RsmA3 modulates RpoS through the RetS-Gac-Rsm signalling pathway in response to H2 O2 stress in the phytopathogen Pseudomonas syringae.

Reactive oxygen species are a fatal challenge to the plant pathogenic bacterium Pseudomonas syringae. In this study, we reveal that the global regulatory protein RsmA3 from the RetS-Gac/Rsm signalling pathway modulates RpoS in the early-log growth phase in the P. syringae wild-type strain MB03, thereby regulating oxidative tolerance to H2 O2 and ultimately affecting pathogenicity to the host plant. Following increased H2 O2 by external addition or endogenous induction by menadione, the resistance of the mutant strain ΔretS to H2 O2 is significantly enhanced due to rapid increases in the transcription of Rsm-related non-coding small RNAs (nc sRNAs), a sigma factor RpoS, and H2 O2 -detoxifying enzymes. Moreover, the ΔretS mutant is significantly less pathogenic in cucumber leaves. Seven Rsm-related nc sRNAs (namely, rsmZ, rsmY and rsmX1-5 ) show functional redundancy in the RetS-Gac-Rsm signalling pathway. External addition of H2 O2 stimulates increases in the transcription of both rsmY and rsmZ. Thus, we propose a regulatory model of the RetS-Gac-Rsm signalling pathway in P. syringae MB03 for the regulation of H2 O2 tolerance and phytopathogenicity in the host plant.

Carbon Metabolism of a Soilborne Mn(II)-Oxidizing Escherichia coli Isolate Implicated as a Pronounced Modulator of Bacterial Mn Oxidation.

Mn(II)-oxidizing microorganisms are generally considered the primary driving forces in the biological formation of Mn oxides. However, the mechanistic elucidation of the actuation and regulation of Mn oxidation in soilborne bacteria remains elusive. Here, we performed joint multiple gene-knockout analyses and comparative morphological and physiological determinations to characterize the influence of carbon metabolism on the Mn oxide deposit amount (MnODA) and the Mn oxide formation of a soilborne bacterium, Escherichia coli MB266. Different carbon source substances exhibited significantly varied effects on the MnODA of MB266. A total of 16 carbon metabolism-related genes with significant variant expression levels under Mn supplementation conditions were knocked out in the MB266 genome accordingly, but only little effect on the MnODA of each mutant strain was accounted for. However, a simultaneous four-gene-knockout mutant (namely, MB801) showed an overall remarkable MnODA reduction and an initially delayed Mn oxide formation compared with the wild-type MB266. The assays using scanning/transmission electron microscopy verified that MB801 exhibited not only a delayed Mn-oxide aggregate processing, but also relatively smaller microspherical agglomerations, and presented flocculent deposit Mn oxides compared with normal fibrous and crystalline Mn oxides formed by MB266. Moreover, the Mn oxide aggregate formation was highly related to the intracellular ROS level. Thus, this study demonstrates that carbon metabolism acts as a pronounced modulator of MnODA in MB266, which will provide new insights into the occurrence of Mn oxidation and Mn oxide formation by soilborne bacteria in habitats where Mn(II) naturally occurs.

Crystal Structure of α-Galactosidase from Thermus thermophilus: Insight into Hexamer Assembly and Substrate Specificity.

α-Galactosidase catalyzes the hydrolysis of a terminal α-galactose residue in galacto-oligosaccharides and has potential in various industrial applications and food processing. We determined the crystal structures of α-galactosidase from the thermophilic microorganism Thermus thermophilus (TtGalA) and its complexes with pNPGal and stachyose. The monomer folds into an N-terminal domain, a catalytic (ß/α)8 barrel domain, and a C-terminal domain. The domain organization is similar to that of the other family of 36 α-galactosidases, but TtGalA presents a cagelike hexamer. Structural analysis shows that oligomerization may be a key factor for the thermal adaption of TtGalA. The structure of TtGalA complexed with stachyose reveals only the existence of one -1 subsite and one +1 subsite in the active site. Structural comparison of the stachyose-bound complexes of TtGalA and GsAgaA, a tetrameric enzyme with four subsites, suggests evolutionary divergence of substrate specificity within the GH36 family of α-galactosidases. To the best of our knowledge, the crystal structure of TtGalA is the first report of a quaternary structure as a hexameric assembly in the α-galactosidase family.

Glossogyne tenuifolia (Hsiang-ju) extract suppresses T cell activation by inhibiting activation of c-Jun N-terminal kinase.

BACKGROUND: Glossogyne tenuifolia (GT) (Hsiang-ju) is a Chinese herbal medicine previously exhibited an anti-inflammatory activity. This study aimed to investigate the effect of GT ethanol extract (GTE) on T cell-mediated adaptive immunity. METHODS: Human peripheral blood mononuclear cells (PBMCs) and Jurkat T cells were activated by phytohemagglutinin in the presence of various doses (3.13-50 µg/mL) of GTE. The effect of GTE on T cell activation was examined by a proliferation assay of activated PBMCs and the level of the activation marker CD69 on the surface of activated Jurkat T cells. Apoptosis was determined by propidium iodide staining in hypotonic solution. Signaling pathway molecules were assessed by western blotting. RESULTS: Glossogyne tenuifolia ethanol extract was demonstrated to inhibit T cell activation, not only in the proliferation of human PBMCs at the concentrations of 12.5, 25 and 50 µg/mL (P = 0.0118, 0.0030 and 0.0021) but also in the CD69 expression in Jurkat cells, which was not due to the cytotoxicity of GTE. The presence of GTE did not change the activity of nuclear factor kappa-light-chain-enhancer of activated B cells or extracellular signal-regulated kinase upon T cell activation. In addition, GTE significantly reduced activation of c-Jun N-terminal kinase (JNK) (P = 0.0167) and p38 (P = 0.0278). Furthermore, decreased JNK activation mediated the preventive effect of GTE on T cell activation-induced cell death (AICD). CONCLUSION: Glossogyne tenuifolia ethanol extract inhibited T cell activation of Jurkat cells and freshly prepared human PBMCs due to suppression of JNK activity. Furthermore, GTE inhibited AICD by blocking prolonged JNK phosphorylation in activated T cells. Taken together, the anti-inflammatory effects exerted by GTE were mediated via suppression of JNK phosphorylation in T cell activation.

Phytochemicals from Tradescantia albiflora Kunth Extracts Reduce Serum Uric Acid Levels in Oxonate-induced Rats.

BACKGROUND: Tradescantia albiflora (TA) Kunth (Commelinaceae) has been used for treating gout and hyperuricemia as folklore remedies in Taiwan. Therefore, it is worthwhile to study the effect of TA extracts on lowering uric acid activity. The hypouricemic effects of TA extracts on potassium oxonate (PO)-induced acute hyperuricemia were investigated for the first time. MATERIALS AND METHODS: All treatments at the same volume (1 ml) were orally administered to the abdominal cavity of PO-induced hyperuricemic rats. One milliliter of TA extract in n-hexane (HE), ethyl acetate (EA), n-butanol (BuOH), and water fractions has 0.28, 0.21, 0.28, and 1.03 mg TA, respectively; and the plasma uric acid (PUA) level was measured for a consecutive 4 h after administration. RESULTS: All four fractions' extracts derived from TA were observed to significantly reduce PUA compared with the PO group. The EA-soluble fraction (TA-EA) exhibited the best xanthine oxidase (XO) inhibitory activity. Following column chromatography, 12 phytochemicals were isolated and identified from the EA fraction. The IC50 values of isolated phytochemicals indicated that bracteanolide A (AR11) showed the remarkable XO inhibitory effect (IC50 value of 76.4 µg/ml). These findings showed that the in vivo hypouricemic effect in hyperuricemic rats was consistent with in vitro XO inhibitory activity, indicating that TA extracts and derived phytochemicals could be potential candidates as hypouricemic agents. SUMMARY: Tradescantia albiflora extracts possess in vivo hypouricemic action in hyperuricemic ratsT. albiflora extracts exhibited strong inhibitory activity against xanthine oxidase (XO)Butenolide may play an important role in XO inhibitionThe extract bracteanolide A was demonstrated potent XO inhibitory activity in vitro. Abbreviations used: TA: Tradescantia albiflora, PO: potassium oxonate, HE: n-hexane, EA: ethyl acetate, BuOH: n-butanol, PUA: plasma uric acid, XO: xanthine oxidase, MeOH: methanol, IP: intraperitoneal.

Physicochemical and functional property changes in soy protein isolates stored under high relative humidity and temperature.

The effects of water activity (aw) and temperature during storage on the physicochemical characteristics and functional properties of soy protein isolate (SPI) were investigated. SPI was stored with two different temperatures (25, 45 °C) and two levels of water activity (0.25, 0.75) for 224 days. During the 224-day storage period, all the samples showed decreases in gel hardness, emulsifying stability, foaming properties, viscosity, solubility, and color alteration, but increased in surface hydrophobicity (RSo). These alterations were stronger when stored at 45 °C than at 25 °C and in 0.75 aw than 0.25 aw, and most pronounced at 45 °C and 0.75 aw. Our results revealed that storage conditions - temperature and water activity - will indeed affect the functional properties of soy protein isolates.

Effects of Antrodia camphorata extracts on anti-oxidation, anti-mutagenesis and protection of DNA against hydroxyl radical damage.

BACKGROUND: Antrodia camphorata is a geographically special fungus and is one of the precious traditional medicines of Taiwan. A lot of reports have addressed its antioxidant activities and anticancer activities. In order to understand whether these protection effects were resulted from its ability of preventing DNA against hydroxyl radical damage, the A. camphorata extract was used to examine its antioxidant, antimutagenic and DNA-protective activities. METHODS: A. camphorata extract was prepared by extracting the lyophilized powder of A. camphorata mycelium with distilled water. The antioxidative activity of this A. camphorata extract was then evaluated by 2,2-diphenyl-1-picrylhydrozyl (DPPH) radical-scavenging assay, and the antimutagenic activities of the extract against direct mutagen 4-nitroquinoline N-oxide (4NQNO) and indirect mutagen benzo[a]pyrene (B[a]P) were evaluated by Ames test. The effects of the A. camphorata extract in terms of DNA protection against hydroxyl radical damage were also investigated. RESULTS: It was found that the higher the concentration of A. camphorata extracts, the higher the DPPH radical-scavenging effect. A. camphorata extract at concentrations between 0.625 and 10 mg/ml was found to be neither toxic nor mutagenic. However, the higher A. camphorata concentration (10 mg/ml) used in the test showed higher inhibitory effects on 4NQNO in a dose-dependent manner. The A. camphorata extract also showed reducing and scavenging activities against superoxide anion radical and also exhibited protective effects on DNA against hydroxyl radical-induced damage. CONCLUSIONS: Results suggested that A. camphorata is a non-toxic and novel material with antioxidant, antimutagenic and DNA-protective activities and could be developed into health foods.

Danggui buxue tang inhibits 2,4-dinitrochlorobenzene: induced atopic dermatitis in mice.

Danggui Buxue Tang (DBT) is a herbal decoction that has been used in Chinese medicine to enhance qi and blood circulation. Previously, we found that DBT can suppress allergy-related asthma in mice, leading us to hypothesize that DBT might ameliorate allergy disease. In this study, we evaluated whether DBT can attenuate atopic dermatitis (AD) symptoms and have an anti-inflammatory effect on AD-like mice. The dorsal skin of female mice was shaved and sensitized cutaneously (skin smear) with 1-chloro-2,4-dinitrobenzene. Mice were then given various doses of DBT from days 14 to 29 cutaneously. DBT treatment suppressed ear swelling and skin inflammation and decreased mast cell and eosinophil infiltration into skin and ear tissue. DBT also inhibited levels of IgE and Th2-associated cytokine levels in serum. These results demonstrate that cutaneous administration of DBT reduced the development of AD-like skin lesions in mice.

Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus.

Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni(2+)-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80 Šon the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.3, b = 86.1, c = 118.2 Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.52 Å(3) Da(-1) and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement.

Crystal structures and molecular dynamics simulations of thermophilic malate dehydrogenase reveal critical loop motion for co-substrate binding.

Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60 °C and specific activity of about 2,600 U/mg with optimal temperature of 90 °C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme's activity.

Structural characterization of sialic acid synthase by electrospray mass spectrometry--a tetrameric enzyme composed of dimeric dimers.

Sialic acid synthase (NeuB) encoded by the neuB gene catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form N-acetylneuraminic acid. The enzyme is essential for the biosynthesis of polysialic acid, a capsular sugar polymer functioning as a virulent factor and antiphagocytic barrier. This report demonstrates the first characterization on the quaternary structure of NeuB from Escherichia coli (EcNeuB) and Streptococcus agalactiae (SaNeuB) by nanoflow electrospray ionization mass spectrometry (ESI-MS). Under non-denaturing conditions, Tris buffer was observed to induce a higher ratio of tetramer/dimer of NeuB in the ESI mass spectra, providing supportive evidence for the existence of a "structurally-specific" tetramer. The instrument parameters were found to significantly affect the ratio of detected tetramer/dimer in ESI mass spectra. The harshest conditions, including high desolvation voltages and pressure in the collision cell, led to enhanced detection of the 160 kDa tetramer. The prevalence of dimeric form is likely the cause in loss of tetramer stability in gas-phase arising from insufficient collisional cooling, which implies an asymmetric assembly, possibly composed of dimeric dimers. Most interestingly, the hypothesis was further supported by chemical cross-linking of SaNeuB, in which the reaction of shorter linker yielded mainly the dimer whereas that of longer linker produced both dimer and tetramer. Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis.

The structural interpretations of residue Ser297 in catalytic efficiency of Escherichia coli phenylalanine aminotransferase.

Escherichia coli phenylalanine aminotransferase (ecPheAT) catalyzes the biosynthesis of phenylalanine and tyrosine. The crystal structure of ecPheAT was determined in our previous study. The comparison of the 3-D structure of several aminotransferases revealed that the residue at position 297 plays an important role in enzyme function. Analysis of activities and kinetic parameters of wild type and mutant ecPheATs suggested that the residue Ser(297) was structurally selected for better catalytic efficiency. Computational modeling of ecPheAT mutants further suggested that Ser in position 297 could make ecPheAT easy with change of conformation from open form to closed form.

Structural characterization of Escherichia coli sialic acid synthase.

Sialic acid synthase encoded by the neuB gene of Escherichia coli catalyzes the condensation of N-acetylmannosamine and phosphoenolpyruvate to form N-acetylneuraminic acid. This report demonstrates the first structural information on sialic acid synthase by CD, MALDI-TOF, and chemical cross-linking studies. Also, a specific cleavage by endogenous protease(s) has been identified at Lys(280) of the enzyme (40 kDa) by LC-MS and N-terminal sequencing analyses. The cleavage results in the formation of two inactive fragments of 33 and 7 kDa. The structural analysis indicates that the fragmentation is associated with a significant change of the enzyme from a tetrameric to trimeric form, and alterations in both secondary and native quaternary structures.