Impact of the genes UGT1A1, GSTT1, GSTM1, GSTA1, GSTP1 and NAT2 on acute alcohol-toxic hepatitis

Alcohol metabolism causes cellular damage by changing the redox status of cells. In this study, we investigated the relationship between genetic markers in genes coding for enzymes involved in cellular redox stabilization and their potential role in the clinical outcome of acute alcohol-induced hepatitis. Study subjects comprised 60 patients with acute alcohol-induced hepatitis. The control group consisted of 122 healthy non-related individuals. Eight genetic markers of the genes UGT1A1, GSTA1, GSTP1, NAT2, GSTT1 and GSTM1 were genotyped. GSTT1 null genotype was identified as a risk allele for alcohol-toxic hepatitis progression (OR 2.146, P=0.013). It was also found to correlate negatively with the level of prothrombin (β= ?11.05, P=0.037) and positively with hyaluronic acid (β=170.4, P=0.014). NAT2 gene alleles rs1799929 and rs1799930 showed opposing associations with the activity of the biochemical markers γ-glutamyltransferase and alkaline phosphatase; rs1799929 was negatively correlated with γ-glutamyltransferase (β=?261.3, P=0.018) and alkaline phosphatase (β= ?270.5, P=0.032), whereas rs1799930 was positively correlated with Γ-glutamyltransferase (β=325.8, P=0.011) and alkaline phosphatase (β=374.8, P=0.011). Enzymes of the glutathione S-transferase family and NAT2 enzyme play an important role in the detoxification process in the liver and demonstrate an impact on the clinical outcome of acute alcohol-induced hepatitis.     

Cucurbits, Sanskrit, and the Indo-Aryas 1

Vedic and early post-Vedic Sanskrit works describe Indo-Aryan life in northern India during 2000–200 B.C. Wild and cultivated plants of the Cucurbitaceae played an important role in Indo-Aryan food, medicine, and culture; over 300 words describing cucurbits are found in the Sanskrit texts. These words, their etymological relationships, and the geographies of the cucurbit taxa were compared. Results indicate that the Indo-Aryas knew only a handful of cucurbits before entering the Indian subcontinent from the west. They learned of at least 11 more species from their neighbors in India, who were speakers of Dravidian or Munda languages.     

Chromosome localization of the loci for PEPA,PEPB, PEPS, 1DH1, GSR,MPI, PGM1, NP,SOD1, and ME1 in the common shrew (Sorex araneus)

This report extends the genetic map of the common shrew (Sorex araneus) by adding chromosome assignments for ten genes to the seven already mapped (Pack et al. 1995). A somatic cell hybrid panel was used for the mapping. The genes for peptidase A (PEPA) and isocitrate dehydrogenase-1 (IDH1) map to chromosome de; the genes for phosphoglucomutase-1 (PGM1), superoxide dismutase-1 (SOD1), and mannosephosphate isomerase (MPI) are located on chromosome af; the genes for nucleoside phosphorylase (NP) and glutathione reductase (GSR) are on chromosome ik; and the genes for peptidase S (PEPS), malic enzyme-1 (ME1), peptidase B (PEPB) are found on chromosomes jl, go, and mp respectively. Received: 2 October 1995 / Accepted: 21 November 1995     

Structures of the potassium-saturated, 2:1, and intermediate, 1:1, forms of a quadruplex DNA

Potassium can stabilize the formation of chair- or edge-type quadruplex DNA structures and appears to be the only naturally occurring cation that can do so. As quadruplex DNAs may be important in the structure of telomere, centromere, triplet repeat and other DNAs, information about the details of the potassium–quadruplex DNA interactions are of interest. The structures of the 1:1 and the fully saturated, 2:1, potassium–DNA complexes of d(GGTTGGTGTGGTTGG) have been determined using the combination of experimental NMR results and restrained molecular dynamics simulations. The refined structures have been used to model the interactions at the potassium binding sites. Comparison of the 1:1 and 2:1 potassium:DNA structures indicates how potassium binding can determine the folding pattern of the DNA. In each binding site potassium interacts with the carbonyl oxygens of both the loop thymine residues and the guanine residues of the adjacent quartet.     

DISC1, PDE4B, and NDE1 at the centrosome and synapse

Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependant Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.     

Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.     

Cannabinoid-based drugs targeting CB1 and TRPV1, the sympathetic nervous system,and arthritis

Chronic inflammation in rheumatoid arthritis (RA) is accompanied by activation of the sympathetic nervous system, which can support the immune system to perpetuate inflammation. Several animal models of arthritis already demonstrated a profound influence of adrenergic signaling on the course of RA. Peripheral norepinephrine release from sympathetic terminals is controlled by cannabinoid receptor type 1 (CB₁), which is activated by two major endocannabinoids (ECs), arachidonylethanolamine (anandamide) and 2-arachidonylglycerol. These ECs also modulate function of transient receptor potential channels (TRPs) located on sensory nerve fibers, which are abundant in arthritic synovial tissue. TRPs not only induce the sensation of pain but also support inflammation via secretion of pro-inflammatory neuropeptides. In addition, many cell types in synovial tissue express CB₁ and TRPs. In this review, we focus on CB₁ and transient receptor potential vanilloid 1 (TRPV1)-mediated effects on RA since most anti-inflammatory mechanisms induced by cannabinoids are attributed to cannabinoid receptor type 2 (CB₂) activation. We demonstrate how CB₁ agonism or antagonism can modulate arthritic disease. The concept of functional antagonism with continuous CB₁ activation is discussed. Since fatty acid amide hydrolase (FAAH) is a major EC-degrading enzyme, the therapeutic possibility of FAAH inhibition is studied. Finally, the therapeutic potential of ECs is examined since they interact with cannabinoid receptors and TRPs but do not produce central side effects.     

Multiple roles for the Na,K-ATPase subunits, Atp1a1 and Fxyd1, during brain ventricle development

Formation of the vertebrate brain ventricles requires both production of cerebrospinal fluid (CSF), and its retention in the ventricles. The Na,K-ATPase is required for brain ventricle development, and we show here that this protein complex impacts three associated processes. The first requires both the alpha subunit (Atp1a1) and the regulatory subunit, Fxyd1, and leads to formation of a cohesive neuroepithelium, with continuous apical junctions. The second process leads to modulation of neuroepithelial permeability, and requires Atp1a1, which increases permeability with partial loss of function and decreases it with overexpression. In contrast, fxyd1 overexpression does not alter neuroepithelial permeability, suggesting that its activity is limited to neuroepithelium formation. RhoA regulates both neuroepithelium formation and permeability, downstream of the Na,K-ATPase. A third process, likely to be CSF production, is RhoA-independent, requiring Atp1a1, but not Fxyd1. Consistent with a role for Na,K-ATPase pump function, the inhibitor ouabain prevents neuroepithelium formation, while intracellular Na(+) increases after Atp1a1 and Fxyd1 loss of function. These data include the first reported role for Fxyd1 in the developing brain, and indicate that the Na,K-ATPase regulates three aspects of brain ventricle development essential for normal function: formation of a cohesive neuroepithelium, restriction of neuroepithelial permeability, and production of CSF.     

Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis

During apoptosis, the mitochondrial network fragments. Using short hairpin RNAs for RNA interference, we manipulated the expression levels of the proteins hFis1, Drp1, and Opa1 that are involved in mitochondrial fission and fusion in mammalian cells, and we characterized their functions in mitochondrial morphology and apoptosis. Down-regulation of hFis1 powerfully inhibits cell death to an extent significantly greater than down-regulation of Drp1 and at a stage of apoptosis distinct from that induced by Drp1 inhibition. Cells depleted of Opa1 are extremely sensitive to exogenous apoptosis induction, and some die spontaneously by a process that requires hFis1 expression. Wild-type Opa1 may function normally as an antiapoptotic protein, keeping spontaneous apoptosis in check. However, if hFis1 is down-regulated, cells do not require Opa1 to prevent apoptosis, suggesting that Opa1 may be normally counteracting the proapoptotic action of hFis1. We also demonstrate in this study that mitochondrial fragmentation per se does not result in apoptosis. However, we provide further evidence that multiple components of the mitochondrial morphogenesis machinery can positively and negatively regulate apoptosis.     

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1⁺ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1.     

Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects Are Enzyme and Substrate Dependent

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K _m, increasing its V _max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K _m are concerned. In the cases of V _max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V _max increases. Additionally, the BSA effects may be UGT subfamily dependent since K _m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V _max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.     

HWP1 Functions in the Morphological Development of Candida albicans Downstream of EFG1, TUP1, and RBF1

The morphological plasticity of Candida albicans is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent. HWP1, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of HWP1 resulted in a medium-conditional defect in hyphal development. HWP1 expression was blocked in a Deltaefg1 mutant, reduced in an Deltarbf1 mutant, and derepressed in a Deltatup1 mutant. Therefore, HWP1 functions downstream of the developmental regulators EFG1, TUP1, and RBF1. Mutation of CPH1 had no effect on HWP1 expression, suggesting that the positive regulators of hyphal development, CPH1 and EFG1, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene, ECE1, was similarly regulated by EFG1. Since ECE1 is not required for hyphal development, the regulatory role of EFG1 apparently extends beyond the control of cell shape determinants. However, expression of ECE1 was not influenced by TUP1, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.     

Curcumin-mediated oxidative stress resistance in Caenorhabditis elegans is modulated by age-1, akt-1, pdk-1, osr-1, unc-43, sek-1, skn-1, sir-2.1, and mev-1

Abstract

Curcumin (diferuloylmethane), a pharmacologically active substance derived from turmeric, exhibits anti-inflammatory, anticarcinogenic, and antioxidant properties. We examined the modulation of oxidative-stress resistance and associated regulatory mechanisms by curcumin in a Caenorhabditis elegans model. Our results showed that curcumin-treated wild-type C. elegans exhibited increased survival during juglone-induced oxidative stress compared with the control treatment. In addition, curcumin reduced the levels of intracellular reactive oxygen species in C. elegans. Moreover, curcumin induced the expression of the gst-4 and hsp-16.2 stress response genes. Lastly, our findings from the mechanistic study in this investigation suggest that the antioxidative effect of curcumin is mediated via regulation of age-1, akt-1, pdk-1, osr-1, unc-43, sek-1, skn-1, sir-2.1, and mev-1. Our study elucidates the diverse modes of action and signaling pathways that underlie the antioxidant activity exhibited by curcumin in vivo.     

Common polymorphisms in CYP1A1, GSTM1, GSTT1, GSTP1 and XPD genes and endogenous DNA damage

Endogenous DNA damage levels were analyzed in relation to polymorphisms in genes encoding phase I detoxifying enzyme—CYP1A1, phase II detoxifying enzymes—GSTM1, GSTT1, GSTP1 and enzyme involved in nucleotide excision repair-XPD. The study group consisted of 220 healthy non-smoking volunteers; 90 men and 130 woman, 25–60 years old (44 ± 10 years). The level of DNA damage (% DNA in tail) was evaluated by alkaline comet assay. The genetic variants were determined by restriction fragment length polymorphism PCR. The highest level of DNA damage (6.7%) was found in carriers of both: AA variant of XPD gene and M1 null variant of GSTM1 gene. The lowest level of DNA breaks (3.7%) was associated with the genotype GSTP1-AA/GSTM1 (+).