Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

BACKGROUND: Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. METHODS: Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. RESULTS: Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. CONCLUSIONS: These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

BACH1 regulates erythrophagocytosis and iron-recycling in ß-thalassemia.

Macrophages play essential roles in erythrophagocytosis and iron recycling. ß-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of ß-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in ß-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with ß-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.

Vibrio alginolyticus influences quorum sensing-controlled phenotypes of acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus.

BACKGROUND: Acute hepatopancreatic necrosis syndrome (AHPND) caused by Vibrio parahaemolyticus strain (VPAHPND) impacts the shrimp industry worldwide. With the increasing problem of antibiotic abuse, studies on quorum sensing (QS) system and anti-QS compounds bring potential breakthroughs for disease prevention and treatment. METHODS: In this study, the cell-free culture supernatant (CFCS) and its extract of V. alginolyticus BC25 were investigated for anti-QS activity against a reporter bacteria, Chromobacterium violaceum DMST46846. The effects of CFCS and/ or extract on motility, biofilm formation and extracellular polymeric substances (EPSs) of VPAHPND PSU5591 were evaluated. Moreover, the effects of V. alginolyticus BC25 on virulence of VPAHPND PSU5591 were investigated by shrimp challenge test. The potentially active anti-QS compounds presented in the extract and effect on gene expression of VPAHPND PSU5591 were identified. RESULTS: The CFCS of V. alginolyticus BC25 and its extract showed a significant anti-QS activity against the reporter bacteria as well as swimming and swarming motilities, biofilms, and EPSs production by VPAHPND PSU5591. Transcriptome analysis revealed that V. alginolyticus BC25 extract significantly reduced the flagella genes involved in biofilm formation and iron-controlled virulence regulatory gene of VPAHPND PSU5591. Whereas, the LuxR family transcriptional regulator gene, c-factor, a cell-cell signaling gene, and capsular polysaccharide were up-regulated. The potentially active anti-QS compounds identified in extract were Cyclo-(L-Leu-L-Pro), and Cyclo-(L-Phe-L-Pro). Furthermore, V. alginolyticus BC25 enhanced disease resistance against VPAHPND PSU5591 in tested shrimp larvae. CONCLUSION: These findings suggest that V. alginolyticus BC25 could provide natural anti-QS and anti-biofilms compounds and has great ability to be used as biocontrol agent against VPAHPND infection in shrimp aquaculture.

Non-Invasive Colorimetric Magneto Loop-Mediated Isothermal Amplification (CM-LAMP) Method for Helicobacter pylori Detection.

More than half the world's population is thought to be infected with Helicobacter pylori. Although the majority of infected people are asymptomatic, H. pylori infection may cause gastric ulcers and deadly gastric cancer. Owing to the difficulty and invasiveness of current routine culture and diagnostic methods, a highly sensitive and specific noninvasive assay for H. pylori is of interest. This study highlighted the design and performance of a colorimetric magneto loop-mediated isothermal amplification (CM-LAMP) assay to detect H. pylori in spiked saliva samples. LF primers were coated on magnetic nanoparticles by carbodiimide-induced immobilization and functionally used for solidphase amplification. During the LAMP reaction at 66°C, biotin-tagged FIPs were incorporated into LAMP amplicons. The colorimetric signal developed after the addition of NeutrAvidin horseradish peroxidase conjugate (NA-HRP) and ABTS. None of the tested microorganisms, including closely related bacteria, was shown positive by the CM-LAMP assay except H. pylori isolates. This novel platform was highly specific and 100-fold more sensitive (40 CFU/ml or 0.2 CFU per reaction) than the PCR and conventional LAMP assays for the detection of H. pylori in spiked saliva. Our results demonstrated the feasibility of using this noninvasive molecular diagnostic test to detect H. pylori in saliva samples.

Indole signaling decreases biofilm formation and related virulence of Listeria monocytogenes.

Bacterial communication system known as quorum sensing (QS) is a pivotal system for bacterial survival, adaptation and pathogenesis. Members in the multicellular community may synthesize or acquire a signaling molecule in order to elicit downstream cellular processes. Roles of indole and derivatives, a new class of quorum-sensing signal molecules, in various bacterial physiologies and virulence have been reported recently. Indole is normally found in mammal gastrointestinal tract as a metabolite of tryptophan metabolism by microbiota. Therefore, interspecies connection via indole signaling among commensal bacteria and enteric pathogens could be anticipated. Effects of indole exposure on the virulence of Listeria monocytogenes were investigated by phenotypic and molecular approaches. Results demonstrated that synthetic indole and indole-rich conditioned medium significantly diminished biofilm formation and related virulence of L. monocytogenes including motility, cell aggregation and exopolysaccharide production. Transcript levels of virulence-associated (pssE, dltA, flaA, fliI, motB, agrA and hly) and regulatory genes (codY, sigB, prfA and gmaR) were substantially downregulated in indole-treated cells. Only mogR gene encoding for a repressor of motility genes was upregulated after indole exposure. Our findings raise the possibility that L. monocytogenes may acquire indole signaling from gut microbiota for resource-effective adaptation upon transition to new environment.

Indole and Derivatives Modulate Biofilm Formation and Antibiotic Tolerance of Klebsiella pneumoniae.

Intercellular communication is a crucial process for the multicellular community in both prokaryotes and eukaryotes. Indole has been recognized as a new member of the signal molecules which enables the regulated control of various bacterial phenotypes. To elucidate the inter-species relationship among enteric microorganisms via indole signaling, Klebsiella pneumoniae (KP) culture was treated with indole solution and examined for the pathogenicity by using various phenotypic tests. Both synthetic and naturally-produced indole preparations had no deteriorating effect on growth and autoaggregative capacity of KP. The results showed that biofilm formation of carbapenem-susceptible K. pneumoniae (KP-S) was clearly induced by indole exposure (≈ 2-10 folds), whereas no significant difference was observed in the resistant counterpart. In addition, the tolerance to ß-lactam antibiotics of KP was altered upon exposure to indole and/or derivatives assessed by Kirby-Bauer disk diffusion test. Taken together, our finding indicates the functional role of indole in changing or modulating pathogenic behaviors of other bacteria.

Role of Indole Production on Virulence of Vibrio cholerae Using Galleria mellonella Larvae Model.

Cell to cell communication facilitated by chemical signals plays crucial roles in regulating various cellular functions in bacteria. Indole, one such signaling molecule has been demonstrated to control various bacterial phenotypes such as biofilm formation and virulence in diverse bacteria including Vibrio cholerae. The present study explores some key factors involved in indole production and the subsequent pathogenesis of V. cholerae. Indole production was higher at 37 °C than at 30 °C, although the growth at 37 °C was slightly higher. A positive correlation was observed between indole production and biofilm formation in V. cholerae. Maximum indole production was detected at pH 7. There was no significant difference in indole production between clinical and environmental V. cholerae isolates, although indole production in one environmental isolate was significantly different. Both growth and indole production showed relevant changes with differences in salinity. An indole negative mutant strain was constructed using transposon mutagenesis and the direct effect of indole on the virulence of V. cholerae was evaluated using Galleria mellonella larvae model. Comparison to the wild type strain, the mutant significantly reduced the mortality of G. mellonella larvae which regained its virulence after complementation with exogenous indole. A gene involved in indole production and the virulence of V. cholerae was identified.

Characterization of tryptophanase from Vibrio cholerae.

Tryptophanase (Trpase) is a pyridoxal phosphate (PLP)-dependent enzyme responsible for the production of indole, an important intra- and interspecies signaling molecule in bacteria. In this study, the tnaA gene of Vibrio cholerae coding for VcTrpase was cloned into the pET-20b(+) vector and expressed in Escherichia coli BL21(DE3) tn5:tnaA. Using Ni(2+)-nitrilotriacetic acid (NTA) chromatography, VcTrpase was purified, and it possessed a molecular mass of ∼49 kDa with specific absorption peaks at 330 and 435 nm and a specific activity of 3 U/mg protein. The VcTrpase had an 80 % homology to the Trpase of Haemophilus influenzae and E. coli, but only around 50 % identity to the Trpase of Proteus vulgaris and Porphyromonas gingivalis. The optimum conditions for the enzyme were at pH 9.0 and 45 °C. Recombinant VcTrpase exhibited analogous kinetic reactivity to the EcTrpase with K m and k cat values of 0.612 × 10(-3) M and 5.252 s(-1), respectively. The enzyme catalyzed S-methyl-L-cysteine and S-benzyl-L-cysteine degradation, but not L-phenylalanine and L-serine. Using a site-directed mutagenesis technique, eight residues (Thr52, Tyr74, Arg103, Asp137, Arg230, Lys269, Lys270, and His463) were conserved for maintaining enzyme catalysis. All amino acid substitutions at these sites either eliminated or remarkably diminished Trpase activity. These sites are thus potential targets for the design of drugs to control the V. cholerae Trpase and to further investigate its functions.

Occurrence of potentially pathogenic vibrios and related environmental factors in Songkhla Lake, Thailand.

Vibrios are halophilic bacteria that are ubiquitous in marine environments. Their occurrence in tropical lakes has rarely been investigated. In this study, the predominance and diversity of Vibrio spp. was investigated over a 12-month period in a coastal lagoon, Songkhla Lake, in southern Thailand. Water samples were collected at 2 stations in the estuary near Yor Island in Songkhla Lake. The predominant vibrios were detected by a culture-based method, using thiosulfate-citrate-bile salt-sucrose agar and CHROMagar Vibrio. The diversity of Vibrio spp. was evaluated using denaturant density gradient electrophoresis (DGGE). The highest numbers of total vibrios and Vibrio parahaemolyticus in both areas were observed during the summer. There was no significant correlation between the numbers of vibrios, including V. parahaemolyticus, and either the water temperature or plankton density. Variations in Vibrio species were observed with changes in salinity. Vibrio parahaemolyticus and V. cholerae non-O1/non-O139 were detected during the rainy season when the salinity dropped to nearly 0 parts per thousand. In both areas, V. alginolyticus was the most prominent species detected by the culture method, whereas Vibrio parahaemolyticus was detected by DGGE, every month. Other Vibrio spp. of potential public health concern were also detected by the culture method; they included V. vulnificus , V. fluvialis , and V. mimicus .

Biochemical and cellular investigation of Vitreoscilla hemoglobin (VHb) variants possessing efficient peroxidase activity.

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in distal pocket of peroxidases have successfully been introduced into that of the VHb. Fifteen-fold increase in catalytic constant (k(cat)) was obtained in P54R variant, which was presumably attributable to the lower rigidity and higher hydrophilicity of distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either H(2)O(2) or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring T29H mutation apparently demonstrated the spectral shift in both ferric and ferrous forms (406-408 to 411 nm and 432 to 424-425 nm, respectively). All VHb proteins in ferrous state had lambda(soret) peak at approximately 419 nm following the carbon monoxide (CO) binding. Expression of P54R mutant mediated the down-regulation of iron superoxide dismutase (FeSOD) as identified by 2-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived H2O2, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of Fe(2+)-ferric uptake regulator protein (Fe(2+)-Fur), an inducer of FeSOD expression.

Metal ion accessibility of histidine-modified superfolder green fluorescent protein expressed in Escherichia coli.

Green fluorescent protein (GFP) is frequently utilized for metal ion detection and quantification. To improve the metal binding potential of GFP, three residues (N146, F165, and L201) were substituted to histidines. Each variant responded differently upon interaction with metal ions. More than 80% of N146H, having the most accessible surface area, could bind to immobilized metal ions. However, only F165H exhibited significant differences in quenching by soluble metal ions (22% fluorescence decrease) in comparison with the template protein (12%). These findings can be utilized for designing GFP variants for metal binding and sensor applications.

Rational design of analyte channels of the green fluorescent protein for biosensor applications.

A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His(6)GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His(6)GFPuv/H148G and His(6)GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 A, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn(2+) and Cu(2+) than those of the template protein (His(6)GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (K(d)) to metal ions were in the order of 4.88 x 10(-6) M, 16.67 x 10(-6) M, 25 x 10(-6) M, and 33.33 x 10(-6) M for His(6)GFPuv/F165G, His(6)GFPuv, His(6)GFPuv/H148G/F165G and His(6)GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.

Prediction of GFP spectral properties using artificial neural network.

The prediction of the excitation and the emission maxima of green fluorescent protein (GFP) chromophores were investigated by a quantitative structure-property relationship study. A data set of 19 GFP color variants and an additional data set consisting of 29 synthetic GFP chromophores were collected from the literature. Artificial neural network implementing the back-propagation algorithm was employed. The proposed computational approach reliably predicted the excitation and the emission maxima of GFP chromophores with correlation coefficient exceeding 0.9. The usefulness of quantum chemical descriptors was revealed by a comparative study with other molecular descriptors. Assignment of appropriate protonation state of the chromophore for the GFP color variants data set was shown to be necessary for good predictive performance. Results suggest that the confinement of the GFP chromophore has no significant influence on the predictive performance of the data set used. A comparative investigation with the traditional modeling methods, particularly multiple linear regression and partial least squares, reveals that artificial neural network is the most suitable modeling approach for the GFP spectral properties. It is anticipated that this methodology has great potential in accelerating the design and engineering of novel GFP color variants of scientific or industrial interest.

Chimeric antibody-binding Vitreoscilla hemoglobin (VHb) mediates redox-catalysis reaction: new insight into the functional role of VHb.

Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric Vitreoscilla hemoglobin (VHb). Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain) were successfully constructed and expressed in E. coli strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 10(7) and 49.1 x 10(7) per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (approximately 7.0) in the presence of high concentration (20-40 mM) of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of Vitreoscilla hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications.