Potential of Azadirachtin and Neem (Azadirachta indica) Based Saponins as Biopesticides for In vitro Insect Pests Cellulase (Beta-1,4-Endoglucanase) Enzyme Inhibition and In vivo Repellency on Tribolium castaneum.

Aims: The work was undertaken to identify the role of Neem derived compounds (saponins and azadirachtin) on the digestive cellulose hydrolyzing enzyme activity of red flour beetle (T. castaneum), rice grasshopper (Oxya chinensis) and red pumpkin beetle (Aulacophora foveicolis). Place and Duration of Study: The work was carried out at the Institute of Biochemistry and Biotechnology University of the Punjab Lahore Pakistan. Methodology: Total cellular proteins were isolated from the insect’s gut and salivary glands and were tested for cellulose hydrolyzing activity on substrate agar plates. Saponins and Azadirachtin were isolated from Azadirachta indica tissues and used for enzyme inhibition studies. Repellancy test was performed for T. Castaneum, using saponins and Azadirachtin. For computational studies sequence of endoglucanase gene was identified from T. castaneum genome and protein structure was deduced. Results: Saponins were able to inhibit beta-1,4-endoglucanase enzyme activity, present in all the three insect pests. A computational dissection of T. castaneum endoglucanase enzyme, deduced from T. castaneum genome, showed that there were five cys involved in the formation of disulphide bridges in the molecule. The disulfide bridges did not provide any protection to endoglucanase active site. Azadirachtin had no effect on cellulase activity of Oxya chinensis and Aulacophora foveicolis, while beta-1,4- endoglucanase activity of T. castaneum was inhibited. Repellency test for T. castaneum revealed that each group of compounds (Saponins and Azadirachtin) was able to repel the insect. Conclusion: Neem derived compounds had a considerable inhibitory effect on the digestive cellulose hydrolyzing enzyme of T. castaneum.

Spectrum of K ras mutations in Pakistani colorectal cancer patients

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

Aetiology of shigellosis in northern Pakistan.

People of northern Pakistan face health hazards because of poor sanitation practices. Bacterial gastrointestinal infections are very common, and sometimes outbreaks occur. The present study was aimed at evaluating and analyzing infestation of Shigella spp. in patients with suspected gastroenteritis and ascertaining the status of antibiotic therapy. Five hundred and eighty-five faecal samples of patients with suspected gastroenteritis, referred to the District Headquarter Hospital Gilgit, were investigated for common enteropathogenic bacteria from July 1997 to September 1999. Seventy-seven (13.2%) of the faecal specimens were infected with different strains of Shigella spp., 61% of which were Shigella dysenteriae, 15.6% were S. flexneri, and 23.4% were Shigella sp. All Shigella strains were sensitive to ceftriaxone, cefotaxime, ciprofloxacin, and enoxacin. Sixty-one percent of the strains were resistant to both ampicillin and chloramphenicol, and 3.9% to ampicillin and nalidixic acid, while 10.4% were resistant to ampicillin alone and 14.3% to chloramphenicol only. Only 10.4% of the strains were sensitive to all the antibiotics tested. Sixty strains of Shigella spp. were processed for isolation of plasmids, and 58 (97%) of these antibiotic-resistant bacteria harboured at least one plasmid. The number of plasmids varied from 1 to 9. Escherichia coli C600 were transformed with the isolated plasmids. Transformants, containing 23-kb plasmid, resisted growth in media containing antibiotics, thereby indicating that antibiotic resistance is plasmid-borne. Based on the findings of the study, it is concluded that the infestation of Shigella spp. is high in northern Pakistan, the aetiological agents are highly resistant to chloramphenicol and ampicillin, and the antibiotic resistance is mediated by the 23-kb plasmid.

Effect of exercise on transplanted extensor digitorum longus muscle in Sprague Dawley rats.

Effect of exercise has been studied on intact and transplanted extensor digitorum longus (EDL) muscle in rats. Majority of muscle fibres hypertrophied and a few showed hyperplasia in intact and transplanted EDL muscle after exercise. The weight, dimensions and diameter of muscle fibres increased, while total muscle area, number of muscle fibres and the number and diameter of nuclei decreased after exercise in all the experimental groups. The DNA, RNA and protein contents were however increased after exercise.

Regeneration following orthotopic transplantation of entire plantaris muscle in Sprague Dawley rats.

Regeneration of rat plantaris muscle has been studied without predeneration. The original muscle fibres were completely degenerated within one week, but were completely regenerated at the end of experiment. The weight of regenerate showed 51% recovery. The breadth, length and total area of muscle and number of muscle fibres was less by 42, 37, 26 and 29% respectively, compared to control muscle at the end of experiment. The diameter of regenerated fibres was almost normalized at the end of experiment. The number and diameter of nuclei in the regenerate was more than that of control which coincides with the increase in DNA and RNA contents. The protein contents after an initial loss started increasing and continued to do so till the end of the experiment. The better regeneration of the plantaris muscle seems to be due to the presence of supporting gastrocnemius muscle.

Effect of mercuric chloride on the activities of some hepatic enzymes of regenerating rabbit liver following partial hepatectomy

Mercuric chloride (HgCl2) administered at a dose of 1mg/kg body wt/day for 5 days decreased hepatic lactate dehydrogenase (LDH) activity (63

) and increased isocitrate dehydrogenase (ICDH) activity (127

). After withdrawal of HgCl2 treatment for 10 days, the LDH and glutamate pyruvate transaminase (GPT) activities showed 56

decrease, respectively, while alkaline phosphatase (AkP) activity increased 4.12 fold. The ICDH activity got normalized. Glutamate oxaloacetate transaminase (GDT) was not affected at all. The hepatic LDH, ICDH and GPT activities decreased 58

. At the end of 15 days after PH, the hepatic ICDH activity increased 4.27 fold, while GPT and GOT activities decreased 67

, respectively. The hepatic ICDH activity of PH-rabbit increased 53

, after 5 days of HgCl2 treatment post-PH, while GPT and AkP activities decreased 89

, 10 days after the last dose, while all other enzymes showed normal values. The total body growth rate and relative liver weight decreased under all experimental conditions.