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N Saxena

Publications and source records attributed to N Saxena.

At least 73 records · Page 4Linked to original sources
PubMed1986

Immunofluorescence antigen localization on boar sperm plasma membranes: monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during sperm maturation and at ejaculation.

Purified boar sperm plasma membranes (PM) and PM proteins were used as antigens to produce 58 monoclonal antibodies against surface antigens. Fluorescence labelling (biotin-avidin-FITC) was used to determine the distribution of antigens in caput and cauda epididymal and in ejaculated spermatozoa with hybridoma supernatants and/or 1:100 diluted ascites fluid after subcloning. Sixteen areas (subdomains) of apparent restricted antigen mobility were identified and significant differences in the localization of most antigens in caput, cauda, and ejaculated PM were recognized. While localization patterns were highly reproducible with a given protocol for sample preparation and immunolabelling, localization patterns were markedly affected by changes in protocols. Fluorescence patterns were affected by the manner in which sperm were labelled (live sperm or sperm labelled at various steps), by washing, and by temperature or by addition of seminal plasma. These results indicate that the dynamic properties of the sperm PM or the surrounding fluids can easily mask or unmask or reconfigure binding sites for highly site-specific monoclonal antibodies and that antigen distribution is probably under-estimated when these labelling techniques are used. Such changes in the accessibility of antigenic sites to monoclonal antibodies limited determining the extent of distribution of a given antigen on epididymal sperm. However, the reproducibility of patterns when a given protocol is used and the large number of antibodies (39/42) displaying marked differences in localization on caput, cauda, and ejaculated PM suggest that changes in the organization of the PM constituents, whether by addition or subtraction of antigen or through configurational changes in proteins, are a major consequence of sperm maturation in the epididymis.

Acrosome↗
PubMed1986

Microfilaments appear in boar spermatozoa during capacitation in vitro.

Boar spermatozoa were incubated in a capacitation medium and examined for the presence of filamentous actin by using the fluorescent probe NBD-phallacidin. F-actin was not observed in uncapacitated sperm, but developed in most regions of the cell during the capacitation period. Fluorescent staining was most intense in the flagellum. When fresh seminal plasma was added to capacitated sperm and the sperm was further incubated, F-actin was no longer observed. In view of previous experiments which indicated that plasma membrane proteins (PMPs), including a major integral PMP, move out of the sperm head into the flagellum during capacitation and that this movement is inhibited by the microfilament poison cytochalasin D (Peterson, Saxena, Saxena, and Russell: Biol. Reprod., in press, '86), we suggest that actin-PMP interactions play a major role in capacitating boar spermatozoa.

Actin Cytoskeleton↗
PubMed1986

Intramural activation of the human ventricle: findings in normal and abnormal myocardial segments in patients with coronary artery disease.

To define the intramural electrical activation of normal and abnormal myocardial segments in patients with coronary artery disease, mapping was performed using a plunge electrode with 10 electrodes 1 mm apart. Five transmural bipolar electrograms (40 to 500 Hz) were recorded from 79 sites in 13 patients at the time of open-heart surgery. The results were correlated with ventriculography. In 64 sites with normal contraction, activation spread from endocardium to epicardium in 20.5 +/- 2 msec, and no electrograms were abnormal. Activation time did not differ between the different anatomic segments. In the eight hypokinetic sites, activation spread from endocardium to epicardium in 22.2 +/- 2 msec, and no electrograms were abnormal. In the seven sites with akinesis or dyskinesis, abnormal electrograms were noted and were concentrated towards the endocardium. In four patients, the 40% to 80% of electrograms closest to the endocardium were abnormal, while in three patients all transmural electrograms were abnormal. The variable depth of electrically abnormal tissue should be considered in operations and ablative procedures for ventricular arrhythmias.

Adult↗
PubMed1986

Characterization of lactate dehydrogenase of Plasmodium knowlesi.

Some properties of purified lactate dehydrogenase, (EC. 1.1.1.27) from schizonts of Plasmodium knowlesi are described. The plasmodial enzyme migrated as single entity on polyacrylamide gel, and resembled rabbit muscle (M4) lactate dehydrogenase in its electrophoretic mobility. The P. knowlesi enzyme consisted of four identical subunits of 31 kDa. Purified lactate dehydrogenase was inhibited almost completely when incubated with 100 microM p-chloromercuribenzoate, Ag+ or Hg+ and such inhibition could be reversed by the addition of beta-mercaptoethanol or L-cysteine. Metal chelators did not show any remarkable effect. Oxalic acid is a competitive inhibitor of pyruvate reduction by this enzyme with apparent Ki of approximately equal to 0.41 mM.

Animals↗
PubMed1986

Purification and properties of lactate dehydrogenase from Plasmodium knowlesi and erythrocytes of the rhesus monkey.

Lactate dehydrogenase, E.C.1.1.1.27 (LDH) from the simian malarial parasite, Plasmodium knowlesi, and from normal rhesus monkey erythrocytes has been purified using Blue Sepharose affinity chromatography, and the properties of the purified enzyme from these two sources have been compared. The enzyme from the host and parasite were different in their kinetic properties, viz., substrate and pH optima, thermostability and isoenzymic behaviour. Partially purified LDH of the erythrocytes resolved into three isomeric bands on polyacrylamide gel electrophoresis, whereas the parasite LDH moved as a single enzyme band of different mobility from the host LDH. The molecular mass of the parasite enzyme was estimated as 117,500 daltons.

Animals↗
PubMed1986

Increase in the concentration of major boar sperm surface proteins during maturation in the epididymis.

High-resolution two-dimensional polyacrylamide gel electrophoresis analyses have indicated that several major boar sperm plasma membrane polypeptides (PMPs) increased in concentration during maturation in the epididymis. To investigate this further, monoclonal antibodies (MAbs) to two of these PMPs/glycoproteins referenced as 4.85 and 5.0 and polyclonal antisera (PCA) raised against PMP 5.0 were used to quantify these changes. ELISA assays for both PMPs, using solubilized plasma membrane (PM) and intact PM as antigens, indicated that both PMPs were present in greater concentration in cauda PM than in caput PM. ELISA assay using PCA against 5.0 and whole sperm from the cauda and caput epididymis also showed an increase in the concentration of this protein during transit through the epididymis. Indirect FITC fluorescence microscopy showed that MAbs reacted specifically with the luminal aspect of secretory cells of the corpus epididymis and, with greater intensity, with secretory cells of the cauda segment. When MAb tags were used the fluorescence technique indicated that PMP 5.0 was restricted to a narrow area of PM overlying the principal segment of the head of caput sperm but fluorescence extended further into the principal segment and into the flagellum of cauda PM. Since MAb binding sites appear to be partially masked, PCA were also used to localize antigenic sites. PCA to PMP 5.0 showed antigen localization in both head and flagellar PM of cauda and caput sperm. The fluorescence of cauda sperm PM, however, was significantly more intense than that of caput sperm PM. MAb to PMP 4.85 also bound to antigen more extensively on cauda sperm. The sum of these analyses suggests that major sperm PMPs are secreted by the epididymis and absorbed by sperm. The magnitude of surface protein changes occurring during epididymal transit in boar sperm appears to be greater than heretofore recognized.

Animals↗
PubMed1986

Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies.

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.

Animals↗
PubMed1985

Isolation of major proteins from boar sperm plasma membranes by preparative gel electrophoresis and localization of a major polypeptide using specific monoclonal antibodies.

A preparative procedure using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is described for isolating major boar sperm plasma membrane polypeptides (PMPs) in soluble form. Proteins were first separated on 6 mm diameter gels using a pH gradient expanded in the acidic region. The second dimension used 6 mm thick, 10% acrylamide gels. Major proteins identified by Coomassie staining were excised and electroeluted. The procedure was applied to the isolation of a group of proteins in the molecular weight range 40K-50K which comprise a major fraction of the total integral membrane protein in these cells (groups 4 and 5). Yields of electrophoretically pure soluble polypeptides from these groups were between 0.3 mg - 0.5 mg from the processing of 16 gels per week. Electroeluted proteins were also used to elicit monoclonal antibodies to major proteins. Monoclonal antibodies to the major plasma membrane protein referenced as 4.85 were isolated and shown to be specific to this protein by transblotting procedures. This protein was primarily localized over the anterior portion of the principal segment of ejaculated sperm by indirect FITC fluorescence microscopy. The ability to isolate 60-100 mg of plasma membranes per week from the cauda epididymides of boars also permitted developing a procedure for the rapid fractionation of large amounts of detergent solubilized plasma membranes by isoelectric focusing in flatbeds of Biogel P200. For the first time, individual proteins of sperm surface proteins can be isolated in large enough amounts to begin detailed biochemical characterization, localization, and functional testing.

Animals↗
PubMed1981

The case of the two-timing pacemaker.

A 71-year-old man with a permanent transvenous pacemaker had recent history of lightheadedness and was brought to the hospital emergency room complaining of shortness of breath. His blood pressure was 80/50, pulse rate 148. Administration of a pressor drug restored blood pressure and coincidentally the heart rate returned to 70. His condition was caused by formation of a dendritic spur in the substrate of the programming circuit in his implanted pacemaker, with consequent intermittent doubling of the impulse frequency. Another pacemaker was substituted surgically, and he recovered uneventfully.

Aged↗
PubMed1981

Perineal anal transplant in low anorectal anomalies.

Data on 67 female infants who received perineal and transplants for for treatment of low anorectal anomalies over a period of 12 years are presented. The procedure is considered to be safe in neonates and infants. When performed in the first year of life, it does not require a preliminary colostomy and gives good functional and cosmetic results. In older children it should be preceded by a diversion colostomy. The immediate complications are few and are not life threatening; the late results are satisfactory.

Anal Canal↗