Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida.

Abstract Cobalamin (vitamin B(12)) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B(12). A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B(12)-binding protein precursor of P. profundum, 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida, but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida, the recombinant protein was expressed with a C-terminal His(6)-tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 +/- 2 microm.

Different prelamin A forms accumulate in human fibroblasts: a study in experimental models and progeria.

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects.We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.

Different prelamin A forms accumulate in human fibroblasts: a study in experimental models and progeria.

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.

Endogenous oxidative stress induces distinct redox forms of tumor necrosis factor receptor-1 in melanoma cells.

Receptors of the TNFR superfamily possess abundant thiols in their extracellular domains, which makes them susceptible to redox modulation by prooxidant agents and processes. Previous studies from our laboratory have documented that membrane gamma-glutamyltransferase (GGT) activity can originate reactive oxygen species in the extracellular milieu, during the GGT-mediated metabolism of extracellular glutathione. The present study was aimed thus to verify a possible redox-modulating effect of GGT activity on TNFR1 receptors. The thiol-specific probe maleimide-polyethylene glycol was used to selectively label the reduced thiol groups in proteins of cell lysates; fractions corresponding to TNFR1 were then identified by immunoblot. In human melanoma Me665/2 cells, expressing varying GGT levels, at least five distinct forms of TNFR1 have been thus identified. The more oxidized forms appear to be prevalent in the 2/60 clone, expressing higher GGT levels, as compared to clone 2/21. Stimulation of GGT activity in the latter induced an increase of the oxidized TNFR1 forms. It is conceivable that different redox states of TNFR1 may correspond to different binding affinity and/or changes in the transducing function of the receptor. As GGT is frequently expressed by malignant tumors, the described phenomena might concur to alter the sensitivity of cancer cells to agents targeted on activation of TNF-alpha-dependent signaling pathways.

Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL.

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freund's adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freund's adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.

Gamma-glutamyltranspeptidase-dependent glutathione catabolism results in activation of NF-kB.

gamma-glutamyltranspeptidase (GGT) is a key enzyme implicated in the homeostasis of intracellular reduced glutathione (GSH) and hence in the regulation of the cellular redox state. Besides, the extracellular cleavage of GSH by GGT leads to reactive oxygen species (ROS) production, depending on the generation and enhanced reactivity of cysteinylglycine (CysGly). Using a model cell line, the V79 GGT, which highly expresses a human GGT transgene, we examined whether the GGT induced oxidant stress could modulate intracellular transcription factors. For the first time, we show that GGT-dependent ROS production induces the NF-kB-binding and transactivation activities. This induction mimicked the one observed by H(2)O(2) and was inhibited by catalase, suggesting the involvement of H(2)O(2) in the NF-kB activation.

Membrane gamma-glutamyl transpeptidase activity of melanoma cells: effects on cellular H(2)O(2) production, cell surface protein thiol oxidation and NF-kappa B activation status.

The metabolism of glutathione by membrane-bound &ggr;-glutamyl transpeptidase (GGT) has been recently recognized as a basal source of hydrogen peroxide in the extracellular space. Significant levels of GGT activity are expressed by malignant tumours, and in melanoma cell lines they were found to correlate with the malignant behaviour. As hydrogen peroxide and other oxidants can affect signal transduction pathways at several levels, the present study was aimed to verify: (i) the occurrence of GGT-dependent production of hydrogen peroxide in melanoma cells; (ii) the effects of GGT-dependent prooxidant reactions on known redox-sensitive cellular targets, i.e. protein thiols, the nuclear transcription factor NF-kappa B and p53. Two melanoma Me665/2 cell clones, exhibiting traces of (clone 2/21) or high (clone 2/60) GGT activity, were studied. The occurrence of GGT-dependent production of hydrogen peroxide was apparent in 2/60 cells, in which it was accompanied by lower levels of cell surface protein thiols. In 2/60 cells, GGT expression was also associated with higher levels of NF-kappa B activation, as compared to GGT-poor 2/21 cell clone. Indeed, stimulation or inhibition of GGT activity in 2/60 cells resulted in progressive activation or inactivation of NF-kappa B, respectively. An analysis of the p53 gene product indicated lack of protein expression in 2/60 cells, whereas a mutant protein was highly expressed in 2/21 cells. Taken together, these results indicate that the expression of GGT activity can provide melanoma cells with an additional source of hydrogen peroxide, and that such prooxidant reactions are capable to modify protein thiols at the cell surface level. In addition, GGT expression results in an up-regulation of the transcription factor NF-kappa B, which could explain the higher metastatic behaviour reported for GGT-rich melanoma cells as compared to their GGT-poor counterparts.

The visualization of oxidant stress in tissues and isolated cells.

Many studies have implicated the role of oxidant stress in a wide range of human diseases and have led to the rapid expansion of research in this area. With many experimental approaches a direct detection of the production of reactive oxygen species (ROS) and free radicals is not possible. Free radicals are very reactive, short-lived and react in a non-specific way, so that ongoing oxidative damage is generally analyzed by measurement of secondary products e.g. H2O2, "oxidized" proteins, peroxidized lipids and their break-down products, "oxidized" DNA or by fluorographic analysis in combination with fluorescent dyes e.g. dichlorofluorescin (DCFH). The histochemical visualization of selected molecular markers for oxidative phenomena can often provide valuable information concerning the distribution of oxidative processes in vivo. A number of biochemical methods are available for the monitoring of almost all oxidant stress-related processes, although their applicability in vivo is limited. This review summarizes the biochemical methods currently available for histochemical detection and indirect visualization of an excess of free radicals and ROS. The cited methods are discussed and the results obtained from their application are critically evaluated.

Erythremic myelosis (AML6er) in a cat.

A 4-year-old male European domestic cat was presented with dysorexia, weakness and depression. Normocytic normochromic non-regenerative anaemia, leucopaenia and thrombocytopenia were detected. Rubriblasts were detected both in the blood and in the bone marrow. Tests of blood chemistry revealed no alterations of renal and hepatic function and a positive reaction to FeLV antigen was detected in the cat's serum. Neoplastic cells did not show positive to cytochemical reactions against granulocytes, monocytes and lymphocytes. According to haematological and bone marrow cytological findings, a diagnosis of erythremic myelosis (AML6er) was made. Histopathology showed extramedullary haematopoiesis in the liver, spleen, kidney and lymph nodes and chronic nephropathy and degenerative signs in the liver.

Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of gamma-glutamyl transpeptidase-dependent H2O2 production and S-thiolation.

The expression of gamma-glutamyl transpeptidase (GGT), a plasma membrane ectoenzyme involved in the metabolism of extracellular reduced glutathione (GSH), is a marker of neoplastic progression in several experimental models, and occurs in a number of human malignant neoplasms and their metastases. Because it favors the supply of precursors for the synthesis of GSH, GGT expression has been interpreted as a member in cellular antioxidant defense systems. However, thiol metabolites generated at the cell surface during GGT activity can induce prooxidant reactions, leading to production of free radical oxidant species. The present study was designed to characterize the prooxidant reactions occurring during GGT ectoactivity, and their possible effects on the thiol redox status of proteins of the cell surface. Results indicate that: (i) in U937 cells, expressing significant amounts of membrane-bound GGT, GGT-mediated metabolism of GSH is coupled with the extracellular production of hydrogen peroxide; (ii) GGT activity also results in decreased levels of protein thiols at the cell surface; (iii) GGT-dependent decrease in protein thiols is due to sulfhydryl oxidation and protein S-thiolation reactions; and (iv) GGT irreversible inhibition by acivicin is sufficient to produce an increase of protein thiols at the cell surface. Membrane receptors and transcription factors have been shown to possess critical thiols involved in the transduction of proliferative signals. Furthermore, it was suggested that S-thiolation of cellular proteins may represent a mechanism for protection of vulnerable thiols against irreversible damage by prooxidant agents. Thus, the findings reported here provide additional explanations for the envisaged role played by membrane-bound GGT activity in the proliferative attitude of malignant cells and their resistance to prooxidant drugs and radiation therapy.

Gamma-glutamyl transpeptidase-dependent iron reduction and LDL oxidation--a potential mechanism in atherosclerosis.

BACKGROUND: gamma-Glutamyl transpeptidase (gamma-GT) is found in serum and in the plasma membranes of virtually all cell types. Its physiologic role is to initiate the hydrolysis of extracellular glutathione (GSH), a tripeptide in which cysteine lies between alpha-glycine and gamma-glutamate residues. Cysteine and other thiol compounds are known to promote LDL oxidation by reducing Fe(III) to redox active Fe(II); therefore, we sought to determine whether similar reactions can be sustained by GSH and influenced by gamma-GT. METHODS: Fe(III) reduction and LDL oxidation were studied by monitoring the formation bathophenanthroline-chelatable Fe(II) and the accumulation of thiobarbituric acid-reactive substances, respectively. Human atheromatous tissues were examined by histochemical techniques for the presence of oxidized LDL and their colocalization with cells expressing gamma-GT activity. RESULTS: A series of experiments showed that the gamma-glutamate residue of GSH affected interactions of the juxtaposed cysteine thiol with iron, precluding Fe(III) reduction and hence LDL oxidation. Both processes increased remarkably after addition of purified gamma-GT, which acts by removing the gamma-glutamate residue. GSH-dependent LDL oxidation was similarly promoted by gamma-GT associated with the plasma membrane of human monoblastoid cells, and this process required iron traces that can be found in advanced or late stage atheromas. Collectively, these findings suggested a possible role for gamma-GT in the cellular processes of LDL oxidation and atherogenesis. Histochemical analyses confirmed that this may be the case, showing that gamma-GT activity is expressed by macrophage-derived foam cells within human atheromas, and that these cells colocalize with oxidized LDL. CONCLUSIONS: Biochemical and histochemical correlates indicate that gamma-GT can promote LDL oxidation by hydrolyzing GSH into more potent iron reductants. These findings may provide mechanistic clues to the epidemiologic evidence for a possible correlation between persistent elevation of gamma-GT and the risk of fatal reinfarction in patients with ischemic heart disease.

Modulation of human T lymphocyte proliferation by 4-hydroxynonenal, the bioactive product of neutrophil-dependent lipid peroxidation.

The proliferative capacity of immune cells is known to be sensitive to conditions of oxidative stress and lipid peroxidation. We tested the hypothesis that activated neutrophils can induce peroxidation in extracellular lipid substrates, and evaluated the effects of 4-hydroxy-2,3-trans-nonenal (4-HNE)--the most reactive aldehydic product of lipid peroxidation--on mitogen-induced proliferation of human T lymphocytes. Neutrophils activated in the presence of extracellular lipid substrates (liposomes, cellular membranes) induced lipid peroxidation. By means of cytoimmunofluorescent labeling and confocal microscopy, the binding of 4-HNE to surface and cytoplasmic proteins of activated neutrophils was observed. Short (20 min) pre-treatment of cells with low concentrations of 4-HNE were able to dose-dependently decrease the proliferation of human peripheral blood lymphocytes challenged with PHA or anti-CD3 monoclonal antibody OKT3, as well as the proliferation of a tetanus specific human T-cell line challenged with tetanus toxoid. In these conditions, the binding of 4-HNE to surface and cytoplasmic proteins of lymphocytes was also observed. When the proliferative capacity of peripheral blood lymphocytes was monitored over several days after 4-HNE treatment and PHA challenge, a recovery and a rebound in cell proliferation was observed. Data reported indicate that the lipid peroxidation promoted by activated neutrophils can exert modulatory effects on the responsivity of human T cells, through the action of its most reactive product, 4-HNE.

Selective colocalization of lipid peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of gamma-glutamyltranspeptidase activity.

A number of studies indicate that cell proliferation can be modulated by changes in the redox balance of (soluble and protein) cellular thiols. Free radical processes, including lipid peroxidation (LPO), can affect such a balance, and a role for LPO in multistage carcinogenesis has been envisaged. The present study was aimed to assess the relationships between the protein thiol redox status and the LPO process in chemically induced preneoplastic tissue. The Solt-Farber's initiation-promotion model of chemical carcinogenesis in the rat liver was used. In fresh cryostat sections, preneoplastic lesions were identified by the reexpression of gamma-glutamyltranspeptidase (GGT) activity. In serial sections, different classes of protein thiols were stained; in additional sections, LPO was elicited by various prooxidant mixtures and determined thereafter by the hydroxynaphthoic hydrazide-Fast Blue B procedure. The incubation of sections in the presence of chelated iron plus substrates for GGT activity leads to the development of LPO in selected section areas closely corresponding to GGT-positive lesions, indicating the ability of GGT activity to initiate LPO. Protein-reactive thiols, as well as total protein sulfur, were decreased by 20-25% in cells belonging to GGT-positive preneoplastic nodules, suggesting the occurrence of oxidative conditions in vivo. The incubation of additional adjacent sections with the prooxidant mixture H2O2 plus iron(II), in order to induce the complete oxidation of lipid present in the section, showed a decreased basal concentration of oxidizable lipid substrate in GGT-rich areas. The decreased levels of both protein thiols and lipid-oxidizable substrate in GGT-positive nodules suggest that the observed GGT-dependent pathway of LPO initiation can be chronically operative in vivo during early stages of chemical carcinogenesis, in cells expressing GGT as part of their transformed phenotype.

Single-cell investigation by laser scanning confocal microscopy of cytochemical alterations resulting from extracellular oxidant challenge.

Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound 4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2, 4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow "redox phenotyping" of isolated cells, which would provide an efficient tool in selected experimental models.

Kinetics of excretion in milk of some antimastitis drugs.

A study on the kinetics of excretion in milk of some antimastitis drugs is described. The five antibiotic molecules used were detected by confronting two methods: the Galesloot-Hassing method which, even though specific for the detection of penicillin in milk, is officially used for the detection of antibiotic residues in general; and the M. luteus paper disk assay, which uses M. luteus ATCC 9341 as test microorganism. The obtained results show that withdrawal times for the assayed molecules are longer than those declared and that M. luteus is more efficient for the detection of kanamycin residues.

[Reflex therapy in respiratory allergy]. / Riflessoterapie nell'allergia respiratoria.

The immune system and factors probably involved in respiratory allergies in certain subjects examined. Subjects were grouped according to age and sex and treated using both conventional methods, specific acupuncture and a placebo. The statistically evaluated results give an encouraging insight into the application of acupuncture within this delicate and complex field of human pathology.