by means of the standard curve generated by the D-antigen standard dilution series. Both the coating antibody and the radioactively labelled antibody may be freeze dried, and this provides a highly stable assay system with a long shelf-life.
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Hela and Namalwa cells readily attach to, and grow on, the surface of DEAE-substituted cellulose fibres. Subcultivation of these cells can be accomplished either by trypsinisation or by contact transfer of cells from fibre to fibre. Attempts to establish an anchorage-preferring sub-population of Namalwa cells were unsuccessful.
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When fresh epididymal spermatozoa were cold shocked for 10 or 30 min, then warmed to 24 degrees C, sperm motility was normal, but cold shocking ejaculated or capacitated spermatozoa caused a significant decrease in the percentage of motile spermatozoa and, for capacitated spermatozoa, in the rate of motility. The acrosomes of motile fresh epididymal and ejaculated spermatozoa became crenulated after cold shock, and the percentage of spermatozoa with crenulated acrosomes increased with longer periods of cold shock and was higher when spermatozoa were cold shocked in serum than in saline. When epididymal spermatozoa were cold shocked after incubation for 4 h at 37 degrees C, the acrosomes on spermatozoa which had not undergone an acrosome reaction became swollen and elevated instead crenulated. Epididymal spermatozoa which were cold shocked and then incubated for 4 h at 37 degrees C exhibited acrosome reactions and activation of motility, but had reduced fertilizing capacity when tested in vitro. Spermatozoa incubated in serum and cold shocked were able to penetrate zone-free ova even though their tails had been bent through 180 degrees. It is suggested that cold shock decreases the fertilizing capacity of hamster spermatozoa by interfering with the ability of spermatozoa to bind to and/or penetrate the zone pellucida.
Plant lectins have been used to advantage to study carbohydrate-containing cell surface receptors in numerous systems. In this study, a simple, reliable assay was developed to quantitate lectin-induced agglutinability of sperm. This assay was used successfully to compare some of the surface properties of uncapacitated and capacitated guinea pig sperm. Capacitation was induced by incubating sperm in minimum capacitation medium (MCM) or modified Tyrodes solution (T-PL). Control incubations were done in Ham's F-10 or Hank's balanced salt solution which do not support capacitation. At timed intervals during incubation, sperm samples were assessed for pattern and degree of lectin-induced agglutination. Results establish that: (1) soybean agglutinin (SBA) and to a lesser extent concanavalin A (Con A) induced agglutinability of guinea pig sperm increase during in vitro capacitation in MCM; (2) a similar increase in SBA induced agglutinability occurs during capacitation in T-PL, but not in the non-capacitating media; and (3) for sperm incubated in MCM or T-PL, there is a significant increase in tail to tail agglutination after capacitation. The results with SBA demonstrate that D-galactose and/or N-acetyl-D-galactosamine containing receptor sites or the guinea pig sperm surface are affected by capacitation, and this effect occurs, at least in part, in the sperm tail. Possible explanations for the observed increase in agglutinability are discussed. The agglutination assay may prove useful as a direct test for the occurrence of capacitation and may be especially valuable for species having a small acrosome or limited number of eggs.
Hamster sperm extracts contain a polypeptide which comigrates with muscle action on polyacrylamide gel electrophoresis. On double diffusion precipitation plates, sperm extracts form a single precipitin band with an antibody to muscle actin (antiactin) and show a reaction of identity with muscle actin. Indirect immunofluorescent microscopy revealed that antiactin binds along the concave margin and equatorial segment in the acrosomal region, in the connecting piece of the neck, and in the principal piece of the tail. These results are evidence that hamster sperm contain actin. The possible significance of these observations in fertilization is considered.
Capacitated guinea pig sperm are more agglutinable by the lectin soybean agglutinin (SBA) than uncapacitated sperm (Talbot and Franklin, '78). This study demonstrates that uncapacitated guinea pig sperm become as agglutinable by SBA as capacitated sperm when treated with trypsin, but not chymotrypsin. The pattern of lectin induced sperm agglutination after trypsinization resembles that for capacitated sperm. Also, trypsinization specifically increases SBA induced agglutination and does not affect agglutination by RCA-60; similar results are obtained during in vitro capacitation. Taken together, these data may indicate that a trypsin-like enzyme modifies the sperm surface during capacitation.
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The presence of spontaneously disrupted cytomegalovirus capsids has made it possible to visualize a subunit within the herpesvirus capsomere. The pattern resolved agreed well with the theoretical net based on the concept of a structural subunit at each apex of the hexon.
Enveloped virions and dense bodies of human cytomegalovirus have been purified by centrifugation, using combination negative viscosity: positive density gradients. Light-scattering bands of each component were obtained, and when these were examined by immune electron microscopy minimal cross contamination was observed.
Ionophore A23187, in the presence of extracellular Ca+2, induces a morphologically normal acrosome reaction in sperm of the sea urchin and precocious acrosome reaction and activation of guinea pig sperm. Increased membrane permeability to Ca+2 is responsible for initiating the acrosome reaction in both sea urchin and guinea pig sperm. In sea urchin sperm, the permeability change is brought about by egg jelly, whereas in the guinea pig sperm it accompanies capacitation.
The morphology and kinetics of the normal acrosome reaction were examined in vitro using hamster sperm incubated in detoxified sera. The reaction involved either swelling and elevation or crenulation and fragmentation of the acrosomal cap. Swelling and elevation occurred during both normal and degenerative reactions, as reported by others. Crenulation with subsequent fragmentation of the cap was observed during normal reactions. Early crenulation of the acrosome could be induced by cold shock (5 degrees C, 25 minutes), but this did not decrease the incubation time required (at 37 degrees C) for completion of the normal reaction. In appropriate sera, the occurrence of normal and degenerative acrosome reactions in motile sperm was significantly separated in time to study the reactions independently. The duration of the normal reaction, i.e., the time between the first morphological change in the acrosome (initiation)until the actual detachment of the cap (termination) was estimated to be 20 minutes. Saline dilution of these sera delayed initiation of the reaction and increased the duration of the reaction once it had started. Data from cold-shock and serum dilution experiments indicate that the mechanisms which govern the initiation and termination of the normal reaction are independently variable, and further suggest that initiation involves a change in membrane permeability and that termination includes membrane vesiculation.
The role of Ca+2 in the acrosome reaction of echinoid and mammalian sperm was investigated using the Ca+2 transporting ionophore A23187. The ionophore induced morphologically normal acrosome reactions in both types of sperm (as assessed by electron microscopic observation of echinoid sperm and phase contrast microscopic observation of mammalian sperm). In echinoids, these reactions were immediate. In the guinea pig and hamster, ionophore significantly decreased the capacitation interval; early reactions were accompanied by activation of motility. Ionophore induced reactions were affected by sperm, ionophore and Ca+2 concentrations. Since both ionophore induced and natural reactions require extracellular Ca+2, it is suggested that an influx of Ca+2 represents the initial step of the acrosome reaction. Under natural conditions, the permeability change which results in Ca+2 influx may be induced in echinoid sperm by egg jelly and may occur in mammalian sperm during capacitation. Ionophore A23187 should prove an experimentally useful drug for further study of the acrosome reaction since its effect on cells is understood, it induces synchronous reactions in a high percentage of sperm, and it conveniently reduces the capacitation interval in mammalian sperm.
Pig sera were assayed for antibodies to swine vesicular disease virus by (a) the radial immuno-diffusion technique combined with autoradiography and (by serum neutralisation tests. The former was more sensitive and was used for initial screening of sera while the latter was used to obtain estimates of titres of positive sera. In a survey of 1759 sera collected at slaughterhouses there were 14 significant titres from a total of seven premises situated in localities where the disease had been known to occur, and it was concluded that this did not indicate wither widespread undetected disease or the occurence of inapparent infection in the pig population.
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