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V Lefebvre

Publications and source records attributed to V Lefebvre.

52 records · Page 3Linked to original sources
PubMed1995

A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice.

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro alpha 1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a beta-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of beta-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted beta-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no beta-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal beta-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.

Animals↗
PubMed1995

Transgenic mice with deficiencies in cartilage collagens: possible models for gene therapy.

We address three issues that are important when considering somatic gene therapy approaches to osteoarthritis (OA) and related syndromes. First, only those diseases for which a precise molecular etiology has been established should be contemplated for somatic gene therapy. Second, DNA sequences should be identified that restrict expression of correcting genes to chondrocytes; we discuss the use of transgenic mice to identify such sequences. Third, we emphasize the usefulness of establishing animal models that mimic human OA syndromes by genetic manipulations. These transgenic models should be essential for testing gene therapy approaches in vivo.

Animals↗
PubMed1994

Fructose metabolism and cell survival in freshly isolated rat hepatocytes incubated under hypoxic conditions: proposals for potential clinical use.

The protective effect of fructose with regard to hypoxia-induced cell injury was investigated. The addition of fructose (2 to 20 mmol/L) protected hepatocytes against hypoxia-mediated cell lysis in a concentration-dependent way. The intracellular ATP content was initially decreased as a result of fructose-1-phosphate formation, but it remained constant during the hypoxic incubation. Conversely, high initial ATP values observed at low fructose concentrations progressively declined. Cellular protection was observed only when fructose was added before (and not after) the start of hypoxia. In addition, a sufficient amount of fructose-1-phosphate rapidly accumulated before the induction of hypoxia, and the linear production of lactate, during hypoxic incubation, indicated that cells synthesized ATP continuously. The lack of cell protection by fructose added after the onset of the hypoxia may be explained by a lesser fructose-1-phosphate formation and a subsequently low accumulation leading to insufficient glycolytic ATP production. Under aerobic conditions, both glycolysis (lactate formation) and gluconeogenesis (glucose formation) were carried out in fructose-1-phosphate-loaded cells with the same initial rates, whereas under hypoxic conditions glycolysis was the main metabolic event. The fact that protein synthesis activity recovered faster during reoxygenation of previously hypoxic fructose-treated cells than in glucose-treated cells led us to hypothesize that in situ perfusion of liver with fructose, before its removal, would improve its metabolic capacity during the hypoxic cold preservation and subsequent transplantation.

Adenosine Triphosphate↗
PubMed1994

Motor memory and the preselection effect in Huntington's and Parkinson's disease.

Patients with Huntington's disease (HD) and Parkinson's disease (PD) show different patterns of preserved and impaired memory performance. This study investigates explicit memory for movements in HD and PD with a linear positioning apparatus using Dick et al.'s procedure (J. Gerontol. 43, 127-135, 1988). In the first experiment, 12 HD patients were compared to 12 matched-controls. HD patients were more impaired than the controls by the delay between criterion and recall movements, whether the delay was filled or unfilled. Switching the limb between criterion and recall movements did not lead to more effects in HD patients and in controls. In the second experiment, 12 non-demented PD patients were compared to matched-controls. PD patients were more impaired than controls when the recall movement was executed with the contralateral hand, but were not more affected by the delay. In both experiments, HD and PD patients, as well as the controls, recalled self-generated preselected movements better than imposed movements. These results suggest the existence of distinct forms of motor memory impairment in some subcortical neurodegenerative diseases.

Adult↗
PubMed1994

Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice.

Studies on the function of extracellular matrix components of cartilages and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.

Animals↗
PubMed1993

An assay for matrix metalloproteinases and other proteases acting on proteoglycans, casein, or gelatin.

We have set up a quantitative and sensitive enzymatic assay for proteases of different classes acting on proteoglycans, casein, or gelatin. Radiolabeled substrates were covalently attached to insoluble microcarriers and assays were performed in 96-well plates. Protease activities were determined by the release of labeled degradation products. Time- and dose-response curves were linear when the solubilization of labeled substrates did not exceed 15-20% of the initially bound molecules. Results were compared to those from zymographic analyses on proteoglycan-, gelatin-, and casein-polyacrylamide gels, as well as to the results obtained with conventional assays using soluble [3H]-casein and [3H]gelatin. Our assay procedure was more sensitive than other available methods: it detected picogram amounts of trypsin as well as picogram or nanogram amounts of the purified human matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9, depending on the specific activities of these MMPs on the different substrates. Our new procedure was appropriate for assaying the MMPs present in crude culture media conditioned by chondrocytes cultivated under various conditions.

Animals↗
PubMed1993

[Adrenoleukomyeloneuropathy in an adult].

We report a case of adrenomyeloneuropathy in a 35-year-old man. He experienced acute adrenal insufficiency at 29 years of age, followed by psychic disorders and mental deterioration, then spastic paraparesia, dysarthria and peripheral neuropathy. MRI showed high-intensity signal areas in the cerebellum and periventricular white matter. Clinical symptoms, neurophysiological and neuroradiological examinations disclosed both features of adrenoleukodystrophy and adrenomyeloneuropathy. This indicates that adrenoleukodystrophy and adrenomyeloneuropathy probably represent two portions of the same entity.

Adrenal Insufficiency↗
PubMed1993

[Infarction of the red nucleus and crossed cerebellar diaschisis].

We report a case of rubral infarct involving adjacent ipsilateral thalamic structures in a 54-year-old man with hypertension and coronary heart disease. Clinical picture at onset was suggestive of Weber's syndrome leading to a Claude's syndrome with vertical gaze palsy and sleep disturbances. Single photon emission computed tomography using Hm-PaO-Tc revealed a crossed cerebellar diaschisis. This is, to our knowledge, the first case of such crossed cerebellar diaschisis in a patient with rubral infarction.

Cerebellar Diseases↗
PubMed1991

Production of gelatin-degrading matrix metalloproteinases ('type IV collagenases') and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha.

The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. Interleukin-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to 35 kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Modulation by interleukin 1 and tumor necrosis factor alpha of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes.

The actions of interleukin 1 (IL1) and tumor necrosis factor alpha (TNF alpha) on several parameters of the collagen metabolism of rabbit articular chondrocytes were studied by comparing the responses of either differentiated chondrocytes in primoculture or dedifferentiated cells in late passage culture to human recombinant (hr) IL1 alpha, hr-TNF alpha and cytokine-enriched fractions of rabbit macrophage-conditioned media. In response to IL1 or TNF alpha, differentiated chondrocytes (i.e., producing the cartilage-specific collagens, types II and XI, but no type I), sharply reduced their synthesis of collagen, a reduction which involved both types II and XI collagens, without consistently changing their production of non-collagenous proteins; they also incorporated a smaller proportion of collagen into the matrix. Similar levels of response were obtained for hr-IL1 alpha at picomolar and for hr-TNF alpha at nanomolar concentrations. However, the action of TNF alpha, but not of IL1, was manifested only in the presence of serum. Simultaneously, IL1, but not TNF alpha, induced the chondrocyte production of procollagenase (a difference which contrasted with the similar levels of procollagenase induced by both cytokines in synovial and skin fibroblasts) but neither cytokine influenced the accumulation of the collagenase inhibitor TIMP. These effects were not affected by indomethacin and are thus unlikely to be prostaglandin-mediated. During their dedifferentiation in monolayer subcultures, chondrocytes became more sensitive to the procollagenase-inducing ability of IL1 and TNF alpha, but their response to TNF alpha was lower than to IL1. They also increased their production of TIMP, which remained unaffected by the cytokines. Simultaneously, they decreased their production of collagen and substituted progressively the synthesis of fibroblast-specific collagens, types I, III and V, for types II and XI. Acting on dedifferentiated cells, even in the presence of indomethacin, IL1 and TNF alpha further decreased the synthesis of collagen, reducing the production of both typical type I (i.e. [alpha 1(I)]2 x alpha 2(I) molecules) and type V collagens as well as their incorporation into the matrix, but increasing the synthesis of type III collagen. Therefore not only IL1, but also TNF alpha can exert profound influences on the collagen degradation and repair processes occurring in the pathology of articular cartilage.

Animals↗
PubMed1990

Production of collagens, collagenase and collagenase inhibitor during the dedifferentiation of articular chondrocytes by serial subcultures.

Rabbit articular chondrocytes were cultured in monolayer and the progressive loss of their differentiated phenotype was monitored from passage to passage. The cell densities achieved in confluent cultures decreased abruptly between the primoculture and the second or third subculture, and more slowly thereafter, reflecting parallel morphological changes. The synthesis of collagen (but not that of other proteins) decreased sharply, and a smaller proportion of collagen was incorporated into the matrix. Cells in primoculture synthesized mainly the cartilage-specific collagens, types II and XI, which were mostly deposited in the matrix, but no type I nor III collagen. With increasing passages, the synthesis of type II collagen decreased progressively while that of types I and III collagens increased, the latter being almost completely released in the culture medium. Simultaneously, the production of type XI collagen was apparently switched to that of type V. Fully differentiated confluent chondrocytes in primoculture produced the collagenase inhibitor TIMP (tissue inhibitor of metalloproteinases) but no detectable procollagenase; their production of procollagenase was, however, induced by interleukin 1. The production of TIMP increased from passage to passage. A spontaneous production of procollagenase was only occasionally observed in confluent cultures of dedifferentiated chondrocytes. However, interleukin 1 induced an always higher production of procollagenase from dedifferentiated chondrocytes than from cells in primoculture.

Animals↗
PubMed1989

The enzymatic evaluation of procollagenase and collagenase inhibitors in crude biological media.

The validity of the enzymatic assay of procollagenase within crude biological media containing also the collagenase inhibitor TIMP (tissue inhibitor of metalloproteinases) as well as other (pro)metalloproteinases and sometimes, metalloproteinase-TIMP complexes, has been reevaluated. To be enzymatically assayed, procollagenase has to be activated. The standard activation procedures by either trypsin or 4-aminophenylmercuric acetate (APMA) both allow an optimal recovery of collagenase from procollagenase when the media do not contain free TIMP. However, they do not destroy TIMP nor do they reactivate the collagenase present in enzyme-inhibitor complexes. Therefore, the collagenase formed by the activation of procollagenase in the presence of free TIMP is immediately inactivated by binding to the inhibitor. As a result, both the bound collagenase and TIMP can no longer be assayed by enzymatic methods. An optimal recovery of collagenase can, however, be obtained if free TIMP is neutralized by the binding of other tissue metalloproteinases (such as those present in culture media of rabbit bone marrow-derived macrophages) prior to the activation and assay of procollagenase. Similarly, it is possible to recover under an active free form a large part of the TIMP present in collagenase- (or other metalloproteinase-)TIMP complexes by heating the complexes at acid pH under conditions which inactivate the collagenase.

Animals↗
PubMedDate not supplied

Double common bile duct with ectopic drainage of the left lobe into the stomach. Case report and review of the literature.

A peculiar abnormal biliary tract consisting in a double common bile duct, one with a normal drainage into the duodenum and the other providing from the left lobe with an ectopic drainage into the stomach is described. For the first time, this anomaly was discovered by endoscopic retrograde cholangiopancreatography (E.R.C.P.). In the literature, only twelve other cases of abnormal drainage into the stomach are described. This could be related to a random subdivision of the hepatic diverticulum during the first weeks of embryogenesis.

Adult↗