Meiosis of Wheat × Lymegrass Hybrids

Meiosis was examined in pollen mother cells of F1 hybrids made from crosses between wheat (Triticum aestivum) and lymegrass (Leymus arenarius and L. mollis). Fluorescence genomic in situ hybridization detected pairing between wheat and lymegrass chromosomes during prophase I and metaphase I. Such pairing, when resulting in bivalent formation, was likely to yield correct disjunction, and hence intergenomic recombination could be incorporated into the gametes. Bivalents in these hybrids, however, were more frequently formed between chromosomes of the same parental origin. Univalents were common, whereas multivalents were not clearly detected. Meiotic behaviour in some cells was not totally aberrant, and this may have accounted for the presence of normal pollen. The results are discussed in relation to intergenomic pairing, meiotic behaviour in wide-hybrids and genome relationships, including the Leymus genome origin.     

Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization

A reliable method for preparing metaphase chromosomes from plant leaf tissues is described. The chromosomes are suitable for karyotype analysis and gene mapping by fluorescence in situ hybridisation (FISH). The method is based on enzymatic digestion of young leaf tissues (shoot-tips) after which the resulting protoplasts are treated hypotonically before being dropped onto microscopic slides. Compared to root-tip chromosomes, leaf chromosomes tend to be longer, or less condensed, and hence more karyotypically differentiated. Metaphase index in young leaf tissues is also very high. Metaphase spread consists of evenly and well-distributed chromosomes and this allows accurate counting. The plant used to demonstrate this method is birch (Betula L.), a group of tree species that has extremely small chromosomes. Root-tip chromosomes of these plants are difficult to obtain, as cutting does not produce roots readily. Seedling chromosomes do not represent the same genomic constitution as their mother trees due to introgressive hybridisation. Furthermore, sample collection in the field is convenient and actively growing leaf buds are available throughout the growing season. FISH experiments with these leaf chromosomes also give good results comparable to those obtained with root-tip chromosomes or even better as mapping on long or extended chromosomes has high resolution in general. Mapping of the 16S–28S ribosomal genes on birch leaf chromosomes has been shown to differentiate between birch species and therefore can accurately confirm their interspecific hybrids.     

Cytotoxic activities and effects of atractylodin and β-eudesmol on the cell cycle arrest and apoptosis on cholangiocarcinoma cell line

Cholangiocarcinoma (CCA) is the cancer of bile duct with high mortality rate particularly in Thailand. The clinical efficacy of the standard chemotherapeutics remains unsatisfactory, and therefore, discovery and development of the new alternative drugs with high efficacy and tolerability is needed. The aim of the study was to investigate cytotoxic activity as well as the underlying mechanisms through which atractylodin and β-eudesmol exert their activities on CCA cell growth inhibition, cell cycle arrest, and cell apoptosis. Effects of the compounds on cell cytotoxicity, cell cycle arrest, and cell apoptosis were analyzed using MTT assay, BD Cycletest? Plus DNA kit, and FITC Annexin V Apoptosis Detection Kit I, respectively. The cytotoxic activities of both compounds were concentration- and time-dependent. The IC₅₀ [mean (SD)] of atractylodin and β-eudesmol were 41.66 (2.51) and 39.33 (1.15) μg/ml respectively. Both promoted cell cycle arrest at G1 phase, and induced cell apoptosis through activation of caspase-3/7. The highest activity was observed at 48 h of exposure. Results suggest that these mechanisms are at least in part, explain the cell cytotoxic and anti-CCA activity of atractylodin and β-eudesmol shown in vitro and in vivo models.     

Prevalence of malaria and HIV coinfection and influence of HIV infection on malaria disease severity in population residing in malaria endemic area along the Thai–Myanmar border

The objective of the study is to investigate the prevalence of malaria and HIV coinfection and assess the effect of HIV coinfection on malaria disease severity in malaria patients from the endemic area of Thailand along the Thai–Myanmar border. Blood samples were collected from a total of 867 patients with malaria (all species and severity) who attended Mae Tao clinic for migrant workers, Tak Province during 2005–2007 (439 samples), 2008–2010 (273 samples), and 2011–2013 (155 samples). The average prevalence rate of malaria and HIV coinfected cases in this malaria endemic area of the country during the three periods was 1.85%. HIV coinfection was observed only in samples with mono-infection of Plasmodium falciparum or Plasmodium vivax, with similar proportions (0.81 vs. 1.04%). Patients’ admission parasite density, an indicator of disease severity, was significantly higher in cases with HIV coinfection observed during 2008–2010. Anemia was found at a significantly higher frequency in patients coinfected with malaria and HIV observed during 2005–2007 compared with those infected with malaria alone. No association was observed between malaria and HIV coinfection and gender, and infected malaria species during the three observation periods. Patients with malaria and HIV coinfection had a significantly lower hemoglobin level than those with malaria infection alone. In conclusion, the prevalence of malaria and HIV coinfection in population of the malaria endemic area along the Thai–Myanmar border is low. HIV coinfection tended to increase parasite density, an indicator of malaria disease severity.     

Antimalarial activity and toxicity of Garcinia mangostana Linn.

Objective:To investigate the antimalarial activity and toxicity of the crude ethanolic extract of its pericarp both in vitro and in vim.Methods:The antimalarial activity of Gareinja mangostana(G.mangostana)Linn.extract against 3D7 and Kl Plasmodium falciparum(P.falciparum)clone were assessed using SYBR green I-based assay.A 4-day suppressive test of Plasmodium berghei{P.berghei)infected mouse was performed to investigate in vivo antimalarial activity.Results:The in vitro antimalarial activity was seleclive(SI5?and classified as weak and good lo moderate activity against both 3D7 and K1 P.falciparum,clones with median IC_(50)(range)values of 11.12(10.94-11.29)and 7.54(6.80-7.68)μg/mL,respectively.The extract was considered nontoxic to mice.The maximum tolerated doses for acute and subacute toxicity in mice were 5 000and 2 000 mg/kg,respectively.Median(range)parasite density on day 4 of the negative control group(25%Tween-80),mice treated with 250,500,1000,and 2 000 mg/kg body weight of the extract,and 10 mg/kg body weight of chloroquine for 14 d were 12.8(12.2-13.7),11.4(9.49-13.8),11.6(9.9-12.5),11.7(10.6-12.8),10.9(9.4-11.6)and 0(0-0)%respectively.Parasite density on day 4in the control group treated with Tween-80 was higher than the groups treated with chloroquine and all dose levels of the extract.Conclusions:G.mangostana linn,showed weak antimalarial activity of the extract both in vitro and in vivo could be due to limitation of absorption of the active compounds.     

Pharmacokinetics of the four combination regimens of dihydroartemisinin/mefloquine in acute uncomplicated falciparum malaria

The pharmacokinetics of oral dihydroartemisinin and mefloquine were investigated in 40 patients (aged 16-30 y, weighing 45-60 kg) with acute uncomplicated falciparum malaria following the four combination regimens of dihydroartemisinin/ mefloquine [regimen-I: 300 mg dihydroartemisinin (h-0) plus 750 mg mefloquine (h-0); regimen-II: 300 mg dihydroartemisinin (h-0) plus 750 mg mefloquine (h-24); regimen-III: 300 mg dihydroartemisinin (h-0) plus 750 and 500 mg mefloquine (h-24 and 30); regimen-IV: 300 mg dihydroartemisinin (h-0) plus 750 and 500 mg mefloquine (h-0, 24)]. The four combination regimens were well tolerated. Patients in all treatment groups had a rapid initial response. However, 9 patients (4, 4, and 1 cases in regimens-I, II, and IV) had reappearance of parasitemia during the follow-up period. Significant changes in the pharmacokinetic parameters of both mefloquine and dihydroartemisinin were observed in patients with malaria compared with healthy subjects reported in a paralleled study. For mefloquine, Cmax (mg per dose), AUC0-day1 (mg per dose), and AUC0-day7 (mg per dose) were significantly higher in patients. Furthermore, tmax, was prolonged while V/F contracted and tl/2,z, MRT shortened in patients with malaria. For dihydroartemisinin, Cmax AUC, tmax and Vz/F were changed in the same direction as mefloquine, whereas t1/2z and MRT were prolonged. CL/F was also significantly reduced in patients with malaria. Absorption/disposition kinetics of oral dihydroartemisinin were similar among the various regimens. On the other hand, AUC0-day1 (mg per dose) of mefloquine after regimen-III was significantly higher than the other three regimens. Combination regimens with two divided doses of mefloquine (regimens-III and IV) resulted in a significantly delayed tmax (especially regimens-IV) compared with those with single dose regimens (regimens-I and II).     

An alternative liquid chromatography-mass spectrometric method for the determination of azithromycin in human plasma and its application to pharmacokinetic study of patients with malaria

A simple, sensitive, selective and reproducible method based on high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS) was developed for the determination of a macrolide antibiotic azithromycin in human plasma. The internal standard (roxithromycin) was separated from azithromycin on a Hypersil Gold C18 column, with retention times of 10.71 and 13.67 minutes, respectively. The mobile phase consisted of a mixture of 20 mM ammonium acetate buffer (pH 5.2), acetonitrile and methanol (50:40:10, v/ v/v), running through the column at a flow rate of 0.3 ml/minute. Chromatographic analysis was carried out at 25 degrees C. Sample preparation was by liquid-liquid extraction with a mixture of 7:3 (v/v) diethylether:dichloromethane. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 5% (% coefficient of variations: % CV). Good accuracy was observed for both intra-day and inter-day assays. The limit of quantification was acceptable at 0.5 ng using 200 microl plasma samples. The mean recoveries for azithromycin and the internal standard were greater than 85%. The method was applied successfully to the investigation of the pharmacokinetics of azithromycin when given in combination with fosmidomycin as oral doses of 750 mg twelve hourly for 3 days in 5 Thai male patients with acute uncomplicated falciparum malaria.     

Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from Thailand

We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC(50)) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 +/- 88, 784 +/- 662, 95 +/- 87, 2,424 +/- 2,784, and 1,625 +/- 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC(50) values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.     

Comparative assessment of the in vitro sensitivity of Brugia malayi infective larvae to albendazole, diethylcarbamazine and ivermectin alone and in combination

The antifilaricidal drugs ivermectin (IVM), diethylcarbamazine (DEC), and albendazole (ALB), used alone or in combinations against infective third-stage larvae (L3) of nocturnally subperiodic (NSP) Brugia malayi (Narathiwat strain), were tested in vitro for sensitivity, for 7 days. IVM alone reduced larval motility at concentrations of 10(-7), 10(-6), and 10(-5) M on day 3. DEC alone also had this effect at concentrations of 10(-6). 10(-5), and 10(-4) M on day 2. ALB alone did not have this effect throughout the experiment, at various concentrations. However, it had greater effect when used in combination with either DEC or IVM. The result also indicated that DEC or IVM, when used in combination with ALB at concentrations of 10(-6) M/10(-6) M, and 10(-5) M/10(-5) M was effectively better than each drug used alone at those concentrations. When both drug combinations were compared, ALB/DEC seemed to be more effective than ALB/IVM at a concentration of 10(-6) M/10(-6) M on day 3. Although IVM and DEC can reduce larval motility when used alone or in combination with ALB, they cannot kill these larvae in an in vitro cultivation, even at a high concentration (10(-5) M).