Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors

2008 ◽  
Vol 413 (3) ◽  
pp. 493-504 ◽  
Author(s):  
Claudia A. Staab ◽  
Johan Ålander ◽  
Margareta Brandt ◽  
Johan Lengqvist ◽  
Ralf Morgenstern ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Glutathione Transferase ◽  
Cultured Cells ◽  
Hydrolysis Product ◽  
Indirect Evidence ◽  
Dual Function ◽  
Transferase Activity ◽  
Cofactor Recycling ◽  
Km Value

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a kcat/Km value approx. 10 times the kcat/Km value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD+/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1–5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.

scholarly journals Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level.

1996 ◽  
Vol 132 (3) ◽  
pp. 345-357 ◽  
Author(s):  
X M Wang ◽  
J G Peloquin ◽  
Y Zhai ◽  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Posttranslational Modifications ◽  
Glutathione Transferase ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Cellular Level ◽  
Vitro Assay ◽  
Normal Functions ◽  
Projection Domain

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.

CXCR4/SDF-1 Is a Key Regulator for Leukemia Migration and Homing to the BM: Impact of AMD3100 on In Vivo Response to Chemotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 569-569 ◽  
Author(s):  
Bruno Nervi ◽  
Pablo Ramirez ◽  
Matthew Holt ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Survival Benefit ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Dual Function ◽  
Hematopoietic Stem ◽  
Response To Chemotherapy ◽  
The Impact

Abstract Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and interact with stroma cells and extracellular matrix. CXCR4/SDF-1 axis regulates the trafficking of HSC to and from the BM. We utilized a PML-RARα knock-in mouse model of human acute promyelocytic leukemia (APL) to study APL interaction with the normal BM. We have previously shown there is a rapid mobilization of APL cells from the BM into peripheral blood (PB) after administration of AMD3100, a competitive inhibitor of CXCR4. We hypothesize that we can sensitize these tumor cells to chemotherapy by interrupting the interaction between APL and the BM stroma. We transduced banked APL cells with a dual function reporter gene that encodes a fusion protein comprised of Click Beetle Red luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (Luc/EGFP). Upon iv injection into genetically compatible recipients (F1 129/B6 mice), APL rapidly migrated to the BM with increased BLI signal in the femurs, spine, ribs, and skull, at 4 days after injection, followed by spleen infiltration and death due to leukostasis by day 15. 129/B6 F1 mice (n=28) were injected iv with 106 APL cells. By day 12 all mice had ±5% APL cells in PB. 8 mice received AraC (500mg/kg/sq) on days 12 and 13, and another 8 mice received AraC+AMD (5mg/kg/sq) 1 hour before and 3 hours after each AraC injection. 6 mice received only AMD and 6 control mice were observed. Total body BLI signal, WBC, and blasts per μl of blood on days 19 and 23 were higher in AraC versus AraC+AMD (p<0.004). Median survival for control, AMD, AraC and AraC+AMD groups were 18, 19, 23 and 30 days respectively (p<0.0006). Hemoglobin, platelet and granulocyte recovery post-chemotherapy was similar in both groups. We developed an in vitro mouse stroma system to study engraftment, ex vivo mobilization and sensitivity to chemotherapy. In vitro culture of APL cells showed no difference in APL survival between AraC versus AraC+AMD as measured by flow cytometry or BLI. Stroma offered a survival benefit versus no stroma (p<0.0001). We injected 4 genetically compatible mice with 106 APL cells iv and after 14 days mice were sacrificed. Blast percentage in blood, spleen and BM was 47, 58 and 40% respectively. We cultured cells from all three compartments ex vivo with AraC (25ng/ml). After 24 hours APL survival was 25, 80 and 60% respectively (p<0.006). We repeated the same experiment, but we did, in addition, a positive selection for CD34 to purify APL cells away from surrounding cells in the BM and spleen. Survival after ex vivo AraC incubation was 32, 30, 34% respectively (p=NS). In summary, CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. The interaction of APL cells with the BM and splenic microenvironments provides a survival benefit. Rapid mobilization of APL cells in vivo by AMD3100 interrupts APL-stromal interactions and sensitizes APL to chemotherapy. The impact of additional mobilizing agents on APL mobilization on sensitizing APL to chemo and radiotherapy will be presented. Finally, preliminary RNA profiling studies will be presented in an attempt to identify genes in APL cell that are differentially expressed when bound to and released from the BM.

Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase

2002 ◽  
Vol 363 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Tobias MODIG ◽  
Gunnar LIDÉN ◽  
Mohammad J. TAHERZADEH
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aldehyde Dehydrogenase ◽  
Pyruvate Dehydrogenase ◽  
Competitive Inhibition ◽  
Critical Role ◽  
Km Value ◽  
In Vivo Effects

The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated Km value of AlDH for furfural was found to be about 5μM, which was lower than that for acetaldehyde (10μM). For ADH, however, the estimated Km value for furfural (1.2mM) was higher than that for acetaldehyde (0.4mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition.

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

2008 ◽  
Vol 23 (3) ◽  
pp. 391-399 ◽  
Author(s):  
Victor Muleya ◽  
Rose Hayeshi ◽  
Hilary Ranson ◽  
Berhanu Abegaz ◽  
Merha-Tibeb Bezabih ◽  
...  
Keyword(s):  
Natural Products ◽  
Anopheles Gambiae ◽  
Glutathione Transferase ◽  
Transferase Activity ◽  
Plant Natural Products

scholarly journals Molecular Characterization of Prdx Family and Response of Antioxidant Enzymes in Berberine Hydrochloride-Treated Charybdis japonica Infected With Aeromonas hydrophila

2021 ◽  
Vol 8 ◽  
Author(s):  
Mingming Han ◽  
Tianheng Gao ◽  
Yuxin Liu ◽  
Zakaria Zuraini ◽  
Chenxi Zhu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Aeromonas Hydrophila ◽  
Glutathione Transferase ◽  
Control Group ◽  
Transferase Activity ◽  
Berberine Hydrochloride ◽  
Conserved Domain ◽  
Charybdis Japonica

Berberine hydrochloride is an isoquinoline alkaloid, which has antitumoral, antibacterial, and antiviral activities in vivo and in vitro. Charybdis japonica is one of the main economic species of crab in Southeast Asia. We studied the molecular mechanism of oxidative stress in berberine hydrochloride-treated C. japonica infected with Aeromonas hydrophila. C. japonica were infected with A. hydrophila after being submerged in different concentrations (0, 100, 200, and 300 mg/L) of berberine hydrochloride for 48 h. The full-length cDNA of Prx6 and the ORFs of Prx5 and PXL2A were cloned. Prx6 and PXL2A each have one conserved domain, Cys44, and Cys81. The Prx5 conserved domain contains three important Cys loci, Cys75, Cys100, and Cys76. Prx6 was different from Prx5 and PXL2A in the Peroxiredoxin family. The transcription levels of PXL2A infected with A. hydrophila were all higher than the control. The transcription levels of C. japonica were further increased by adding berberine hydrochloride and were increased the highest at a concentration of 300 mg/L. The activities of glutathione peroxidase, superoxide dismutase, and catalase in the hepatopancreas of berberine hydrochloride-treated C. japonica infected with A. hydrophila were significantly increased compared with those only infected with A. hydrophila and the control group. The glutathione transferase activity in the hepatopancreas was significantly increased in berberine hydrochloride-treated C. japonica. The results of this study provide a new understanding of the potential role of berberine hydrochloride on the oxidative stress mechanisms of C. japonica.

Preliminary investigation of the mechanisms of DDT and pyrethroid resistance in Culex quinquefasciatus Say (Diptera: Culicidae) from Saudi Arabia

1989 ◽  
Vol 79 (3) ◽  
pp. 361-366 ◽  
Author(s):  
A. M. Amin ◽  
J. Hemingway
Keyword(s):  
Saudi Arabia ◽  
Culex Quinquefasciatus ◽  
Glutathione Transferase ◽  
Pyrethroid Resistance ◽  
Preliminary Investigation ◽  
Oxidative Degradation ◽  
Cross Resistance ◽  
Transferase Activity ◽  
Metabolic Studies

AbstractHigh levels (>1000-fold) of resistance to DDT, permethrin and deltamethrin were detected in Culex quinquefasciatus Say from Saudi Arabia. Biochemical enzyme and metabolic studies indicated that there is evidence for a metabolic basis to both the organochlorine and pyrethroid resistances. Electrophysiological studies indicated that there is no kdr-type mechanism conferring resistance to the pyrethroid lambda-cyhalothrin neurophysiologically, although there is evidence of cross-resistance between DDT and the pyrethroids by bioassays. There was a change in the oxidase system in both the DDT- and permethrin-selected strains and an increase in glutathione transferase activity in the DDT-selected line. Metabolic studies indicated that both oxidases and glutathione transferases are involved with DDT resistance as DDA and DDE were the predominant metabolites after a 5-h in-vitro incubation period. Permethrin resistance is likely to involve an increase in oxidative degradation, but further metabolic studies are needed to confirm this.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Sign in / Sign up

Export Citation Format

Share Document