- Differences in protein profiles between Malassezia pachydermatis strains obtained from healthy and infected dogs. [Journal Article]
- MMycologia 2019 Jul 19; :1-8
- Malassezia pachydermatis causes infections of the skin and mucous membranes, especially in individuals with metabolic, hormonal, and immunological disorders. The search for M. pachydermatis propertie…
Malassezia pachydermatis causes infections of the skin and mucous membranes, especially in individuals with metabolic, hormonal, and immunological disorders. The search for M. pachydermatis properties that differentiate isolates from healthy and infected animals may result in the identification of typically commensal and potentially pathogenic strains within the entire species. We aimed to determine and compare protein profiles of M. pachydermatis strains isolated from 30 dogs with clinical symptoms of otitis externa and 34 dogs without symptoms of any disease. Two-dimensional gel electrophoresis was applied, and proteins distinguishing the two groups of strains were identified by liquid chromatography coupled with tandem mass spectrometry. Significant differences were found between potentially pathogenic and commensal isolates. The most significant finding was the presence of nicotinamide adenine dinucleotide phosphate (NADP)-dependent mannitol dehydrogenase and ketol-acid reductoisomerase among M. pachydermatis strains obtained from dogs with otitis externa. Nevertheless, it is not clear whether they are associated directly with the pathogenicity or they play the role of fungal allergen. On the basis of these findings, we can conclude that there may be two distinct groups of M. pachydermatis strains-one typically commensal and the other with properties that enhance the infection process. These results may be used for more precise diagnosis and identification of potentially pathogenic strains in the future.
- TIGAR impedes compression-induced intervertebral disc degeneration by suppressing nucleus pulposus cell apoptosis and autophagy. [Journal Article]
- JCJ Cell Physiol 2019 Jul 17
- To investigate whether TP53-induced glycolysis and apoptosis regulator (TIGAR) participates in compression-induced intervertebral disc (IVD) degeneration, and to determine the regulatory effect of TI…
To investigate whether TP53-induced glycolysis and apoptosis regulator (TIGAR) participates in compression-induced intervertebral disc (IVD) degeneration, and to determine the regulatory effect of TIGAR on nucleus pulposus (NP) cell autophagy and apoptosis following compression-induced injuries. IVD tissues were collected from human patients undergoing surgery (n = 20) and skeletally mature Sprague-Dawley rats (n = 15). Initially, the effect of compression on the expression of TIGAR was evaluated with in vivo and in vitro models. In addition, TIGAR was silenced to investigate the regulatory effect of TIGAR on compression-induced intracellular reactive oxygen species (ROS) levels, autophagy, and apoptosis in rat NP cells. Furthermore, the P53 inhibitor pifithrin-α (PFTα) and SP1 inhibitor mithramycin A were employed to detect expression level changes of TIGAR and autophagy-associated target molecules. TIGAR expression of NP cells increased gradually in human degenerative IVDs and in rat NP cells under compression both in vivo and in vitro. TIGAR knockdown enhanced compression-induced intracellular ROS generation and the NADPH/NADP+ and GSH/GSSG ratios. Moreover, TIGAR knockdown amplified the compression-induced caspase-3 activation and the apoptosis rate of rat NP cells. Likewise, knockdown of TIGAR significantly accelerated LC3B expression and autophagosome formation in rat NP cells during compression-induced injuries. The results also established that mithramycin A could inhibit TIGAR expression and autophagy levels in NP cells under compression conditions, while PFTα had no similar effect. Our data demonstrated that TIGAR acted as an important endogenous negative regulator of ROS levels, which might inhibit compression-induced apoptosis and autophagy through SP1-dependent mechanisms.
- Physiological Significance of NAD Kinases in Cyanobacteria. [Review]
- FPFront Plant Sci 2019; 10:847
- Unicellular cyanobacteria are thought to be the evolutionary ancestors of plant chloroplasts and are widely used both for chemical production and as model organisms in studies of photosynthesis. Alth…
Unicellular cyanobacteria are thought to be the evolutionary ancestors of plant chloroplasts and are widely used both for chemical production and as model organisms in studies of photosynthesis. Although most research focused on increasing reducing power (that is, NADPH) as target of metabolic engineering, the physiological roles of NAD(P)(H) in cyanobacteria poorly understood. In cyanobacteria such as the model species Synechocystis sp. PCC 6803, most metabolic pathways share a single compartment. This complex metabolism raises the question of how cyanobacteria control the amounts of the redox pairs NADH/NAD+ and NADPH/NADP+ in the cyanobacterial metabolic pathways. For example, photosynthetic and respiratory electron transport chains share several redox components in the thylakoid lumen, including plastoquinone, cytochrome b6f (cyt b6f), and the redox carriers plastocyanin and cytochrome c6. In the case of photosynthesis, NADP+ acts as an important electron mediator on the acceptor-side of photosystem I (PSI) in the linear electron chain as well as in the plant chloroplast. Meanwhile, in respiration, most electrons derived from NADPH and NADH are transferred by NAD(P)H dehydrogenases. Therefore, it is expected that Synechocystis employs unique NAD(P)(H) -pool control mechanisms to regulate the mixed metabolic systems involved in photosynthesis and respiration. This review article summarizes the current state of knowledge of NAD(P)(H) metabolism in Synechocystis. In particular, we focus on the physiological function in Synechocystis of NAD kinase, the enzyme that phosphorylates NAD+ to NADP+.
- High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains. [Journal Article]
- EMEnzyme Microb Technol 2019; 129:109359
- Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 a…
Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD+- or protein-engineered NADP+-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD+-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30 °C and 40 °C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40 °C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40 °C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402 g/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.
- Aspects of uncertainty in total reactive nitrogen deposition estimates for North American critical load applications. [Journal Article]
- STSci Total Environ 2019 Jun 26; 690:1005-1018
- Determination of the amount of reactive nitrogen (Nr) deposition in excess of the ecosystem critical load (CL) requires an estimate of total deposition. Because the CL exceedance is used to inform po…
Determination of the amount of reactive nitrogen (Nr) deposition in excess of the ecosystem critical load (CL) requires an estimate of total deposition. Because the CL exceedance is used to inform policy decisions, uncertainty in both the CL and the exceedance itself must be understood. In this paper we review the state of the science with respect to the sources of uncertainty in total Nr deposition budgets used for CL assessments in North America and put forth recommendations for research and monitoring to improve deposition measurements and models. In the absence of methods to rigorously quantify uncertainty in total Nr deposition, a simple weighted deposition uncertainty metric (WDUM) is introduced as a tool for scientists and decision makers to use in assessing CL exceedances. Maps of the WDUM applied to National Atmospheric Deposition Program (NADP) Total Deposition (TDep) estimates show greater uncertainty in areas of the U.S. where dry deposition makes a larger contribution to the deposition budget, particularly ammonia (NH3) in agricultural areas and oxidized nitrogen (NOx) in urban areas. Organic N deposition is an important source of uncertainty over much of the U.S. Our analysis illustrates how the WDUM can be used to assess spatial patterns of deposition uncertainty and inform actions to improve deposition budgets for CL assessments at the local scale.
- Coenzyme Binding Site Analysis of an Isopropanol Dehydrogenase with Wide Substrate Spectrum and Excellent Organic Solvent Tolerance. [Journal Article]
- ABAppl Biochem Biotechnol 2019 Jul 12
- NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for t…
NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for the regeneration of coenzyme are expected as costly of the coenzymes. Herein, a solvent-tolerant isopropanol dehydrogenase (IDH) showing lower similarity (37%) with other proteins was obtained and characterized. The enzyme exhibits high catalysis ability of its substrates methanol, ethanol, ethylene glycol, glycerol, isopropanol, n-butanol, isobutanol, and acetone. And it has good adaptability in organic solvents (isopropanol, acetonitrile, acetone, and acetophenone). Interaction force and the corresponding amino acid residues between IDH and NAD+ or NADP+ were parsed by docking. The wide substrate spectrum, excellent organic solvent tolerance, and good biocatalytic activity make the excavated enzyme a promising biocatalyst for the production of chiral compounds industrially and the construction of coenzyme regeneration systems in aqueous organic phase or organic phase.
- C4 photosynthetic enzymes play a key role in wheat spike bracts primary carbon metabolism response under water deficit. [Journal Article]
- PPPlant Physiol Biochem 2019 Jun 13; 142:163-172
- C4 photosynthetic enzymes are present in C3 plants and participate in non-photosynthetic metabolism. Wheat spike bracts had a higher drought tolerance, photosynthesis and senesced later compared to t…
C4 photosynthetic enzymes are present in C3 plants and participate in non-photosynthetic metabolism. Wheat spike bracts had a higher drought tolerance, photosynthesis and senesced later compared to the flag leaves under water deficit. This research was conducted to investigate the different response of primary carbon metabolism induced by C4 photosynthetic enzymes in wheat flag leaves and spike bracts including glumes and lemmas under water deficit. The activities of C4 photosynthetic enzymes and Ribulose bisphosphate carboxylase oxygenase (Rubisco), the expression of related genes and primary carbon metabolism contents were demonstrated in wheat flag leaves and spike bracts exposed to water deficit. Results showed that drought stress strongly inhibited wheat photosynthetic metabolism by decreasing Rubisco activity in flag leaves. The activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), phosphate dikinase (PPDK) and NADP- malic dehydrogenase (NADP-MDH) increased in wheat spike bracts under water deficit. Transcript levels of C4 photosynthetic genes in wheat spike bracts were higher under water deficit than that of control. Furthermore, the results indicated that drought stress induced changes in the contents of primary carbon metabolism including malate, oxaloacetic acid (OAA), citric, fumaric acid were organ-specific. In conclusion, the functions of C4 photosynthetic enzymes appear to be important for wheat spike bracts primary carbon metabolism and defence response under drought stress.
- Crystal Structure and Biochemical Characterization of Ketol-acid Reductoisomerase from Corynebacterium glutamicum. [Journal Article]
- JAJ Agric Food Chem 2019 Jul 12
- L-Valine belongs to the branched-chain amino acids (BCAAs), and is an essential amino acid that is crucial for all living organisms. L-Valine is industrially produced by the nonpathogenic bacterium C…
L-Valine belongs to the branched-chain amino acids (BCAAs), and is an essential amino acid that is crucial for all living organisms. L-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI, and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions depending on the conformational change.
- Serine hydroxymethyltransferase from the silkworm Bombyx mori: Identification, distribution, and biochemical characterization. [Journal Article]
- AIArch Insect Biochem Physiol 2019 Jul 12; :e21594
- Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate. cDNA encoding Bombyx mori SHMT (bmSHMT) was cloned …
Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate. cDNA encoding Bombyx mori SHMT (bmSHMT) was cloned and sequenced. The deduced amino acid sequence consisted of 465 amino acids and was found to share homology with other SHMTs. Recombinant bmSHMT was overexpressed in Escherichia coli and purified to homogeneity. The enzyme showed optimum activity at pH 3.0 and 30°C and was stable under acidic conditions. The Km and kcat /Km values for THF in the presence of Nicotinamide adenine dinucleotide phosphate (NADP+) were 0.055 mM and 0.081 mM-1 s-1 , respectively, whereas those toward NADP+ were 0.16 mM and 0.018 mM-1 s-1 and toward l-serine were 1.8 mM and 0.0022 mM-1 s-1 , respectively. Mutagenesis experiments revealed that His119, His132, and His135 are important for enzymatic activity. Our results provide insight into the roles and regulation mechanism of one-carbon metabolism in the silkworm B. mori.
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- Increased ethanol tolerance associated with the pntAB locus of Oenococcus oeni and Lactobacillus buchneri. [Journal Article]
- JIJ Ind Microbiol Biotechnol 2019 Jul 09
- Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that …
Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.