Parametric analysis of metabolic fluxes of alpha-amylase and protease-producing Bacillus subtilis.

Parametric analysis was applied for a metabolic flux model for the fed-batch culture of Bacillus subtilis producing recombinant alpha-amylase and protease. The metabolic flux model was formulated as a linear programming problem consisting of 49 reactions (decision variables) and 50 metabolites (equality constraints). This study was aimed to determine the response of the metabolic fluxes and objective function value of minimizing the difference between ATP consumption and ATP production (ATP balance). With regard to intracellular metabolite accumulation, the objective function value was least sensitive to variation in succinate and most sensitive to variation in malate. Amongst the variations in the accumulation rates of extracellular metabolites, the objective function value was least sensitive to variation in glutamate and most sensitive to variation in starch hydrolysis and triglyceride synthesis. A 10% variation in metabolite accumulation rates caused a maximum of 13.8% variation (standard error = 3.8%) in the objective function value.

Differentiation of Brugia malayi and Brugia pahangi by PCR-RFLP of ITS1 and ITS2.

Lymphatic filariasis has been targeted by the World Health Organization for elimination by the year 2020. Malayan filariasis, caused by Brugia malayi, is endemic in southern Thailand where domestic cats serve as a major reservoir host. However, in nature, domestic cats also carry B. pahangi infection. In addition to chemotherapy and vector control, control in reservoir hosts is necessary to achieve the elimination of the disease. Therefore, differentiation between B. malayi and B. pahangi in the cat reservoir will help the lymphatic control program to monitor and evaluate the real disease situation. It is difficult to differentiate these two Brugia species by microscopic examination. The technique is also time-consuming and requires expertise. We employed the polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique of internal transcribed spacer regions, ITS1 and ITS2, of ribosomal DNA (rDNA) to differentiate B. malayi from B. pahangi species. Among the restriction enzymes tested, only the PCR product of ITS1 digested with Ase I could differentiate B. malayi from B. pahangi. This PCR-RFLP technique will be useful for lymphatic filariasis control programs for monitoring and evaluating animal reservoirs.

A survey of the infective larvae of Gnathostoma spinigerum in swamp eels bought in a local market in Bangkok, Thailand.

The livers were separated from the viscera of 2738 swamp eels (Monopterus alba) purchased from Klong Toey market, the largest market in Bangkok, between June 1999 and May 2000. When these livers were digested in artificial gastric juice, 524 (19.1%) were found to be infected with the human-infective, third-stage larvae (L3) of Gnathostoma spp. All the identified larvae were confirmed morphologically to be G. spinigerum. Prevalence of the infection varied with season, from a high of 38.3% in September to a low of 7.0% in April, being generally high during the rainy season and winter (June-February). The mean (S.E.) number of L3 recovered/infected liver, which was 3.99 (0.52) overall, also varied with the season, peaking at 5.38 (1.89) in January, but the month-on-month variation was not statistically significant. Although the results of an earlier study had indicated that the prevalence of eel infection decreased in November, after the rainy season, the most abrupt decrease observed in the present study occurred at the beginning of summer (March). However, the period covered by the present study was unusually wet, and the prevalence of eel infection may depend on rainfall more than season.

Glucose-6-phosphate dehydrogenase (G6PD) mutations in Thailand: G6PD Viangchan (871G>A) is the most common deficiency variant in the Thai population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary disorder in humans. Through a population study for G6PD deficiency using a cord blood quantitative G6PD assay in Bangkok, Thailand, we found that the prevalence of G6PD deficiency is 11.1% in Thai male (N=350) and 5.8% in female (N=172) cord blood samples. Among the neonates with hyperbilirubinemia, the prevalence of G6PD deficiency is 22.1% in males (N=140) and 10.1% in females (N=89). We developed a PCR-restriction enzyme-based method to identify G6PD Viangchan (871G>A), and searched for this and 9 other mutations in DNA from G6PD deficient blood samples. G6PD Viangchan (871G>A) was the most common mutation identified (54%), followed by G6PD Canton (1376G>T; 10%), G6PD Mahidol (487G>A; 8%), G6PD Kaiping (1388G>A; 5%), G6PD Union (1360C>T; 2.6%) and "Chinese-5" (1024C>T; 2.6%). Among 20 neonates with hyperbilirubinemia, G6PD Viangchan was also most frequently identified (60%), followed by G6PD Canton (10%), G6PD Mahidol, G6PD Union, and G6PD Kaiping (5% each). G6PD Viangchan appears from this study to be the most common G6PD mutation in the Thai population, bringing into question previous reports that G6PD Mahidol is most prevalent. G6PD Viangchan, together with G6PD Mahidol and G6PD Canton, are responsible for over 70% of G6PD deficiency in this study of Thais. With the data from other Southeast Asian ethnic groups such as Laotians, G6PD Viangchan (871G>A) is probably the most common variant in non-Chinese Southeast Asian population.

Specific IgE antibody responses to somatic and excretory-secretory antigens of third stage G. spinigerum larvae in human gnathostomiasis.

Specific IgE antibody levels in the serum of patients with proven gnathostomiasis and in those with intermittent cutaneous migratory swelling (CMS) were determined by the enzyme-linked immunosorbent assay (ELISA) using somatic extract and excretory-secretory (ES) products of Gnathostoma spinigerum infective larvae as antigens. The third stage larval used were obtained from naturally infected eels. There was an increase in specific IgE antibody to both antigens in these patients. The mean levels of these specific IgE antibodies were significantly higher than that of the healthy control (P<0.01). Comparison between using somatic extract and ES products in the test showed, a positive result in the group of suspected patients with gnathostomiasis or CMS was significantly higher when using ES products (81.81%) than somatic extract (59.09%) as the antigens (P<0.05). However, both somatic and ES antigens cross-reacted with other parasitic sera. The overall sensitivity of the ELISA for these IgE antibodies detection were 71.87 per cent and 87.50 per cent with somatic and ES antigens, respectively. The specificity was 57.53 per cent when somatic antigen was used and increased to 69.86 per cent when ES antigen was used. The positive and negative predictive values of the test were 42.59 per cent and 82.35 per cent by using somatic antigen. Both of these values, were also increased to 56.00 per cent and 92.72 per cent by using the ES antigen. It is obvious that more potential components may be present in ES products than those in the somatic extract. The ES antigen may have to be further purified and may be suitable for evaluation of the effectiveness of chemotherapy. As such, the antibody responses to secreted products are more closely related to active infection than the anti-whole worm antibody that may persist following the death of the parasites. However, in this disease, the effect of the IgE antibody on its pathophysiology it is still not known.

Molecular cloning of phospholipase A2 from a Thai Russell's viper venom gland cDNA library.

Snake venom contains several toxins. Russell's viper (D. russellii, RV) is a venomous snake prevalent in northern and central Thailand. RV bites can cause disseminated coagulation, hemolysis, and edema of the bitten limbs. To identify protein components of RV venom, we made a cDNA library from RV venom glands, and randomly sequenced cloned cDNA. We were able to clone a cDNA encoding RV phospholipase A2 (PLA2). PLA2 is an active enzyme found in several species of snake venom worldwide. PLA2 is thought to be toxic to cell membrane, thereby, can cause local cell and tissue damage, as well as systemic effects in snake bite victims. This PLA2 cDNA clone would facilitate in vivo studies of the pathophysiology of RV bite.

Endemic bancroftian filariasis in Thailand: detection by Og4C3 antigen capture ELISA and the polymerase chain reaction.

Lymphatic filariasis, mainly caused by Wuchereria bancrofti and Brugia malayi, has been targeted for elimination by the World Health Organization by the year 2020. To achieve this goal, highly sensitive and specific diagnostic tests are necessary for close monitoring and evaluation of the control program. We employed an ELISA to detect the Og4C3 antigen and a polymerase chain reaction-based assay for diagnosis of W. bancrofti infection, among the Thai-Karen population in Tak province, Thailand. We found that this endemic area had a microfilarial rate of 10 per cent, while the antigen assay could detect cases about two fold as many (23%). The repeated PCR for the detection of Ssp I of W. bancrofti was positive in 12 per cent of the population under this study. Our data emphasize the need for using highly sensitive and specific assays for assessment of the real burden of the disease.