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Biomedical subjects

F Morel

Publications and source records attributed to F Morel.

At least 37 records · Page 2Linked to original sources
PubMed2000

TIMP-1/MMP-9 imbalance in an EBV-immortalized B lymphocyte cellular model: evidence for TIMP-1 multifunctional properties.

Tissue inhibitors of metalloproteinases (TIMPs) were initially described as agents controlling metalloproteinase activity. The purpose of this study was to investigate the expression and the roles of TIMP-1 secreted by Epstein-Barr-virus (EBV)-immortalized B lymphocytes. TIMP-1 was isolated from conditioned medium of interleukin (IL)-1beta stimulated EBV-B lymphocytes; purified TIMP-1 was identified by mass spectrometry and immunochemistry. TIMP-1-free MMP-9 was quantified after purification by zymography and enzyme-linked immunosorbent assay. EBV-B lymphocyte-secreted TIMP-1 inhibited MMP-9 gelatinolytic activity resulting in decreased B-cell transmigration as measured in vitro. The release of huge amounts of TIMP-1 in proportion to MMP-9 from B lymphocytes after EBV transformation was shown to be correlated with secretion of IL-10 and dependent on culture time. In contrast, there was little TIMP-1 and almost no IL-10 released from native B cells, suggesting a possible IL-10 mediated autocrine regulation mechanism of TIMP-1 synthesis. The MMP-9/TIMP-1 imbalance observed in the culture medium of EBV-B lymphocytes (TIMP-1>MMP-9) and of native B cells (MMP-9>TIMP-1) is suggestive of a new function for TIMP-1. We propose that TIMP-1 acts as a survival factor controlling B-cell growth and apoptosis through an autocrine regulation process involving IL-10 secreted by EBV-B lymphocytes.

Amino Acid Sequence↗
PubMed2000

Protein delivery by Pseudomonas type III secretion system: Ex vivo complementation of p67(phox)-deficient chronic granulomatous disease.

Bacterial type III secretion system drives the translocation of virulence factors into the cystosol of host target cells. In phagocytes and in Epstein-Barr virus immortalized B lymphocytes, NADPH oxidase generates O(-2) through an electron transfer chain the activity of which depends on the assembly of three, p67(phox), p47(phox) and p40(phox) cytosolic activating factors with Rac 1/2 and a membrane redox component, cytochrome b(558). In p67(phox) deficient chronic granulomatous disease (CGD) patients, p67-phox is missing and NADPH oxidase activity is abolished. ExoS is a virulence factor of Pseudomonas aeruginosa which is secreted via the type III secretion system: it was fused with p67(phox). Pseudomonas aeruginosa synthesized and translocated the hybrid ExoS-p67(phox) fusion protein into the cytosol of B lymphocytes via the type III secretion system. Purified ExoS-p67(phox) hybrid protein was as efficient as normal recombinant p67(phox) in cell-free reconstitution of NADPH oxidase activity. Therefore, ExoS-p67(phox) was transferred via the type III secretion system of Pseudomonas aeruginosa into the cytosol of B lymphocytes from a p67(phox)-deficient CGD patient and functionally reconstituted NADPH oxidase activity. In the complementation process, ExoS acted as a molecular courier for protein delivery: the reconstitution of an active NADPH oxidase complex suggests type III secretion system to be a new approach for cellular therapy.

B-Lymphocytes↗
PubMed2000

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy.

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.

Cell-Free System↗
PubMed2000

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells.

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR(+) cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin(-/lo)Sca-1(+) and c-kit(+)Sca-1(+) subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR(+)c-kit(+)Sca-1(+) bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR(+) cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

Animals↗
PubMed2000

Gelatinase isoforms in urine from bladder cancer patients.

Matrix metalloproteinases are involved in tumor invasion and metastasis in many types of human carcinomas, in leukocyte infiltration and inflammatory reactions. Three metalloproteinases with gelatinolytic activity were isolated from the urine of patients with untreated high grade bladder cancer or with functioning renal grafts (control). Urinary proteins were fractionated after concentration by continuous-elution SDS-polyacrylamide gel electrophoresis. Collected fractions were analyzed by gelatin zymography and Western blotting. The one-step purification process isolated the gelatinase species from crude urine samples: (1) a 72 kDa progelatinase A (MMP-2) and its actived 68 kDa form; (2) a 92 kDa progelatinase B (MMP-9); (3) a higher molecular weight (HMW) complex (115 kDa) which was identified as progelatinase B associated with lipocalin, NGAL. A similar marker profile was observed in bladder cancer tissues. The current study demonstrated the efficiency of continuous elution electrophoresis. It offered two main advantages: (1) the separation of latent from active gelatinase isoforms with no interference from the TIMPs and (2) the identification and isolation in a single step of large amounts of urine gelatinase species with both high recovery and significant specific activities. Continuous-elution electrophoresis can be used for correlation with clinical events of bladder cancer diagnosis and prognosis.

Blotting, Western↗
PubMed2000

Structural and functional properties of membrane and secreted IgD.

More than 35 years ago, study of an unknown immunoglobulin (Ig) in the serum from a myeloma patient led to the discovery of IgD. Subsequently, the finding that it also exists as a membrane-bound Ig stimulated a large number of studies during the 70s. Then, the interest on IgD shrank, largely because of the lack of known function of secretory IgD (secIgD) and of a stagnating knowledge of the functions of surface IgD. In the recent years, very significant advances followed the tremendous accumulation of data on the physiology of the B cell receptor, of which IgD is the major component, on the role of secIgD in normal and diseased individuals. This review, which is focused on human IgD but integrates data in the mouse and other species when needed, summarizes present data on the structure, synthesis and functions of both membrane and secIgD, IgD receptors and the involvement of IgD in various diseases, especially the hyperIgD syndrome.

Animals↗
PubMed2000

Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction.

Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.

B-Lymphocytes↗
PubMed2000

Segregation of sex chromosomes in spermatozoa of 46,XY/47,XXY men by multicolour fluorescence in-situ hybridization.

The sex chromosome disomy and diploidy rates on ejaculated spermatozoa from two patients with mosaic Klinefelter's syndrome were estimated, using X/Y/15 multicolour fluorescence in-situ hybridization (FISH). A 8/18 dual fluorescence in-situ hybridization analysis was also carried out. In triple FISH, a total of 1691 (patient 1) and 811 (patient 2) spermatozoa were analysed. Frequencies of cells with hyperhaploidies for sex chromosomes were 2. 01% and 3.45% for patients 1 and 2 respectively, with both patients showing a significantly increased incidence of 24,XY and 24,XX disomies and only patient 2 showing a significantly increased incidence of 24,YY disomy in comparison to the control (P < 0.001). The 46,XX diploidy rate in patient 1 was also significantly higher than the control (P < 0.01). The ratio of X-bearing to Y-bearing spermatozoa differed from the expected 1:1 ratio for only patient 1 (1.18:1). There was no significant difference for chromosomes 8, 15 or 18 disomy frequencies in comparison to those estimated in the control population. These results support the hypothesis that some 47,XXY cells are able to go through meiosis and form spermatozoa with an abnormal gonosomal complement. Thus, there is an increased risk, for these 46,XY/47,XXY men, of producing offspring with a gonosomal abnormality.

Case-Control Studies↗
PubMed2000

Role of metalloproteases and inhibitors in the occurrence and progression of diabetic renal lesions.

Renal remodelling in hyperinsulinic/insulinopenic states is mediated by glucotoxicity, endothelial dysfunction and vascular and nephron collagen turnover. Hypertensive and renal links are renewed by renoprotective interventions of renin-angiotensin. Vasoactive peptide processing and vascular collagen deposition are under the tight control of two zinc metalloproteinase families that regulate vascular tone and trophicity: gluzincins (or vasopeptidases) are convertases of angiotensins, endothelins or atrial natriuretic factors; and metzincins or matrix metalloproteases (MMP, matrixins)] regulate vascular type IV collagen basement membrane proteolysis. Association of natural tissue inhibitors of MMPs, pharmacological inhibitors of vasopeptidases [either conventional (angiotensin-converting enzyme inhibitors) or innovative (omapatrilat)], together with synthetic MMP inhibitors, are currently screened to counteract vascular remodelling and renal scarring. Our studies focused on the 72 kDa (MMP-2) and 92 kDa (MMP-9) matrixin gelatinases and tissue inhibitors involved in basement membrane degradation and rebuilding. Three complementary settings were developed, allowing evaluations from basic to clinical stages. A leucocyte-endothelial transmigration model was designed for transcription and addressing of enzymes and inhibitors, in situ matrix degradation, and blockading by metalloprotease inhibitors (captopril). Insulin-resistant fructose-fed rats showed heavy proteinuria and glomerulosclerosis involving angiotensin II-dependent changes in renal gelatinases and inhibitors. Urinary gelatinolytic profiles from Type 2 diabetic patients with overt nephropathy were compared with those of normal first-degree relatives and age-matched healthy controls. Physiologically, MMP-9 was the primary urinary gelatinolytic enzyme. In Type 2 diabetic proteinuric patients, MMP-9 and MMP-2 releases were significantly increased in the absence of renin-angiotensin blockade, while first-degree relatives showed reduced gelatinase levels suggestive of a genetic control of renal matrix regulation prior to potential glycaemic dysregulation. These preliminary data suggest that local MMP/TIMP imbalance is involved in diabetic renal remodelling. Further studies are needed to define the redundancies and specificities of vasopeptidase and MMP inhibitors, differentiate the antihypertensive effect from target-organ protection, screen for innovative pharmacological compounds, and validate simple, efficient biological markers of renal fibrosis progression and the effect of anti-fibrotic therapeutic interventions.

Animals↗
PubMed1999

Hematologic recovery in mice transplanted with bone marrow stem cells expressing anti-human immunodeficiency virus genes.

We have used a mouse bone marrow transplantation (BMT) model to study the safety of retrovirus-mediated transfer of anti-HIV genes (RevM10 and HIV-1 pol antisense) into hematopoietic stem/progenitor cells (HSPCs). In particular, we have monitored the hematologic recovery post-BMT and transgene expression in myeloid and lymphoid lineages, and analyzed tissue sections for evidence of any transgene-related pathological condition. Expression of anti-HIV genes had no effect on kinetics of hematologic recovery post-BMT. The average time to reach 20% of normal cell counts was 15-17 days for white blood cells and 12-14 days for platelets, and the average time to reach complete recovery was 42-56 days for leukocytes and 104-161 days for platelets. Hematocrit levels were not significantly affected by irradiation and transplantation procedures. Donor chimerism was uniformly > or =90% in all transplanted animals. At 4-5 weeks post-BMT transgene expression was detected in peripheral blood leukocytes in 100% of the animals and ranged from 4.5 to 44.7%. In a majority of the animals the percentage of transgene-expressing cells in circulation decreased over time but remained detectable for the length of the study (>6 months). Expression was detected in all analyzed cell lineages (RBCs, platelets, monocytes, granulocytes, and T and B cells). Relative counts of various leukocytes (Mac1+ monocytes, Gr1+ granulocytes, CD3+ T cells, and B220+ B cells) were normal. There were no treatment-related histopathological changes in a wide range of tissues examined. In addition, there were no treatment effects on differential leukocyte counts, and morphology of peripheral blood and bone marrow brush smears. In summary, transfer and expression of the RevM10 and the HIV-1 antisense genes into hematopoietic stem/progenitor cells in vivo appears safe. We propose that the mouse bone marrow transplantation model could be used to evaluate some safety aspects of HSPC-based gene therapies.

Animals↗
PubMed1999

The prolyl aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus belongs to the alpha/beta hydrolase fold family.

Prolyl aminopeptidase (PepIP) of Lactobacillus delbrueckii subsp. bulgaricus displays the Gly-x-Ser-x-Gly-Gly consensus motif surrounding the catalytic serine of the prolyl oligopeptidases family. Sequence comparison revealed that this motif and two other domains appear well conserved among bacterial PepIPs and members of the alpha/beta hydrolase fold family. Secondary structural predictions of PepIP were performed from amino acid sequence and corroborated by circular dichroism analysis. These predictions well matched the core structure of alpha/beta hydrolases organised in eight beta-sheets connected by alpha-helices. We obtained 26 mutants of PepIP by chemical or site-directed mutagenesis. Most substitutions associated with stable and inactive mutant proteins were mainly located in the three conserved boxes (including the catalytic serine motif). Taken together, our results strongly suggest that PepIP belongs to the alpha/beta hydrolase fold family and that Ser107, Asp246 and His273 constitute the catalytic triad of the enzyme.

Amino Acid Sequence↗
PubMed1999

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox.

NADPH oxidase is an O2*- -generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes.

Actins↗
PubMed1999

Possible role of RAC-GTPase-activating protein in the termination of superoxide production in phagocytic cells.

The mechanism leading to the termination of superoxide production of phagocytes is poorly understood. The aim of the present study was to investigate the involvement of the active (GTP-bound) form of the GTP-binding proteins in maintaining continuous electron transport through the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Activation of the enzyme was carried out under in vitro conditions and a shift from the active to the inactive form of the GTP-binding protein was attained (i) by addition of an excess of GDP to the assembled enzyme complex or (ii) by variation of the Rac-GTPase activating (Rac-GAP) capacity of the constituents of the cell-free system. Significant inhibition of O2*- production was observed when guanine dinucleotides were added after the assembly of the active enzyme complex. The effect was specific for GDP and GDP,S whereas ADP, CDP and UDP were ineffective. GTP was significantly less efficient in inducing superoxide production in a cell-free system containing endogenous GAP activity than in a system devoid of GAP activity. It is suggested that the active, GTP-bound form of Rac is required for sustained catalytic function and Rac-GAP proteins are involved in the downregulation of the oxidase.

Animals↗
PubMed1999

Biochemical and molecular consequences of ethidium bromide treatment on Drosophila cells.

KC167 Drosophila cells were incubated with low concentrations of ethidium bromide (200 ng/ml), causing changes in mitochondrial DNA (mtDNA) content (2-184% of that of controls). SSCP (single strand conformational polymorphism) analysis of mtDNA indicated that the incubation with ethidium bromide also generated mutations. Compared with controls, there were marked reductions in the activities of respiratory complexes III and IV measured in these cells, and in respiration and ATP synthesis capacities measured in isolated mitochondria. These reductions matched that in mtDNA content. In contrast, no link could be demonstrated between mtDNA content and steady-state concentrations of the transcripts of genes COIII and Cyt b.

Adenosine Triphosphate↗
PubMed1999

Differential regulation of canalicular multispecific organic anion transporter (cMOAT) expression by the chemopreventive agent oltipraz in primary rat hepatocytes and in rat liver.

Expression of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump involved in biliary secretion of xenobiotics, was investigated in rat hepatocytes exposed to the chemopreventive agent oltipraz. Northern blotting indicated that this compound increased cMOAT mRNA levels in primary cultured hepatocytes. Such an induction of cMOAT transcripts was demonstrated to be dose-dependent and started as early as 4 h treatment; in addition, western blotting showed increased levels of 190 kDa cMOAT in oltipraz-treated primary rat hepatocytes when compared with their untreated counterparts. In contrast, administration of oltipraz to rats failed to enhance hepatic cMOAT mRNA and protein amounts whereas it was found to induce liver expression of glutathione S-transferase P1, a well-known oltipraz-regulated drug metabolizing enzyme. These data therefore suggest that cMOAT up-regulation occurring in rat hepatocytes in response to oltipraz may be restricted to in vitro situations and is therefore unlikely to be directly involved in the in vivo chemopreventive properties of oltipraz.

Animals↗
PubMed1998

Genomic organization, 5'-flanking region and chromosomal localization of the human glutathione transferase A4 gene.

We have isolated and characterized a human glutathione transferase A4 (hGSTA4) subunit gene from a yeast artificial chromosome containing several other glutathione transferase alpha genes and pseudogenes. The homodimeric protein hGSTA4-4, is involved in the detoxification of 4-hydroxynonenal and other reactive electrophiles produced by oxidative metabolism, and may have a significant role in protecting intracellular components from oxidative damage. The hGSTA4 gene spans nearly 18 kb, contains seven exons, maps onto chromosome 6p12, and lies in close proximity to the 7SK small nuclear RNA gene in a head-to-tail orientation. The intron/exon borders conform to the standard rules, an open reading frame is present beginning at position 154 in exon 2, and the stop codon is at position 822 in exon 7. The transcription initiation site has been determined by primer extension analysis and is located 135 bp upstream of intron 1. Isolation and sequencing of the hGSTA4 gene 5'-flanking region revealed it to be devoid of TATA or CCAAT boxes but it does contain an initiator element overlapping the transcription start site, a GC box and putative binding sites for transcription factors AP1, STAT, GATA1 and NF-kappaB. Reverse transcription-PCR analysis revealed that hGSTA4 mRNA was present in all the tissues tested, although in low amounts, suggesting that this subunit may be ubiquitously expressed.

Amino Acid Sequence↗