Secretory expression of a β‐xylosidase gene from Thermomyces lanuginosus in Escherichia coli and characterization of its recombinant enzyme

Aims: To characterize a β‐xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β‐xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017‐bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β‐xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6‐fold to homogeneity with an estimated mass of 51·6 kDa by SDS‐PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a K_i value of 63 mmol l^?1. Conclusions: In this study, a GH family 43 β‐xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β‐xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.     

Different Roles for Contracture and Calpain in Calcium Paradox-Induced Heart Injury

The Ca²⁺ paradox represents a good model to study Ca²⁺ overload injury in ischemic heart diseases. We and others have demonstrated that contracture and calpain are involved in the Ca²⁺ paradox-induced injury. This study aimed to elucidate their roles in this model. The Ca²⁺ paradox was elicited by perfusing isolated rat hearts with Ca²⁺-free KH media for 3 min or 5 min followed by 30 min of Ca²⁺ repletion. The LVDP was measured to reflect contractile function, and the LVEDP was measured to indicate contracture. TTC staining and the quantification of LDH release were used to define cell death. Calpain activity and troponin I release were measured after Ca²⁺ repletion. Ca²⁺ repletion of the once 3-min Ca²⁺ depleted hearts resulted in almost no viable tissues and the disappearance of contractile function. Compared to the effects of the calpain inhibitor MDL28170, KB-R7943, an inhibitor of the Na⁺/Ca²⁺ exchanger, reduced the LVEDP level to a greater extent, which was well correlated with improved contractile function recovery and tissue survival. The depletion of Ca²⁺ for 5 min had the same effects on injury as the 3-min Ca²⁺ depletion, except that the LVEDP in the 5-min Ca²⁺ depletion group was lower than the level in the 3-min Ca²⁺ depletion group. KB-R7943 failed to reduce the level of LVEDP, with no improvement in the LVDP recovery in the hearts subjected to the 5-min Ca²⁺ depletion treatment; however, KB-R7943 preserved its protective effects in surviving tissue. Both KB-R7943 and MDL28170 attenuated the Ca²⁺ repletion-induced increase in calpain activity in 3 min or 5 min Ca²⁺ depleted hearts. However, only KB-R7943 reduced the release of troponin I from the Ca²⁺ paradoxic heart. These results provide evidence suggesting that contracture is the main cause for contractile dysfunction, while activation of calpain mediates cell death in the Ca²⁺ paradox.     

Reporting Quality of Systematic Reviews/Meta-Analyses of Acupuncture

Background

The QUOROM and PRISMA statements were published in 1999 and 2009, respectively, to improve the consistency of reporting systematic reviews (SRs)/meta-analyses (MAs) of clinical trials. However, not all SRs/MAs adhere completely to these important standards. In particular, it is not clear how well SRs/MAs of acupuncture studies adhere to reporting standards and which reporting criteria are generally ignored in these analyses.

Objectives

To evaluate reporting quality in SRs/MAs of acupuncture studies.

Methods

We performed a literature search for studies published prior to 2014 using the following public archives: PubMed, EMBASE, Web of Science, the Cochrane Database of Systematic Reviews (CDSR), the Chinese Biomedical Literature Database (CBM), the Traditional Chinese Medicine (TCM) database, the Chinese Journal Full-text Database (CJFD), the Chinese Scientific Journal Full-text Database (CSJD), and the Wanfang database. Data were extracted into pre-prepared Excel data-extraction forms. Reporting quality was assessed based on the PRISMA checklist (27 items).

Results

Of 476 appropriate SRs/MAs identified in our search, 203, 227, and 46 were published in Chinese journals, international journals, and the Cochrane Database, respectively. In 476 SRs/MAs, only 3 reported the information completely. By contrast, approximately 4.93% (1/203), 8.81% (2/227) and 0.00% (0/46) SRs/Mas reported less than 10 items in Chinese journals, international journals and CDSR, respectively. In general, the least frequently reported items (reported≤50%) in SRs/MAs were “protocol and registration”, “risk of bias across studies”, and “additional analyses” in both methods and results sections.

Conclusions

SRs/MAs of acupuncture studies have not comprehensively reported information recommended in the PRISMA statement. Our study underscores that, in addition to focusing on careful study design and performance, attention should be paid to comprehensive reporting standards in SRs/MAs on acupuncture studies.     

Magnesium Deficiency Enhances Hydrogen Peroxide Production and Oxidative Damage in Chick Embryo Hepatocyte In Vitro

Magnesium deficiency and oxidative stress have been identified as correlative factors in many diseases. The origin of free radicals correlated with oxidative damage resulting from Mg-deficiency is unclear at the cellular level. To investigate whether hydrogen peroxide (H₂O₂) is associated in the oxidative stress induced by Mg-deficiency, the effect of Mg²⁺ deficiency (0, 0.4, 0.7 mM) on the metabolism of H₂O₂ was investigated in cultured chick embryo hepatocytes. After being cultured in the media with various concentrations of Mg²⁺ for 1, 2, 4, 6 and 10 days, parameters of H₂O₂ production, catalase activity, lipid peroxidation, intracellular total Mg and cell viability were analyzed. Results demonstrated that long-term incubation of chick embryo hepatocyte in extracellular Mg²⁺-deprivative and Mg²⁺-deficient (0.4 mM) states significantly enhanced the production of H₂O₂ (approximately twofold, respectively) and lipid peroxidation in the cell cultures, while decreasing the cell viability. Additionally, the reversing action of Mg²⁺ re-added to 1.0 mM and the partial reversing action of dimethylthiourea suggested that (i) [Mg²⁺]_e deficiency induced the increase of H₂O₂ production, (ii) [Mg²⁺]_e deficiency decreased catalase activity in chick embryo hepatocyte in vitro, subsequently causing oxidative stress and cell peroxidative damage.     

Molecular mapping of stripe rust resistance gene YrCH42 in Chinese wheat cultivar Chuanmai 42 and its allelism with Yr24 and Yr26

Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most devastating diseases in common wheat (Triticum aestivum L.) worldwide. The objectives of this study were to map a stripe rust resistance gene in Chinese wheat cultivar Chuanmai 42 using molecular markers and to investigate its allelism with Yr24 and Yr26. A total of 787 F₂ plants and 186 F₃ lines derived from a cross between resistant cultivar Chuanmai 42 and susceptible line Taichung 29 were used for resistance gene tagging. Also 197 F₂ plants from the cross Chuanmai 42×Yr24/3*Avocet S and 726 F₂ plants from Chuanmai 42×Yr26/3*Avocet S were employed for allelic test of the resistance genes. In all, 819 pairs of wheat SSR primers were used to test the two parents, as well as resistant and susceptible bulks. Subsequently, nine polymorphic markers were employed for genotyping the F₂ and F₃ populations. Results indicated that the stripe rust resistance in Chuanmai 42 was conferred by a single dominant gene, temporarily designated YrCH42, located close to the centromere of chromosome 1B and flanked by nine SSR markers Xwmc626, Xgwm273, Xgwm11, Xgwm18, Xbarc137, Xbarc187, Xgwm498, Xbarc240 and Xwmc216. The resistance gene was closely linked to Xgwm498 and Xbarc187 with genetic distances of 1.6 and 2.3 cM, respectively. The seedling tests with 26 PST isolates and allelic tests indicated that YrCH42, Yr24 and Yr26 are likely to be the same gene.G.Q. Li and Z.F. Li contributed equally to the work.     

DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors

It has been reported that the phosphorylated form of histone variant H2AX (gammaH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using gammaH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce gammaH2AX foci in a 24h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce gammaH2AX foci formation in a time- and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of gammaH2AX foci in a time- and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce gammaH2AX foci formation in FL cells; however, AAF can induce gammaH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce gammaH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce gammaH2AX foci. Cell survival analyses indicated that the induction of gammaH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that gammaH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining gammaH2AX foci formation induced by a specific agent.     

High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genomic hybridization

We have used array comparative genomic hybridization to map DNA copy-number changes in 94 patients with cri du chat syndrome who had been carefully evaluated for the presence of the characteristic cry, speech delay, facial dysmorphology, and level of mental retardation (MR). Most subjects had simple deletions involving 5p (67 terminal and 12 interstitial). Genotype-phenotype correlations localized the region associated with the cry to 1.5 Mb in distal 5p15.31, between bacterial artificial chromosomes (BACs) containing markers D5S2054 and D5S676; speech delay to 3.2 Mb in 5p15.32-15.33, between BACs containing D5S417 and D5S635; and the region associated with facial dysmorphology to 2.4 Mb in 5p15.2-15.31, between BACs containing D5S208 and D5S2887. These results overlap and refine those reported in previous publications. MR depended approximately on the 5p deletion size and location, but there were many cases in which the retardation was disproportionately severe, given the 5p deletion. All 15 of these cases, approximately two-thirds of the severely retarded patients, were found to have copy-number aberrations in addition to the 5p deletion. Restriction of consideration to patients with only 5p deletions clarified the effect of such deletions and suggested the presence of three regions, MRI-III, with differing effect on retardation. Deletions including MRI, a 1.2-Mb region overlapping the previously defined cri du chat critical region but not including MRII and MRIII, produced a moderate level of retardation. Deletions restricted to MRII, located just proximal to MRI, produced a milder level of retardation, whereas deletions restricted to the still-more proximal MRIII produced no discernible phenotype. However, MR increased as deletions that included MRI extended progressively into MRII and MRIII, and MR became profound when all three regions were deleted.     

Precise blood lead analysis using a combined internal standard and standard addition approach with disposable screen-printed electrodes

We report here a highly accurate and efficient method for blood lead analysis (BLA) through the use of a disposable electrode. A new type of mercury-plated preanodized screen-printed carbon electrode, together with a thallium(III) internal standard, simplifies calibration and gives easily quantifiable signals for accurate BLA. A preanodization procedure improves the preconcentration ability of the working electrode through the effect of lead (Pb) complexing with an electrogenerated surface functional group, mainly >C=O. Under optimized conditions, the ratio of the anodic stripping peak currents of Pb and Tl (i.e., i(Pb)/i(Tl)) is linear against [Pb] in the window of 1-300 ppb with a correlation coefficient and detection limit (signal-to-noise ratio=3) of 0.999 and 0.23 ppb, respectively. This approach was used to analyze blood samples (n=55) from workers at a local battery factory. The results are consistent with those obtained from graphite furnace atomic absorption spectroscopy and confirm the applicability of the proposed method.     

Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is the second member of the angiopoietin-like family of proteins previously shown to increase plasma triglyceride (TG) levels in vivo. We recently reported that Angptl4 is a variable-sized oligomer formed by intermolecular disulfide bonds and undergoes regulated proteolytic processing upon secretion. We now show that adenoviral overexpression of Angptl4 potently increases plasma TG levels by a mechanism independent of food intake or hepatic VLDL secretion. We determined that cysteine residues at positions 76 and 80 of Angptl4, conserved among mouse, rat, and human, are required to form higher order structures. By generating adenoviral expression vectors of Angptl4 containing different epitope tags at both N and C termini, we show that loss of oligomerization results in decreased stability of the N-terminal coiled-coil domain of Angptl4 as well as decreased ability to increase plasma TG levels, suggesting that intermolecular disulfide bond formation plays important roles in determining the magnitude of the hyperlipidemic effect of Angptl4. Because Angptl4 is more potent than Angptl3 in increasing plasma TG levels in mice, inappropriate oligomerization of Angptl4 could be associated with disorders of lipid metabolism in vivo.