The crystal structure of Rv0793, a hypothetical monooxygenase from <Emphasis Type="BoldItalic">M.␣tuberculosis</Emphasis>

Mycobacterium tuberculosis infects millions worldwide. The Structural Genomics Consortium for M. tuberculosis has targeted all genes from this bacterium in hopes of discovering and developing new therapeutic agents. Open reading frame Rv0793 from M. tuberculosis was annotated with an unknown function. The 3-dimensional structure of Rv0793 has been solved to 1.6 A resolution. Its structure is very similar to that of Streptomyces coelicolor ActVA-Orf6, a monooxygenase that participates in tailoring of polyketide antibiotics in the absence of a cofactor. It is also similar to the recently solved structure of YgiN, a quinol monooxygenase from Escherichia coli. In addition, the structure of Rv0793 is similar to several structures of other proteins with unknown function. These latter structures have been determined recently as a result of structural genomic projects for various bacterial species. In M. tuberculosis, Rv0793 and its homologs may represent a class of monooygenases acting as reactive oxygen species scavengers that are essential for evading host defenses. Since the most prevalent mode of attack by the host defense on M. tuberculosis is by reactive oxygen species and reactive nitrogen species, Rv0793 may provide a novel target to combat infection by M. tuberculosis.     

Plasma visfatin,associated with a genetic polymorphism −1535C > T,is correlated with C-reactive protein in Chinese Han patients with traumatic brain injury

Visfatin is a newly identified pro-inflammatory adipokine and a genetic polymorphism −1535 C > T located in the visfatin gene promoter has been suggested to be associated with the regulation of visfatin expression in some inflammatory illness. However, there were some conflicting results regarding whether this variant is functional or not. This study aimed to examine the relations of the −1535 C > T single nucleotide polymorphism (SNP) of visfatin gene to the plasma visfatin and C-reactive protein concentrations in traumatic brain injury (TBI). 318 Chinese Han patients with TBI were recruited in this study. Plasma visfatin and C-reactive protein levels were significantly different between the genotypes in the SNP-1535 C > T even after adjustment for age, sex and body mass index. The genotype C–C had the highest plasma visfatin and C-reactive protein concentrations. The plasma visfatin and C-reactive protein concentrations between the variant genotypes C–T and T–T did not differ significantly. Plasma visfatin level was significantly associated with plasma C-reactive protein level using multivariate linear regression. Thus, the SNP-1535 C > T of visfatin gene seemed to be potentially involved in the inflammatory component of TBI through a decreased production of visfatin.     

Mycobacterium tuberculosis Rv1090 and Rv1987 encode functional β-glucan-targeting proteins

Mycobacterium tuberculosis is a facultative intracellular pathogen, and the ability of this bacterium to survive and to grow inside macrophages is central to its virulence. Multiple strategies are employed by M. tuberculosis to ensure survival in macrophages, including secretion of several proteins, which are good candidates to be virulence factors, drug targets for disease intervention, and vaccine antigens. However, some M. tuberculosis secreted proteins do not appear to play any role in the growth or survival of the bacterium in its mammalian host. Among these proteins are three putative cellulose-targeting proteins encoded by the genes Rv0062, Rv1090, and Rv1987. It has been previously shown that Rv0062 encodes an active cellulase. Here we report that Rv1090 and Rv1987 also encode functional proteins. Rv1090 is able to hydrolyze barley β-glucan while Rv1987 displays cellulose-binding activity on filter paper and on microcrystalline cellulose (Avicel). Collectively, these observations point toward a unique unknown relationship between M. tuberculosis and a cellulose-containing host. We hypothesize that amoeba could be such hosts.     

结核分枝杆菌H37Rv新基因Rv2742克隆表达及纯化

Rv2742是本课题组前期基于蛋白质基因组学策略从结核分枝杆菌Mycobacteriumtuberculosis H37Rv中发现、鉴定的遗漏注释基因。文中旨在建立结核分枝杆菌H37Rv漏注释蛋白Rv2742的可溶性诱导表达、纯化体系,为进一步探索Rv2742基因参与的生物学功能奠定基础。前期实验发现构建的pGEX-4T-2-Rv2742、pET-28a-Rv2742、pET-32a-Rv2742及pMAL-c2X-Rv2742原核表达载体均无法实现目的蛋白的诱导表达。但经密码子优化后,仅有pMAL-c2X-Rv2742载体能够实现目的蛋白的可溶性诱导表达。此外,通过比较不同宿主菌、温度及IPTG浓度对目的蛋白表达量的影响,发现目的蛋白在Rosetta (DE3)中,16℃及0.5mmol/LIPTG诱导条件下表达量最高。直链淀粉树脂(Amyloseresin)亲和层析柱纯化获得较纯的产物,经LC-MS/MS验证确认是Rv2742融合蛋白肽段序列。成功获得结核分枝杆菌H37Rv新基因Rv2742的重组蛋白,可进一步开展其潜在相互作用及免疫原性研究工作。     

Characterization of a bifunctional β-lactamase/ribonuclease and its interaction with a chaperone-like protein in the pathogen <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> H37Rv

Most mycobacteria appear to be naturally resistant to β-lactam antibiotics such as penicillin. However, very few β-lactamases and their regulation have been clearly characterized in Mycobacterium tuberculosis H37Rv. In this study, a unique bifunctional protein, Rv2752c, from M. tuberculosis showed both β-lactamase and RNase activities. Two residues, D184 and H397, appear to be involved in Zn²⁺-binding and are essential for the dual functions. Both activities are lost upon deletion of the C-terminal 100 a.a. long Rv2752c tail, which contains an additional loop when compared with the RNase J of Bacillus subtilis. A chaperone-like protein, Rv2373c, physically interacted with Rv2752c and inhibited both activities. This is the first report of characterization of a bifunctional β-lactamase and its regulation in mycobacteria. These data offered important clues for further investigation of the structure and function of microbial β-lactamases. Increased understanding of this protein will provide further insights into the mechanism of microbial drug resistance.     

TLR4 and TLR21 expression,MIF, IFN-β, MD-2, CD14 activation,and sIgA production in chickens administered with EFAL41 strain challenged with <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

The protective effect of Enterococcus faecium EFAL41 on chicken’s caecum in relation to the TLR (TLR4 and TLR21) activation and production of luminal IgA challenged with Campylobacter jejuni CCM6191 was assessed. The activation of MIF, IFN-β, MD-2 and CD14 was followed-up after bacterial infection. Day-old chicks (40) were divided into four groups (n = 10): control (C), E. faecium AL41 (EFAL41), C. jejuni (CJ) and combined E. faecium AL41+C. jejuni (EFAL41+CJ). Relative mRNA expression of TLR4, TLR21 and CD14 was upregulated in the probiotic strain and infected (combined) group on day 4 and 7 post infection (p.i.). The caecal relative MD-2 mRNA expression was upregulated on day 4 p.i. in the EFAL41+CJ and CJ groups. MIF and IFN-β reached the highest levels in the combined groups on day 7 p.i. The concentration of the sIgA in intestinal flush was upregulated in EFAL41+CJ group on day 4 p.i. The results demonstrated that E. faecium EFAL41 probiotic strain can modulate the TLRs expression and modify the activation of MIF, IFN-β, MD-2 and CD14 molecules in the chickens caecum challenged with C. jejuni CCM 6191. The counts of EFAL41 were sufficient and high, similarly the counts of enterococci in both, caecum and faeces but without reduction of Campylobacter counts.     

Leaf δ<Superscript>13</Superscript>C variability with elevation,slope aspect,and precipitation in the southwest United States

Leaves from several desert and woodland species, including gymnosperms and angiosperms with both C₃ and C₄ physiology, were analyzed to detect trends in '¹³C_leaf with elevation and slope aspect along two transects in southeastern Utah and south-central New Mexico, USA. The main difference between the two transects is the steeper elevational gradient for mean annual and summer precipitation in the southern transect. For any given species, we found that isotopic differences between individual plants growing at the same site commonly equal differences measured for plants along the entire altitudinal gradient. In C₃ plants, '¹³C_leaf values become slightly enriched at the lowest elevations, the opposite of trends identified in more humid regions. Apparently, increasing water-use efficiency with drought stress offsets the influence of other biotic and abiotic factors that operate to decrease isotopic discrimination with elevation. For some species shared by the two transects (e.g., Pinus edulis and Cercocarpus montanus), '¹³C_leaf values are dramatically depleted at sites that receive more than 550 mm mean annual precipitation, roughly the boundary (pedalfer-pedocal) at which soils commonly fill to field capacity in summer and carbonates are leached. We hypothesize that, in summer-wet areas, this may represent the boundary at which drought stress overtakes other factors in determining the sign of '¹³C_leaf with elevation. The opposition of isotopic trends with elevation in arid versus humid regions cautions against standard correction for elevation in comparative studies of '¹³C_leaf.     

Histopathology findings in common marmosets (<Emphasis Type="Italic">Callithrix jacchus</Emphasis> Linnaeus, 1758) with chronic weight loss associated with bile tract obstruction by infestation with <Emphasis Type="Italic">Platynosomum</Emphasis> (Loos, 1907)

Chronic weight loss in marmosets is often associated with wasting marmoset syndrome (WMS), an important disease that occurs in callitrichid colonies around the world. Even though its etiology is very difficult to determine, particular variables, such as weight loss, diarrhea and alopecia, associated or not with infestation in the pancreatic ducts with Trichospirura leptossoma (Nematoda: Thelazioidea), seem to be linked with the syndrome. This study investigated the histopathology of the lungs, duodenum, liver, gallbladder, extrahepatic bile ducts and pancreatic ducts of six common marmosets (Callithrix jacchus) suffering from severe non-diarrheic weight loss. Three individuals died naturally and the other three were euthanized. Microscopic findings showed the presence of adult flukes (Platynosomum) in the liver. These flukes, which provoke common infection in cats, were also observed inside the gallbladder as well as in the intra and extrahepatic bile ducts in common marmosets. Portal fibrosis was observed in two animals, which developed chronic fibrosing hepatopathy (biliary pattern, grade 3). The disease progresses without diarrhea and without pancreatic lesions or infestation. With the progression, the animals presented with ascending cholangitis, cholestasis and portal fibrosis, sometimes culminating in secondary biliary cirrhosis. Therefore, this infirmity, associated with chronic weight loss in common marmosets, could be another etiological factor linked with WMS.     

α,α′-trehalose 6,6′-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120-2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir-Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.     

Photosynthesis,water use efficiency,and δ<Superscript>13</Superscript>C in two rice genotypes with contrasting response to water deficit

The effects of water deficit and re-irrigation were studied in glasshouse-grown rice plants (cvs. Cimarrón and Fonaiap 2000) which differ in their susceptibility to water deficit. Relative water content decreased from >90 to 67–69 % and recovered to pre-stress values within 24 h after re-irrigation. The irradiance-saturated rate of photosynthesis (P _sat), transpiration rate (E), and stomatal conductance (g _s) decreased with water deficit. E and g _s decreased similarly in both cultivars, but P _sat was more strongly inhibited in Cimarrón than in Fonaiap 2000. Water deficit increased water use efficiency (WUE_T) over 2-fold in Fonaiap 2000 and by 1.5-fold in Cimarrón. The ratio of intercellular to ambient CO₂ concentration (C _i/C _a) decreased in Fonaiap 2000 during mild stress but increased at severe stress. Contrarily, Cimarrón did not change C _i/C _a with water deficit. After re-irrigation Fonaiap 2000 recovered P _sat to ca. 80 % of control values 24 h after re-irrigation, whereas Cimarrón recovered to 60 % of control values 48 h after re-irrigation. E and g _s recovered to a lesser extent (50 %) than P _sat, after 48 h of re-irrigation in both cultivars. Total aboveground and green (live) biomass were unaffected by water deficit in Fonaiap 2000 but were reduced by 21 and 40 % in Cimarrón, respectively. Dead biomass increased in stressed plants of both cultivars but to a larger extent in Cimarrón than in Fonaiap 2000. Water deficit increased δ¹³C in Fonaiap 2000, whereas Cimarrón was unaffected by water deficit showing lower values than those of Fonaiap 2000. δ¹³C was highly and linearly correlated to the ratio C _i/C _a. WUE_T was also significantly correlated to δ¹³C.     

The genus Brulleia Szépligeti (Hymenoptera,Braconidae, Helconinae) from China,with descriptions?of four new species

The species of the genus Brulleia Szépligeti, 1904 (Hymenoptera, Braconidae, Helconinae) from China are revised. Four new species, namely Brulleia fanjingensis YanandChen, sp. n., Brulleia longipalpis YanandChen, sp. n., Brulleia noncarinata YanandChen, sp. n. andBrulleia punctata Yan andChen, sp. n. are described and illustrated. A key to the Chinese species of the genus Brulleia is included.     

Theoretical study on the substitution reactions of the germylenoid H<Subscript>2</Subscript>GeLiF with SiH<Subscript>3</Subscript>X (X = F,Cl, Br)

The substitution reactions of H₂GeLiF (G) with SiH₃X (X = F, Cl, Br) were investigated using calculations performed at the QCISD/6-311++G (d, p)//B3LYP/6-311+G (d, p) level of theory. The results led to the following conclusions. (i) The substitutions are nucleophilic reactions. There are two substitution paths, I and II, which both lead to the germane H₂GeFSiH₃. The enantiomers of this germane are obtained via these two paths if an H in SiH₃X is replaced with a different group or atom. (ii) Both substitution pathways show the same order of barrier heights (SiH₃F > SiH₃Cl > SiH₃Br). The difference between the bond energies of Li–X and Si–X may explain the precedence among the substitution reactions of G with SiH₃X. Path I has a lower activation barrier than path II, indicating that path I is more favorable. (iii) Comparison between the relevant insertion and substitution reactions shows that substitutions are more favorable and that the substitution product H₂GeFSiH₃ predominates over the insertion product. (iv) The substitution reactions of H₂GeLiF with SiH₃X are exothermic.     

The interaction of CCl<Subscript>4</Subscript> with Ng (Ng = He,Ne, Ar), O<Subscript>2</Subscript>, D<Subscript>2</Subscript>O and ND<Subscript>3</Subscript>: rovibrational energies,spectroscopic constants and theoretical calculations

This investigation generated rovibrational energies and spectroscopic constants for systems of CCl₄ with Ng (Ng?=?He, Ne, Ar), O₂, D₂O and ND₃ from scattering experimental data, and the results presented are of interest for microwave spectroscopy studies of small halogenated molecules. The rovibrational spectra were obtained through two different approaches (Dunham and DVR) within the improved Lennard Jones (ILJ) model. Spectra were also generated within ordinary Lennard Jones and deviations suggest that the ILJ model should be preferred due to interactions beyond dispersion forces presented in these systems. Data from the literature and additional high level quantum mechanical calculations presented in this work show that these systems should not be considered as van der Waals complexes due to halogen bonding (HB) interactions, and this is especially true for the CCl₄–D₂O and CCl₄–ND₃ complexes. The charge displacement from the latter systems are one order of magnitude higher than the values from literature for CCl₄ and He, Ne, Ar and O₂ systems, and show significant deviations between DFT and Hartree-Fock values not previously reported in the literature.     

Theoretical descriptions of novel triplet germylenes M<Subscript>1</Subscript>-Ge-M<Subscript>2</Subscript>-M<Subscript>3</Subscript> (M<Subscript>1</Subscript> = H,Li, Na,K; M<Subscript>2</Subscript> = Be,Mg, Ca; M<Subscript>3</Subscript> = H,F, Cl,Br)

In a quest to identify new ground-state triplet germylenes, the stabilities (singlet–triplet energy differences, ΔE_S–T) of 96 singlet (s) and triplet (t) M₁-Ge-M₂-M₃ species were compared and contrasted at the B3LYP/6–311++G**, QCISD(T)/6–311++G**, and CCSD(T)/6–311++G** levels of theory (M₁?=?H, Li, Na, K; M₂?=?Be, Mg, Ca; M₃?=?H, F, Cl, Br). Interestingly, F-substituent triplet germylenes (M₃?=?F) appear to be more stable and linear than the corresponding Cl- or Br-substituent triplet germylenes (M₃?=?Cl or Br). Triplets with M₁?=?K (i.e., the K-Ge-M₂-M₃ series) seem to be more stable than the corresponding triplets with M₁?=?H, Li, or Na. This can be attributed to the higher electropositivity of potassium. Triplet species with M₃?=?Cl behave similarly to those with M₃?=?Br. Conversely, triplets with M₃?=?H show similar stabilities and linearities to those with M₃?=?F. Singlet species of formulae K-Ge-Ca-Cl and K-Ge-Ca-Br form unexpected cyclic structures. Finally, the triplet germylenes M₁-Ge-M₂-M₃ become more stable as the electropositivities of the α-substituents (M₁ and M₂) and the electronegativity of the β-substituent (M₃) increase.     

Do electromagnetic fields interact with electrons in the Na,K‐ATPase?

The effects of low frequency electric and magnetic fields on several biochemical systems, including the Na,K-ATPase, indicate that electromagnetic (EM) fields interact with electrons. The frequency optima for two enzymes in response to EM fields are very close to their turnover numbers, suggesting that these interactions directly affect reaction rates. Nevertheless, generally accepted ideas about Na,K-ATPase function and ion transport mechanisms do not consider interactions with electrons. To resolve the clash of paradigms, we hypothesize interaction with transient electrons and protons that arise from flickering of H-bonds in the hydrated protein. These transient charges in the enzyme could provide a trigger for the sequence of conformation changes that are part of the ion transport mechanism. If the distributions of transient electrons and protons in the membrane are affected by their concentration and the membrane potential, as expected from electric double layer theory, this can account for the different effects of low frequency electric and magnetic fields, as well as for the observation that membrane hyperpolarization reverses the ATPase reaction to generate ATP.     

Extremozymes — biocatalysts with unique properties from extremophilic microorganisms

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‘Stick with your own kind,or hang with the locals?’ Implications of shoaling strategy for tropical reef fish on a range‐expansion frontline

Range shifts of tropical marine species to temperate latitudes are predicted to increase as a consequence of climate change. To date, the research focus on climate‐mediated range shifts has been predominately dealt with the physiological capacity of tropical species to cope with the thermal challenges imposed by temperate latitudes. Behavioural traits of individuals in the novel temperate environment have not previously been investigated, however, they are also likely to play a key role in determining the establishment success of individual species at the range‐expansion forefront. The aim of this study was to investigate the effect of shoaling strategy on the performance of juvenile tropical reef fishes that recruit annually to temperate waters off the south east coast of Australia. Specifically, we compared body‐size distributions and the seasonal decline in abundance through time of juvenile tropical fishes that shoaled with native temperate species (‘mixed’ shoals) to those that shoaled only with conspecifics (as would be the case in their tropical range). We found that shoaling with temperate native species benefitted juvenile tropical reef fishes, with individuals in ‘mixed’ shoals attaining larger body‐sizes over the season than those in ‘tropical‐only’ shoals. This benefit in terms of population body‐size distributions was accompanied by greater social cohesion of ‘mixed’ shoals across the season. Our results highlight the impact that sociality and behavioural plasticity are likely to play in determining the impact on native fish communities of climate‐induced range expansion of coral reef fishes.