Expression and characterization of two new alkane-inducible cytochrome P450s from Trichoderma harzianum.

n-Dodecane and fatty acids were good inducers of cytochrome P450 (CYP) and the ω-hydroxylase of lauric acid, which is a marker for ω-hydroxylation of n-alkanes, in Trichoderma harzianum. A cDNA, containing an ORF of 1520 bp, encoding a CYP52 of 520 amino acids, was isolated by RACE. Another n-alkane-inducible CYP was identified by LLC-MS/MS analysis of a microsomal protein band induced by n-dodecane in a library of T. harzianum. This suggests that T. harzianum has a CYP-dependent conversion of alkanes to fatty acids allowing their incorporation into lipids.

Spontaneous chromosome loss and colcemid resistance in lymphocytes from patients with myotonic dystrophy type 1.

Myotonic Dystrophy type 1 (DM1) is one of the many inherited human diseases whose molecular defect is the expansion of a trinucleotide DNA sequence. DM1 shares with fragile X syndrome (FMR1), another "unstable triplet syndrome", several molecular features not present in the remaining triplet diseases. As FMR1 is also characterised by chromosome instability at the site of the expanded triplet, lymphocytes from DM1 patients and healthy donors were cultured for micronucleus (MN) analysis, in order to verify if DM1 is also prone to chromosome instability. A FISH analysis was also carried out to detect the presence of centromeric sequences in the observed MN. The data indicate that DM1 patients present a percentage of centromere-positive MN significantly higher than controls, suggesting that chromosome loss is the main mechanism underlying the origin of the increased spontaneous instability. To further assess the proneness to instability of cells of DM1 patients, cultures from patients and controls were treated in vitro with growing concentrations of two different mutagens: colcemid, a "pure" aneugen compound whose target is tubulin, and mytomicin C, a strong clastogen. The results show that the patient group is significantly less sensitive to colcemid. These data, together with FISH analysis, suggest the presence, in DM1 patients, of an already damaged tubulin, which becomes no more sensitive to the effect of colcemid and which could be the main defect underlying the aneugenic effects in DM1.

An HLA-DRB alpha-helix motif shared by DR11 and DR8 alleles is implicated in the pluriallelic restriction of peptide-specific T-cell lines.

The T-cell recognition of HLA-DR-peptide complexes is generally restricted by the polymorphism of the DRB molecules but pluriallelic restriction has been described. The molecular basis of restriction and promiscuity of such peptide-specific responses is poorly understood. We isolated a panel of T-cell lines specific for the tetanus toxin peptide p2 (TT830-843) exhibiting pluriallelic restriction by DR11 and DR8 alleles. Fine restriction specificity of the T-cell lines was examined in functional assays against DR oligotyped APCs expressing different variants of DR11 and DR8 alleles. Our results show that (a) polymorphisms between serologically related alleles are relevant in terms of restriction of the peptide-specific T-cell response; in some instances, a single amino acid substitution can determine the restriction of a T-cell line; (b) different patterns of restriction are not the result of specific differences in DR-p2 binding as p2 peptide binds to all DR11 and DR8 alleles tested (DRB1* 1101, -1102, -1103, -1104, 110X, -0801, -0802, -0803, and -0806); and (c) pluriallelic restriction of the peptide-specific T-cell response correlates with the presence of a DRB1 alpha-helix motif (67-71-86) shared by some DR11 and DR8 alleles. Possible implications of pluriallelic restriction of peptide-specific T-cell response in autoimmune disorders associated with DR11 and DR8 are discussed.

Alteration of beta-cell constitutive NO synthase activity is involved in the abnormal insulin response to arginine in a new rat model of type 2 diabetes.

We have previously obtained a new type 2 diabetic syndrome in adult rats given streptozotocin and nicotinamide, characterized by reduced beta-cell mass, partially preserved insulin response to glucose and tolbutamide and excessive responsiveness to arginine. We have also established that the neuronal isoform of constitutive NO synthase (nNOS) is expressed in beta-cells and modulates insulin secretion. In this study, we explored the kinetics of glucose- and arginine-stimulated insulin release in perifused isolated islets as well as the effect of N-omega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, to get insight into the possible mechanisms responsible for the arginine hypersensitivity observed in vitro in this and other models of type 2 diabetes. A reduced first phase and a blunted second phase of insulin secretion were observed upon glucose stimulation of diabetic islets, confirming previous data in the isolated perfused rat pancreas. Exposure of diabetic islets to 10 mM arginine, in the presence of 2.8 mM glucose, elicited a remarkable monophasic increment in insulin release, which peaked at 639 +/- 31 pg/islet/min as compared to 49 +/- 18 pg/islet/min in control islets (P << 0.01). The addition of L-NAME to control islets markedly enhanced the insulin response to arginine, as expected from the documented inhibitory effect exerted by nNOS activity in normal beta-cells, whereas it did not further modify the insulin secretion in diabetic islets, thus implying the occurrence of a defective nNOS activity in these islets. A reduced expression of nNOS mRNA was found in the majority but not in all diabetic islet preparations and therefore cannot totally account for the absence of L-NAME effect, that might also be ascribed to post-transcriptional mechanisms impairing nNOS catalytic activity. In conclusion, our results provide for the first time evidence that functional abnormalities of type 2 experimental diabetes, such as the insulin hyper-responsiveness to arginine, could be due to an impairment of nNOS expression and/or activity in beta-cells.

A constitutive nitric oxide synthase modulates insulin secretion in the INS-1 cell line.

We provide immunocytochemical evidence that the neuronal isoform of constitutive NO synthase (cNOS) is expressed in the rat insulinoma cell line INS-1. Furthermore, using N omega-nitro-L-arginine methyl ester (L-NAME), a pharmacological inhibitor of cNOS activity, we show that this enzyme is implicated in the modulation of insulin secretion in INS-1 cells. Indeed, in the presence of 2.8 mM glucose, L-NAME induced a specific and dose-dependent increase in insulin release, suggesting that cNOS exerts an inhibitory tone on basal insulin secretion. Moreover, L-arginine, the physiological substrate of cNOS, significantly reduced the marked enhancing effect of L-NAME on insulin release and to a lesser extent, at low concentrations, that of 10 mM KCl. L-NAME also potentiated the insulin secretion stimulated by 5.5 and 8.3 mM glucose, but in this case, its effect was not reduced by L-arginine. In conclusion, our data show that the neuronal isoform of cNOS exerts a negative modulation on insulin secretion in INS-1 cells, confirming the previous results obtained in the isolated perfused rat pancreas or pancreatic islets.

Human GM-CSF interaction with the alpha-chain of its receptor studied using surface plasmon resonance.

A surface plasmon resonance (SPR) based biosensor has been used for studying the interaction of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) with genetically engineered alpha-chain subunits of its specific receptor (GM-Ralpha). Western blot analysis of GM-Ralpha confirmed the correct size (80 kDa) and reactivity of these proteins against anti-GM-Ralpha polyclonal or monoclonal antibodies. GM-CSF was immobilized, using standard amine coupling methods, to the dextran-modified gold biosensor surface in order to capture GM-Ralpha subsequently injected over the sensing layer. GM-Ralpha were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of GM-Ralpha with dilutions of soluble GM-CSF before injection of the mixture over the GM-CSF matrix, prevented ligand binding in a dose dependent manner. In contrast, unrelated soluble cytokines or serum proteins (e.g. G-CSF, albumin, etc.) were found to exert no inhibition. Complexes formation blockage by pre-incubation of constant amounts of GM-Ralpha with dilutions of neutralizing anti-GM-Ralpha antibodies was concentration dependent, further assessing the specificity of the interaction. To investigate the possibility of relating the effect on binding affinity of critical conformational changes at the contact site, experiments of multisite binding were performed, flowing a set of neutralizing monoclonal antibodies reacting to different epitopes on GM-CSF over the GM-CSF matrix, before injecting GM-Ralpha. The results indicated that antibody interaction with helix D and helix A of GM-CSF markedly inhibited GM-CSF binding to GM-Ralpha. Comparable results were obtained using the biosensor technology and enzyme-linked immunoassays, in representative experiments performed with the same reagents. These experiments demonstrate that SPR can be successfully used for studying complementary interactions between GM-CSF and its receptor alpha-chains in solution without using labels or secondary tracers and, compared with conventional immunoanalysis methods, significantly saving time.

BQ-123 inhibits both endothelin 1 and endothelin 3 mediated C6 rat glioma cell proliferation suggesting an atypical endothelin receptor.

The mitogenic action of endothelins (ETs) 1 and 3 was studied on C6 rat glioma cells in serum-free culture conditions. In order to characterize the ET receptor subtype involved in this effect, BQ-123, and ETA receptor selective antagonist was used. Our results confirmed that both ET-1 and ET-3 are mitogenic peptides for C6 cells and demonstrated for the first time that the ETA receptor antagonist BQ-123 inhibits the proliferative effect of both ET-1 and ET-3 in this cellular system, providing evidence of an atypical ET receptor on C6 cells.

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

An immunodominant epitope in a functional domain near the N-terminus of human granulocyte-macrophage colony-stimulating factor identified by cross-reaction of synthetic peptides with neutralizing anti-protein and anti-peptide antibodies.

We produced polyclonal and monoclonal antibodies (MAbs) against recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and performed studies of epitope mapping by ELISA, using five synthetic peptides corresponding to sequences along this molecule. Additionally, anti-peptide MAbs were generated. The antibody ability to inhibit rhGM-CSF activity was determined using as bioassay the MO7e cell line, which is dependent on hGM-CSF for growth in vitro. An immunodominant epitope able to induce the highest neutralization antibody titers was identified near the N terminus of hGM-CSF. A synthetic peptide 14-24, homologous to a sequence including part of the first alpha-helix of the molecule, was recognized by neutralizing anti-protein antibodies. Similarly, MAbs anti- 14-24 cross-reacted with rhGM-CSF and specifically blocked its function. Replacement of Val16 or Asn17 with alanine greatly reduced the antibody-binding capacity to peptide 14-24, whereas substitution of Gln20 or Glu21 was less critical. Monoclonal antibodies generated against residues 30-41 (corresponding to an intrahelical loop) and 79-91 (homologous to a sequence including part of the third alpha-helix) or its analog [Ala88](79-91)beta Ala-Cys, were conformation dependent and nonneutralizing: they failed to react or bound poorly to rhGM-CSF in ELISA, but readily recognized the homologous sequence in the denatured protein, by Western blotting.

The aryl receptor inhibitor epigallocatechin-3-gallate protects INS-1E beta-cell line against acute dioxin toxicity.

The aim of this research was to investigate the mechanism(s) underlying the acute toxicity of dioxin in pancreatic beta cells and to evaluate the protective effects of epigallocatechin-3-gallate (EGCG), the most abundant of the green tea's catechins and a powerful inhibitor of the aryl hydrocarbon receptor (AhR). Using the insulin-secreting INS-1E cell line we have explored the effect of 1h exposure to different concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), alone or in the presence of EGCG, on: (a) cell survival; (b) cellular ultrastructure; (c) intracellular calcium levels; (d) mitochondrial membrane potential; (e) glucose-stimulated insulin secretion and (f) activation of MAP kinases. Our results demonstrate that TCDD is highly toxic for INS-1E cells, suggesting that pancreatic beta cells should be considered a relevant and sensitive target for dioxin acute toxicity. EGCG significantly protects INS-1E cells against TCDD-induced toxicity in terms of both cell survival and preservation of cellular ultrastructure. The mechanism of this protective effect seems to be related to: (a) the ability of EGCG to preserve the mitochondrial function and thus to prevent the TCDD-induced inhibition of glucose-stimulated insulin secretion and (b) the ability of EGCG to inhibit the TCDD-induced activation of selected kinases, such as e.g. ERK 1/2 and JNK. Our results clearly show that EGCG is able to protect pancreatic beta cells against dioxin acute toxicity and indicate the mitochondrion as the most likely target for this beneficial effect.

Structural influence of isothiocyanates on expression of cytochrome P450, phase II enzymes, and activation of Nrf2 in primary rat hepatocytes.

Primary cultures of rat hepatocytes were used to investigate whether and how eight isothiocynates (ITCs) with different chemical structures (the aromatic benzyl, 4-hydroxybenzyl, phenethyl isothiocyanates and the aliphatic allyl, napin, iberin, raphasatin isothiocyanates and sulforaphane) derived from hydrolyzed glucosinolates, were able to modulate cytochrome P450 (CYP) and antioxidant/detoxifying enzymes and to activate the Nrf2 transcription factor. The aromatic ITCs at 40 µM markedly increased the transcription of CYP1A1 and 1A2 mRNA and increased the associated ethoxyresorufin O-deethylase (EROD) activity after 24 h of treatment. By contrast, the aliphatic ITCs (40 µM) decreased CYP1A1 and 1A2 transcription, together with the corresponding EROD activity. The same treatment also caused a striking and similar transcriptional repression of CYP3A2, and the corresponding benzyloxyquinoline debenzylase activity in response to all the ITCs tested. In the same culture conditions, most of the antioxidant/detoxifying enzymes were significantly up-regulated by 40µM ITCs. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different levels, at transcriptional, protein and/or activity levels by all the ITCs. However, glutathione S-transferase activity was not induced by the allyl, benzyl, and 4-hydroxybenzyl ITCs, glutathione reductase activity was not induced by benzyl, and 4-hydroxybenzyl ITCs and catalase activity was not induced by allyl ITC. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus was revealed by immunoblotting after 1h of treatment for all the ITCs tested. The ability of ITCs to induce the antioxidant and phase II enzymes did not appear to be affected by their hydrophilicity or other structural factors. Taken together, these results show that these ITCs are effective inducers of ARE/Nrf2-regulated antioxidant/detoxifying genes and have the potential to inhibit, at least in rat liver, the bioactivation of carcinogens dependent on CYP3A2 catalysis.

Cell division does not affect Sendai virus genome replication in persistently infected BHK cells.

The extent of Sendai virus genome replication in persistently infected BHK cells actively growing or at confluence was followed by estimation of the [3H]uridine incorporated into intracellular nucleocapsid RNA. First, we showed that, in the presence of actinomycin D, actively growing persistently infected cells were taking up threefold more [3H]uridine than resting cells. This higher uptake exhibited by growing cells was observed neither in persistently infected cells in the absence of actinomycin D, nor in acutely infected cells in the presence of actinomycin D. Assuming that the cellular pool of unlabelled uridine stays constant, we used a correction factor for this difference in [3H]uridine uptake and estimated [3H]uridine incorporation in nucleocapsid RNA, normalizing the data either to the amount of cell or of viral template. Results showed that the viral genome replication, expressed either way, was not significantly influenced by cell growth conditions.

Three variations in the cell surface expression of the haemagglutinin-neuraminidase glycoprotein of Sendai virus.

The fate of the haemagglutinin-neuraminidase glycoprotein (HN) of Sendai virus in three types of infection was studied by measuring its sensitivity to endoglycosidase H and its rate of appearance and turnover at the cell surface. HN behaved differently in the three types of infection. When highly expressed at the surface, as in a lytic standard virus infection, HN accumulated at the surface in a stable form (half-life of disappearance from the surface much greater than 10 h). When moderately expressed, as in a non-lytic standard virus plus defective interfering virus infection, HN reached the membrane normally, but turned over rapidly (half-life about 2 h) and was re-internalized. When poorly expressed, as in long-term persistent infection, HN did not reach the cell surface and appeared to be degraded before reaching it. In contrast to HN, the other viral glycoprotein, F0, exhibited a similar turnover rate at the cell surface in the three situations. However, when compared to surface expression in standard virus-infected cells under standardized conditions, F0 surface expression in persistently infected cells was reduced. This reduction correlates with a decreased maturation rate in these cells.

Restriction of cell surface expression of Sendai virus hemagglutinin-neuraminidase glycoprotein correlates with its higher instability in persistently and standard plus defective interfering virus infected BHK-21 cells.

To gain an understanding of the mechanism(s) by which Sendai virus generates a persistent infection, the expression of the hemagglutinin-neuraminidase (HN) and fusion (Fo) glycoproteins at the surfaces of BHK-21 cells infected with standard virus, a mixture of standard and defective interfering (DI) particles (mixed virus infection), and during persistent infection was investigated. The expression of HN and Fo was measured on the surfaces of infected cells by the binding of anti-HN and anti-Fo monoclonal antibodies. The results show that HN expression was restricted relative to Fo during mixed virus and persistent infections. The decreased levels of HN were investigated further by pulse-chase experiments which revealed that HN has an increased turnover rate in persistently infected cells and, to a lesser extent, in mixed virus infected cells. In analyzing the [35S]methionine-labeled protein composition of virus particles produced during the pulse-chase experiments, the increased turnover of newly synthesized HN was found to correlate with its decreased incorporation into virus particles. Interestingly, the poor HN incorporation also correlates with less efficient incorporation of the matrix M protein into virus particles.

Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents.

It has been shown that the ethylating agent diethylsulphate (DES) induces centromere-containing micronuclei with kinetics suggesting that molecules other than DNA could be targets. In quiescent Chinese hamster fibroblasts CHEF/18, O6-alkylated bases inhibit ribosomal protein S6 kinase (S6K1), the terminal member of a kinase cascade responsible for an increased rate of protein synthesis, but not extracellular signal-activated kinases (ERK1/2) or terminal kinases of a second cascade which activates transcription. The inhibition correlates with the appearance of abnormal metaphases at the following mitosis, suggesting that alkylation of the nucleotide pool and inhibition of S6K1 could be one of the mechanisms leading to chromosome loss by alkylating agents. To clarify the role of protein kinases in chromosome loss induced by alkylating agents, we have studied the effects of DES and methylnitrosourea (MNU) on S6K1 and ERK1/2 activation by growth factors. The alkylating agents were studied in a battery of Chinese hamster fibroblasts (CHEF/18, CHO and ClB) with normal and mutated p53 to control for DNA damage-induced activation of p53, which could indirectly inhibit protein kinases. The role of repair in induction of micronuclei was studied in mismatch repair-proficient CHO and repair-deficient ClB cells. Our results indicate that DES induced micronuclei in a mismatch repair-independent manner, within 8 h of treatment, in agreement with a role for S6K1 inhibition in micronucleus formation. MNU induced centromere-containing micronuclei only in CHO cells, one cell cycle after treatment, without any detectable influences on either kinase cascade, suggesting a role for mismatch repair in chromosome loss.

Functional dissection of the serological DR LYGUE and genotypic DRB1*1303 specificities using a tetanus toxin-specific T-cell clone.

The DRB1 sequence of the homozygous cell line HAG (DR13-DwHAG-DQ7) represents a new DRB allele assigned DRB1*1103, whereas its DRB3 sequence corresponds to the previously described DRB3*0101 (DR52a) allele. The DRB1*1303 gene product is undetectable by current sera used in routine serology typing. We report here direct evidence that the MHC molecule encoded by the DRB1*1303 gene is functional in antigen presentation and in T-cell restriction. We describe a T-cell clone specific for tetanus toxin whose restriction pattern strictly follows the DRB1*1303 allele, as defined by oligonucleotide typing. It also follows the serologic reactivity with the serum LYGUE and also the DwHAG MLC-defined specificity pattern, with one exception. The potential functional sites for the DRB1*1303 gene product involved in T-cell restriction were deduced from sequence comparisons between DRB1*1303 and closely related DRB1 alleles. The relevant as substitutions were located within close proximity to each other on the HLA class II structural model. Our results demonstrate that 1) DRB1*1303 is functional in antigen presentation and T-cell restriction 2) the functional region involved in antigen presentation and T-cell restriction by DRB1*1303 can be defined structurally.

Binding of human GM-CSF to synthetic peptides of the alpha subunit of its receptor.

We have synthesized a series of peptides corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit (hGM-CSFR alpha). The sequences were chosen according to the homology between hGM-CSFR alpha and the growth hormone receptor (GHR) and correspond to the regions reported to form the binding site of the latter receptor. The peptides were examined for their binding activity to hGM-CSF by affinity chromatography on resin-immobilized hGM-CSF and by a solid phase binding assay. For peptides endowed with hGM-CSF binding activity were identified and the postulated homology between the binding sites of hGM-CSFR alpha GHR was confirmed.