Stimulation of Ca2+ uptake in the human liver fluke Opisthorchis viverrini by praziquantel

⁴⁵Ca²⁺ uptake by the human liver fluke Opisthorchis viverrini is enhanced by praziquantel. The drug-induced ⁴⁵Ca²⁺ uptake was dependent on the presence of Ca²⁺ and was attenuated in the presence of 10 mM Mg²⁺. La³⁺ and vanadate at concentration of 1mM partially reduced the amount of ⁴⁵Ca²⁺ uptake into the liver fluke in response to praziquantel treatment. The stimulating effect of praziquantel was eliminated in the presence of 10 μM verapamil. These findings suggest that praziquantel increases the permeability of the liver fluke tegument to Ca²⁺ probably by interfering with the mechanism that regulates Ca²⁺ binding or trnasport across the tegumental membrane.     

Screening and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici)

Yeasts antagonistic to Colletotrichum capsici were isolated from Thai fruits and vegetables. Four antagonists (R13, R6, ER1, and L2) were found that inhibited C. capsici growth with biocontrol efficacies of 93.3%, 83.1%, 76.6%, and 66.4%, respectively. Identification by 26S rDNA, and ITS region sequence together with physiological and morphological characteristics, showed them to be Pichia guilliermondii, Candida musae, Issatchenkia orientalis, and Candida quercitrusa, in order of their efficacy. P. guilliermondii strain R13 showed efficacy in reducing disease incidence on C. capsici infected chilli fruits to as low as 6.5%. Lower disease incidence was observed at lower storage temperature. The application of P. guilliermondii is more effective for preserving chilli fruits than conventional preservation with chlorinated water.     

Induction of defense response against Colletotrichum capsici in chili fruit by the yeast Pichia guilliermondii strain R13

Pichia guilliermondii strain R13, a yeast isolated from Thai rambutan, has been shown to suppress the fungal pathogen Colletotrichum capsici in harvested chili. Its multiple modes of action include nutrient competition, tight attachment to the fungus, and hydrolytic enzyme secretion. This study investigated the ability of the P. guilliermondii strain R13 to induce resistance against C. capsici in chili fruit. The pretreatment of chili with the yeast antagonist, physically separated from the fungus by known distances, significantly reduced disease incidence and lesion diameter caused by C. capsici. Compared to the controls, the yeast treatment also significantly enhanced the activities of phenylalanine ammonia-lyase, chitinase, and β-1,3-glucanase, and the accumulation of capsidiol phytoalexin in chili tissue. Scanning electron micrographs showed that the morphology of C. capsici spores and hyphae were abnormal and that the pathogen had restricted growth on chili tissue adjacent to the yeast-inoculated sites. The results indicate that the induction of resistance may be another mechanism by which the yeast antagonist suppresses C. capsici.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Acidic protease from human seminal plasma. Purification and some properties of active enzyme and of proenzyme.

A procedure to purify to homogeneity the active form as well as the proenzyme form of the acidic protease of human seminal plasma is described. This involved precipitation with ammonium sulfate, chromatography on diethylaminoethylcellulose, Sephadex G-200, and Sephadex G-100. The molecular weights of the active form and of the proenzyme were determined by electrophoresis and gel filtration to be 35,000 and 42,000, respectively. The proenzyme was more stable than the active form in alkaline solution and can be converted into the active enzyme under acidic conditions. The active form of the acidic protease can hydrolyze hemoglobin, N,N'-dimethylcasein, N-acetyl-L-phenylalanyl-L-diiodotyrosine, and N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine, but cannot hydrolyze bovine serum albumin, ovalbumin, N-benzyloxycarbonyl-L-glutamyl-L-tyrosine. The active form was also inhibited by p-bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane.     

Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei

Pattanakitsakul S. and Ruenwongsa P. 1984. Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei. International Journal for Parasitology14: 513–520. Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) from Plasmodium berghei were copurified by Sephacryl S-300 and Sephadex G-200 column chromatography and found to have an apparent mol. wt of 132,000. Electrophoresis of the partially purified enzyme under non-denaturing conditions showed the comigration of TS and DHFR. The mol. wt of TS was estimated to be 65,000 on SDS-gel electrophoresis. Both enzymes exhibit a broad pH optimum in the range of 6.5–8.0. Urea, NaCl and KC1 inhibit TS but activate DHFR. For TS, the apparent K_m for dUMP and methylene-tetrahydrofolate have been found to be 71.4 and 312.5 μM, respectively. For DHFR, the apparent K_m for dihydrofolate and NADPH have been found to be 4.4 and 12.5 μM, respectively. Inhibition of DHFR by pyrimethamine, methotrexate and trimethoprim are competitive with dihydrofolate with K_is of 0.63, 0.5 and 1.88 nM, respectively. FdUMP inhibition of TS is competitive with dUMP with K_is of 0.05 μM, but inhibition by methotrexate is uncompetitive with dUMP and MTHF with K_ii of 103 and 23 μM, respectively.     

Molecular and morphological evidence supports the species status of the Mahachai fighter Betta sp. Mahachai and reveals new species of Betta from Thailand

Two regions of mitochondrial (mt) DNA, cytochrome c oxidase subunit 1 (COI) and 16S rRNA, were sequenced in nine species of Betta from Thailand and Indonesia. Most species showed little intraspecific COI variation (adjusted mean = 0·48%) including the putative species Betta sp. Mahachai, but one species (Betta smaragdina) included three lineages showing much greater divergence (7·03–13·48%) that probably represent overlooked species. These findings were confirmed by maximum likelihood analysis and Bayesian inference, which revealed well‐supported corresponding monophyletic clades. Based on these results and morphological differences, the putative species Betta sp. Mahachai from central Thailand is a species distinct from other members of the B. splendens group and represents a new and hitherto undescribed species. Furthermore, this study also demonstrated the probable existence of two overlooked Betta species found in the Khorat plateau basin, illustrating the utility of mitochondrial genetic markers in the revelation of overlooked diversity.     

Impairment of acetylcholine synthesis in thiamine deficient rats developed by prolonged tea consumption

The synthesis of whole brain acetylcholine is reduced in thiamine deficient rats produced by prolonged administration of tea. In those rats fed a normal diet and given tea (1:50, w/v) instead of drinking water for 20 weeks, the conversion of [14C] pyruvate to [14C]acetylcholine decreased by 35%. However, no neurological symptoms were observed. Administration of tea to rats fed a thiamine half-deficient diet for 7-8 weeks caused not only 60% decrease in acetylcholine synthesis but also neurological symptoms. This decreased synthesis of acetylcholine is related to a decline in pyruvate dehydrogenase activity. The results suggest that prolonged administration of tea to rats cause an impairment of acetyl CoA production resulting in a deficit in acetylcholine synthesizing capacity.     

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant Plasmodium chabaudi

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant smodium chabaudi. International Journal for Parasitology16: 483–490. Thymidylate synthetase from cloned strains of pyrimethamine-sensitive and resistant P. chubaudi were partially purified and characterized. The enzyme from both strains have equal mol. wt of 120,000 as estimated by Sephadex G-200 column chromatography. The enzyme from drug-sensitive parasites has an optimum pH of 6.5–7.5 and is stable at pH 4–11 while that from drug-resistant strain has an pH optimum of 7.0–8.0 and is stable at pH 5–10. The K_m for methylenetetrahydrofolate are 206 ± 6 and 495 ± 5 μm for the enzyme from drug-resistant and sensitive parasites, respectively. The K_m for dUMP of the enzyme from drug-resistant and sensitive parasites are 42 ± 1 and 49 ± 6 μm, respectively. Inhibition of the enzyme from both strains by FdUMP are competitive with dUMP; however,the K_is for the enzyme from drug-resistant strain (0.043 ± 0.005 μm) is less than that from drug-sensitive strain (0.11 ± 0.007 μm) by a factor of 2.5. The K_ii for methotrexate with respect to methylenetetrahydrofolate of the enzyme from drug-resistant parasites (58 ± 3 μm) is 3 times larger than that from drug-sensitive parasites (17 ± 1 μm).