Exploitation of sperm-Escherichia coli interaction at the receptor-ligand level for the development of anti-receptor antibodies as the vaginal contraceptive.

In an earlier work performed in our laboratory, we have been able to isolate a sperm receptor (SR) from human spermatozoa playing critical role in fertilization using sperm--E. coli interactions at the receptor-ligand level. It has been hypothesized that for the development of an immunocontraceptive, antibodies generated against the SR should have the ability to impair sperm parameters. In this league, an attempt was made to generate anti-SR antibodies and their effect on sperm parameters such as motility, viability, Mg(2+) -dependent ATPase activity, acrosome status, and apoptotic index was examined. Loss of sperm motility was observed with 100% agglutination. Interaction of anti-SR antibodies with spermatozoa resulted in reduced Mg(2+) -dependent ATPase activity (1020 ± 0.53%), premature acrosomal loss (61.5 ± 0.67%) as well as induced apoptosis (58.76 ± 2.5%). The induction of sperm damage was specifically because of anti-SR polyclonal antibodies as it could be mitigated by the addition of purified SR. Further, when in vivo efficacy of anti-SR antibodies was checked, results showed that a single intravaginal administration with anti-SR antibodies in female BALB/c mice led to the failure of conception. However, simultaneous administration of SR with anti-SR polyclonal antibodies resulted in sustenance of fertility. Infertility induced by anti-SR antibodies did not show any other tissue pathology; hence the present work highlights the potential of anti-SR polyclonal antibodies to be used as a vaginal contraceptive.

Receptor mediated amelioration of the detrimental effects of sperm agglutinating factor on sperm parameters.

Sperm agglutinating factor (SAF) isolated from Staphylococcus aureus immobilizes spermatozoa by agglutination and causes sperm death. This interaction of SAF with spermatozoa is receptor mediated and this receptor has been isolated and purified from human spermatozoa. In this study we attempt to study whether the receptor could ameliorate the detrimental effects of SAF on sperm parameters. Receptor was evaluated against SAF mediated compromised sperm parameters such as Mg(2+) dependent ATPase activity, acrosome status and apoptosis, in vitro using fluorescent microscopy and flow cytometry as well as in vivo by studying the impact on fertility in mice. Incubation of SAF (80 µg) with spermatozoa resulted in reduced Mg(2+) dependent ATPase activity and premature acrosomal loss whereas a higher concentration (100 µg), induced apoptosis. However, in the presence of receptor a dose dependent blockage of SAF induced inhibition of Mg(2+) dependent ATPase activity was observed. At higher concentrations 100 and 125 µg, receptor could inhibit both the premature acrosomal loss and apoptosis. In vivo studies showed that receptor (50 µg) could alleviate SAF induced infertility in female Balb/c mice following a single intravaginal application before mating. The work highlights the efficacy of the receptor as a corrective measure against negative influence of SAF on functional parameters of spermatozoa as well as fertility and presents receptor as a potential therapeutic intervention against SAF induced infertility.

Posterior urethral injury in male children: long-term follow up.

OBJECTIVE: Owing to the immature pelvis and the relative intra-abdominal position of the child's bladder, children with a posterior urethral injury differ from adults. We report our experience in the management of such injuries in male children. PATIENTS AND METHODS: Children with suspected urethral injury underwent retrograde urethrography once their clinical condition was stable. Children with complete urethral injury underwent primary urethral realignment either endoscopically or by open surgical technique. Suprapubic cystostomy was performed in other children who were unfit to undergo primary realignment or in whom the management of other injuries took precedence over that of urethral injury. Children referred from elsewhere for further management of urethral injury and those with initial suprapubic cystostomy underwent delayed urethroplasty. RESULTS: Twenty-two children with mean age of 11.3 years were treated at our centre for urethral injury. Seven children underwent primary endoscopic urethral realignment, five open surgical realignment and 10 initial suprapubic cystostomy followed by delayed urethroplasty. Six of the 12 children undergoing primary urethral realignment required additional endoscopic urethrotomy for managing the stricture, and three of these six children eventually underwent urethroplasty. Of the 10 children undergoing delayed urethroplasty, three required additional sessions of endoscopic urethrotomy and two of these required further correction graft urethroplasty. CONCLUSION: Most male children with posterior urethral injuries need immediate realignment to prevent long-term complications.

Contraceptive effect of sperm-agglutinating factor isolated from Staphylococcus aureus in mouse.

OBJECTIVE: To investigate the contraceptive effect of sperm-agglutinating factor (SAF) isolated from Staphylococcus aureus in mouse. DESIGN: Experimental study performed with LACA mice obtained from the Central Animal House, Panjab University, Chandigarh, India. SETTING: In vivo studies conducted in the Department of Microbiology, Panjab University, Chandigarh, India. POPULATION: Sixty female and 18 male mice were used for the studies. METHODS: Mice sperm-S. aureus agglutination, scanning electron microscopy (SEM), in vivo studies in mice. MAIN OUTCOME MEASURE: Contraceptive efficacy of SAF. RESULTS: S. aureus readily adhered to and agglutinated mouse spermatozoa. By SEM, S. aureus adherence was observed on sperm head as well as sperm tail. SAF was found to be causing 100% fertility inhibition in mouse with no effect on vaginal epithelium. CONCLUSIONS: Sperm-agglutinating factor has been found to have a potential as a contraceptive agent.

Genetic transformation in Aspergilli: tools of the trade.

DNA-mediated transformation is a powerful tool that allows the introduction of specific genetic changes in an organism. Transformation of Aspergilli, acclaimed for their wide use in the industry, has been possible for about two decades now. Several basic and applied problems related to fungal biology have been addressed using this technique. Nonetheless, new markers and strategies for transformation are still being developed for these filamentous fungi. Different methods and markers that are currently available for the transformation of Aspergilli are summarized here. The review also brings out the importance of these transformation systems in analyzing fungal gene function. Aspects of Aspergillus niger transformation are selectively emphasized.

Changes in brain and plasma amino acids of mice intoxicated with methyl isocyanate.

Precolumn derivatization with OPA was used for the analysis of brain and plasma free amino acids of mice after the administration of different doses (0.25 LD50, 0.5 LD50 and LD50) of methyl isocyanate (MIC) for different durations (45 min, 4 h, 4 days and 7 days). In general, there were a dose-dependent decrease in brain free amino acid content with the exception of glycine and arginine (increased above the control level with 0.25 LD50 and 0.5 LD50 doses), and taurine which increased with 0.5 LD50 and LD50 doses in 45 min. All the amino acids from plasma were increased with all the three doses, with the exception of arginine which decreased at the 0.25 LD50 dose in 45 min. With increase in duration of observation to 4 h, 4 days and 7 days, the brain amino acid content was still below the control levels and plasma levels were higher as compared to the respective controls. The only exceptions were serine, histidine, alanine and arginine, which decreased on the 7th day. This study suggests that MIC produced an imbalance of both the brain and plasma amino acids, suggesting neurotoxic and systemic effects.

Optimization of 7 alpha-hydroxysteroid dehydrogenase production by Escherichia coli 080.

7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.159) production by Escherichia coli strain 080 was highest when the organism was grown in brain heart infusion broth at pH 6.5 for 72-96 h with shaking at 37 degrees C. The oxygen consumption rate had a strong effect on the production of this constitutive enzyme. Glucose and lactose at 0.2-0.4%, detergents, and ethylenediaminetetra-acetic acid were found to increase the enzyme production.

Purification of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli strain 080.

Purification studies of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 degrees C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54,000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera.

Kinetic properties of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli 080.

Results on the kinetics of 7 alpha-hydroxysteroid dehydrogenase 7 alpha-HSDH showed that this enzyme could oxidize all bile acids having an -OH group at the C-7 position. Lineweaver-Burk plots showed Michaelis constant (Km) values of 0.83 and 0.12 mM for cholic acid and chenodeoxycholic acid, respectively. The effect of enzyme concentration on the reaction velocity showed a constant increase in the enzyme activity with increase in enzyme-protein concentration. 7 alpha-HSDH was activated by Na+, K+, Ca2+, and Mn2+ ions and by reducing agents having a thiol group (dithiothreitol, 2-mercaptoethanol). Co2+, Hg2+, Fe3+, Mg2+, Zn2+, Ba2+, and Cu2+ ions, chelating agents (potassium oxalate, heparin, EDTA) oxidizing agents (sodium perchlorate, sodium periodate, sodium persulphate), and detergents (Tween 20, Tween 40, Tween 80, Triton X-100, sodium lauryl sulphate) were inhibitory to 7 alpha-HSDH activity.

Serum and tissue positivity for hepatitis B virus markers in histopathologically proven glomerulonephropathies.

To assess the pathogenic significance of hepatitis B virus (HBV) in glomerulonephritis (GN), 98 patients with histopathologically proven glomerulonephropathies were screened for HBV markers, complement components and levels of circulating immune complexes (CICs); and renal biopsies from 31 of them were examined for the presence of hepatitis B surface antigen (HBsAg), and its location, by immunoperoxidase staining. The HBsAg positive rate in the patients (who came from a population with 10% HBsAg positivity) ranged from 51.9% in minimum change nephrotic syndrome (MCNS) to 81.8% in patients with proliferative glomerulonephritis (PGN). Whereas 24.5% of the cases were positive for HBsAg only, 10.2% had anti-HBcIgM with HBsAg, 13.3% had HBeAg with HBsAg and 9.2% had HBsAg, HBeAg and anti-HBcIgM. Complement component C3 levels were decreased in all groups of GN studied, but C4 levels varied. CIC levels were significantly increased (p less than 0.01) only in HBsAg-positive MCNS, focal glomerulosclerosis (FGS) and membranous glomerulonephritis (MGN). Of the 31 renal biopsies examined for the deposition of HBsAg, 4 (12.9%) were found to be positive for HBsAg in situ; 64.5% of biopsied patients were seropositive for HBsAg and 77.4% had CICs. All the four in-situ HBsAg-positive cases were seropositive for HBsAg, HBeAg and anti-HBcIgM with significantly high CIC levels (p less than 0.01). HBsAg deposition was intracytoplasmic in the mesangial cells of the glomeruli, in the glomerular basement membrane or in the tubules, or in a combination of these sites.