Primary Alcohol-Activated Human and Mouse Hepatic Stellate Cells Share Similarities in Gene-Expression Profiles.

Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins and formation of a fibrous scar. Hepatic stellate cells (HSCs) are the major source of collagen type 1 producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol-induced activation of human and mouse HSCs is not fully understood. We compared the gene-expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n = 3) or without underlying liver disease (n = 4) using RNA-sequencing analysis. Furthermore, the gene-expression profile of ALD hHSCs was compared with that of alcohol-activated mHSCs (isolated from intragastric alcohol-fed mice) or CCl4-activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, in addition to alcohol-activated and CCl4-activated mHSCs, share the expression of common HSC activation (Col1a1 [collagen type I alpha 1 chain], Acta1 [actin alpha 1, skeletal muscle], PAI1 [plasminogen activator inhibitor-1], TIMP1 [tissue inhibitor of metalloproteinase 1], and LOXL2 [lysyl oxidase homolog 2]), indicating that a common mechanism underlies the activation of human and mouse HSCs. Furthermore, alcohol-activated mHSCs most closely recapitulate the gene-expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely up-regulated in primary cultured alcohol-activated hHSCs and freshly isolated mHSCs, which include CSF1R (macrophage colony-stimulating factor 1 receptor), PLEK (pleckstrin), LAPTM5 (lysosmal-associated transmembrane protein 5), CD74 (class I transactivator, the invariant chain), CD53, MMP9 (matrix metallopeptidase 9), CD14, CTSS (cathepsin S), TYROBP (TYRO protein tyrosine kinase-binding protein), and ITGB2 (integrin beta-2), and other genes (compared with CCl4-activated mHSCs). Conclusion: We identified genes in alcohol-activated mHSCs from intragastric alcohol-fed mice that are largely consistent with the gene-expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol-induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD

Cooperativity of Glucocorticoid Response Elements Located Far Upstream of the Tyrosine Aminotransferase Gene

Two glucocorticoid response elements (GREs) located 2.5 kb upstream of the transcription initiation site of the tyrosine aminotransferase gene were identified by gene transfer experiments and shown to bind to purified glucocorticoid receptor. Although the proximal GRE has no inherent capacity by itself to stimulate transcription, when present in conjunction with the distal GRE, this element synergistically enhances glucocorticoid induction of gene expression. Cooperativity of the two GREs is maintained when they are transposed upstream of a heterologous promoter. An oligonucleotide of 22 bp representing the distal GRE is sufficient to confer glucocorticoid inducibility. As evidenced by the mapping of DNAase I hypersensitive sites, local alterations in the structure of chromatin at the GREs take place as a consequence of hormonal treatment

Functional Genomic Investigation of Aromatic Aminotransferases Involved in Ephedrine Alkaloid Biosynthesis in Ephedra Sinica (Stapf)

Ephedra sinica (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This medicinal plant accumulates large amounts of the ephedrine alkaloids, including (S)-cathinone, (1R,2S)-norephedrine, (1R,2S)-ephedrine and (1S,2S)-pseudoephedrine in its aerial tissues. These analogues of amphetamine mimic adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the biological mechanisms by which they are synthesized remains largely unknown. A functional genomics platform was established in order to investigate alkaloid biosynthesis. RNA was extracted from Ephedra sinica stems and sequenced by Illumina HiSeq2000 next-generation sequencing. Candidate biosynthetic enzymes were obtained from this EST collection based on similarity to characterized enzymes with similar functions. This portion of the collaborative study is focused on identifying aminotransferase enzymes involved in alkaloid biosynthesis. Two functional aromatic aminotransferase enzymes, possibly involved in ephedrine biosynthesis have been identified. One exhibiting much greater enzymatic activity was chosen for characterization

Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferases

<p>Abstract</p> <p>Background</p> <p>Kynurenine aminotransferase (KAT) catalyzes the transamination of kynunrenine to kynurenic acid (KYNA). KYNA is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of N-methyl-D-aspartate receptors. Four KAT enzymes, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase, have been reported in mammalian brains. Because of the substrate overlap of the four KAT enzymes, it is difficult to assay the specific activity of each KAT in animal brains.</p> <p>Results</p> <p>This study concerns the functional expression and comparative characterization of KAT I, II, III, and IV from mice. At the applied test conditions, equimolar tryptophan with kynurenine significantly inhibited only mouse KAT I and IV, equimolar methionine inhibited only mouse KAT III and equimolar aspartate inhibited only mouse KAT IV. The activity of mouse KAT II was not significantly inhibited by any proteinogenic amino acids at equimolar concentrations. pH optima, temperature preferences of four KATs were also tested in this study. Midpoint temperatures of the protein melting, half life values at 65°C, and pKa values of mouse KAT I, II, III, and IV were 69.8, 65.9, 64.8 and 66.5°C; 69.7, 27.4, 3.9 and 6.5 min; pH 7.6, 5.7, 8.7 and 6.9, respectively.</p> <p>Conclusion</p> <p>The characteristics reported here could be used to develop specific assay methods for each of the four murine KATs. These specific assays could be used to identify which KAT is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of KAT-involved human diseases.</p

A Study of Abscisic Acid Regulated Enzymes and Histone Binding Proteins in Plants

Plant hormone abscisic acid (ABA) plays a main role in coordinating various stress signals in plants. ABA regulates the expression of genes and activities of enzymes in response to various stress conditions. In the following studies we were able to study the ABA mediated regulation of enzymes in plants. Using in-gel activity analysis we identified that ABA regulates the activity of aspartate aminotransferase (AAT), an enzyme involved in nitrogen assimilation and carbohydrate metabolism. Our results indicate that phosphorylation of AAT by SnRK2.2 and 2.3 kinases results in down regulation of AAT2 and AAT3 isozyme activities in Arabidopsis. AAT was identified as a negative regulator of drought stress and aat mutant plants showed improved survival following drought conditions. Using in-gel staining method we were able to visualize sugar phosphatases like fructose 1-6 bisphosphatase family, sedoheptulase-1,7-bisphosphatase, inositol mono phosphatases; protein serine/threonine phosphatases, protein tyrosine phosphatases and studied their response to ABA and drought stress. Fructose-1-6 bisphosphatase family of phosphatases were identified to be induced by ABA in Arabidopsis and rice. N-acetylglucosamine (GlcNAc) is present on glycoproteins and as post translational modification (PTM) in cytoplasmic and nuclear proteins. N-acetylglucosamine is removed from target proteins by hexosaminidases. Little is known about the hexosaminidases in plants. Using in-gel activity analysis we were able to identify an ABA induced Beta-hexosaminidase with a neutral pH optimum in soybean. The nuclear DNA in chromatin is associated with basic proteins called histones. The N-terminal tails of histones contain different PTMs including methylation, phosphorylation, ubiquitination, acetylation, ADP-ribosylation and glycosylation. The histone lysine methylation can serve as a binding site or repel/disrupt the histone binding proteins. The effector/reader proteins specifically recognize the post translational modifications and responsible for the downstream process. Many histone methyl modification effector proteins have been characterized but very few proteins whose binding was disrupted by the presence of a PTM were identified. Using peptide pulldown analysis, far western analyses we identified a WD-40 domain containing histone binding (HB01) protein as direct interactor of unmodified histone. The presence of post translational modifications disrupts HB01 binding to histone H3