[Image of the month. Complex stenosis of the anterior interventricular artery treated by angioplasty and the placement of a long coronary stent].
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Aged↗
Biomedical subjects
C Martinez
Publications and source records attributed to C Martinez.
Crystallization and preliminary X-ray study of the 8-amino-7-oxopelargonate synthase from Bacillus sphaericus.
The 8-amino-7-oxopelargonate synthase (AOPS) cloned from Bacillus sphaericus, overproduced in Escherichia coli, has been crystallized in the pyridoxal 5'-phosphate (PLP)-bound form at pH 7.5, using polyethylene glycol as the precipitant. One crystal form corresponds to a tetragonal space group, with unit-cell dimensions a = b = 66, c = 181 A. These crystals do not diffract beyond 5 A, with conventional X-ray sources and cannot be used in the structure elucidation. A second crystal form is obtained when crystallization conditions are varied slightly by the addition of 0.2 M ammonium sulfate. The space group is P2(1)2(1)2(1), with unit-cell dimensions a = 68.9, b = 85.5, c = 125.9 A, indicating the presence of two molecules in the asymmetric unit (V(m) = 2.26 A(3) Da(-1); 46% water). These crystals diffract X-rays up to 3.2 A using in-house facilities and a preliminary data set has been collected. A second data set using the synchrotron radiation source W32 at LURE (Paris) has shown the crystals to diffract to at least 3 A, resolution, with good statistics. The structure determination of AOPS will provide a structural framework for the other alpha-amino ketone synthases for which no three-dimensional structure is yet available.
Journal Article↗
Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptides (PACAP27) and PACAP38) protect CD4+CD8+ thymocytes from glucocorticoid-induced apoptosis.
In the present study, the effects of vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptides, PACAP27 and PACAP38, in a concentration range from 10(-13) to 10(-6) mol/L were studied in vitro on the spontaneous and dexamethasone (DEX)-induced apoptosis in rat thymocytes. The results show that VIP and both PACAPs inhibit significantly and in a similar way the DNA fragmentation characteristic of glucocorticoid-induced apoptosis and increase the cell survival of thymocytes, with a maximal effect observed at 10(-8) to 10(-9) mol/L. This study showed the ability of the VIP-receptor (VIP-R) antagonist [N-Ac-Tyr1,D-Phe2]-GRF(1-29) amide to partially reverse the inhibitory effect of VIP and both PACAPs on DEX-induced apoptosis, providing evidence for a specific VIP1-R-mediated response and supporting the involvement of a single receptor for the three neuropeptides. Phenotypic analysis showed that VIP, PACAP27, and PACAP38 protect predominantly CD4+CD8+ thymocytes from glucocorticoid-induced apoptosis. These findings suggest that these neuropeptides could be involved in intrathymic T-cell maturation.
Animals↗
Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide-38 inhibit IL-10 production in murine T lymphocytes.
Vasoactive intestinal peptide (VIP), a neuropeptide present in the peptidergic innervation of lymphoid organs and expressed in thymocytes and peripheral lymphocytes has been previously reported to modulate cytokine expression in T lymphocytes. In this study, we investigated the effects of VIP and of the structurally related neuropeptide PACAP-38 on the expression of IL-10 in murine lymphocyte cultures. Both neuropeptides inhibit IL-10 production by spleen cells or thymocytes activated via the TCR-associated CD3 complex in a similar dose-response manner. The inhibition is specific, presumably mediated through the VIP-R1, and maximum inhibitory levels are achieved within the first 5 to 15 min of exposure to VIP or PACAP-38. CD4+ T cells function as direct cellular targets for the two neuropeptides. The fact that VIP, PACAP-38, and forskolin, all known cAMP inducers, also inhibit IL-10 production, suggests the participation of cAMP in signal transduction. VIP and PACAP-38 regulate transcriptional expression of IL-10, since IL-10 steady state mRNA levels are significantly reduced by treatment with the two neuropeptides. These results expand the range of neuroendocrine-regulated cytokines and support the idea that neuropeptides such as VIP and PACAP, which are released or produced in the local lymphoid microenvironment and specifically modulate the expression of various cytokines, may participate in the intricate cytokine network controlling local immune responses.
Animals↗
Characterization of gene expression of VIP and VIP1-receptor in rat peritoneal lymphocytes and macrophages.
In the present report we show the gene expression pattern of VIP and VIP1 receptor in two peritoneal cell populations, macrophages and lymphocytes by reverse transcription (RT) and polymerase chain reaction (PCR). Only in the lymphoid cells we have obtained a specific VIP cDNA product of 458 bp identical in size to the one obtained from cerebral cortex. On the other hand, we have obtained in both peritoneal populations lymphocytes and macrophages, a specific VIP1 receptor cDNA product of 311 bp identical in size to that obtained from lung. These results have been confirmed by Southern blot hybridization. Our findings suggest an autocrine/paracrine action of VIP in peritoneal microenvironment, supporting an immunoregulatory role for this neuropeptide.
Animals↗
Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.
The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.
Base Sequence↗
Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.
Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.
Amino Acid Sequence↗
Dynamics of Fusarium solani cutinase investigated through structural comparison among different crystal forms of its variants.
In characterizing mutants and covalently inhibited complexes of Fusarium solani cutinase, which is a 197-residue lipolytic enzyme, 34 variant structures, crystallizing in 8 different crystal forms, have been determined, mostly at high resolution. Taking advantage of this considerable body of information, a structural comparative analysis was carried out to investigate the dynamics of cutinase. Surface loops were identified as the major flexible protein regions, particularly those forming the active-site groove, whereas the elements constituting the protein scaffold were found to retain the same conformation in all the cutinase variants studied. Flexibility turned out to be correlated with thermal motion. With a given crystal packing environment, a high flexibility turned out to be correlated with a low involvement in crystal packing contacts. The high degree of crystal polymorphism, which allowed different conformations with similar energy to be detected, made it possible to identify motions which would have remained unidentified if only a single crystal form had been available. Fairly good agreement was found to exist between the data obtained from the structural comparison and those from a molecular dynamics (MD) simulation carried out on the native enzyme. The crystallographic approach used in this study turned out to be a suitable tool for investigating cutinase dynamics. Because of the availability of a set of closely related proteins in different crystal environments, the intrinsic drawback of a crystallographic approach was bypassed. By combining several static pictures, the dynamics of the protein could be monitored much more realistically than what can be achieved on the basis of static pictures alone.
Carboxylic Ester Hydrolases↗
Variability in estimation of coronary dimensions from 6F and 8F catheters.
To investigate the suitability of diagnostic 6F catheters for coronary angiographic measures in the clinical setting, we determined the relative accuracy and reproducibility of the measures obtained with these catheters as scaling devices in 59 stenoses. Comparison was made with duplicate injections, obtained before angioplasty, using an 8F guiding catheter as scaling device. Intra- and interobserver variability was evaluated in 15 stenoses. The coefficient of variation averaged 18.3% for the minimal lumen diameter, 10.4% for the percent stenosis, and only 7.4% for the reference diameter. Reproducibility of angiographic measures done with the 6F catheter was similar to that obtained with the 8F catheter, although accuracy was lower with the 6F for the measurement of reference diameter. Thus, quantitative coronary angiography (QCA) measures derived from routine diagnostic angiograms may be suitable for determination of reference diameter, allowing enough precision for determination of the size of a coronary device for intervention, but these measures may lack accuracy for precise determination of minimum diameter and percent stenosis, making their use questionable in studies looking at individual changes in coronary stenosis dimensions.
Coronary Angiography↗
The output of the hippocampus is inhibited during social behavior in the male rat.
During sexual behavior in the male rat, peptidergic cells in the medial amygdaloid nucleus become active and release a vasopressin-like peptide. The present experiments were designed to examine hippocampal changes as a result of this peptide's action during sexual behaviors. Chronic field-potential recordings from the hippocampus of male rats were acquired in a wide variety of social and nonsocial circumstances. Hippocampal responses that resemble the known action of the vasopressin-like peptide were seen only with social stimuli such as sexual stimuli and stimuli that led to aggressive behavior between males. The results show that the occasions of peptide action in the hippocampus correlate with the occasions of peptide release as determined by recording from the peptidergic cell bodies. The results are interpreted to indicate that the amygdala projection to the hippocampus has a special role to play in social behavior.
Animals↗
Use of human vessels and human vascular smooth muscle cells in pharmacology.
Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, but these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.
Calcium↗
Restraint stress reversibly enhances spatial memory performance.
The effects of restraint stress on performance of a spatial memory task, the eight arm radial maze, was examined in rats. When stress was given for 6 h/day for 7 days and performance evaluated days 10-13 post stress, no effect on performance was noted; however, daily restraint stress for 13 days caused a small, but significant, enhancement of performance days 10-13 post stress. Stressed rats performed better than controls: their number of correct choices in the first 8 visits was higher than the controls, and stressed rats took fewer total choices to finish the maze than controls. Stress-dependent, enhanced performance does not appear permanent since further maze testing on days 14 and 15 post stress showed no differences between the groups. Performance of the stressed rats significantly correlated with their stress-induced, serum corticosterone levels measured after 6 h of restraint on the last day of restraint, day 13 (r = -0.63, P < 0.05); rats with higher levels of CORT took fewer choices to finish the task. Examination of hippocampal CA3c pyramidal neurons with Golgi techniques showed no effect of stress on the basal or apical dendritic arbors. Since our previous study showed that 21 days of restraint stress is associated with impaired spatial memory performance (10), these results suggest that the duration of stress may differentially affect learning/memory with shorter periods of stress serving an adaptive function while longer durations causing maladaptive changes.
Animals↗
Hydrophobic derivatives of chitosan: characterization and rheological behaviour.
Chitosans which are substituted with alkyl chains having a minimum of six carbon atoms demonstrate hydrophobic interactions in solution. The chemical structure of synthetized polymers is determined from NMR spectroscopy and microanalysis and the distribution between the different types of substituted units is obtained. In relation to the rheological behaviour different parameters are studied such as the nature of the hydrophobic chain and the substitution degree, the polymer concentration, the temperature and the ionic content of the polymeric solution. All the observations tend to prove the hydrophobic nature of the interaction mechanism.
Biocompatible Materials↗
Differential expression of vasoactive intestinal peptide receptors 1 and 2 (VIP-R1 and VIP-R2) mRNA in murine lymphocytes.
Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, modulates cytokine expression and affects T cell proliferation. Recent molecular studies identified two VIP receptors. VIP-R1 and VIP-R2, primarily in nonlymphoid cells. In this study, we investigate the expression of VIP-R1 and VIP-R2 mRNA in unstimulated and stimulated lymphocytes and thymocytes, and in various lymphocyte subpopulations. In contrast to VIP-R1 which is constitutively expressed, the expression of VIP-R2 is induced only following stimulation through the TCR-associated CD3 complex. Both CD4+ and CD8+ T cells express VIP-R1 and VIP-R2. Two T cell lines, EL-4.IL-2 and D10.G4.1 express exclusively VIP-R2. VIP induces the expression of the VIP-R2 gene in the absence of additional stimuli. Differential expression and regulation of the two VIP receptors in T lymphocytes suggests different physiological roles in mediating the immunomodulatory activities of VIP and related neuropeptides.
Animals↗
Murine T-lymphocytes express vasoactive intestinal peptide receptor 1 (VIP-R1) mRNA.
Vasoactive intestinal peptide (VIP), a neuropeptide present in primary and secondary lymphoid organs has been previously reported to inhibit IL-2 and IL-4 production as well as the proliferation of mitogen- or antigen-stimulated T-cells. Binding studies suggested that the immunoregulatory effects of VIP are mediated through specific VIP-binding sites present on lymphocyte subpopulations. Here we report on the expression of VIP-R1 mRNA in various murine lymphocyte subpopulations. By using RT-PCR. RNase protection assay, cDNA cloning, and sequence analysis, we show that stimulated and unstimulated murine spleen cells, thymocytes. CD4+ and CD8+ T-cells express VIP-R1. The VIP-R1 fragment amplified from murine brain, thymocytes, spleen cells and CD4+ T-cells share identical nucleotide sequences, and a high degree of homology with the corresponding nonlymphoid rat and human VIP-R1 sequences. The expression of VIP-R1 in thymocytes and peripheral lymphocytes, and especially in the CD4+ T-cell subset supports the idea that VIP produced or released locally in the lymphoid microenvironment could directly affect cytokine production and proliferation of T-lymphocytes.
Animals↗
Acyl glycerol hydrolases: inhibitors, interface and catalysis.
The last five years have witnessed the solution of a large number of lipase structures, which has led, among other insights, to the structural interpretation of the interfacial activation phenomenon in terms of 'lid' opening. This interpretation has been extended this year to include phospholipase A2. Recent structural studies on lipases have provided data on the detailed mechanisms underlying the behaviour of lipases: how they bind to inhibitors or substrates, and what interactions occur between their hydrophobic face and hydrophobic molecules, for example. In addition, studies on cutinase point mutants have shed some light on the role of the oxyanion hole in lipolytic catalysis.
Animals↗
Blood manganese correlates with brain magnetic resonance imaging changes in patients with liver disease.
BACKGROUND: Chronic liver failure is associated with high signal abnormalities in the basal ganglia on T1-weighted magnetic resonance imaging of the brain. These abnormalities are strikingly similar to those seen following manganese intoxication. As dietary manganese is normally cleared by the liver, we hypothesize that hepatic dysfunction could lead to manganese overload and account for the MRI abnormalities seen in patients with chronic liver disease. METHODS: We measured blood manganese concentrations in eleven patients with biopsy-proven hepatic cirrhosis and eleven healthy age and sex-matched controls. We also performed semi-quantitative measures of T1 signal abnormalities on MRI in the patients with chronic liver disease. RESULTS: Patients with cirrhosis had significantly higher blood manganese concentrations (20.6 +/- 10.2 mcg/L) than controls (7.2 +/- 2.7, p = .0013). In addition, semi-quantitative scores of T1-weighted signal hyperintensity on MRI correlated with blood manganese concentration in patients with cirrhosis (r = .65, p = .029). CONCLUSIONS: These findings demonstrate that chronic liver disease is associated with manganese overload and suggest that manganese is responsible for the T1-weighted signal hyperintensity seen on MRI of patients with liver disease. As manganese intoxication is known to cause parkinsonism and an encephalopathy similar to those which occur with chronic liver disease, it is possible that manganese toxicity contributes to the development of these symptoms in liver damaged patients and that therapies which prevent or reduce manganese overload may have clinical benefit.
Adult↗
The multicentre south European study 'Helios'. I: Skin characteristics and sunburns in basal cell and squamous cell carcinomas of the skin.
The aim of this study was to investigate constitutional and environmental determinants of non-melanocytic skin cancer among different populations from south Europe. Between 1989 and 1993 we interviewed incident cases and a random population sample of controls from five centres where a cancer registry was operating, whereas we selected a sample of hospital-based cases and controls from three other centres. Controls were stratified according to the age and sex distribution of cases. In all, 1549 cases of basal cell carcinoma (BCC), 228 of squamous cell carcinoma (SCC) and 1795 controls were interviewed. Both cancers affected primarily sun-exposed sites such as face, head and neck, but the prevalence of BCC on the trunk was higher than for SCC. Pigmentary traits such as hair and eye colour as well as tendency to sunburn were strong and independent indicators of risk for both BCC and SCC. In SCC, adjusted odds ratios (ORs) ranged from 1.6 for fair hair colour to 12.5 for red hair. Light-blonde hair entailed a risk of about 2 for BCC. Pale eye colour was associated with a risk of 1.8 for SCC and 1.4 for BCC. Subjects who always burn and never tan showed an adjusted OR of 2.7 for BCC and 2.0 for SCC. A history of sunburns and a young age at first sunburn were associated with an increased risk for BCC only (OR 1.7). Pigmentary traits and sun sensitivity of the skin confirmed their role as risk indicators. The effect of sunburns, as an indicator of both exposure and sun sensitivity of the skin, is less clear. Nevertheless, its association with BCC suggests, by analogy with melanoma, a relationship with intense sun exposure. Conversely, SCC would require prolonged exposure to sunlight.
Adolescent↗