Metabolism studies on prim‐O‐glucosylcimifugin and cimifugin in human liver microsomes by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Prim‐O‐glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti‐inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O‐glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP‐glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular genetics ofPhanerochaete chrysosporium

InPhanerochaete chrysosporium ME446 lignin degradation is a secondary metabolic event triggered by carbon and nitrogen limitation. It is therefore possible to study lignin degradation at the level of gene expression by comparing mRNA populations produced during primary and secondary growth in both wild-type strains and in strains mutant in lignin degradation. We have isolated mutants deficient in phenol oxidase activity. These mutants fall into three phenotypic categories with respect to lignin degradation: (1) negative, (2) delayed onset after nitrogen starvation, (3) enhanced. Polyacrylamide gel electrophoresis of rabbit reticulocyte polypeptide translation products ofPhanerochaete mRNA shows differences between populations from primary and secondary growth. Differences in the range of polypeptides (and therefore of mRNA) have also been demonstrated between a mutant and its wild-type progenitor under identical conditions. A gene bank has been prepared from P.chrysosporium strain ME446 genomic DNA using a bacteriophage λ vector. This gene bank is being screened with labeled mRNA from secondary growth mycelium in the presence of excess competing cold RNA from primary growth mycelium. Using this method (and/or using labeled cDNA probes), we hope to isolate clones carrying sequences expressed only during lignin degradation. A gene bank has also been constructed fromSporotrichum pulverulentum (Novobranova), which is on morphological criteria considered to be the imperfect form ofP. chrysosporium. DNA probes from randomly chosen clones of both gene banks have been hybridized to restricted and electrophoresed total DNA of the two “gene bank” strains and of two other isolates ofP. chrysosporium on Southern blots. We found very strong DNA homology between the two “gene bank” strains, but far less homology between these two strains and the two others. These degrees of relationships were supported by the analysis of mitochondrial DNA from the four strains. We thank the Agricultural Research Council and the British Petroleum Venture Research Unit for support.     

Suppressive Subtractive Hybridization Approach Revealed Differential Expression of Hypersensitive Response and Reactive Oxygen Species Production Genes in Tea (Camellia sinensis (L.) O. Kuntze) Leaves during Pestalotiopsis thea Infection

Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of ??-actin gene. A total of 377 and 720 clones with insert size >250?bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59?%) or hypothetical functions (23 and 18?%) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.     

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.     

Volatile profiling reveals intracellular metabolic changes in <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>: <Emphasis Type="Italic">veA</Emphasis> regulates branched chain amino acid and ethanol metabolism

Background  

Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes.     

A Novel Porcine Gene-<Emphasis Type="Italic">erlin2</Emphasis>, Differentially Expressed in the Liver Tissues from Meishan and Large White Pigs

The messenger RNA differential display technique was performed to investigate the differences of gene expression in the liver tissues from Meishan and Large White pigs. A fragment of one differentially expressed gene was isolated and sequenced. A complete complementary DNA (cDNA) sequence was obtained using the rapid amplification of cDNA end method. Nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 339 amino acids which have high homology with those of the ER lipid-raft-associated 2 isoform 2 (ERLIN2) of eight species—human (97%), rhesus monkey (97%), rat (96%), horse (97%), cattle (97%), mouse (97%), dog (95%), and red jungle fowl (90%)—so that it can be defined as the swine erlin2 gene. The phylogenetic tree analysis revealed that the swine erlin2 gene has a closer genetic relationship with the erlin2 genes of human and rhesus monkey. The tissue expression profile analysis indicated that the swine erlin2 gene is differentially expressed in detected tissues from Meishan and Large White pigs. Our experiment suggested that the swine erlin2 gene might play an important role in the superabundant fat deposition of Chinese pigs.     

Expressed Sequence Tag Analysis of the Dinoflagellate Lingulodinium polyedrum During Dark Phase¶

To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light‐dark cycle. A total of 4324 5′‐end sequence tags were isolated. The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database. Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae. We also isolated three bioluminescence‐related (luciferase and two luciferinbinding proteins [LBP]) and 37 photosynthesis‐related genes. Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene. These cDNA clones and EST sequence data should provide a powerful resource for future genome‐wide functional analyses for uncharacterized genes.     

Global expression analysis of CESA and CSL genes in Arabidopsis

We have used Affymetrix gene chips to measure the expression of 10 CESA and 29 CSL genes of Arabidopsis in different developmental stages or organs. These measurements reveal that many of the genes exhibit different levels of expression in the various organs. While several CESA genes are highly expressed in all the tissues examined, very few CSL genes approach such high levels of expression. This suggests that the CSL genes either encode enzymes for the synthesis of minor components of cell walls or are expressed only in specific cell types. The expression data also highlights the potential importance of the CESA genes for primary and secondary cell wall formation during different developmental stages and in the different organs examined.     

Characterization of heterologous and native enzyme activity profiles in metabolically engineered Zymomonas mobilis strains during batch fermentation of glucose and xylose mixtures

Zymomonas mobilis has been metabolically engineered to broaden its substrate utilization range to include d-xylose and l-arabinose. Both genomically integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose d-xylose have been created by incorporating four genes: two genes encoding xylose utilization metabolic enzymes (xylA/xylB) and two genes encoding pentose phosphate pathway enzymes (talB/tktA). We have characterized the activities of the four newly introduced enzymes for xylose metabolism, along with those of three native glycolytic enzymes, in two different xylose-fermenting Z. mobilis strains. These strains were grown on glucose-xylose mixtures in computer-controlled fermentors. Samples were collected and analyzed to determine extracellular metabolite concentrations as well as the activities of several intracellular enzymes in the xylose and glucose uptake and catabolism pathways. These measurements provide new insights on the possible bottlenecks in the engineered metabolic pathways and suggest methods for further improving the efficiency of xylose fermentation.     

Molecular Cloning and Expression Analysis of an <Emphasis Type="Italic">Mn</Emphasis>-<Emphasis Type="Italic">SOD</Emphasis> Gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>

A rapid amplification cDNA end (RACE) assay was established to achieve the complete sequence of mitochondrial manganese-superoxide dismutase (Mn-SOD) cDNA in Nelumbo nucifera. The obtained full-length cDNA of Mn-SOD was 926 bp and contained a 699-bp open reading frame encoding an Mn-SOD precursor of 233 amino acids. The recombinant of Mn-SOD expressed by PET-32a vector in Escherichia coli BL21 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting assays. A 3D structural model of the Mn-SOD was constructed by homology modeling. Real-time polymerase chain reaction analysis revealed that Mn-SOD mRNA was expressed in young leaves, blossom, stems, and terminal buds during reproductive stage but with the highest expression in young leaves. This significant difference demonstrated the differential expression of Mn-SOD in various organs of N. nucifera.     

Functional Annotation of <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 Carbohydrate Active Enzymes

Fibrobacter succinogenes is a cellulolytic bacterium that degrades plant cell wall biomass in ruminant animals and is among the most rapidly fibrolytic of all mesophilic bacteria. The complete genome sequence of Fisuc was completed by the DOE Joint Genome Institute in late 2009. Using new expression tools developed at Lucigen and C5-6 Technologies and a multi-substrate screen, 5,760 random shotgun expression clones were screened for biomass-degrading enzymes, representing 2× genome expression coverage. From the screen, 169 positive hits were recorded and 33 were unambiguously identified by sequence analysis of the inserts as belonging to CAZy family genes. Eliminating duplicates, 24 unique CAZy genes were found by functional screening. Several previously uncharacterized enzymes were discovered using this approach and a number of potentially mis-annotated enzymes were functionally characterized. To complement this approach, a high-throughput system was developed to clone and express all the annotated glycosyl hydrolases and carbohydrate esterases in the genome. Using this method, six previously described and five novel CAZy enzymes were cloned, expressed, and purified in milligram quantities.     

CHEMISTRY OF BIOLOGICALLY ACTIVE SULFUR COMPOUNDS

Abstract

All living organisms require sulfur for various metabolic processes. Sulfur occurs in enzymes, in structural proteins of cells, and in a wide variety of naturally occurring compounds which often play key roles in metabolism. The chemistry and biochemistry of sulfur compounds has been reviewed.^1–3.     

Sirtuins are crucial regulators of T cell metabolism and functions

It is well known that metabolism underlies T cell differentiation and functions. The pathways regulating T cell metabolism and function are interconnected, and changes in T cell metabolic activity directly impact the effector functions and fate of T cells. Thus, understanding how metabolic pathways influence immune responses and ultimately affect disease progression is paramount. Epigenetic and posttranslational modification mechanisms have been found to control immune responses and metabolic reprogramming. Sirtuins are NAD⁺-dependent histone deacetylases that play key roles during cellular responses to a variety of stresses and have recently been reported to have potential roles in immune responses. Therefore, sirtuins are of significant interest as therapeutic targets to treat immune-related diseases and enhance antitumor immunity. This review aims to illustrate the potential roles of sirtuins in different subtypes of T cells during the adaptive immune response.Subject terms: Acetylation, T cells     

Sterol Glycosyltransferases—The Enzymes That Modify Sterols

Sterols are important components of cell membranes, hormones, signalling molecules and defense-related biotic and abiotic chemicals. Sterol glycosyltransferases (SGTs) are enzymes involved in sterol modifications and play an important role in metabolic plasticity during adaptive responses. The enzymes are classified as a subset of family 1 glycosyltransferases due to the presence of a signature motif in their primary sequence. These enzymes follow a compulsory order sequential mechanism forming a ternary complex. The diverse applications of sterol glycosides, like cytotoxic and apoptotic activity, anticancer activity, medicinal values, anti-stress roles and anti-insect and antibacterial properties, draws attention towards their synthesis mechanisms. Many secondary metabolites are derived from sterol pathways, which are important in defense mechanisms against pathogens. SGTs in plants are involved in changed sensitivity to stress hormones and their agrochemical analogs and changed tolerance to biotic and abiotic stresses. SGTs that glycosylate steroidal hormones, such as brassinosteroids, function as growth and development regulators in plants. In terms of metabolic roles, it can be said that SGTs occupy important position in plant metabolism and may offer future tools for crop improvement.     

Heterologous Expression of Legumin Gene in <Emphasis Type="Italic">E. coli</Emphasis> Isolated from cDNA Clones of Immature Seeds of Pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L.)

Proteins are one of the targets for improving the nutritional quality, and attempts are being made through manipulation of its native gene(s). Pigeonpea (Cajanus cajan L.) is one of the nutritionally important legumes of tropical and subtropical regions of the world, and studies of the structure of seed storage proteins and their interactions have been limited by the difficulty of isolating single-protein subunits in large amounts from a complex mixture of the seed endosperm. One way to overcome this problem is the expression of seed storage protein-encoded gene(s) in heterologous systems that have additional advantages wherein specific gene modifications can be made and the new gene constructs can quickly be expressed. Legumin protein was extracted from pigeonpea seeds of different developmental stages (5th to 25th day after flowering [DAF]) and characterized. The legumin gene (leg) of size 1.482 kb was screened, using the deoxygenin-labeled legumin probe, from the complementary deoxyribonucleic acid (cDNA) library, constructed from 18-day-old (DAF) immature seeds of pigeonpea and sequenced (accession no. AF3555403). The legumin gene was further characterized by DNA blotting, and its probable secondary structure was predicted using online ExPASy server. Significant Protein Data Bank (PDB) alignment of the deduced legumin protein by BLASTP was observed with proglycinin of soybean. Comparative 3D structural homology was predicted by Cn3D software, and the legumin protein showed the 3D structure alignment and interaction homology with proglycinin chain 1FXZA (PDB no. 1FXZ). The legumin gene was subcloned in vector pET-24a driven by the bacterial promoter, and its expression was detected in Escherichia coli by immunoblotting using polyclonal antibodies, raised against the purified legumin protein.