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Labile iron pool (LIP) is intracellular nonprotein bound iron that can generate oxygen radicals via the Fenton reaction resulting in oxidative cell damage. Therefore, quantitative measurement of LIP will be helpful for detecting and monitoring the toxic iron status in iron overloaded patients. This study demonstrated LIP level and its correlation to oxidative stress status in β‐thalassemic erythrocytes. LIP and reactive oxygen species (ROS) level, numbers of erythrocyte vesicles and apoptosis were assayed by flow cytometric methods in 30 blood samples from β‐thalassemia/hemoglobin E patients and 17 blood samples from healthy volunteers with normal hemoglobin type. β‐thalassemic erythrocytes showed higher LIP level, defined as the difference in calcein fluorescent intensity of the cells treated with or without deferiprone, than normal erythrocytes (mean ± 2SD as 62.39 ± 39.58 versus 44.65 ± 35.86, P =0.003). The LIP level above 67, a cutoff value of LIP level obtained from receiver operating characteristic curve analysis, had a significant positive correlation with oxidative stress status for ROS level (r= 0.90, P< 0.001) and also the amount of erythrocyte vesicles (r= 0.79, P= 0.002). In contrast, the LIP level showed a significant negative correlation with the patients' hemoglobin level (r=−0.66, P= 0.028). The LIP assay is suggested as an alternative test to monitor the magnitude of iron overload and its consequent oxidative stress in β‐thalassemia. LIP level may also be used as a marker for therapeutic response to iron chelation treatment. © 2018 International Clinical Cytometry Society

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and thalassemia are genetically independent hemolytic disorders. Co-inheritance of both disorders may affect red blood cell pathology to a greater extent than normally seen in either disorder alone. This study determines the prevalence and evaluates hematological changes of G-6-PD deficiency and thalassemia co-inheritance.

α-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of α-globin protein. Couples who both carry the α-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the α-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study, a new and accurate method for α-thalassemia diagnosis was developed by genotyping α-thalassemia-1, the Southeast Asian type (−−SEA) and Thai type (−−THAI) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments, the −−SEAand −−THAIdeletions and two normal fragments, ψζ- and α2-globin gene. The primers were capable of distinguishing α-thalassemia 1 heterozygotes from α-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the −−THAI, −−SEA, ψζ-globin, and α2-globin gene fragments were 79.9 ± 0.2, 89.4 ± 0.5, 92.8 ± 0.2, and 85.0 ± 0.2°C, respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis, and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process, which is laborious, and allows high throughput screening suitable for large population screening for prevention and control of thalassemia.

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.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.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.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. © 2006 International Society for Analytical Cytology

Analysis of the nucleotide sequence downstream from the Xanthomonas oryzaepv. oryzae recAgene reveals two orfs designated orfXand recX. The former has the potential to code for a 5.6 kDa protein of unknown function while the latter encodes for a putative 14.6 kDa protein with homology to RecX from various bacteria. Northern blot analysis and RT‐PCR results show that recA‐orfX‐recXare co‐regulated and arranged in an operon. A recXmutant was constructed. The mutant has no obvious growth defects or stress response defects, except that it cannot support high‐level expression of recAfrom an expression vector. Introduction of the plasmid containing recAinto the recXmutant resulted in reduced transformation efficiency and all transformants tested had mutations with reduced RecA levels. Moreover, the recXmutant has reduced basal levels of RecA. This has not been observed in other bacteria. When inactivated recXwas complemented in trans, both changes were reversed. recXmutation has no effect on the regulation of the recApromoter, suggesting that its effect on the RecA level could be post‐transcriptional.

Oxidant induced protection against peroxide killing was investigated in a prawn bacterial pathogen, Vibrio harveyi. Exposure to 250 μM H2O2induced adaptive protection against subsequent exposure to killing concentrations of H2O2. In addition, 200 μM t‐butyl hydroperoxide (tBOOH) induced cross protection to H2O2killing. On the other hand, peroxide pretreatment did not induce protection against tBOOH killing. Peroxide induced adaptive and cross protection responses required new protein synthesis and were abolished by addition of a protein synthesis inhibitor. Pretreatments of V. harveyiwith 250 μM H2O2and 200 μM tBOOH induced an increase in peroxide scavenging enzymes, catalase and alkyl hydroperoxide reductase subunit C. In addition, stationary phase cells of V. harveyiwere more resistant to H2O2and iodoacetamide killing but highly susceptible to tBOOH killing compared to exponential phase cells. Many aspects of the oxidative stress response of V. harveyiare different from those of other bacteria and these factors may be important for bacterial survival in the environment and during interactions with host shrimp.

Pretreatment of Xanthomonas campestrispv. phaseoliwith low inducing concentrations of thiol reagents such as N‐ethylmaleimide (NEM) and diamide induced resistance to H2O2killing. In addition, these compounds were moderate inducers of peroxide detoxification enzymes such as catalase and alkyl hydroperoxide reductase. For both cases, thiol reagent induced responses required a functional redox sensor/transcription activator oxyRand were absent in an oxyRmutant. By contrast, NEM pretreatment enhanced the killing effects of organic peroxide. The observed Xanthomonasphysiological responses to thiol reagent pretreatment, and subsequent challenge with peroxide stress, differed from other bacteria.

Several versions of broad host range (BHR), L‐arabinose‐inducible expression vectors were constructed. These expression vectors were based on a high copy number BHR pBBR1MCS‐4 replicon that could replicate in both enteric and non‐enteric Gram‐negative bacteria. Two versions of expression cassettes containing multiple cloning sites either with or without a ribosome binding site were placed under transcriptional control of the Escherichia coliBAD promoter and araCgene. Three versions of vectors containing ampicillin or kanamycin or tetracycline resistance genes as selectable markers were constructed. In all six new L‐arabinose‐inducible BHR expression vectors containing many unique cloning sites, selectable markers were made to facilitate cloning and expression of genes in various Gram‐negative bacteria. A Tn9 chloramphenicol acetyl transferase (cat) gene was cloned into an expression vector, resulting in pBBad18Acat that was used to establish optimal expression conditions (addition of 0.02%L‐arabinose to mid‐exponential phase cells for at least 1 h) in a Xanthomonas campestrispv. phaseoli. Comparison of the Cat enzyme activities between uninduced and a 180‐min L‐arabinose‐induced culture showed a greater than 150‐fold increased Cat specific activity. In addition, L‐arabinose induction of exponential phase cells harboring pBBad18Acat gave a higher amount of Cat than similarly treated stationary phase cells. The usefulness of the expression vector was also demonstrated in both enteric and non‐enteric Gram‐negative bacteria.

Dihydroartemisinin and other artemisinin derivatives are relatively ineffective against Plasmodium falciparuminfecting α-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 μm, and for Hb A,Bmax= 0.74 ± 0.18 mol/mol Hb andKd= 224 ± 15 μm. It is concluded that preferential binding of dihydroartemisinin to Hb H over Hb A accounts partly for the higher accumulation capacity of the α-thalassemic erythrocytes, which leads to its antimalarial ineffectiveness.

Hydroxyl radical, a product of H2O2metabolism in the Fenton reaction, is a major reactive oxygen species involved in H2O2killing of Xanthomonas campestrispv. phaseoli(Xp). Compounds which absorbed hydroxyl radicals protected Xanthomonasfrom H2O2killing, but not from killing by a superoxide generator or an organic peroxide. Addition of iron potentiated H2O2killing. On the other hand, pre‐treatment of Xpwith an iron chelator showed no protective effects and H2O2killing was actually enhanced. Unexpectedly, resuspension of Xpin either water or phosphate buffer induced high‐level resistance to H2O2killing. The unknown protection mechanism was independent of new protein synthesis.

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.

In Xanthomonas oryzaepv. oryzae, exposure to a sub‐lethal concentration of H2O2conferred protection to subsequent challenges with a killing concentration of H2O2(30 mM, adaptive response). No adaptive response to tert‐butylhydroperoxides (tBOOH) or the superoxide generator menadione was observed. However, exposure to sub‐lethal concentrations of tBOOH or N‐ethylmaleimide (a thiol reagent) induced cross‐protection to low 30 mM lethal levels of H2O2but not to organic peroxides. Pre‐treatment with a superoxide generator conferred cross‐protection against a high lethal concentration (150 mM) of H2O2and an organic hydroperoxide (tBOOH). The levels of protection against H2O2killing caused by various inducers were correlated with their ability to induce catalase. There was no cross‐regulation between oxidative stress and heat shock. Both adaptive and cross‐protection responses required de novo protein synthesis. Many aspects of these responses in X. oryzaepv. oryzaeare unlike responses previously observed in other microbes and they may have important physiological roles.

Thalassemia and malaria are present at high frequency in tropic and subtropic regions. Impaired growth of Plasmodium falciparumhas been demonstrated in vitro in both α-and β- thalasemic erythrocytes. In this study, we investigated the effects of thalassemic sera on Plasmodium falciparumdevelopement including 5 α-thalasemia1/α-thalasemia2 (HbH) sera, 4 a-thalasemia1/ hemoglobin Constant Springs (HbH/HbCS) sera and 7 β-thalassemia/ HbE sera compared to 7 normal sera. Study on malarial growth in medium containing 10% various sera for 4 days revealed that HbH and b-thalasemia/ HbE sera was significantly inhibited (p<0.05, by Mann-Whitney Utest) the parasite growth comparing to normal serum. Mean percentages of the parasites grown in HbH (2.24+0.75%) and β-thalassemia/ HbE (1.69+0.97%) sera were about 2 times less than that in normal serum (4.55+2.40%). The mean parasitemia cultured in HbH/HbCS serum (2.38+1.54%) was also lower than that in normal serum but it was non-significantly different. Investigation on stage changing of the parasite among the starting, 1st, 2nd, 3rd and 4th day showed that all thalassemic sera retarded the parasite maturation from stage to stage. Decrease in percent ring stage productionwas observed in culture with HbH/HbCS and β-thalassemia/HbE sera. This might be resulted from reduction of merozoite population in mature schizont or failure of re-invasion of the merozoite. Therefore, not only cellular factors but also serum factors of thalassemic patients inhibited P. falciparumdevelopment which may be related to the protective mechanism of thalassemic patients from P. falciparuminfection

Analysis of the nucleotide sequence downstream from the Xanthomonas oryzae pv. oryzae recA gene reveals two orfs designated orfX and recX. The former has the potential to code for a 5.6 kDa protein of unknown function while the latter encodes for a putative 14.6 kDa protein with homology to RecX from various bacteria. Northern blot analysis and RT-PCR results show that recA-orfX-recX are co-regulated and arranged in an operon. A recX mutant was constructed. The mutant has no obvious growth defects or stress response defects, except that it cannot support high-level expression of recA from an expression vector. Introduction of the plasmid containing recA into the recX mutant resulted in reduced transformation efficiency and all transformants tested had mutations with reduced RecA levels. Moreover, the recX mutant has reduced basal levels of RecA. This has not been observed in other bacteria. When inactivated recX was complemented in trans, both changes were reversed. recX mutation has no effect on the regulation of the recA promoter, suggesting that its effect on the RecA level could be post-transcriptional.

In Xanthomonas oryzae pv. oryzae, exposure to a sub-lethal concentration of H2O2 conferred protection to subsequent challenges with a killing concentration of H2O2 (30 mM, adaptive response). No adaptive response to tert-butylhydroperoxides (tBOOH) or the superoxide generator menadione was observed. However, exposure to sub-lethal concentrations of tBOOH or N-ethylmaleimide (a thiol reagent) induced cross-protection to low 30 mM lethal levels of H2O2 but not to organic peroxides. Pre-treatment with a superoxide generator conferred cross-protection against a high lethal concentration (150 mM) of H2O2 and an organic hydroperoxide (tBOOH). The levels of protection against H2O2 killing caused by various inducers were correlated with their ability to induce catalase. There was no cross-regulation between oxidative stress and heat shock. Both adaptive and cross-protection responses required de novo protein synthesis. Many aspects of these responses in X. oryzae pv. oryzae are unlike responses previously observed in other microbes and they may have important physiological roles.