Following the expiry of patent protection of the innovator product Berenil®, there has been an influx of substandard generic substitutes of the veterinary trypanocide in international commerce, which have been implicated as being a major contributor to the emergence of drug resistance. This situation has necessitated the development of analytical techniques, which would help safeguard the quality and efficacy of generic formulations of diminazene. A selective, accurate, precise and simple reverse-phase isocratic HPLC method for the simultaneous assay of diminazene aceturate and antipyrine (excipient) in pharmaceutical formulations has been developed and validated. The degradation and manufacturing impurities of diminazene have been identified by electrospray ionization mass spectrometry and characterized by NMR spectroscopy of the synthetic compounds. The developed method has been applied for the quality evaluation of over one hundred generic samples of diminazene obtained from Sub -Saharan Africa. The results give an indication that the quality of generic formulations of diminazene on the African market is compromised. Changes in the metabolism of a drug can lead to altered pharmacokinetics, resulting in an increase or decrease in drug plasma concentration, leading to toxification or therapeutic failure. The metabolism of diminazene and pentamidine in isolated rat and pig hepatocytes have been investigated. Diminazene was not metabolized in either rat or pig hepatocytes. While there were no obvious qualitative differences in the metabolic profiles of pentamidine in either of the animals, the rate of metabolism in rats appeared to be faster. Pretreatment of rats with either 3-methylcholanthrene (3-MC), phenobarbitone (PB) or deltamethrin (DM) caused inhibition of pentamidine metabolism to different extents. There were significant differences between the profiles of the three major metabolites of pentamidine in DM and 3-MC pretreated rats compared to the control group, whereas etreatment with PB did not result in any significant changes in profiles of metabolites.

No description available.

Culex mosquitoes, as well as being vectors of filariasis and Japanese encephalitis, are a world wide biting nuisance. Organophosphorus insecticides (OPs) have been widely used to control Culex populations. Resistance to OPs has occurred and is typically mediated by the increase in non-specific esterase activity. The two esterases involved are classified as 'A' and 'B' esterases with respect to their preference for the substrates α- or β- naphthyl acetate. The commonest phenotype involves two elevated esterases, A2 and B2, which occur in complete linkage disequilibrium. The over expression of esterase B1 is due to gene amplification. Initially, in order to further study the molecular biology of OP resistance, full length cDNAs coding for both A2 and B2 esterases were isolated and sequenced from an OP resistant Sri Lankan strain of Culex quinquefasciatus, PelRR. The B2 esterase cDNA was isolated with PCR using primers sharing homology with the B1 esterase cDNA and has 97.4% homology with esterase B1 at the amino acid level. This confirmed that the B esterases belong to an allelic series. Partial genomic sequences of B2 esterase from PelRR and four other OP resistant Culex strains were identical. This suggests that the initial B2 esterase amplification has occurred only once. However, the cDNA sequence of a B1 esterase cDNA isolated from an OP resistant Cuban strain of Culex quinquefasciatus, MRES, was different to that of the previously published B1 esterase gene sequence. At the genomic level, the haplotype of the Cuban B1 esterase gene, based on EcoRI endonuclease analysis, was also different, suggesting that the initial B1 esterase gene amplification event has occurred at least twice. AB esterase cDNA from an OP susceptible strain, PelSS, has also been partially sequenced. PelSS was derived from the same origins as PelRR but its B esterase cDNA sequence and haplotype of the gene are different. Thus, the B2 esterase gene conferring OP resistance, as well as being amplified, is only found in the resistant strain, PelRR. The A2 esterase cDNA was isolated by screening a PelRR cDNA expression library with an anti-A2 antiserum. The cDNA coded for a protein of 540 amino acids (the same as B2 esterase) and shared 47% amino acid homology with B2 esterase. This strongly suggests that the two genes arose from a duplication of an ancestral counterpart. Furthermore, screening of a PelRR genomic library with A2 and B2 esterase gene probes suggests that the two esterase genes, A2 and B2 are situated in tandem within the genome. PCR was used to amplify the coding region of the PelRR A2 esterase cDNA and this was co-transfected into the baculovirus expression system. The recombinant virus expressed an active A esterase.

Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D are associated with extraintestinal strains. The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogeny and its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibiotic resistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvian children <1 year of age were determined by a Clermont triplex PCR and Kirby-Bauer method, respectively. The distribution of the 369 E. coli strains among the 4 phylogenetic groups was A (40%), D (31%), B1 (21%), and B2 (8%). DEC-control strains were more associated with group A while DEC-diarrhea strains were more associated with group D (𝑃 < 0.05). There was a tendency (𝑃 = 0.06) for higher proportion of persistent diarrhea (≥14 days) among severe groups (B2 and D) in comparison with nonsevere groups (A and B1). Strains belonging to group D presented significantly higher percentages of multidrug resistance than the rest of the groups (𝑃 > 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.

Revisión por pares / joruiz@clinic.ub.es / Furazolidone-resistant mutants were obtained from four clinical isolates of diarrhoeagenic Escherichia coli. The stability of the resistance and the frequency of mutation were established. The minimal inhibitory concentration of furazolidone, nitrofurantoin, nalidixic acid, ampicillin, chloramphenicol and tetracycline was established both in the presence and absence of the efflux pump inhibitor Phe-Arg-β-Naphtylamyde. The presence of mutations in the nitroreductase genes nfsA and nfsB was analysed by PCR; sequencing and their enzymatic activity was assessed by a spectrophotometric assay. Alterations in outer membrane proteins were studied by SDS-PAGE. The frequency of mutation ranged from <9.6 × 10-10 to 9.59 × 10-7 . Neither an effect on efflux pumps inhibited by Phe-Arg-β-Naphtylamyde nor cross-resistance with the antibiotics studied was observed. Nineteen mutants (52.94%) presented mutations in the nitroreductase-encoding genes: 17 in the nfsA gene (15 mutants with an internal stop codon, 2 with amino acid changes), 2 in the nfsB (all amino acid changes). Alterations in the outer membrane proteins OmpA and OmpW were also observed. Although more studies are necessary to find other resistance mechanisms, present data showed the low potential of selecting furazolidone-resistant mutants, together with the lack of cross-resistance with unrelated antimicrobial agents.

The purpose of this investigation was to study the effects of decreasing barometric pressure upon the pressure-flow relationships in a peripheral vascular bed in an attempt at better delineation of the autoregulatory mechanisms. A decrease in barometric pressure does influence the transmural pressure and could theoretically affect smooth muscle tone. An evaluation of the extent of the transmural effect is essential to understanding vascular dynamics at altitudes.

Doctor of Philosophy / Department of Plant Pathology / Harold N. Trick / Bread wheat (Triticum aestivum L.) is an important staple crop for 35% of the world’s population. One economically important pathogen of wheat is Puccinia triticina, the causal agent of leaf rust, can cause up to 50% yield loss during epidemics. Despite the lack of an alternate host to complete the sexual stages, P. triticina still has variation within the population, which can make achieving durable resistance difficult. This study aims to gain a better understanding of the P. triticina-wheat interaction by identifying wheat genes that are induced by individual and multiple races. Six P. triticina races were evaluated on a susceptible variety of wheat at six days post inoculation. RNA was sequenced and 63 wheat genes were identified that showed varying expression in response to the six P. triticina races. Fifty-four wheat genes were characterized during the first seven days of infection using real-time PCR. Race specific gene expression was found in three wheat genes with race differences on Lr2A, Lr2C, and Lr17A. Wheat genes that had similar expression in response to all six races were also identified. Seven of the characterized genes were then silenced using RNAi hairpin constructs. The transgenic plants were molecularly characterized and inoculated with a virulent P. triticina race in the T₂ generation. However, the endogenous genes were not silenced and the transgenic plants maintained susceptibility. A mutation approach was also used to identify wheat genes involved in infection. A mutant population of 3780 wheat plants was created using EMS. Fifteen hundred mutants from the M1 population were screened for plants with a different infection phenotype compared to the non-mutated control and 570 were selected. After two additional generations of selection, eight resistant mutants were obtained. The gene expression of the seven previously identified genes were evaluated and one mutant showed reduced expression of an ER molecular chaperone gene. This research uses a forward and reverse genetics approach to identify and evaluate the function of wheat genes in the wheat-P. triticina interaction. Although RNAi could not determine the gene function, the knockout mutant shows that the identified genes may have a crucial role in infection.

No description available.

M.Tech. (Homeopathy) / Insulin resistance (IR) is a metabolic derangement and a documented clinical feature of the metabolic syndrome. It is an important risk factor in the development of cardiovascular disease and Type 2 Diabetes mellitus. Insulin resistance is often characterized by an increased Homeostasis Model Assessment (HOMA) index and hyperinsulinaemia, but it may also be present without increased insulin levels. Metabolic syndrome is a cluster of risk factors characterized by visceral adiposity (a girth exceeding 102cm in men and 88cm in women), dyslipidaemia (low HDL and raised triglycerides levels), hypertension and dysglycaemia, particularly raised fasting blood glucose levels, predisposing individuals to cardiovascular disease and Type 2 Diabetes mellitus. Diaglukon™ Dr Reckeweg R40 is formulated as an adjunct in the treatment of type 2 diabetes mellitus to assist in lowering the blood glucose (hyperglycaemia). The aim of the research was to evaluate and document its efficacy in the treatment of insulin resistance. A cohort of forty five participants between the ages of nineteen to forty five years was randomized into a double blind placebo controlled, 16 week, clinical study. Participants were matched according to age, race and gender. Anthropometric evaluation consisted of weight, height, BMI, waist circumference and blood pressure readings; these were recorded at 4 weekly intervals for sixteen weeks. Metabolic data included fasting insulin, glucose and a full lipogram at baseline (Week 0) and at study conclusion (Week 16). The insulin and glucose was used to calculate the HOMA index as a measure of insulin resistance (IR). Non parametric statistical analysis was conducted on all parameters using the SPSS statistical programme...

MSc Pharmacology, Faculty of Health Sciences, University of the Witwatersrand / Natural remedies from medicinal plants are considered to be effective and safe alternative treatment for diabetes mellitus. The aim of this study was to demonstrate the hypoglycaemic and antidiabetic activity of the aqueous extract of Icacina tracantha (tuber) (fam Icacinaceae)Ananas cosmos (fam. Bromeliaceae)and Uraria picta (leaves) (fam leguminosae) on an animal model of insulin resistance, a condition which predisposes to type 2 diabetes. The plants have a long history of use as anti-diabetic agents in western Nigeria. Method: 120 male Sprague-Dawley rats were assigned into two major groups. One group was fed on normal rat chow with the other group fed on a high calorie diet for four months a period sufficient for the animals to be fed to attain insulin resistance. The animals were then randomly assigned into different groups (each containing 6 male rats). The plant crude extracts were made by weighing specific dried quantities of each plant, boiling in distilled water for about 2 hours, cooling overnight and separating solid from liquid by filtration. The solution was then poured into preweighed 250 ml beakers and allowed to dry in an oven at a temperature of 60oC. The dried, crude extracts were then weighed out and required doses prepared from the extracts. A non-treated group of animals was used as the control. The mixed dose of extract was administered at 300 mg/kg. Over a 3 week period, all the animals were orally dosed with the different doses of plant extracts daily while metformin was administered through the animals’ drinking water, blood was collected from the tail vein of each rat prior to dosing and thereafter weekly, plasma was preserved and 6 analysed for glucose, insulin, free fatty acid concentrations and calculation of HOMA values to determine insulin sensitivity. During this period, the animals were weighed weekly and food intake was measured every three days. An oral glucose tolerance test (OGTT) was performed after the dosing period and fasting, 0, 30, 60 and 120 minute blood samples were taken and assayed for glucose concentration. Animals were terminated and blood analysed. Statistical analysis: The results were tabulated as mean ± standard deviation and percentage median ± quartile range. The statistical analysis for other parameters was carried out via ANOVA (between groups) and Student’s paired T test (within groups). Only data from percentage median and quartile range was used because of the observed variation in glucose concentration between groups even at baseline values. Statistica software (StatSoft, Tulsa, OK, USA) was used for the analysis. Results: All plant extracts in the study showed differing concentration of significant difference in their effect on the plasma glucose, insulin and free fatty acid concentrations in the rat. The most significant effect was observed on the insulin concentration in the normal rat chow and high calorie diet fed animals. The plant extracts were observed to improve insulin sensitivity in most of the groups. This effect was more significant in the normal rat chow fed rats. The effect of the plant extracts on the weight, food consumed glucose and free fatty acid was minimal and in most of the groups was not significant. Conclusion: In conclusion, the results obtained suggest that the plant extracts may be used to improve insulin resistance in the management of diabetes mellitus.