Papain-Catalyzed Hydrolysis of -Benzoyl-arginine--nitroanilide in an Aqueous-Organic Medium.

Enzyme-based biosensors have emerged as an effective alternative, providing simplicity, high sensitivity, and the capability to detect multiple residues. However, despite their widespread use, limited studies have examined how organic solvents inhibit these sensors. This study investigates the enzymatic reactions and structure of the selected model enzyme, papain, a protease derived from , in the presence of various organic solvents.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94.

Objective: To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE).

Methods: Proteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software.

Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand.

The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002-2004. The pfcrt 76T allele was identified in 109 isolates (99.

[Genotyping of Plasmodium falciparum isolates by amplification of glutamate-rich protein gene using polymerase chain reaction].

Aim: To develop a genotyping method based on amplifying glutamate-rich protein (GLURP) gene for the diagnosis and identification of Plasmodium falciparum.

Methods: Two pairs of primers specific for GLURP gene of P. falciparum were designed and synthesized.

Cryptic Plasmodium falciparum parasites in clinical P. vivax blood samples from Thailand.

Polymerase chain reaction detection revealed cryptic Plasmodium falciparum infections in 21 of 160 samples collected from Thai patients diagnosed (by microscopy) with vivax malaria. The clinical and biological significance of these mixed infections is discussed in the context of chloroquine resistance and the low inoculation rates which characterize malaria epidemiology in Thailand.

Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine.

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred.

Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand.

Plasmodium falciparum isolates were obtained from Thai patients attending a malaria clinic on the Thai-Kampuchean border over 4 cross-sectional surveys carried out at 3-monthly intervals. The genetic structure of the parasite populations was determined by nested polymerase chain reaction (PCR) amplification of polymorphic regions of 3 P. falciparum antigen genes: msp1, msp2 and glurp.

Antiparasite adherence activity in Thai individuals living in a P. falciparum endemic area.

Two types of antimalaria antibodies in the serum of 54 villagers living in a malaria endemic area of Thailand were determined by indirect immunofluorescence assay in order to define the status of malaria immunity within the group. Antibodies to parasite-derived antigens in the membrane of ring stage-infected erythrocytes were very high (> or = 1:1,250) in 44%, moderate to low (< or = 1:250) in 37% of the sera, and the rest did not have the antibody. However, all the sera had antibodies to antigens of the intraerythrocytic mature parasites, showing a very high level in 65% and moderate to low levels in 37% of the sera.

An integrated system using immunomagnetic separation, polymerase chain reaction, and colorimetric detection for diagnosis of Plasmodium falciparum.

An integrated system for sample preparation and DNA detection of the malaria parasite using immunomagnetic separation in combination with the polymerase chain reaction (PCR) and colorimetric analysis is described. A cocktail of three monoclonal antibodies towards merozoite surface antigen-1 was used for magnetic capture of parasites from microliter amounts of whole blood. A sensitivity down to a parasitemia of 10(-6)% was achieved using cultured parasites as a model.

Interallelic recombination in the merozoite surface protein 1 (MSP-1) gene of Plasmodium vivax from Thai isolates.

The merozoite of Plasmodium vivax possesses a high molecular mass surface protein called Pv-merozoite surface protein 1, PvMSP-1, which exhibits antigenic diversity among isolates. In this study, the extent of sequence variation in the polymorphic region and the flanking interspecies conserved blocks (ICBs) 5 and 6 of the PvMSP-1 gene was analyzed using the polymerase chain reaction to amplify the DNA fragment encompassing these regions, followed by sequencing. Twenty different alleles were obtained from 15 Thai isolates.

Flow cytometric assessment of hydroxypyridinone iron chelators on in vitro growth of drug-resistant malaria.

The resurgence of drug-resistant malaria makes urgent the evaluation of new antimalarial agents. This study describes a flow cytometric method (FCM) for testing in vitro drug susceptibility of Plasmodium falciparum malaria to several orally active hydroxypyridinone (CP) iron chelators and to the parenteral iron chelator desferrioxamine (DF). After exposure of parasites to various concentrations of iron chelating agents, aliquots of cultures were fixed with glutaraldehyde.

The variation of Pf155/RESA gene in field isolates of Plasmodium falciparum from Thailand.

Pf155/RESA, an antigen found on the surface of Plasmodium falciparum red blood cell membrane was once a proposed malarial vaccine candidate. The complete sequence of Pf155/RESA gene from one strain and partial sequence from two other isolates revealed that the gene is well conserved. But polymorphism of other antigenic encoded regions occurs with high frequency among isolates especially those collected from the field.

4-aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum.

We have synthesized several 4-aminoquinolines with shortened side chains that retain activity against chloroquine-resistant isolates of Plasmodium falciparum malaria (W. Hofheinz, C. Jaquet, and S.

In vivo study of the response of Plasmodium falciparum to standard mefloquine/sulfadoxine/pyrimethamine (MSP) treatment among gem miners returning from Cambodia.

An in vivo study of the response of P. falciparum to the combination drug, MSP, was conducted among gem miners who contracted malaria from Cambodia in 1991-1992. High level resistance (RII, RIII responses) was observed in 22.

Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies.

The epidemiology of malaria results from the interactions of three gene pools--parasite, human, and mosquito vector--with one another and with their environment. Methods are being developed for characterizing the genetics of human populations at risk and of potential vectors. The characterization of natural populations of Plasmodium and knowledge of their distribution within the human and insect hosts in any given area under study would also greatly enhance understanding of the epidemiology, pathology and biology of this parasite, particularly when combined with simultaneous human and vector studies.

Epidemiological studies of malaria at Pong Nam Ron, eastern Thailand.

Malaria is still a serious health problem in Thailand. Present attempts at controlling the disease by drug treatment and other means remain unsatisfactory. Thus, development of vaccination against malaria is a major research goal of malaria immunology.

Lymphocyte responses to Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA) peptides in individuals with naturally acquired Plasmodium falciparum malaria.

Antibody titers and lymphocyte responses to synthetic peptides corresponding to repeated amino acid sequences of the 3' and 5' regions of Pf155/ring-infected erythrocyte surface antigen (RESA) were studied in two groups of Thai subjects, soldiers (Rangers), and villagers who differed in their history of malaria exposure. The frequency of Pf155/RESA seropositivity was similar in the two groups while the frequency of high titer antibody was significantly greater in villagers than in Rangers. Lymphocyte responsiveness in vitro to all Pf155/RESA peptides was infrequent for both groups although half of the subjects studied responded to crude Plasmodium falciparum asexual blood stage malaria antigen (MA).

Immunomagnetic purification to facilitate DNA diagnosis of Plasmodium falciparum.

The detection of pathogens by polymerase chain reaction (PCR) in clinical samples, such as blood, urine, or feces, requires initial sample preparation to remove polymerase inhibitors and to concentrate the target DNA. Here we show for the first time that immunomagnetic separation can be used to recover pathogens from whole blood and then used for PCR analysis. With antibodies to the merozoite surface protein (MSP1), the malaria-causing parasite Plasmodium falciparum was purified and concentrated from clinical samples.

Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections.

Genus- and species-specific sequences are present within the small subunit ribosomal RNA genes of the four human malaria parasites. Oligonucleotide primer pairs specific to each species were designed for specific amplification by the Polymerase Chain Reaction (PCR), to detect each malaria species. DNA equivalent to 5 microliters of blood was sufficient for the detection of each of the species.

Amplification of pfmdr 1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand.

Drug resistance in Plasmodium falciparum is an expanding problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum.

Flow cytometric screening of blood samples for malaria parasites.

An automated method for the detection and estimation of malaria parasites in blood samples using flow cytometry is presented. In a single-step procedure 50 microliters of blood sample was collected in 1 ml of lysis solution containing formaldehyde, causing red blood cells to lyse while parasites and white blood cells are preserved. Thus prepared, samples could be transported and remained stored in lysis solution until flow cytometric analysis was performed.

Malaria in a rural area of eastern Thailand: baseline epidemiological studies at Bo Thong.

Malaria is still a serious health problem in Thailand. Present attempts at controlling the disease by drug treatment and other means remain unsatisfactory. Thus, development of vaccination against malaria is a major research goal of malaria immunology.

Seroepidemiologic studies of humoral immune response to the Plasmodium falciparum antigens in Thailand.

We have investigated seroreactivity against Plasmodium falciparum crude parasite antigens, the P. falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), as well as against two synthetic peptides (EENV)6 and (EENVEHDA)3 that represent important epitopes of Pf155/RESA. The study population consisted of 421 children and adult Thais living in an area with moderate malaria transmission.