Genetic variation between hybrid (Labeo rohita ♂, Cirrhinus mrigala ♀ and Labeo rohita) by RAPD marker.

The present work was to study the genetic variability between the major carps Labeo rohita and Cirrhinus mrigala and their hybrids of L. rohita (male♂) and C. mrigala (female♀). Genetic variability was studied by employing RAPD molecular markers. 25 samples of each target species having different sizes with the same age group for the determination of interspecific variation were collected. The morphometric parameters such as body weight, total length, tail length, and lengths of dorsal and anal fins of each individual were recorded and results showed that wet body weight, total length, dorsal fin, anal fin, and tail fin length are positively correlated and then the DNA was extracted using the inorganic salt-based method and conformed by Gel electrophoresis. Twenty-four arbitrary decamer primers were used to get species-specific RAPD analysis Distinct and highly reproducible RAPD profiles with significant genetic variability was detected among species. Only five primers showed amplification. The RAPAD primer OPB-05 produced a total of seven bands out of these 5 monomorphic and 2 polymorphic, so in this case, the percentage polymorphism was 28.57%. The Hybrid show more than a 50% difference from the Labeo rohita. This shows that the Hybrid more resembles C.mrigala. Phylogenetic analysis demonstrated that hybrid (L. rohita ♂ X Cirrhinus mrigala ♀) is the closest to C. mrigala and the farthest from L. rohita. Overall data are presented concerning the applications of RAPD markers for hybrid identification, genetic diversity assessment, and studying taxonomic relationships at a molecular level.

Dark-coloured semen in nonobstructive azoospermia: a report of four cases.

Dark-coloured semen is rarely observed in men with nonobstructive azoospermia in absence of genital tract bleeding. Dark-coloured semen is also observed rarely in men with spinal cord injury with or without genital tract bleeding. However, this condition has not been reported in the literature in absence of genital tract bleeding or spinal cord injury. Here, we report men with nonobstructive azoospermia with dark-coloured semen and without genital tract bleeding or spinal cord injury. The study included four subjects with dark-coloured semen. All the samples first investigated for the presence of red blood cells as well as haemoglobin. All the cases were examined in details with seminal, hormonal and genetic parameters. In addition, an elemental electron microscopy and inductively coupled plasma-atomic emission spectrometry evaluation for heavy metals was performed. The semen parameters showed normal volume, pH, leucocyte count and azoospermia or oligoazoospermia. Dark-coloured semen specimens contained neither red blood cells nor haeme pigment. Electron microscopy showed presence of platinum in all the cases, whereas inductively coupled plasma-atomic emission spectrometry revealed increased levels of lead, manganese and nickel in serum as compared to controls. The results indicate dark-coloured semen may be linked with heavy metals found in seminal cells.

Evolution in the practice of primary angioplasty: effect of adjunctive coronary stenting and glycoprotein IIb/IIIa inhibitors on long-term outcomes.

Primary angioplasty strategies have evolved dramatically, including increasing adjunctive use of stents and glycoprotein (GP) IIb/IIIa inhibitors. The purpose of this study was to examine the specific effects of these adjunctive therapies on long-term outcomes after primary angioplasty. From 1996 to 1998, 257 unselected, consecutive patients underwent primary PTCI at our institution. In-hospital mortality was 5.4% (2.9% for patients without cardiogenic shock). The remaining 243 patients were followed for 2.0 +/- 0.7 years. Adjunctive stenting was associated with increased late mortality (8.7% vs. 2.3%, P = 0.02). GP IIb/IIIa inhibitors were associated with reduced late mortality among patients receiving stents (6.9% vs. 21.4%, P = 0.07), but not in those patients treated with balloon angioplasty alone (2.9% vs. 0%, P > 0.20). Coronary stenting remained a significant predictor of late mortality (hazard ratio 5.6, 95% CI 1.5-21.2) after adjustment for other established risk factors. In this unselected series, adjunctive coronary stenting was associated with higher late mortality among hospital discharge survivors. Concomitant GP IIb/IIIa inhibitors partially corrected for this increase. These results are limited by the small sample size and retrospective design of this study. Additional long-term studies are required to test these findings and evaluate for possible mechanisms.

Characteristics of Pakistani women seeking abortion and a profile of abortion clinics.

A study of the characteristics of Pakistani women seeking abortion and a profile of abortion clinics was conducted in 32 abortion clinics in three provincial capitals of the country. All 452 women who had their pregnancies terminated between October and December 1997 were interviewed. Except for 39 women (8.6%), all study subjects were married. A majority of the women (36.6%) were aged >35 years, 61.0% had given birth to > or =5 children, and 40.2% were illiterate. The predominant reasons for abortion were "too many children" (64.4%), contraceptive failure (20.3%), premarital affairs (8.6%), medical reasons (5.4%), and extramarital affairs (1.3%). Nearly two thirds of the abortions were induced by inadequately trained persons. Only 22% of the abortion clinics met the World Health Organization (WHO) standards required for safe termination of pregnancy. At all these clinics, the procedure used to terminate the pregnancy was dilatation and curettage (D&C). Only one clinic was using manual vacuum aspiration (MVA). Induced abortion seems to be fairly common among married women of high parity, advanced age, and low educational status. Keeping in view the large number of terminations, new medical and surgical techniques of pregnancy termination should be introduced to those already providing abortion services.

Norplant: reasons for discontinuation and side-effects.

OBJECTIVE: To study the side-effects and reasons for the discontinuation of Norplant. METHODS: The study was based on the analysis of the medical records of 265 Norplant users, who obtained Norplant from two clinics of the Family Planning Association of Pakistan and who were followed for 5 years. RESULTS: The most common reason (29.4%) for the discontinuation of Norplant was menstrual disturbances, followed by medical problems (4.1%), desire for more children (3.1%) and family problems (3.1%). The major side-effects were weight gain, breast tenderness, headache and dizziness. Only 7.7% of users mentioned any change in sexual behavior and 69.6% of the complaints leading to Norplant removal developed within 6 months of insertion. CONCLUSION: The extent and nature of the side-effects experienced by Pakistani women using Norplant are similar and comparable to those reported from other countries.

Efficacy and continuation rates of Norplant in Pakistan.

A total of 265 women who had the Norplant system inserted were followed for 5 years. The present study is based on 11,435 women-months of use, describing the continuation rates and efficacy of Norplant among these women. The 5-year cumulative continuation rate was 45.7 per 100 continuing users. The continuation rates were age-dependent. The women > or = 35 years of age consistently maintained higher continuation rates at all time intervals as compared with those of younger women. During 5 years of follow-up, five women became pregnant. Three pregnancies occurred in year 2, one in year 3, and one in year 5. The 5-year cumulative pregnancy rate was 2.5 per 100 continuing users. The weight of the user did not influence the pregnancy rate. The continuation rates, as well as the pregnancy rates, are comparable to those reported from other countries in the region.

PIP: This article documents a 5-year follow-up of Norplant acceptors. The study was based on the medical records of 265 women who obtained Norplant from the Family Planning Association of Pakistan clinics in Lahore and Karachi during 1988-89 to examine the efficacy and continuation rates of Norplant in Pakistan. The studied sample participated in the introductory trial that were followed up for 5 years. Results revealed that the cumulative continuation rate during the 5-year period was 45.7 per 100 continuing users. The continuation rates were age-dependent, wherein women aged 35 years or older consistently maintained higher continuation rates at all time intervals as compared with younger women. During the 5-year follow-up, 5 women became pregnant (3 in year 2, 1 in year 3, and 1 in year 5). Furthermore, the 5-year cumulative pregnancy rate was 2.5 per 100 continuing users. The user¿s weight did not influence the pregnancy rate. Lastly, the continuation rates, as well as the pregnancy rates, were comparable to those reported from other countries in the region.

Norplant: users' perspective in Pakistan.

Five hundred and eighteen Norplant acceptors (260 ever-users and 258 current users) were interviewed to assess their perceptions about Norplant. The mean age of the acceptors was 32.6+/-5.7 years (mean +/- SD). The mean parity was 4.3 and many of the users (40.2%) were illiterate. The most common reason to choose Norplant was its long duration of action (70.1%) followed by doctor's advice (10.4%) and use by other women (10.1%). Norplant was recommended by family planning workers in 35.3% cases, doctors in 29.2% cases and friends in 17.4% cases. Advertisement did not play any role in the women's choice of Norplant. In 77.3% cases, the decision to use Norplant was a joint decision. Only 15% of the users had fears/anxieties before insertion. Most of these women (44%) were concerned about possible ill-effects of Norplant on their health rather than efficacy. The social acceptance of Norplant was very high (76%) and more than half of the users (52.5%) were satisfied with the method. Among current users, 83.9% wanted to continue Norplant for 5 years. Only 39 users (15.1%) intended to discontinue. The main reason for discontinuation was menstrual disturbance (69.2%), followed by weight gain (12.7%). The study suggests that long duration of effective action and high social acceptance are likely to make Norplant a popular method among Pakistani women.

Expression of a male-specific cytochrome P450 isozyme (CYP2C11) in fa/fa Zucker rats: effect of phenobarbital treatment.

The present study determined the effect of genetic obesity and phenobarbital (PB) treatment on the expression and regulation of the hepatic cytochrome P450 enzyme (CYP2C11) in Fa/? and fa/fa Zucker rats. Hepatic CYP2C11 levels as determined by Western immunoblotting and associated enzymatic activity (testosterone oxidation at the 2 alpha position) were significantly lower in untreated fa/fa Zucker rats compared with that observed in Fa/? Zucker rats. There was no significant difference in the constitutive CYP2C11 steady-state mRNA level hybridizable to the cDNA (P450 16 alpha) or specific oligonucleotide probe (Northern and slot blot analyses) between fa/fa and Fa/? Zucker rats. The depressed constitutive CYP2C11 protein levels in fa/fa rats may be attributed to their low plasma testosterone and growth hormone levels; however, lack of differences in CYP2C11 steady-state mRNA suggest post-transcriptional regulatory mechanism(s). Treatment with PB further suppressed hepatic CYP2C11 protein levels and activities in both fa/fa and Fa/? Zucker rats in comparison with that seen in controls. The level of CYP2C11 steady-state mRNA was significantly higher after treatment with PB in Fa/? Zucker rats, while no change was observed in fa/fa animals. The mechanism by which PB treatment fails to increase CYP2C11 steady-state mRNA levels in the fa/fa Zucker rat is unknown; however, it may share a common molecular basis with the defect in nuclear transcription rate previously observed with CYP2B1/2B2.

Acetaminophen hepatotoxicity: influence of phenobarbital and beta-naphthoflavone treatment in obese and lean Zucker rats.

The effect of phenobarbital (PB) and beta-naphthoflavone (beta-NF) on acetaminophen (APAP)-induced hepatotoxicity was evaluated in obese and lean Zucker rats. In addition, the consequences of APAP overdose on hepatic CYP2B1/2B2 enzyme activities following PB treatment were assessed. A single oral dose of APAP 3 g/kg (total body weight) was administered to both littermates 24 hr after the last dose of PB or beta-NF. Histologic evidence of hepatocellular necrosis and serum hepatic aminotransferase enzymes 48 hr after APAP administration was utilized to evaluate hepatic damage. Hepatic microsomal total cytochrome P450 concentrations, alkoxyresorufin O-dealkylase activities, and 16 beta-testosterone hydroxylase activities were determined to evaluate the effect of APAP overdose on cytochrome P450 enzyme-substrate activities in the presence and absence of PB treatment. APAP overdose produced lower hepatotoxicity in the obese Zucker rat compared to lean controls. A similar trend was observed in animals treated with PB prior to APAP administration. In contrast, beta-NF treatment produced potentiation of APAP toxicity and/or death of both obese and lean Zucker rats. Generally, APAP overdose produced reduction of hepatic cytochrome P450 enzyme-substrate activities. However, obese Zucker rats retained a higher percentage of their pre-APAP-treated enzyme activities which is consistent with the observation that obese Zucker rats are less affected by the hepatotoxic effects of APAP overdosage.

Cytochrome P450 2B enzyme (CYP2B) induction defect following phenobarbital treatment in the fa/fa Zucker rat: molecular characterization.

The present study describes the mechanism of the dampened induction of the CYP2B1 and CYP2B2 genes following phenobarbital treatment in the phenotypically obese fa/fa Zucker rat. The fa/fa Zucker rat demonstrated a threefold lower level of CYP2B1/2B2 enzyme induction, as indicated by reduced testosterone oxidation at the 16 beta position and resorufin formation from pentoxy- and benzyloxyresorufin, protein concentration (Western blot analysis), and steady-state mRNA levels (Northern and slot blot analyses) following in vivo treatment with phenobarbital than the Fa/? littermate controls. A primary hepatocyte cell culture system was used to determine if the dampened induction of the CYP2B1/2B2 enzyme is dependent on hormonal influences. Phenobarbital-treated (0.75 mM) hepatocytes from fa/fa Zucker rats showed approximately a three-fold lower induction response based on measurements of CYP2B1/2B2 (R-17 cDNA probe) and CYP2B1 (oligo probe) mRNAs. In order to evaluate whether this dampened response was at the level of transcriptional activation or initiation, as opposed to altered message stability, we measured the rate of transcription of CYP2B1/2B2 genes in nuclei from cultured hepatocytes during run-off experiments. Compared to Fa/? controls, the fa/fa Zucker rat had a greater than threefold lower nuclear transcription rate of CYP2B1/2B2 mRNA. These results suggest that the defective induction of the CYP2B1 and CYP2B2 genes exists at the transcriptional level in the mutant obese fa/fa Zucker rat. These data provide strong evidence that at least two genes are involved. Multiple gene involvement would suggest that the defect is not due to a mutation of the CYP2B gene cis-acting sequence. Instead, the lack of binding of a trans-acting factor, the presence of a repressor, or a defect in transcriptional activation is more likely the molecular mechanism(s) for this enzyme induction defect.

Effect of genetic obesity and phenobarbital treatment on the hepatic conjugation pathways.

The effect of genetic obesity and phenobarbital treatment on hepatic conjugation pathways was evaluated in the obese Zucker rat. Acetaminophen pharmacokinetic parameters were examined in vivo after a 30-mg/kg acetaminophen intravenous bolus dose in the presence and absence of phenobarbital treatment. Glucuronidation and glutathione conjugation pathways were studied in vitro in obese and lean Zucker rats after phenobarbital treatment. Obese Zucker rats demonstrated a higher glucuronidation capacity as evidenced by a higher formation clearance of acetaminophen glucuronide and greater UDP-glucuronosyltransferase (UDPGT) activity toward acetaminophen and p-nitrophenol compared with lean controls. Sulfate and glutathione conjugation pathways were not affected by genetic obesity. Obese Zucker rats possessed a higher total hepatic glutathione content due to greater liver weight. Phenobarbital treatment enhanced glucuronidation of acetaminophen and structurally related compounds (i.e., p-nitrophenol) similarly in both phenotypes, but the treatment failed to induce morphine UDPGT in the obese Zucker rat. No effect of phenobarbital was observed on sulfate conjugation, gamma-glutamyl cysteine synthetase activity or hepatic glutathione content in obese or lean Zucker rats. Similar increases in glutathione transferase activities were observed in animals of both phenotypes after phenobarbital treatment. This study demonstrates that glucuronidation is enhanced in genetically obese rats, whereas phenobarbital causes normal induction of several enzymes of the glucuronidation and glutathione conjugation pathways in the obese Zucker rat. However, morphine UDPGT was not induced by phenobarbital, suggesting that obese Zucker rats may possess a defect in the induction of this enzyme similar to that already described for the CYP2B gene in this strain.

The effects of liver and renal disease on stereoselective serum binding of flurbiprofen.

Stereoselectivity in the serum binding of flurbiprofen, a non-steroidal anti-inflammatory drug which is highly bound to albumin, was studied in patients with liver and renal disease. Subjects with renal disease or liver disease with ascites had significantly lower serum albumin concentrations than normals, resulting in higher free fractions of both the R(-) and S(+) enantiomers of flurbiprofen as determined by equilibrium dialysis. The ratio (+/- s.d.) of R/S-flurbiprofen free fractions was lower in the subjects with ascites (0.714 +/- 0.298) than in those without ascites (0.796 +/- 0.090) (P < 0.05).