Rapid genotyping of loci involved in antifolate drug resistance in Plasmodium falciparum by primer extension

Current methods used to genotype point mutations in Plasmodium falciparum genes involved in resistance to antifolate drugs include restriction digestion of PCR products, allele-specific amplification or sequencing. Here we demonstrate that known point mutations in dihydrofolate reductase and dihydropteroate synthase can be scored quickly and accurately by single-nucleotide primer extension and detection of florescent products on a capillary sequencer. We use this method to genotype parasites in natural infections from the Thai-Myanmar border. This approach could greatly simplify large-scale screening of resistance mutations of the type required for evaluating and updating antimalarial drug treatment policies. The method can be easily adapted to other P. falciparum genes and will greatly simplify scoring of point mutations in this and other parasitic organisms. © 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Resistance to Fiji disease in sugar cane: Role of cultivar preference by planthopper vector Perkinsiella saccharicida (Homoptera : Delphacidae)

Fiji disease (FD) of sugar cane caused by Fiji disease virus (FDV) is transmitted by the planthopper Perkinsiella saccharicida Kirkaldy (Hemiptera: Delphacidae). FD is effectively managed by using resistant cultivars, but whether the resistance is for the vector or for the Virus is Unknown. This knowledge would help develop a rapid and reliable glasshouse-based screening method for disease resistance. Sugar cane cultivars resistant, intermediate, and susceptible to FD were screened in a glasshouse, and the relationship between vector preferences and FD incidence was studied. Cultivar preference by nymphs increased with an increase in cultivar susceptibility to FD, but the relationship between adult preference and FD resistance was not significant. There was a positive correlation between the vector population and FD incidence, and the latent period for symptom expression declined with the increase in the vector populations. FD incidence in the glasshouse trial reflected the field-resistance status of sugar cane cultivars with known FD-resistance scores. The results suggest that resistance to FD in sugar cane is mediated by cultivar preference of the plant-hopper vector.

The effect of heat transfer mode on heart rate responses and hysteresis during heating and cooling in the estuarine crocodile Crocodylus porosus

The effect of heating and cooling on heart rate in the estuarine crocodile Crocodylus porosus was studied in response to different heat transfer mechanisms and heat loads. Three heating treatments were investigated. C. porosus were: (1) exposed to a radiant heat source under dry conditions; (2) heated via radiant energy while half-submerged in flowing water at 23degreesC and (3) heated via convective transfer by increasing water temperature from 23degreesC to 35degreesC. Cooling was achieved in all treatments by removing the heat source and with C. porosus half-submerged in flowing water at 23degreesC. In all treatments, the heart rate of C. porosus increased markedly in response to heating and decreased rapidly with the removal of the heat source. Heart rate during heating was significantly faster than during cooling at any given body temperature, i.e. there was a significant heart rate hysteresis. There were two identifiable responses to heating and cooling. During the initial stages of applying or removing the heat source, there was a dramatic increase or decrease in heart rate ('rapid response'), respectively, indicating a possible cardiac reflex. This rapid change in heart rate with only a small change or no change in body temperature (

A foliar rating system for comparing the resistance of banana cultivars grown as tissue-cultured plantlets in the laboratory to Fusarium wilt

A foliar rating system was developed to assess the progress of Fusarium wilt ( Panama disease) caused by Fusarium oxysporum f. sp. cubense in seven banana cultivars differing in their resistance to race 1 of the pathogen. Plantlets were transplanted into unamended soil naturally infested with the pathogen, soil amended with urea and soil amended with aged chicken manure. A corm invasion score was also developed to assess the accuracy of the foliar symptom score as an indicator of cultivar resistance. On the basis of foliar symptom scores alone, the response of five of the seven cultivars in the chicken manure treatment corresponded to their known field response. However, the response of the other two cultivars, both susceptible to the pathogen in the field, fell into two categories. One had a high foliar symptom score and a correspondingly high corm invasion score, whereas the other had a low foliar symptom score and a high corm invasion score. Breeders need to be aware of the two categories of susceptible response, if inferior breeding material is to be rejected early on in a breeding program.

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved, effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.

Heat transfer and flow behind a step in high enthalpy superorbital flow

Heat transfer levels have been investigated behind a rearward-facing step in a superorbital expansion tube. The heat transfer was measured along a flat plate and behind 2 and 3mm steps with the same length to step height ratio. Results were obtained with air as the test gas at speeds of 6.76kms(-1) and 9-60kms(-1) corresponding to stagnation enthalpies of 26MJ/kg and 48MJ/kg respectively. A laminar boundary layer was established on the flat plate and measured heat transfer levels were consistent with classical empirical correlations. In the case of flow behind a step, the measurements showed a gradual rise in heat transfer from the rear of the step to a plateau several step heights downstream for both flow conditions. Reattachment distance was estimated to be approximately 1.6 step heights downstream of the 2mm step at the low enthalpy condition through the use of flow visualisation.

Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes

Ageing results in a progressive, intrinsic and generalised imbalance of the control of regulatory systems. A key manifestation of this complex biological process includes the attenuation of the universal stress response. Here we provide the first global assessment of the ageing process as it affects the heat shock response, utilising human peripheral lymphocytes and cDNA microarray analysis. The genomic approach employed in our preliminary study was supplemented with a proteomic approach. In addition, the current study correlates the in vivo total antioxidant status with the age-related differential gene expression as well as the translational kinetics of heat shock proteins (hsps). Most of the genes encoding stress response proteins on the 4224 element microarray used in this study were significantly elevated after heat shock treatment of lymphocytes obtained from both young and old individuals albeit to a greater extent in the young. Cell signaling and signal transduction genes as well as some oxidoreductases showed varied response. Results from translational kinetics of induction of major hsps, from 0 to 24 It recovery period were broadly consistent with the differential expression of HSC 70 and HSP 40 genes. Total antioxidant levels in plasma from old individuals were found to be significantly lower by comparison with young, in agreement with the widely acknowledged role of oxidant homeostasis in the ageing process. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Caenorhabditis elegans mutants resistant to phosphine toxicity show increased longevity and cross-resistance to the synergistic action of oxygen

Phosphine (hydrogen phosphide, PH3) is the fumigant most widely used to protect stored products from pest infestation. Despite the importance of this chemical, little is known about its mode of action. We have created three phosphine-resistant lines (pre-1, pre-7, pre-33) in the model organism C. elegans, with LC50 values 2, 5, and 9 times greater than the fully susceptible parental strain. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a nonlethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. We take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, all three mutants have an extended average life expectancy of from 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Because the wild-type and mutant nematodes develop at the same rate, the longevity is unlikely to be caused by a clk-type reduction in oxidative metabolism, a potential alternative mechanism of phosphine resistance.

Systematic characterization of mutations in yeast acetohydroxyacid synthase: Interpretation of herbicide-resistance data

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.

Increased expression of the SNARE accessory protein Munc18c in lipid-mediated insulin resistance

Fatty acids inhibit insulin-mediated glucose metabolism in skeletal muscle, an effect largely attributed to defects in insulin-mediated glucose transport. Insulin-resistant mice transgenic for the overexpression of lipoprotein lipase (LPL) in skeletal muscle were used to examine the molecular mechanism(s) in more detail. Using DNA gene chip array technology, and confirmation by RT-PCR and Western analysis, increases in the yeast Sec1p homolog Munc18c mRNA and protein were found in the gastrocnemius muscle of transgenic mice, but not other tissues. Munc18c has been previously demonstrated to impair insulin-mediated glucose transport in mammalian cells in vitro. Of interest, stably transfected C2C12 cells overexpressing LPL not only demonstrated increases in Munc18c mRNA and protein but also in transcription rates of the Munc18c gene. jlr To confirm the relevance of fatty acid metabolism and insulin resistance to the expression of Munc18c in vivo, a 2-fold increase in Munc18c protein was demonstrated in mice fed a high-fat diet for 4 weeks. Together, these data are the first to implicate in vivo increases in Munc18c as a potential contributing mechanism to fatty acid-induced insulin resistance.

Ovary colonization by Claviceps africana is related to ergot resistance in male-sterile sorghum lines

Ergot, caused by Claviceps africana, has emerged as a serious threat to sorghum hybrid seed production worldwide. In the absence of gene-for-gene-based qualitative resistance in commercial cultivars, varieties with high pollen production that can escape ergot infection are preferred. Recent demonstration of differences in ergot susceptibility among male-sterile lines has indicated the presence of partial resistance. Using chitin-specific fluorescin-isothiocyanate-conjugated wheat germ agglutin and callose-specific aniline blue, this study investigated the process of sorghum ovary colonization by C. africana. Conidia germinated within 24 h after inoculation (a.i.); the pathogen was established in the ovary by 79 h a.i., and at least half of the ovary was converted into sphacelial tissue by 120 h a.i. Changes in fungal cell wall chitin content and strategic callose deposition in the host tissue were associated with penetration and invasion of the ovary. The rate of ovary colonization differed in three male-sterile lines that also differed in ergot susceptibility. This work demonstrates a possible histological basis for partial resistance in male-sterile sorghum lines that could lay the foundation for variety improvement through further breeding and selection.

Heat-induced changes in UHT milks - Part 1

Raw milk was stored for 0, 2 and 4 days and processed in a UHT pilot plant by either direct or indirect heating. The unstored raw milk was also pasteurised. The thermally induced changes resulting from these treatments were investigated by examining a number of indices of heat damage. Lactulose, furosine, total and free hydroxymethylfurfural (HMF) and acid-soluble beta-lactoglobulin were analysed by high performance liquid chromatography (HPLC) while soluble tryptophan was examined by fluorescence spectroscopy. The directly heated UHT milk showed less heat damage than the indirectly heated milk, while the pasteurised milk displayed the least heat damage. During storage of the UHT milk for 12 weeks at similar to20degreesC, the levels of lactulose remained constant, while the furosine concentration increased. Both the total HMF and undenatured beta-lactoglobulin contents showed a general decrease during storage; however free HMF values initially rose but then decreased after four weeks' storage. As the age of the milk at the time of UHT processing increased, the levels of some of the indicators decreased. It is concluded that lactulose is the most reliable index of heat treatment, as it is virtually unaffected by refrigerated storage of the milk before or ambient storage after UHT processing. Reliance on other indicators may give misleading information on the heat load that UHT milk has received during processing.