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1.
Oncoimmunology ; 12(1): 2170095, 2023.
Article in English | MEDLINE | ID: mdl-36733497

ABSTRACT

Indoleamine 2,3 dioxygenase 1 (IDO1), a leader tryptophan-degrading enzyme, represents a recognized immune checkpoint molecule. In neoplasia, IDO1 is often highly expressed in dendritic cells infiltrating the tumor and/or in tumor cells themselves, particularly in human melanoma. In dendritic cells, IDO1 does not merely metabolize tryptophan into kynurenine but, after phosphorylation of critical tyrosine residues in the non-catalytic small domain, it triggers a signaling pathway prolonging its immunoregulatory effects by a feed-forward mechanism. We here investigated whether the non-enzymatic function of IDO1 could also play a role in tumor cells by using B16-F10 mouse melanoma cells transfected with either the wild-type Ido1 gene (Ido1WT ) or a mutated variant lacking the catalytic, but not signaling activity (Ido1H350A ). As compared to the Ido1WT -transfected counterpart (B16WT), B16-F10 cells expressing Ido1H350A (B16H350A) were characterized by an in vitro accelerated growth mediated by increased Ras and Erk activities. Faster growth and malignant progression of B16H350A cells, also detectable in vivo, were found to be accompanied by a reduction in tumor-infiltrating CD8+ T cells and an increase in Foxp3+ regulatory T cells. Our data, therefore, suggest that the IDO1 signaling function can also occur in tumor cells and that alternative therapeutic approach strategies should be undertaken to effectively tackle this important immune checkpoint molecule.


Subject(s)
Melanoma, Experimental , Tryptophan , Mice , Humans , Animals , CD8-Positive T-Lymphocytes/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Immune Checkpoint Proteins , Melanoma, Experimental/genetics , Signal Transduction
4.
Contraception ; 65(4): 301-4, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12020783

ABSTRACT

The lipid metabolism in sperm cells is important both for energy production and for cell structure. A special composition of membrane phospholipids, rich in polyunsaturated fatty acids (PUFA), and the different composition of sperm and immature germ cell membrane are described and discussed. Testis germ cells as well as epididymal maturing spermatozoa are endowed with enzymatic and non-enzymatic scavenger systems to prevent lipoperoxidative damage. Catalase, superoxide dismutase, and glutathione-dependent oxidoreductases are present in variable amounts in the different developmental stages. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity and roles in caput and cauda epididymal sperm cells are discussed. Also seminal plasma has a highly specialized scavenger system that defends the sperm membrane against lipoperoxidation and the degree of PUFA insaturation acts to achieve the same goal. Systemic predisposition and a number of pathologies can lead to an anti-oxidant/pro-oxidant disequilibrium. Scavengers, such as glutathione can be used to treat these cases as they can restore the physiological constitution of PUFA in the cell membrane.


Subject(s)
Cell Membrane/chemistry , Fatty Acids, Unsaturated/analysis , Glutathione Peroxidase/physiology , Glutathione/physiology , Spermatozoa/ultrastructure , Animals , Free Radical Scavengers/analysis , Glutathione/therapeutic use , Humans , Infertility, Male/metabolism , Infertility, Male/therapy , Lipid Peroxidation , Male , Phospholipid Hydroperoxide Glutathione Peroxidase , Semen/chemistry , Spermatozoa/chemistry
5.
Gut ; 49(4): 506-11, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11559647

ABSTRACT

BACKGROUND: It has been suggested that serological screening for coeliac disease (CD) should be performed in patients with chronic unexplained hypertransaminasaemia. AIMS: To evaluate the specificity for CD diagnosis of serum IgA antitissue transglutaminase (tTG) determination in consecutive patients with chronic hypertransaminasaemia using the most widely utilised ELISA based on tTG from guinea pig as the antigen. PATIENTS AND METHODS: We studied 98 patients with chronic hypertransaminasaemia, evaluated for the first time in a hepatology clinic. Serum anti-tTG and antiendomysial (EmA) assays were performed. Patients positive for EmA and/or anti-tTG were proposed for intestinal biopsy. Finally, all sera were reassayed for anti-tTG using an ELISA based on human recombinant tTG as the antigen. RESULTS: A total of 94/98 hypertransaminasaemic patients were positive for hepatitis virus markers, with 82/98 (83%) positive for anti-hepatitis C virus. Liver histology showed that most patients had mild or moderate chronic hepatitis while severe fibrosis or overt liver cirrhosis was found in 20/98. CD screening showed that 15/98 (16%) hypertransaminasaemic subjects had anti-tTG values in the same range as CD patients; however, IgA EmA were positive in only 2/98 (2%). Distal duodenal biopsy, performed in nine patients, showed subtotal villous atrophy in the two EmA+/anti-tTG+ patients but was normal in 7/7 EmA-/anti-tTG+ subjects. The presence of anti-tTG+ values in EmA- patients was unrelated to particular gastrointestinal symptoms, other associated diseases, severity of liver histology, or distribution of viral hepatitis markers. There was a significantly higher frequency of positive serum autoantibodies (antinuclear, antimitochondrial, antismooth muscle, and anti-liver-kidney microsomal antibodies) in anti-tTG+/EmA- patients than in the other subjects (9/13 v 10/83; p<0.003). Also, a correlation was found between serum gamma globulin and anti-tTG values (p<0.01). When sera were tested with the ELISA based on human tTG as the antigen, no false positive results were observed: only the two EmA+ patients with atrophy of the intestinal mucosa were positive for anti-tTG while all others were negative, including those false positive in the ELISA based on guinea pig tTG as the antigen. CONCLUSIONS: In patients with elevated transaminases and chronic liver disease there was a high frequency of false positive anti-tTG results using the ELISA based on tTG from guinea pig as the antigen. Indeed, the presence of anti-tTG did not correlate with the presence of EmA or CD. These false positives depend on the presence of hepatic proteins in the commercial tTG obtained from guinea pig liver and disappear when human tTG is used as the antigen in the ELISA system. We suggest that the commonly used tTG ELISA based on guinea pig antigen should not be used as a screening tool for CD in patients with chronic liver disease.


Subject(s)
Celiac Disease/enzymology , Enzyme-Linked Immunosorbent Assay/methods , Hepatitis, Viral, Human/enzymology , Liver Cirrhosis/enzymology , Transaminases/blood , Transglutaminases/immunology , Adolescent , Adult , Animals , Autoantibodies/immunology , Celiac Disease/complications , Chronic Disease , False Positive Reactions , Female , Guinea Pigs , Hepatitis, Viral, Human/complications , Humans , Immunoglobulin A/immunology , Linear Models , Liver Cirrhosis/complications , Male , Middle Aged , Predictive Value of Tests , Sensitivity and Specificity , Statistics, Nonparametric
6.
J Pediatr Gastroenterol Nutr ; 32(2): 178-81, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11321389

ABSTRACT

BACKGROUND: Several proinflammatory cytokines are involved in the pathogenesis of inflammatory bowel diseases. A significant role has been given to tumor necrosis factor alpha (TNF-alpha) as a guide proinflammatory cytokine. Thalidomide selectively reduces TNF-alpha production by inflammatory cells. The aim of the study was to assess the efficacy of thalidomide to induce and maintain remission in refractory Crohn disease. METHODS: The decision to administer thalidomide was made on the basis of patient intolerance or resistance to conventional medical treatment or as the last medical resort before surgical intervention. Only 5 of 96 patients with inflammatory bowel disease satisfied these criteria. All five patients had Crohn disease (male: mean age, 17 years). Thalidomide was administered at night at a dose of 1.5-2 mg/kg/day. The Pediatric Crohn Disease Activity Index, modified Harvey-Bradshaw scores, and steroids reduction were used to assess clinical response. RESULTS: Disease activity decreased consistently in four patients with a reduction of mean Pediatric Crohn Disease Activity Index from 36,9 to 2,5 and the mean Harvey-Bradshaw from 8.5 to 0.75 after 3 months of treatment. Steroid treatment (mean dose, 35 mg/day before treatment) was tapered and then discontinued, in four patients, within 1-3 months. Four patients are in remission after 19-24 months of treatment. The fifth patient discontinued thalidomide after 1 week because of distal paresthesia. CONCLUSION: Thalidomide seems to be an effective and safe treatment in patients with refractory Crohn disease. This is the first report of long-term use of thalidomide in refractory Crohn disease in pediatric patients.


Subject(s)
Crohn Disease/drug therapy , Immunosuppressive Agents/therapeutic use , Thalidomide/therapeutic use , Tumor Necrosis Factor-alpha/biosynthesis , Adolescent , Adult , Humans , Immunosuppressive Agents/pharmacology , Male , Recurrence , Remission Induction , Safety , Thalidomide/pharmacology , Time Factors , Treatment Outcome , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/drug effects
7.
J Mol Microbiol Biotechnol ; 2(4): 575-9, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11075934

ABSTRACT

We have previously demonstrated that the alphaMbeta2 integrin (known as CR3 or Mac-1) expressed on neutrophils (PMNs) and/or on CHO Mac-1 transfected cells,in the presence of serum complement binds B. burgdorferi and promotes an increased non -opsonic adhesion, in the presence of serum complement. In this study we demonstrate that: 1) living motile B. burgdorferiand recombinant lipidated OspA and OspC, up-regulate CR3 expression on PMNs; 2) in the absence of serum, B. burgdorferi induces increased adhesion of CHO cells expressing CR3 to fibronectin, an extracellular matrix protein. Both the I-domain and the lectin-like domain of CR3 are involved in the binding recognition and activation because mAb anti I-domain and N-acetyl-glucosamine inhibit cell adhesion to fibronectin. These data indicate that B. burgdorferi whole cells, but not Osps, activate CR3 integrin; since this receptor plays a key role in priming neutrophils to important inflammatory events, the interaction of B. burgdorferi with neutrophils via the CR3 may enhance their role both in defence and in disease.


Subject(s)
Antigens, Bacterial , Borrelia burgdorferi Group/physiology , Fibronectins/physiology , Macrophage-1 Antigen/genetics , Neutrophils/microbiology , Neutrophils/physiology , Animals , Antibodies, Monoclonal/pharmacology , Antigens, Surface/metabolism , Bacterial Adhesion , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , Binding Sites , CHO Cells , Cell Adhesion/physiology , Cricetinae , Gene Expression Regulation , Humans , In Vitro Techniques , Lipoproteins/metabolism , Lyme Disease Vaccines/metabolism , Macrophage-1 Antigen/physiology , Recombinant Proteins/metabolism , Transfection , Up-Regulation
8.
Front Biosci ; 5: E1-E15, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10702376

ABSTRACT

The lipid metabolism in sperm cells is important both as one of the main sources for energy production and for cell structure. The double leaflets of the membrane should be considered not simply as a passive lipid film, but as a very specialized structure. The complete maturation of the sperm cell membrane is attained after testicular lipid biosynthetic processes and after passage through the epididymis. A special composition of membrane phospholipids, rich in polyunsaturated fatty acids (PUFA), and the different composition of sperm and immature germ cell membrane are described and discussed. Testis germ cells as well as epididymal maturing spermatozoa are endowed with enzymatic and non-enzymatic scavenger systems to prevent lipoperoxidative damage. Catalase, superoxide dismutase and GSH-dependent oxidoreductases are present in variable amounts in the different developmental stages. Phospholipid hydroperoxide GSH peroxidase (PHGPx) activity and alpha tochopherol of epididymal spermatozoa are considered in detail. Their distribution and roles in caput and cauda epididymal sperm cells are discussed. Seminal plasma also has a highly specialized scavenger system that defends the sperm membrane against lipoperoxidation and the degree of PUFA insaturation acts to achieve the same goal. Systemic predisposition and a number of pathologies can lead to an anti-oxidant/pro-oxidant disequilibrium. Scavengers, such as GSH, can be used to treat these cases as they can restore the physiological constitution of PUFA in the cell membrane. The results of GSH therapy are presented and discussed.


Subject(s)
Fatty Acids, Unsaturated/metabolism , Membrane Lipids/metabolism , Phospholipids/metabolism , Spermatozoa/metabolism , Animals , Cell Membrane/metabolism , Free Radical Scavengers/metabolism , Glutathione/metabolism , Glutathione/therapeutic use , Humans , Infertility, Male/drug therapy , Infertility, Male/metabolism , Male , Reactive Oxygen Species/metabolism
9.
Biol Reprod ; 59(4): 753-8, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9746722

ABSTRACT

Mammalian caput and cauda epididymidal spermatozoa exhibit diverse stages of maturation, and their plasma membrane shows diverse composition and stability levels, thus enabling these spermatozoa to undergo the acrosomal reaction after transit through the epididymis. As a result, the study of antiperoxidative mechanisms is quite relevant, since epididymal spermatozoa must be properly protected against agents such as reactive oxygen species, which can impair the complex maturation process. We considered activities of certain enzymes (glutathione peroxidase [GPx], phospholipid hydroperoxide glutathione peroxidase [PHGPx], glutathione reductase [GR], superoxide dismutase [SOD], and catalase [CAT]) and the vitamin E content in isolated rat caput and cauda epididymidal spermatozoa. The results indicate that caput epididymidal sperm have significantly greater PHGPx (3.5x), GPx (2.4x), and SOD (1.7x) activities, as well as a greater amount of vitamin E (3.8x). There were no detectable differences in the GR and CAT activities of caput and cauda epididymidal spermatozoa. The substantial drop in PHGPx activity during epididymal transit is discussed in relation to an additional function of this enzyme: the use of caput sperm protamines as a sulfhydryl substrate. In vitro peroxidation of the two sperm populations by the free radical generator (azo-initiator) 2,2'-azobis(2-amidinopropane) dihydrochloride revealed that only about 13% of the vitamin E content of the caput epididymidal spermatozoa was consumed, which contrasts with the greater consumption (about 70%) of the vitamin in cauda epididymidal spermatozoa. Selective inhibition of PHGPx, SOD, or CAT did not change this picture. The higher susceptibility of cauda epididymidal spermatozoa to radicals is discussed in relation to the diverse enzymatic activities, vitamin E content, and peroxidative response. These factors are correlated with the different stages of sperm cell maturation, which are characterized-from caput to cauda epididymidis-by progressive destabilization of the plasma and acrosomal membranes.


Subject(s)
Antioxidants/metabolism , Epididymis/metabolism , Spermatozoa/metabolism , Animals , Epididymis/enzymology , Free Radicals/metabolism , In Vitro Techniques , Lipid Peroxides/metabolism , Male , Rats , Rats, Wistar , Spermatozoa/enzymology , Vitamin E/metabolism
10.
Biol Reprod ; 57(6): 1502-8, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9408261

ABSTRACT

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) is present, in both free and membrane-bound form, in several mammalian tissues. It utilizes thiols such as glutathione to specifically scavenge phospholipid hydroperoxides. The testis exhibits the highest PHGPx-specific activity so far measured, and interest in the presence and function of the enzyme in this tissue has recently grown. Here we report the localization of PHGPx in rat epididymal spermatozoa and its distribution in subfractions obtained by sucrose density gradient centrifugation. Immunochemical evidence and enzymatic activity revealed for the first time that PHGPx is present in sperm heads and tail midpiece mitochondria. The binding of the enzyme to spermatozoa, head, and mitochondria was barely affected by ionic strength or thiols or detergents, as compared to the detachment of PHGPx obtained from testis nuclei. Moreover, we demonstrated that pure PHGPx exhibits a higher thiol-oxidase activity toward isolated epididymal caput protamines than toward protamines from epididymal cauda. These results suggest a role for the enzyme in the maturation of spermatozoa through the metabolism of hydroperoxides and sperm thiol oxidation, in addition to its serving as an antioxidant protector.


Subject(s)
Epididymis/cytology , Glutathione Peroxidase/analysis , Glutathione Peroxidase/metabolism , Spermatozoa/enzymology , Animals , Cell Fractionation , Cell Nucleus/enzymology , Centrifugation, Density Gradient , Detergents/pharmacology , Male , Microscopy, Electron , Mitochondria/enzymology , Osmolar Concentration , Phospholipid Hydroperoxide Glutathione Peroxidase , Rats , Sperm Head/enzymology , Sperm Tail/enzymology , Spermatozoa/ultrastructure , Sulfhydryl Compounds/pharmacology , Testis/enzymology , Testis/ultrastructure
11.
Biochem Mol Med ; 59(2): 118-24, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8986633

ABSTRACT

In rat testis nuclei the activity of the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) is much higher than in other tissues and subcellular compartments, with the sole exception of mitochondria. In nuclei, the bound enzyme is solubilized by DNase I treatment, thus suggesting a binding to chromatin. Treatment with ionic strength releases about 70% of bound PHGPx, suggesting that electrostatic bonds are involved. Immunogold electron microscopy indicates the association of PHGPx with chromatin structures in isolated nuclei. A possible interpretation of these data is a PHGPx protective role against DNA peroxidative damage. Furthermore, in agreement with kinetic and structural information, PHGPx-chromatin binding could suggest an hypothetical thiol oxidase activity toward specific thiol bearing proteins which could substitute for GSH as alternative donor substrates. Such activity could give to the enzyme a new important function which is not only protective but also has a specific regulatory function in chromatin condensation.


Subject(s)
Cell Nucleus/enzymology , Chromatin/metabolism , Glutathione Peroxidase/metabolism , Testis/enzymology , Animals , Cell Nucleus/metabolism , Male , Microscopy, Immunoelectron , Phospholipid Hydroperoxide Glutathione Peroxidase , Protein Binding , Rats , Rats, Wistar , Static Electricity , Subcellular Fractions/enzymology , Subcellular Fractions/metabolism
12.
Biochem Mol Med ; 58(2): 221-6, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8812743

ABSTRACT

Rat testis mitochondria contain large amounts of both seleno-enzyme phospholipid hydroperoxide glutathione peroxidase (EC 1.11.1.12, PHGPx) and alpha-tocopherol. The scavenger role of vitamin E consists of transforming the lipoperoxyl radicals into lipid hydroperoxides, thus interrupting the peroxidative cascade. These hydroperoxides are in turn substrates of the PHGPx, which is considered one of the most important specific enzymes capable of protecting, in situ, the membranes from lipid peroxidation. A connection or synergism could, therefore, be envisaged between vitamin and enzyme opposing lipid damage in the mitochondria. Here we present data concerning the HPLC evaluation of vitamin E consumption in rat testis mitochondria and mitochondrial membranes, under different conditions of PHGPx activity, after Fe2+-induced lipid peroxidation. We have found that the enzyme activity, under the conditions tested, does not spare vitamin E from its peroxidation, therefore indicating that the postulated synergism between PHGPx and alpha-tocopherol can be excluded in rat testis mitochondria.


Subject(s)
Glutathione Peroxidase/metabolism , Iron/pharmacology , Lipid Peroxidation/drug effects , Testis/metabolism , Vitamin E/metabolism , Age Factors , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Chromatography, High Pressure Liquid , Glutathione/metabolism , Glutathione Peroxidase/antagonists & inhibitors , Iodoacetates/pharmacology , Iodoacetic Acid , Male , Membrane Lipids/metabolism , Mercaptoethanol/metabolism , Mitochondria/enzymology , Mitochondria/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase , Phospholipids/metabolism , Rats , Rats, Inbred Strains , Testis/ultrastructure
13.
Biochim Biophys Acta ; 1191(1): 147-50, 1994 Apr 20.
Article in English | MEDLINE | ID: mdl-8155669

ABSTRACT

The distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat testis mitochondria was investigated, using a reverse sucrose density gradient centrifugation procedure for the separation of the inner and outer membranes and the contact sites between the two membranes. The results indicate that PHGPx is largely localized in the contact sites fraction. This finding might therefore suggest that the enzyme has more than just an antioxidant function.


Subject(s)
Glutathione Peroxidase/metabolism , Mitochondria/enzymology , Testis/enzymology , Animals , Brain/enzymology , Intracellular Membranes/enzymology , Liver/enzymology , Male , Phospholipid Hydroperoxide Glutathione Peroxidase , Rats
14.
Neurosci Lett ; 169(1-2): 167-70, 1994 Mar 14.
Article in English | MEDLINE | ID: mdl-7519336

ABSTRACT

The inside out configuration of the patch-clamp technique was used to study single-channel anionic currents from purified hippocampal synaptosomes fused into liposomes to form giant proteoliposomes. At least six different anionic channels with unitary conductances of 22-150 pS were found. The most frequently observed was the 32-pS conductance channel. This was voltage-dependent; the open probability increased from 0.20 at -40 mV to 0.46 at 40 mV. This channel may be involved in the repolarization of nerve terminal membranes after an action potential, thus, limiting the duration of the spike and the transmitter release.


Subject(s)
Hippocampus/metabolism , Ion Channels/metabolism , Synaptosomes/metabolism , Animals , Anions/metabolism , Electrophysiology , Hippocampus/ultrastructure , In Vitro Techniques , Liposomes/metabolism , Membranes/metabolism , Rats , Rats, Wistar
15.
Eur Biophys J ; 20(6): 311-9, 1992.
Article in English | MEDLINE | ID: mdl-1373115

ABSTRACT

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM], derived from mouse liver, showed the presence of currents corresponding to low conductances (less than 50 pS), as well as of four distinct conductances of 99 pS, 152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).


Subject(s)
Intracellular Membranes/metabolism , Ions , Mitochondria/metabolism , Animals , Brain/ultrastructure , Guinea Pigs , Intracellular Membranes/physiology , Ion Channels/metabolism , Ion Channels/physiology , Liposomes , Membrane Potentials/physiology , Mice , Mitochondria/physiology , Mitochondria, Liver/metabolism , Mitochondria, Liver/physiology , Permeability
16.
FEBS Lett ; 290(1-2): 35-7, 1991 Sep 23.
Article in English | MEDLINE | ID: mdl-1915888

ABSTRACT

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated, using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.


Subject(s)
Brain/enzymology , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , Mitochondria/enzymology , Animals , Brain/ultrastructure , Intracellular Membranes/enzymology , Male , Rats , Subcellular Fractions/enzymology
17.
FEMS Microbiol Immunol ; 3(1): 33-8, 1991 Feb.
Article in English | MEDLINE | ID: mdl-1711876

ABSTRACT

SDS-PAGE and Western immunoblot profiles have been determined for different strains of Borrelia burgdorferi. Major proteins of 60 kDa, 41 kDa corresponding to flagellin, 34-36 kDa and 30-31 kDa corresponding to OspB and OspA respectively, and 18-20 kDa corresponding to 'pC' fractions were detected. A "rough" lipopolysaccharide which we called lipooligosaccharide (LOS) of 8-11 kDa appeared to be present, being detected by specific silver staining, as in crude Borrelia lysates as in proteinase K digested Borrelia strains, quite similar in shape among the different strains examined. The LOS reacted in Western blotting with immune anti-B. burgdorferi rabbit serum and also with sera collected from humans affected by Lyme borreliosis. The LOS did not react with sera positive for syphilis or leptospirosis, and their immunological specificity is discussed.


Subject(s)
Antigens, Bacterial/immunology , Borrelia burgdorferi Group/immunology , Lipopolysaccharides/immunology , Animals , Antibody Specificity , Bacterial Outer Membrane Proteins/immunology , Blotting, Western , Borrelia Infections/immunology , Electrophoresis, Polyacrylamide Gel , Endopeptidase K , Epitopes , Flagellin/immunology , Humans , Leptospirosis/immunology , Lyme Disease/immunology , Rabbits , Serine Endopeptidases/metabolism , Serotyping , Syphilis/immunology
18.
Biochim Biophys Acta ; 1035(3): 300-5, 1990 Sep 14.
Article in English | MEDLINE | ID: mdl-2207125

ABSTRACT

H2O2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H2O2 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H2O2 generated by monoamine oxidase. The H2O2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not alpha-ketoglutarate + NH4+. These results suggest that the removal of H2O2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H2O2 generated by monoamine oxidase enhances Ca2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca2+ efflux.


Subject(s)
Brain/metabolism , Calcium/metabolism , Glutathione/metabolism , Hydrogen Peroxide/metabolism , Mitochondria/metabolism , Monoamine Oxidase/metabolism , Animals , Brain/enzymology , Glutathione/pharmacology , Kinetics , Mitochondria/drug effects , Mitochondria/enzymology , NADP/metabolism , Rats
19.
J Biol Chem ; 265(2): 908-13, 1990 Jan 15.
Article in English | MEDLINE | ID: mdl-1688556

ABSTRACT

From morphological and biochemical studies it has been recognized that the regions where the outer and inner membranes of mitochondria come in close contact (contact sites) can be the route mechanism through which mitochondria interact directly with the cytoplasm. We have studied these regions electrophysiologically with the patch clamp technique, with the aim of understanding if this direct interaction is mediated by high conductance ion channels similar to the channel already detected in the inner membrane of mitochondria (Sorgato M. C., Keller, B. U., and Stühmer, W. (1987) Nature 330, 498-500). Contact sites isolated from rat brain mitochondria were thus incorporated into liposomes subsequently enlarged sufficiently to be patch clamped. This study shows that these particular fractions contain ion channels with conductances ranging from approximately 5 picosiemens to 1 nanosiemens (in symmetrical 150 mM KCl). Most of these channels are not voltage-dependent and can be open at physiological potentials sustained by respiring mitochondria. The lack of voltage sensitivity seems not to be the outcome of methodological artifacts, as voltage-gated channels are detected in giant liposomes containing either the outer mitochondrial membrane or a partially purified fraction of the inner mitochondrial membrane. These data therefore indicate that channels present in mitochondrial contact sites have properties which render them amenable to perform several of the functions hypothesized for these regions, particularly that of translocating macromolecules from the cytoplasm to the matrix of mitochondria.


Subject(s)
Brain/physiology , Ion Channels/physiology , Mitochondria/physiology , Animals , Cattle , In Vitro Techniques , Ion Channel Gating , Liposomes , Membrane Potentials , Mitochondria, Heart/physiology , Rats
20.
Zentralbl Bakteriol Mikrobiol Hyg A ; 269(3): 277-83, 1988 Nov.
Article in English | MEDLINE | ID: mdl-2464258

ABSTRACT

Purified preparations of lipopolysaccharides (LPS) extracted from two different strains of Leptospira interrogans have been electrophoretically analyzed in order to determine their location at the level of outer envelope (OE). Evidence has been collected for the presence of some LPS fractions in the OE, suggesting that a part of this molecule is embedded in the membrane structure. The serological specificity of the LPS has been in addition tested by means of monoclonal antiserovar antibodies (Moabs); the results indicated that the LPS structure is endowed of the immunodeterminants of the serovar. The remarkable relevance of this finding for the Leptospira taxonomy is discussed.


Subject(s)
Leptospira interrogans/analysis , Lipopolysaccharides/analysis , Antibodies, Monoclonal/immunology , Antibody Specificity , Cell Membrane/analysis , Electrophoresis, Polyacrylamide Gel , Epitopes , Leptospira interrogans/classification , Leptospira interrogans/ultrastructure , Lipopolysaccharides/immunology , Precipitin Tests
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