Search

Cadmium is an important heavy metal pollutant. For this study, we investigated the effects of cadmium exposure on the oxidative stress responses of Xanthomonas campestris, a soil and plant pathogenic bacterium. The exposure of X. campestris to low concentrations of cadmium induces cross-protection against subsequent killing treatments with either H2O2 or the organic hydroperoxide tert-butyl hydroperoxide (tBOOH), but not against the superoxide generator menadione. The cadmium-induced resistance to peroxides is due to the metal's ability to induce increased levels of peroxide stress protective enzymes such as alkyl hydroperoxide reductase (AhpC), monofunctional catalase (KatA), and organic hydroperoxide resistance protein (Ohr). Cadmium-induced resistance to H2O2 is dependent on functional OxyR, a peroxide-sensing transcription regulator. Cadmium-induced resistance to tBOOH shows a more complex regulatory pattern. The inactivation of the two major sensor-regulators of organic hydroperoxide, OxyR and OhrR, only partially inhibited cadmium-induced protection against tBOOH, suggesting that these genes do have some role in the process. However, other, as yet unknown mechanisms are involved in inducible organic hydroperoxide protection. Furthermore, we show that the cadmium-induced peroxide stress response is mediated by the metal's ability to predominately cause an increase in intracellular concentrations of organic hydroperoxide and, in part, H2O2. Analyses of various mutants of peroxide-metabolizing enzymes suggested that this increase in organic hydroperoxide levels is, at least in part, responsible for cadmium toxicity in Xanthomonas.

katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA-ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like sigma70 -35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.

Arsenic has been shown to mediate its toxicity through induced generation of reactive oxygen species. Here, we examined the role of oxidative stress-inducible genes (katA, ahpC and ohr) and their regulators (oxyR and ohrR) in the response to arsenic treatment in a plant pathogenic bacterium, Xanthomonas campestris pv. phaseoli (Xp). Overproduction of peroxide-scavenging enzymes (KatA, AhpCF and Ohr) did not enhance arsenic tolerance in wild-type Xp. Furthermore, inactivation of katA, ahpC, ohr, and ohrR genes had no effect on the level of arsenic resistance. By contrast, an oxyR mutant (Xp oxyR) showed increased sensitivity to both pentavalent arsenate and, to a greater extent, trivalent arsenite. The resistance of cells to arsenite treatment was significantly affected by the level of iron. Cells were 10-fold more sensitive to arsenite killing in the presence of excess iron, while removal of iron by an iron chelator (2,2'-dipyridyl) protected Xanthomonas from arsenite toxicity. The arsenite-sensitive phenotype of Xp oxyR could be complemented by the expression of functional OxyR from a plasmid vector, but not by the expression of other known OxyR-regulated peroxide-scavenging enzymes such as KatA and AhpCF, Ohr and OhrR. The data suggested that as yet unidentified, OxyR-regulated gene(s) are involved in conferring arsenic resistance in Xp. To our knowledge, this is the first report showing that the peroxide-sensing regulator OxyR is involved in arsenic resistance.

Agrobacterium tumefaciens is an aerobic plant pathogenic bacterium that is exposed to reactive oxygen species produced either as by-products of aerobic metabolism or by the defense systems of host plants. The physiological function of the bifunctional catalase-peroxidase (KatA) in the protection of A. tumefaciens from reactive oxygen species other than H(2)O(2) was evaluated in the katA mutant (PB102). Unexpectedly, PB102 was highly sensitive to the superoxide generator menadione. The expression of katA from a plasmid vector complemented the menadione-hypersensitive phenotype. A. tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE. Neither inactivation nor high-level expression of the catE gene altered the menadione resistance level. Moreover, heterologous expression of the catalase-peroxidase-encoding gene katG from Burkholderia pseudomallei, but not the monofunctional catalase gene katE from Xanthomonas campestris could restore normal levels of menadione resistance to PB102. A recent observation suggests that the menadione resistance phenotype involves increased activities of organic peroxide-metabolizing enzymes. Heterologous expression of X. campestris alkyl hydroperoxide reductase from a plasmid vector failed to complement the menadione-sensitive phenotype of PB102. The level of menadione resistance shows a direct correlation with the level of peroxidase activity of KatA. This is a novel role for KatA and suggests that resistance to menadione toxicity is mediated by a new, and as yet unknown, mechanism in A. tumefaciens.

Induced adaptive and cross-protective responses to peroxide stress are important strategies used by bacteria to survive stressful environments. We have shown that exposure to low levels of peroxide (adaptive) and superoxide anions (cross-protection) induced high levels of resistance to peroxide killing in Agrobacterium tumefaciens. The mechanisms and genes involved in these processes have not been identified. Here, the roles played by peroxide (oxyR) and superoxide (soxR) global regulators and a catalase gene (katA) during these responses were investigated. H2O2-induced adaptive protection was completely abolished in both the oxyR and katA mutants. Superoxide generator (menadione)-induced cross-protection to H2O2 killing was observed in a soxR mutant, but not in either an oxyR or a katA mutant. In vivo analysis of the katA promoter, using a katA::lacZ transcriptional fusion, revealed that it could be induced by menadione in an oxyR-dependent manner. These results lead us to conclude that H2O2 and superoxide anions directly or indirectly oxidize OxyR and it is the resulting activation of katA expression that is responsible for the induced protection against lethal concentrations of H2O2.

The gene for Agrobacterium tumefaciens OxyR, a peroxide sensor and transcriptional regulator, was characterized. Phylogenetic analysis of bacterial OxyR showed that the protein could be divided into four clades. The A. tumefaciens OxyR grouped in clade III that consists primarily of OxyRs of Alphaproteobacteria displayed the highest homology to OxyR from Rhizobium leguminosarum. oxyR is located next to, and is divergently transcribed from, a bifunctional catalase-peroxidase gene (katA). An A. tumefaciens oxyR mutant was constructed and shown to be hyper-sensitive to H2O2, but not to the superoxide generator, menadione, or an organic hydroperoxide. Exposure of A. tumefaciens to H2O2 resulted in induction of the catalase-peroxidase enzyme. This induction was abolished in the oxyR mutant. In vivo analysis of a katA::lacZ promoter fusion confirmed the results of enzyme assays and indicated that induction of the katA promoter by H2O2 was dependent on functional OxyR. We also examined the regulation of oxyR in A. tumefaciens. Exposure to H2O2 did not induce expression of the gene but simply changed OxyR from a reduced to an oxidized form. The in vivo oxyR promoter analysis showed that the promoter was auto-regulated and that transcription was not induced by H2O2.

Vibrio harveyi is a causative agent of destructive luminous vibriosis in farmed black tiger prawn (Penaeus monodon). V. harveyi peroxide and superoxide stress responses toward elevated levels of a superoxide generated by menadione were investigated. Exposure of V. harveyi to sub-lethal concentrations of menadione induced high expression of genes in both the OxyR regulon (e.g., a monofunctional catalase or KatA and an alkyl hydroperoxide reductase subunit C or AhpC), and the SoxRS regulon (e.g., a superoxide dismutase (SOD) and a glucose-6-phosphate dehydrogenase). V. harveyi expressed two detectable, differentially regulated SOD isozymes, [Mn]-SOD and [Fe]-SOD. [Fe]-SOD was expressed constitutively throughout the growth phase while [Mn]-SOD was expressed at the stationary phase and could be induced by a superoxide generator. Physiologically, pre-treatment of V. harveyi with menadione induced cross-protection against subsequent exposure to killing concentrations of H(2)O(2). This induced cross-protection required newly synthesized proteins. However, the treatment did not induce significant protection against exposures to killing concentrations of menadione itself or cross-protect against an organic hydroperoxide (tert-butyl hydroperoxide). Unexpectedly, growing V. harveyi in high-salinity media induced protection against menadione killing. This protection was independent of SOD induction. Stationary-phase cells were more resistant to menadione killing than exponential-phase cells. The induction of oxidative stress protective enzymes and stress-altered physiological responses could play a role in the survival of this bacterium in the host marine crustaceans.

We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyR(Xp)). The oxyRR2(Xp) mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H(2)O(2) and tert-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2(Xp) mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2(Xp) mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyR(Xp) mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris.

Alkyl hydroperoxide reductase (ahpC) and organic hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl hydroperoxide (tBOOH) while the former was more efficient at degrading cumene hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.

We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert-butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR-regulated P1 promoter. H2O2 was a weak and non-physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert-butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR-regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert-butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC-AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.

Here we report the study on BRCA1 and BRCA2 mutations in 12 Thai breast and/or ovarian cancer families and 6 early-onset breast or breast/ovarian cancer cases without a family history of cancer. Five distinct rare alterations were identified in each gene: four introducing premature stop codons, one in-frame deletion, two missense changes, two intronic alterations and one silent rare variant. The BRCA1 or BRCA2 truncating mutations were detected in four of seven patients with familial or personal history of breast and ovarian cancer, in one of four isolated early onset breast cancer cases and in none of seven breast cancer site specific families. The BRCA1 and BRCA2 mutation yield in Thai patients is consistent with that reported from Europe and North America in similar groups of patients, being particularly high in individuals with personal or family history of breast and ovarian cancer. The BRCA1 and BRCA2 alterations found in this series are different from those identified in other Asian studies, and all but two have never been reported before. We report at least three novel deleterious mutations, the BRCA1 3300delA, BRCA1 744ins20 and BRCA2 6382delT. One in-frame deletion was also found, the BRCA2 5527del9, which seggregated within family members of breast-only cancer patients and was thought to be a cancer-related mutation. BRCA1 3300delA and Asp67Glu alterations were detected each in at least two families and thus could represent founder mutations in Thais., (Copyright 2002 Wiley-Liss, Inc.)

Plasmodium falciparum infecting alpha-thalassemic erythrocytes are resistant to artemisinin and its derivatives. Binding of the drug to hemoglobin H resulting in drug inactivation was previously demonstrated. We now show that an additional host factor, membrane heme, significantly accounted for decreased antimalarial activity of artemisinin. The antimalarial activity of dihydroartemisinin in the presence of normal and thalassemic erythrocyte membranes showed a correlation with the heme content of the membrane (r(2)=0.466, P<0.01). The correlation was more clearly seen when the drug effectiveness was correlated with the heme content of alpha-thalassemic membrane (r(2)=0.636, P<0.01). However, the drug effectiveness showed no correlation to ferrozine-reactive (free or non-heme) iron content (r(2)=0.0001, P>0.05). alpha-Thalassemic erythrocytes contained higher amounts of membrane heme (11.04+/-8.96 nmol/mg membrane protein) than those from normal and beta-thalassemia/HbE erythrocytes (2.68+/-1.28 and 3.98+/-3.98 nmol/mg membrane protein, respectively, P<0.01). Loss of drug effectiveness was also correlated with increment of heme content in membrane prepared from normal erythrocytes treated with phenylhydrazine. It is concluded that heme in both normal and thalassemic erythrocyte membranes is an important factor in drug inactivation.

ohrR encodes a novel organic peroxide-inducible transcription repressor, and we have demonstrated that ohrR is regulated at the transcriptional and the post-transcriptional levels. Primer extension results show that ohrR transcription initiates at the A residue of the ATG translation initiation codon for the ohrR coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1) has high homology to the consensus sequence for Xanthomonas promoters, which is reflected in the high in vivo promoter activity of P1. Deletion of a 139 bp fragment containing the P1 promoter showed that the sequences upstream of -35 regions were required for neither the promoter activity nor OhrR autoregulation. In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands. The protected regions include the -35 and -10 regions of P1. We suggest that OhrR represses gene expression by blocking RNA polymerase binding to the promoter. There are two steps in the post-transcriptional regulation of ohrR, namely differential stability and inefficient translation of the mRNA. The bicistronic ohrR-ohr mRNA was highly labile and underwent rapid processing in vivo to give only stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore, the ohrR mRNA was inefficiently translated. We propose that, in uninduced cells, the concentration of OhrR is maintained at low levels by the autoregulation mechanism at the transcriptional levels and by the ohrR mRNA instability coupled with inefficient translation at the post-transcriptional level. Upon exposure to an organic peroxide, the compound probably interacts with OhrR and prevents it from repressing the P1 promoter, thus allowing high-level expression of the ohrR-ohr operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic per-oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.

Xanthomonas campestris Ohr (a protein involved in organic peroxide protection) and Escherichia coli OsmC (an osmotically inducible protein of unknown function) are related proteins. Database searches and phylogenetic analyses reveal that Ohr and OsmC homologues cluster into two related subfamilies of proteins widely distributed in both Gram-negative and Gram-positive bacteria. To determine if these two subfamilies are functionally distinct, ohr and osmC in Pseudomonas aeruginosa (a bacterium with one representative from each subfamily) were analysed. Only ohr mutants are hypersensitive to organic peroxide, and this phenotype can be restored by complementation with ohr but not osmC. In addition, expression of ohr was highly induced only by organic peroxides, and not by other oxidants or stresses. In contrast, osmC was induced by ethanol and osmotic stress. A similar pattern of regulation was observed for Ohr and OsmC homologues in the Gram-positive bacterium Deinococcus radiodurans, though uninduced expression was much higher and induction lower in this species. These data clearly support the conclusion that Ohr and OsmC define two functionally distinct subfamilies with distinct patterns of regulation.

The ability of XANTHOMONAS: campestris pv. phaseoli to protect itself against lethal concentrations of man-made (N:-ethylmaleimide, NEM) and endogenously produced (methylglyoxal, MG) electrophiles was investigated. Pretreatment of X. c. pv. phaseoli with a low concentration of NEM induced protection against lethal concentrations of NEM and MG. MG pretreatment weakly induced protection against NEM but not against MG itself. NEM-induced protection against electrophile killing required new protein synthesis and was abolished by the addition of a protein synthesis inhibitor. By contrast, MG-induced protection against NEM killing was independent of de novo protein synthesis. X. c. pv. phaseoli harbouring an expression vector carrying a catalase gene was over 100-fold more resistant to MG and NEM killing. High expression levels of genes for other peroxide-protective enzymes, such as those for alkyl hydroperoxide reductase (ahpC and ahpF) and ohr, failed to protect against electrophile killing. Thus, catalase appears to have a novel protective role(s) against electrophile toxicity. This finding suggests that in X. c. pv. phaseoli NEM and MG toxicity might involve accumulation and/or increased production of H(2)O(2). This idea was supported by the observation that addition of 10 mM sodium pyruvate, a compound that can react chemically with peroxide or hydroxyl radical scavengers (DMSO and glycerol), was found to protect XANTHOMONAS: from electrophile killing. The protective role of catalase and the role of H(2)O(2) in electrophile toxicity are novel observations and could be generally important in other bacteria. In addition, unlike other bacteria, XANTHOMONAS: in stationary phase was more susceptible to electrophile killing compared to cells in exponential phase.

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H(2)O(2) killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1, 000-fold; conversely, treatment of cells with menadione plus H(2)O(2) resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H(2)O(2) and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H(2)O(2) and tert-butyl hydroperoxide plus H(2)O(2). These data suggest that H(2)O(2) is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H(2)O(2)-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.

A good laboratory practice is the heart of clinical laboratory quality. One must establish a standard system in order to achieve the quality. However, standard system is not only the technical but also a state of the art. The socioeconomic and culture are the influence factors. At present, technique of reporting the blood smear examination is still a nation controversy. We surveyed the blood smear examination reporting system of the public hospital in Thailand. There were 77 hospitals participated in this study. A questionnaire comprised of 23 questions was distributed to 105 clinical laboratory staffs of the public health hospitals. Results showed that there was a diversity of blood smear examination reporting system. Generally, there were 2 ways of blood smear review. Every smeared slide and only abnormal slides were re-examined by the conventional technique. When an abnormal white blood cell blood picture was observed. The presence of either blast cell or atypical lymphocyte has clinical significance. A majority of laboratory would report in form "counting number of abnormal cell within 100% of the differential count". For other abnormalities of white blood cell; i.e. the neutrophils with toxic granules, most of laboratory reported as "presence". Interestingly, the red blood cell reporting system varied from laboratory to laboratory. There was a total of nine reporting patterns. Results indicated that pattern 4 (few or some, 1+,2+,3+,4+ are 5-10%, 11-25%, 26-50%, 51-75% and 51-100% cell/oil field, respectively) was the most popular one in reporting anisocytosis and poikilocytosis. However, the reporting pattern on red blood cell staining was difference. Pattern 4 and pattern 7 (grading by the size of central pallor) obtained a same popularity on the consideration of hypochromia. But the pattern 9 (few or some, 1+,2+,3+ are 0-1, 1-3, 4-6 and >6 cell/oil field, respectively) was the most frequently used in reporting of polychromasia. Fortunately we found that the reporting system for platelet was not complicated. A majority of laboratory chose the qualitative pattern (reporting as adequate, increased and decreased). Our study indicated that Thai clinical laboratory encounter a diversity of blood smear reporting system. Every clinical laboratory should pay a great attention to this circumstance. Since a good laboratory practice is a knot of the knot-bolt system of healthcare service, therefore, a nation standard system must be established in the near future.

Nowadays, Thailand encounters a serious economic crisis. A clear consensus has been made that a cost-saving system must be the important tool. Both private and government organizations are engaged in this situation. We studied the cost-saving in the clinical laboratory. A questionnaire was distributed to 45 hospital laboratories located in Bangkok. Results showed that efforts to control the cost are the essential policy. There was a variety of factors contributing to the cost-saving process. The usage of public utility, non-recycle material and unnecessary utility were reconsidered. Besides, capital cost (wages and salary) personnel incentive are assessed. Forty three of the 45 respondents had attempted to reduce the cost via curtailing the unnecessary electricity. Eliminating the needless usage of telephone-call. water and unnecessary material was also an effective strategy. A reduction of 86.9%, 80 % and 80.0% of the mentioned factors respectively, was reported. An inventory system of the reagent, chemical and supplies was focused. Most of the laboratories have a policy on cost-saving by decreased the storage. Twenty eight of the 45 laboratories considered to purchase the cheaper with similar quality reagents instead. And some one would purchase a bulky pack when it is the best bargain. A specific system "contact reagent with a free rent instrument" has been used widely (33.3%). Finally, a new personnel management system has been chosen. Workload has rearranged and unnecessary extra-hour work was abandoned.

Dihydroartemisinin and other artemisinin derivatives are relatively ineffective against Plasmodium falciparum infecting alpha-thalassemic erythrocytes, namely hemoglobin (Hb) H or HbH/Hb Constant Spring erythrocytes, as compared with those infecting genetically normal erythrocytes. The variant erythrocytes accumulate radiolabeled dihydroartemisinin to a much higher extent than the normal ones, and the accumulated drug was retained after extensive washing, in contrast to the drug in normal erythrocytes which was mostly removed. At initial drug concentration of 1 mM, most (82-88%) of the drug was found in the cytosol fraction of both variant and normal erythrocytes. Binding of the drug to hemoglobins accounted for 40-70% of the total uptake. Hb H accounted for 10.9 +/- 2.7% and 12.4 +/- 6.2% of total protein in HbH and HbH/Hb Constant Spring erythrocytes. HbH bound with 28.7 +/- 6.7% of the drug, whereas HbH/Hb Constant Spring erythrocytes bound with 21.8 +/- 8.3% of the drug. Binding experiments showed that Hb H had 5-7 times the drug-binding capacity of Hb A. For Hb H, the maximum binding capacity (Bmax) = 1.67 +/- 0.17 mol/mol Hb, and the dissociation constant (Kd) = 66 +/- 17 microM, and for Hb A, Bmax = 0.74 +/- 0.18 mol/mol Hb and Kd = 224 +/- 15 microM. It is concluded that preferential binding of dihydroartemisinin to Hb H over Hb A accounts partly for the higher accumulation capacity of the alpha-thalassemic erythrocytes, which leads to its antimalarial ineffectiveness.

Drug resistance in tuberculosis (TB) has become a major public health threat, particularly when the disease cannot be 100% controlled by BCG vaccination. In Thailand, resistance to rifampicin, a major component of multidrug regimens of treatment, is the common cause of tuberculosis recurrence. The mechanism of rifampicin resistance involves alterations of the RNA polymerase subunit beta (rpo B) gene. Mutations in rpo B gene were often found to cluster within a region of 23 amino acids starting from amino acid residue 511 to residue 533. Direct PCR sequencing was utilized to compare base changes in rpo B gene in three rifampicin resistant phenotypes of M. tuberculosis isolated from Thai patients. The sequences showed one base substitution at codon 531 resulting in an amino acid change from serine (TCG) to leucine (TTG) in a multidrug resistant isolate compared to that of a sensitive isolate, whereas a point mutation at codon 516 causing a change from aspartic acid (GAC) to tyrosine (TAC) was detected in a multidrug resistant isolate from a HIV positive patient. In an isolate resistant only to rifampicin a double mutation at codon 531 changing serine (TCG) to phenylalanine (TTT) was found. No mutations were observed in the same region in streptomycin, ethambutol or isoniazid resistant isolates. This finding reports two new types of mutation (GAC to TAC at codon 516 and TCG to TTT at codon 531) and confirms a direct correlation between rpo B gene alteration and rifampicin resistant phenotype in M. tuberculosis.

Thirty-two unconcentrated urines were electrophoresed on cellogels in 0.05 M barbital buffer, pH 8.6, at 280 volts for 35 minutes. Instead of concentrating 100 fold, the urine samples were directly applied on the gel in volumes from 1.2 to 3.6 microliters and Coomassie dye was used for staining. This makes it possible to observe the protein patterns at trace concentration by reagent strips (about 0.05-0.2 g/l). This method is simple, convenient and suited for routine services.