Biomedical subjects

P Wilairat

Publications and source records attributed to P Wilairat.

At least 19 recordsLinked to original sources

PubMed2006

Cytoadherence between endothelial cells and P. falciparum infected and noninfected normal and thalassemic red blood cells.

BACKGROUND: Cytoadhesion of P. falciparum infected red blood cells (RBCs) to endothelial cells (ECs) is an important phenomenon that causes cerebral malaria in man. Reduced adhesion especially in thalassemia and hemoglobinopathies may be related to a protective mechanism against malaria in such people. METHODS: The cytoadherence assay was performed using both conventional and floating conditions between ECs (ECV 304) and P. falciparum infected and noninfected RBCs from both normal and thalassemia subjects. In floating condition, RBC was fluorescently labeled with anti-glycophorin A antibody, whereas EC was identified by surface expression of PECAM-1, CD-36, ICAM-1, and E-selectin. The condition of floating EC was similar to the condition for subcultivation as they can adhere or bind to any surface. The phosphatidylserine (PS) exposure was also determined by using flow cytometer. RESULTS: The adhesion of noninfected heterozygous thalassemic RBCs (all genotypes) to ECs was significantly increased as compared with normal RBCs (P < 0.02). Interestingly, after P. falciparum infection, the number of normal RBCs bound to ECs was significantly increased as compared with noninfected RBCs (P < 0.01), whereas heterozygous thalassemic RBCs infected by P. falciparum showed no significant difference compared with noninfected RBCs. In addition, we found a similar level of PS exposure in normal and thalassemic infected RBCs, which was related to the cytoadherence phenomenon. CONCLUSION: The reduced adhesion between heterozygous thalassemic RBCs infected by P. falciparum to ECs provides an explanation for their protective mechanism against malaria, as increased adhesion is a high risk for cerebral malaria and nonbinding infected RBCs can be removed by the reticuloendothelial system and other mechanism(s) in vivo.

Animals↗

PubMed2005

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility.

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.

Amino Acid Sequence↗

PubMed2002

Stage specificity of Plasmodium falciparum telomerase and its inhibition by berberine.

Telomerase activity in synchronized Plasmodium falciparum during its erythrocytic cycle was examined using the TRAP assay. Telomerase activity was detected at all stages of the parasite intraerythrocyte development, with higher activity in trophozoite and schizont stages compared with ring form. Berberine, extracted from Arcangelisia flava (L.) Merr., inhibited telomerase activity in a dose-dependent manner over a range of 30-300 microM, indicating that P. falciparum telomerase might be a potential target for future malaria chemotherapy.

Animals↗

PubMed2001

Antimalarial alkoxylated and hydroxylated chalcones [corrected]: structure-activity relationship analysis.

Chalcones with 2',3',4'-trimethoxy, 2',4'-dimethoxy, 4'-methoxy, 4'-ethoxy, 2',4'-dihydroxy, and 4'-hydroxy groups on ring B were synthesized and evaluated in vitro against Plasmodium falciparum (K1) in a [3H] hypoxanthine uptake assay. The other ring A was quinoline, pyridine, naphthalene, or phenyl rings with electron-donating or electron-withdrawing substituents of varying lipophilicities. Trimethoxy 6 and 27, dimethoxy 7, 8, 29, and methoxy 31 analogues had good in vitro activities (IC(50) < 5 microM). 3-Quinolinyl ring A derivatives were well represented among the active compounds. Hydroxylated chalcones were less active than the corresponding alkoxylated analogues. When evaluated in vivo, 8 and 208 were comparable to chloroquine in extending the lifespan of infected mice. Multivariate data analysis showed that in vitro activity was mainly determined by the properties of ring B. Quantitative structure-activity relationship models with satisfactory predictive ability were obtained for various B ring chalcones using projections to latent structures. A model with good predictability was proposed for 19 active chalcones. Size and hydrophobicity were identified as critical parameters.

Animals↗

PubMed2001

Inhibitory effects of 9-anilinoacridines on Plasmodium falciparum gametocytes.

Two gametocyte-producing isolates of Plasmodium falciparum, KT1 arid KT3, were cultivated in vitro. On day 11 of cultivation, pure gametocytes containing stage II, III and IV were used to test the gametocytocidal activity of 9-anilinoacridines that had previously demonstrated their activity against the asexual stage of the parasite. After drug exposure for 48 h, gametocytes were maintained without drugs for another 2 days before thin films were prepared for parasite counting. Gametocytocidal activities of 13 analogs of 9-anilinoacridine were observed with 50% inhibitory concentrations in the range of 0.6 microM to> 100 microM The most active compound was 1'-CH2NMe2-9-anilinoacridine. Anilinoacridine derivatives with 3,6-diamino substitution had reduced gametocytocidal activity in contrast to their enhancing effect against the asexual forms. Morphological abnormalities of gametocytes were observed following drug exposure.

Acridines↗

PubMed2001

A novel two-protein component flavoprotein hydroxylase.

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetobacter baumannii and shown to be a two-protein component enzyme. The small component (C1) is the reductase enzyme with a subunit molecular mass of 32 kDa. C1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation of HPA. C1 is a flavoprotein with FMN as a native cofactor but can also bind to FAD. The large component (C2) is the hydroxylase component that hydroxylates HPA in the presence of C1. C2 is a tetrameric enzyme with a subunit molecular mass of 50 kDa and apparently contains no redox centre. FMN, FAD, or riboflavin could be used as coenzymes for hydroxylase activity with FMN showing the highest activity. Our data demonstrated that C2 alone was capable of utilizing reduced FMN to form the product 3,4-dihydroxyphenylacetate. Mixing reduced flavin with C2 also resulted in the formation of a flavin intermediate that resembled a C(4a)-substituted flavin species indicating that the reaction mechanism of the enzyme proceeded via C(4a)-substituted flavin intermediates. Based on the available evidence, we conclude that the reaction mechanism of HPAH from A. baumannii is similar to that of bacterial luciferase. The enzyme uses a luciferase-like mechanism and reduced flavin (FMNH2, FADH2, or reduced riboflavin) to catalyse the hydroxylation of aromatic compounds, which are usually catalysed by FAD-associated aromatic hydroxylases.

Acinetobacter↗

PubMed2001

Determination of lidocaine in dental pulp by high-performance liquid chromatography.

The purpose of this study was to develop a method for lidocaine detection in dental pulp by high-performance liquid chromatography. The amounts of lidocaine in dog pulps were quantitated after local injection to evaluate lidocaine recovery from pulp tissue with this technique. Comparison was also made between the amount of lidocaine found in upper and lower canines. The high-performance liquid chromatography system was shown to be a reliable and reproducible tool for lidocaine determination. Lidocaine extraction from the tissue showed recovery of 90%. The amount of lidocaine recovered from the upper canine (0.21 microg/mg) was higher than the lower canine (0.17 microg/mg).

Algorithms↗

PubMed2001

Molecular defect of PKD1 gene resulting in abnormal RNA processing in a Thai family.

Autosomal dominant polycystic kidney disease (ADPKD) is a common human autosomal disorder caused mainly by mutations of the PKD1 gene. In analysis of PKD1 transcripts by long RT-PCR and nested PCR procedures, we observed PKD1-cDNA fragments from three ADPKD siblings from the same family with a size approximately 250 base pairs (bp) shorter than normal. Further investigations showed that the PKD1 transcripts from these patients had been abnormally processed, the nucleotide sequence of exon 43 containing 291 nt was missing from the transcripts, which would result in an abnormal polycystin-1 with an in-frame deletion of 97 amino acids. This splicing defect did not result from a mutation that disrupted the splice donor or acceptor sites adjacent to exon 43 or the branch sites in flanking introns but was most likely due to 20-bp deletion observed in intron 43. The intronic deletion was present in 8 affected members but absent in 11 unaffected members, corresponding with the results of genetic linkage analysis using 5 polymorphic markers in the PKD1 region. Molecular diagnosis of PKD1 in this family could, therefore, be carried out by genomic DNA amplification to directly detect the PKD1 intronic deletion.

Humans↗

PubMed2001

Partial purification of mitochondrial DNA topoisomerase II from Plasmodium falciparum and its sensitivity to inhibitors.

Mitochondria of Plasmodium falciparum (K1 strain) were isolated by differential centrifugation. Mitochondrial DNA topoisomerase II from P. falciparum was partially purified using fast protein liquid chromatography(FPLC). Parasite mitochondria contained approximately 8% of DNA topoisomerase II activity compared with its nuclear fraction. The effects of fluoroquinolones, inhibitors of bacterial DNA topoisomerase II or DNA gyrase, against partially purified P. falciparum mitochondrial DNA topoisomerase II were investigated using a decatenation assay. Minimum inhibitory concentrations (MIC) of ofloxacin, ciprofloxacin and norfloxacin were > 1, 10 and 100 mM, compared with that of >0.5 and 10 mM for eukaryotic DNA topoisomerase II inhibitor etoposide (VP-16) and amsacrine, respectively. The results indicate that partially purified mitochondrial DNA topoisomerase II was insensitive to fluoroquinolones and it is suggested that their inhibitory effects on P. falciparum growth may be directed against plastid DNA topoisomerase II.

Animals↗

PubMed2000

Inactivation of artemisinin by thalassemic erythrocytes.

Plasmodium falciparum infecting alpha-thalassemic erythrocytes (Hb H or Hb H/Hb Constant Spring) is resistant to artemisinin derivatives. Similar resistance, albeit at a much lower level, is shown by the parasite infecting beta-thalassemia/Hb E erythrocytes. The resistance is due to host-specific factors, one of which is the higher uptake of the drugs by thalassemic erythrocytes than normal erythrocytes, due to binding with Hb H. In addition to higher drug binding, incubation of artemisinin with alpha-thalassemic erythrocytes resulted in preferential inactivation of the drug. Both thalassemic and normal erythrocytes have the capability to inactivate the drug. Addition of serum can protect against inactivation by normal erythrocytes, but not by thalassemic erythrocytes. Incubation with either the hemolysate or the membrane fraction from these erythrocytes also resulted in preferential inactivation of the drug. The drug was also inactivated by purified Hb H. It is concluded that the ineffectiveness of artemisinin derivatives against P. falciparum infecting thalassemic erythrocytes is due partly to competition of the host cell components for binding with the drugs, and partly to inactivation of the drugs by the cell components.

Animals↗

PubMed2000

Cleavage of DNA induced by 9-anilinoacridine inhibitors of topoisomerase II in the malaria parasite Plasmodium falciparum.

Due to resistance by Plasmodium falciparum, the most virulent strain of the four species of human malaria parasites, to most currently used antimalarial drugs, development of new effective antimalarials is urgently needed. Derivatives of 9-anilinoacridine, an antitumor drug, have been shown to inhibit P. falciparum growth in culture and to inhibit parasite DNA topoisomerase II activity in vitro. Using KCl-SDS precipitation assay to detect the presence of protein-DNA complexes within parasite cells, an indicator of DNA topoisomerase II inactivation, derivatives containing 3,6-diNH(2) substitutions with 1'-electron donating (NMe(2), CH(2)NMe(2), NHSO(2)Me, OH, OMe), and 1'-electron withdrawing (SO(2)NH(2)) groups produced protein-DNA complexes. However, the antimalarial pyronaridine, 9-anilinoazaacridine, did not generate protein-DNA complexes, although it was capable of inhibiting P. falciparum DNA topoisomerase II activity in vitro. These results should prove useful in future designs of novel antimalarial compounds directed against parasite DNA topoisomerase II.

Amsacrine↗

PubMed2000

A novel splice-acceptor site mutation (IVS13-2A>T) of polycystic kidney disease 1 (PKD1) gene resulting in an RNA processing defect with a 74-nucleotide deletion in exon 14 of the mRNA transcript.

Autosomal dominant polycystic kidney disease (ADPKD) occurs mainly from mutations of polycystic kidney disease 1 (PKD1) gene. A novel mutation of the PKD1 gene due to a nucleotide substitution in splice-acceptor site of IVS13 (AG->TG) was identified by analyses of PKD1-cDNA and genomic DNA. The IVS13-2A>T substitution resulted in an inactivation of this splice site and utilization of cryptic splice acceptor site in exon 14, causing a 74-nucleotide deletion of this exon in the PKD1-mRNA transcript. The abnormal transcript was present ectopically in the patients' lymphocytes. The partial deletion of PKD1-mRNA leads to frameshift translation and introduces a termination signal at codon 1075. The truncated protein with about one quarter of the full-length polycystin-1 is most likely inactive. Thus, the effect of this mutation would be "loss-of-function" type. Allele specific amplification (ASA) was developed to detect the mutation in DNA samples of other family members. The mutation was present in 11 affected but absent in 13 unaffected family members, corresponding to the results of linkage analysis. In addition, it was not observed in DNA samples of 57 unrelated healthy individuals. Hum Mutat 15:115, 2000.

Amino Acid Sequence↗

PubMed2000

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum.

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a gamma-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-beta-D-arabinofuranosyladenine-5'-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2',3'-dideoxythymidine-5'-triphosphate (IC(50)>400 microM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase gamma. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC(50)>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).

Animals↗

PubMed2000

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro.

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.

Amsacrine↗

PubMed2000

Immunophenotypic discrepancies between granulocytic and erythroid lineages in peripheral blood of patients with paroxysmal nocturnal haemoglobinuria.

In paroxysmal nocturnal haemoglobinuria (PNH), somatic mutation of the PIG-A gene is thought to result in altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins. This study was performed to determine if there were any heterogeneities of cellular phenotypes between two major peripheral blood cells, erythrocytes and granulocytes. Using CD59-based immunocytometry, the patterns of CD59 expression were shown to be conserved in the circulating erythroid cells (reticulocytes and mature erythrocytes) in all 29 patients with PNH. Twenty-one patients had distinct combinations of PNH type I, II, and III cells in different lineages. Only eight patients exhibited similar patterns of CD59 expression between the two lineages. Approximately one third of the patients had PNH type II cells in either or both of the two lineages indicating variable lineage involvement. The proportion of abnormal granulocytes was higher than those of abnormal reticulocytes and erythrocytes. In patients with appropriate erythropoietic responses to haemolysis (RPI > 2.0), shift reticulocytes display predominantly PNH phenotypes. These immature erythroid cells with altered expression of GPI-anchored proteins may dominate the peripheral blood during periods of increased marrow activity resulting in greater phenotypic mosaicism in such patients. Discrepancies in expression of GPI-anchored proteins in PNH which are highly variable between the two lineages may be the result of their different life spans and the influence of complement-mediated cytolysis. The phenomena also indicated the possible occurrence of more than one PNH clones with variable clonal dominance.

Adolescent↗

PubMed2000

Relative quantitation of mRNA in beta-thalassemia/Hb E using real-time polymerase chain reaction.

beta-Thalassemia and Hb E patients, with seemingly identical genotypes, have a remarkable variability in severity. Reduction in red cell survival in beta-thalassemia is correlated with the amount of intracellular unmatched alpha-globin chains. However, it was only recently realized that mRNA, whose translation is prematurely terminated, is also unstable. No systematic attempts have been made to investigate mRNA stability in beta-thalassemia arising from nonsense mutations located upstream from the normal termination codon. In this study, one-step real-time polymerase chain reaction has been employed to compare the levels of alpha- and beta-globin mRNA in reticulocytes from beta-thalassemia/Hb E subjects. The results showed the highest alpha/beta-globin mRNA ratio (median = 5.70, n = 13) in frameshift codons 41/42 (-TTCT)/Hb E individuals compared to normal subjects (median = 1.02, n = 6), or those with Hb E trait (median = 2.15, n = 8). In addition, there was a concomitant increase in the alpha/beta-globin mRNA ratio with decrease in hemoglobin level, i.e., increase in severity. The difference in the ratio among beta-thalassemia/Hb E patients with the same genotype may be attributed to individual variations of efficiency in betaE-globin mRNA splicing and in the destruction of prematurely terminated mRNA.

Adult↗

PubMed1999

Distal renal tubular acidosis and high urine carbon dioxide tension in a patient with southeast Asian ovalocytosis.

Southeast Asian ovalocytosis (SAO) is the best-documented disease in which mutation in the anion exchanger-1 (AE1) causes decreased anion (chloride [Cl-]/bicarbonate [HCO3-]) transport. Because AE1 is also found in the basolateral membrane of type A intercalated cells of the kidney, distal renal tubular acidosis (dRTA) might develop if the function of AE1 is critical for the net excretion of acid. Studies were performed in a 33-year-old woman with SAO who presented with proximal muscle weakness, hypokalemia (potassium, 2.7 mmol/L), a normal anion gap type of metabolic acidosis (venous plasma pH, 7. 32; bicarbonate, 17 mmol/L; anion gap, 11 mEq/L), and a low rate of ammonium (NH4+) excretion in the face of metabolic acidosis (26 micromol/min). However, the capacity to produce NH4+ did not appear to be low because during a furosemide-induced diuresis, NH4+ excretion increased almost threefold to a near-normal value (75 micromol/L/min). Nevertheless, her minimum urine pH (6.3) did not decrease appreciably with this diuresis. The basis of the renal acidification defect was most likely a low distal H+ secretion rate, the result of an alkalinized type A intercalated cell in the distal nephron. Unexpectedly, when her urine pH increased to 7.7 after sodium bicarbonate administration, her urine minus blood carbon dioxide tension difference (U-B Pco2) was 27 mm Hg. We speculate that the increase in U-B Pco2 might arise from a misdirection of AE1 to the apical membrane of type A intercalated cells.

Acidosis, Renal Tubular↗

PubMed1999

Autosomal recessive distal renal tubular acidosis associated with Southeast Asian ovalocytosis.

BACKGROUND: A defect in the anion exchanger 1 (AE1) of the basolateral membrane of type A intercalated cells in the renal collecting duct may result in a failure to maintain a cell-to-lumen H+ gradient, leading to distal renal tubular acidosis (dRTA). Thus, dRTA may occur in Southeast Asian ovalocytosis (SAO), a common AE1 gene abnormality observed in Southeast Asia and Melanesia. Our study investigated whether or not this renal acidification defect exists in individuals with SAO. METHODS: Short and three-day NH4Cl loading tests were performed in 20 individuals with SAO and in two subjects, including their families, with both SAO and dRTA. Mutations of AE1 gene in individuals with SAO and members of the two families were also studied. RESULTS: Renal acidification in the 20 individuals with SAO and in the parents of the two families was normal. However, the two clinically affected individuals with SAO and dRTA had compound heterozygosity of 27 bp deletion in exon 11 and missense mutation G701D resulting from a CGG-->CAG substitution in exon 17 of the AE1 gene. Red cells of the two subjects with dRTA and SAO and the family members with SAO showed an approximate 40% reduction in sulfate influx with normal 4,4'-di-isothiocyanato-stilbene-2,2'-disulfonic acid sensitivity and pH dependence. CONCLUSION: These findings suggest that compound heterozygosity of abnormal AE1 genes causes autosomal recessive dRTA in SAO.

Acidosis, Renal Tubular↗