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Biomedical subjects

B Hainque

Publications and source records attributed to B Hainque.

At least 55 records · Page 3Linked to original sources
PubMed1998

PCR based mutation screening of the laminin alpha2 chain gene (LAMA2): application to prenatal diagnosis and search for founder effects in congenital muscular dystrophy.

Classical congenital muscular dystrophy with merosin deficiency is caused by mutations in the laminin alpha2 chain gene (LAMA2). Extended sequencing of the introns flanking the 64 LAMA2 exons was carried out and, based on these sequences, oligonucleotide primers were designed to amplify the coding region of each exon separately. By PCR-SSCP analysis, we identified eight new mutations in nine families originating from various countries. All induced a premature truncation of the protein, either in the short arm or in the globular C-terminal domain. A 2 bp deletion in exon 13, 2098delAG, was found in three French non-consanguineous families and a nonsense mutation of exon 20, Cys967stop, in two other non-consanguineous families originating from Italy. Determination of rare intragenic polymorphisms permitted us to show evidence of founder effects for these two mutations suggesting a remote degree of consanguinity between the families. Other, more frequent polymorphisms, G to A 1905 (exon 12), A to G 2848 (exon 19), A to G 5551 (exon 37), and G to A 6286 (exon 42), were used as intragenic markers for prenatal diagnosis. This study provides valuable methods for determining the molecular defects in LAMA2 causing merosin deficient congenital muscular dystrophy.

Child↗
PubMed1997

Mechanism of adenovirus improvement of cationic liposome-mediated gene transfer.

Substantial effort has been focused on the development of highly efficient gene transfer strategies. Although viral and non-viral methods have been elaborated, mechanisms of gene delivery are still poorly understood. We exploited our recent observation that replication-deficient type 5 adenovirus dramatically enhances lipofectAMINE-mediated gene transfer (lipoadenofection) in differentiated cells to elucidate the mechanism of adenovirus action in this process. Heat-induced denaturation of viral capsid abolishes adenovirus action whereas inactivation of viral genome by short treatment with UV has no effect. Electron microscopic observations reveal the formation of a complex containing adenovirus and lipofectAMINE which probably carries DNA into cells via endocytosis. Anti-adenovirus antiserum or monoclonal anti-alpha(v)beta3 integrin antibody inhibits lipoadenofection, at least partially. Neutralization of endosomal compartments with chloroquine, ammonium chloride or monensin does not prevent adenovirus improvement of gene transfer. Hence, adenovirus-lipofectAMINE-DNA complexes in which viral particles are each encompassed by three lipid layers, penetrate cells via an endocytic pathway involving probably the adenovirus receptor and alpha(v)beta3 integrin. The resulting efficient transfer and expression of plasmid DNA proceeds from a mechanism in which adenoviral endosomolytic activity appears to be required while viral genome is not essential.

Adenoviridae↗
PubMed1997

Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy.

Cardiac myosin binding protein C (MyBP-C) is a sarcomeric protein belonging to the intracellular immunoglobulin superfamily. Its function is uncertain, but for a decade evidence has existed for both structural and regulatory roles. The gene encoding cardiac MyBP-C (MYBPC3) in humans is located on chromosome 11p11.2, and mutations have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). Detailed characterization of the MYBPC3 gene is essential for studies on gene regulation, analysis of the role of MyBP-C in cardiac contraction through the use of recombinant DNA technology, and mutational analyses of FHC. The organization of human MYBPC3 and screening for mutations in a panel of French families with FHC were established using polymerase chain reaction, single-strand conformation polymorphism, and sequencing. The MYBPC3 gene comprises > 21,000 base pairs and contains 35 exons. Two exons are unusually small in size, 3 bp each. We found six new mutations associated with FHC in seven unrelated French families. Four of these mutations are predicted to produce truncated cardiac MyBP-C polypeptides. The two others should each produce two aberrant proteins, one truncated and one mutated. The present study provides the first organization and sequence for an MyBP-C gene. The mutations reported here and previously in MYBPC3 result in aberrant transcripts that are predicted to encode significantly truncated cardiac MyBP-C polypeptides. This spectrum of mutations differs from the ones previously observed in other disease genes causing FHC. Our data strengthen the functional importance of MyBP-C in the regulation of cardiac work and provide the basis for further studies.

Base Sequence↗
PubMed1997

Penetrance of familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant cardiac disease for which the penetrance remains a much-debated issue. Since the recent identification of the genes involved in the disease, the penetrance of FHC has not been reassessed in a large genotyped population. The aim of our study was therefore to evaluate it, according to age and sex, in ten families with previously identified mutations. Among 178 individuals we studied, 90 were genetically affected (9 different mutations in 3 genes). We found that penetrance, assessed by classical echocardiographic and electrocardiographic criteria, was (1) incomplete: 69%; (2) age-related: 55% between 10 and 29 years old, 75% between 30 and 49 y. and 95% over 50 y.; (3) greater in males than in females: 77% vs 58%, age-adjusted odds ratio: 3.98, CI 95%: 1.34 to 11,48; (4) similar for the genes analyzed. The consequences of these results for genetic counseling and linkage analyses are discussed.

Adolescent↗
PubMed1996

Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy is a phenotypically and genetically heterogeneous disease. In some families, the disease is linked to the CMH2 locus on chromosome 1q3, in which the cardiac troponin T gene (TNNT2) has been identified as the disease gene. The mutations found in this gene appear to be associated with incomplete penetrance and poor prognosis. Because mutational hot spots offer unique possibilities for analysis of genotype-phenotype correlations, new missense mutations that could define such hot spots in TNNT2 were looked for in unrelated French families with familial hypertrophic cardiomyopathy. METHODS AND RESULTS: Family members were genotyped with microsatellite markers to detect linkage to the four known disease loci. In family 715, analyses showed linkage to CMH2 only. To accurately position potential mutations on TNNT2, its partial genomic organization was established. Screening for mutations was performed by single-strand conformation polymorphism analysis and sequencing. A new missense mutation, Arg102Leu, was identified in affected members of family 715 because of a G-->T transversion located in the 10th exon of the gene. Penetrance of this new mutation is complete; echocardiographic data show a wide range of hypertrophy; and there was no sudden cardiac death in this family. CONCLUSIONS: The codon 102 of the TNNT2 gene is a putative mutational hot spot in familial hypertrophic cardiomyopathy and is associated with phenotypic variability. Analysis of more pedigrees carrying mutations in this codon is necessary to better characterize the clinical and prognostic implications of TNNT2 mutations.

Adult↗
PubMed1996

Efficient transfer of regulated genes in adipocytes and hepatoma cells by the combination of liposomes and replication-deficient adenovirus.

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepatoma cells with the lipofectAMINE/pSV2-CAT complex in the presence of replication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50-fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykinase gene fused to the CAT gene (pPL1-CAT). This construct was shown previously to be cAMP-responsive after calcium-phosphate-mediated transfection of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in which pPL1-CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2-3-fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.

3T3 Cells↗
PubMed1995

Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by a ventricular hypertrophy predominantly affecting the interventricular septum and associated with a large extent of myocardial and myofibrillar disarray. It is the most common cause of sudden death in the young. In the four disease loci found, three genes have been identified which code for beta-myosin heavy chain, cardiac troponin T and alpha-tropomyosin. Recently the human cardiac myosin binding protein-C (MyBP-C) gene was mapped to chromosome 11p11.2 (ref. 8), making this gene a good candidate for the fourth locus, CMH4 (ref. 5). Indeed, MyBP-C is a substantial component of the myofibrils that interacts with several proteins of the thick filament of the sarcomere. In two unrelated French families linked to CMH4, we found a mutation in a splice acceptor site of the MyBP-C gene, which causes the skipping of the associated exon and could produce truncated cardiac MyBP-Cs. Mutations in the cardiac MyBP-C gene likely cause chromosome 11-linked hypertrophic cardiomyopathy, further supporting the hypothesis that hypertrophic cardiomyopathy results from mutations in genes encoding contractile proteins.

Amino Acid Sequence↗
PubMed1995

Regulation of glucose transporters in cultured rat adipocytes: synergistic effect of insulin and dexamethasone on GLUT4 gene expression through promoter activation.

A triggering effect of insulin on GLUT4 expression in adipocytes is consistently observed in vivo, whereas GLUT1 is roughly unaffected. However, in cultured rat adipocytes, insulin increases GLUT1 but fails to increase GLUT4, suggesting that additional factors are involved in vivo. This prompted us to evaluate the potential role of glucocorticoids as coregulators with insulin of glucose transporter expression using 3T3-F442A adipose cells and primary cultured rat adipocytes. In both systems, insulin increased and dexamethasone decreased GLUT1 messenger RNA (mRNA) and protein, an effect inhibited by the glucocorticoid antagonist RU 38486. When the two hormones were added together, the effect of dexamethasone was dominant in 3T3-F442A cells, but was totally antagonized in rat adipocytes. Moreover, in rat adipocytes, the GLUT1 gene transcription rate (run-on) was identical in the absence or presence of the two hormones. With regard to GLUT4 expression, neither insulin nor dexamethasone alone had any significant effect after 2 days of treatment. In contrast, the combined hormones markedly increased GLUT4 mRNA (+550% in rat adipocytes; +130% in 3T3-F442A cells) and protein (+164% in rat adipocytes; +79% in 3T3-F442A cells) with a 24- to 48-h delay after mRNA induction. Studies of the molecular mechanism(s) showed that exposure of rat adipocytes to dexamethasone plus insulin did not affect GLUT4 mRNA stability, but increased the GLUT4 gene transcription rate 3-fold. Transient transfections of rat adipocytes with the 5'-flanking 2.2-kilobase sequence of the rat GLUT4 gene fused to luciferase demonstrated that promoter activity was unchanged by insulin, increased 50% by dexamethasone, and increased 3-fold in the presence of both. These data show that insulin elicits an increase in GLUT4 gene expression provided glucocorticoids are present. Our results indicate that the synergism between insulin and glucocorticoids on GLUT4 gene transcription is mediated through GLUT4 promoter activation.

Adipocytes↗
PubMed1995

Are free fatty acids related to plasma plasminogen activator inhibitor 1 in android obesity?

Plasminogen activator inhibitor 1 (PAI-1) levels are elevated in obese insulin-resistant subjects. However the mechanism underlying increased PAI-1 levels is unknown. To determine the impact of diabetes on PAI-1 levels and its possible relationship to insulin resistance, hyperinsulinemic euglycemic clamp studies were performed in nine lean control subjects, nine non-diabetic obese subjects and eight obese patients with NIDDM. Fasting plasma PAI-1 levels were 4.0 to 4.7 fold higher in the two obese groups than in the control group. During the 40 mU/m2 x min insulin infusion, suppression of FFA concentration was correlated with fasting plasma PAI-1 levels in both obese non-diabetic and obese NIDDM subjects. It is concluded that (1) obesity rather than diabetes itself plays a major role for the increased PAI-1 levels in NIDDM; (2) resistance to the antilipolytic effect of insulin, resulting in increased FFA concentrations, may participate in producing elevated PAI-1 levels in android obese subjects.

Adult↗
PubMed1995

Sensitive northern blot hybridization using digoxigenin RNA probes for the mRNA detection of two glucose transporter isoforms.

Diseases involving glucose metabolism disorders are more and more prevalent. Therefore the question of glucose transporter gene expression is being addressed in experimental and clinical studies. Radioactive probes are generally used to assess glucose transporter mRNA levels, but these probes are short-lived, costly and harmful to the environment. Alternative methods that do not present these disadvantages, for example digoxigenin (DIG) labelled probes, might prove to be very interesting for the study of glucose transporter mRNA. The aim of the present work was to compare DIG-labelled cRNA probes to 32P-labelled cRNA probes in order to see whether or not the non-radioactive method can be used to assess glucose transporter gene expression. This work shows that DIG-labelled glucose transporter (GLUT1 and GLUT4) cRNAs are suitable probes for the assessment of these gene expressions. We have found that the DIG system offers a much higher sensitivity than the 32P system for both GLUT1 and GLUT4 mRNA detection. This represents a decisive advantage in human studies where tissue quantity is a limiting factor. In addition, stability, safety, time saving and cost reduction are other considerations that make DIG-labelled GLUT1 and GLUT4 cRNAs very attractive.

Animals↗
PubMed1990

Long term regulation of glucose transporters by insulin in mature 3T3-F442A adipose cells. Differential effects on two glucose transporter subtypes.

The question of a long term regulatory role of insulin on adipocyte glucose transporter content was addressed using the differentiating or fully mature 3T3-F442A adipocytes. Glucose transport was measured in intact cells. Glucose transporter content in plasma membranes and low density microsomes (LDM) was assessed by cytochalasin B binding and Western analysis. In insulin- versus spontaneously differentiated adipocytes, glucose transport and glucose transporters content of plasma membranes and LDM were increased 5-, 4-, and 2-fold, respectively. Insulin deprivation for 24 h induced a redistribution of glucose transporters in those cells which then displayed 2-fold higher glucose transport and glucose transporter content in plasma membranes than spontaneously differentiated cells and 3-fold more glucose transporters in LDM. When fully insulin-differentiated adipocytes were insulin-deprived for 4 days, there was a marked decrease in glucose transporters in both membrane fractions that was fully reversible by reexposing the cells to insulin for 4 days. Glucose uptake changes were closely proportionate to changes in glucose transporter content of plasma membranes as assessed by an antiserum to the C-terminal peptide of the erythrocyte/HepG2/brain-type glucose transporter. When Western blots were immunoblotted with 1F8 monoclonal antibody, specific for glucose transporter in insulin responsive tissues, an abundant immunoreactive protein was detected in both plasma membranes and LDM but the amount of this glucose transporter did not change with insulin exposure in any membrane fractions. In conclusion, insulin plays a long term regulatory role on cultured adipocyte glucose transporter content through a selective effect on the erythrocyte/HepG2/brain-type glucose transporter.

Adipose Tissue↗
PubMed1990

Analysis of gene expression during adipogenesis in 3T3-F442A preadipocytes: insulin and dexamethasone control.

In the present study, we have investigated dexamethasone and insulin regulation of the expression of adipose-specific mRNA, namely, glycerophosphate dehydrogenase (G3PDH) and adipsin, at different stages of differentiation. During adipose conversion, insulin promotes an accumulation of G3PDH mRNA which is linked to cell differentiation; in fully differentiated cells, insulin is not required to maintain G3PDH gene expression. Differentiating cells in serum deprived medium already exhibit, at day 1, a maximal amount of mRNA encoding for adipsin, which is tenfold decreased by 10 nM of insulin; insulin also exerts a negative effect on the abundance of adipsin mRNA in mature cells. This result indicates that adipsin appears to be a very early marker of adipose conversion, the gene expression of which is down-regulated by the presence of insulin. Dexamethasone (DEX) decreases the G3PDH message at all stages of adipose conversion, while it promotes the accumulation of adipsin mRNA mainly in differentiating cells. In DEX-treated adipocytes, the transcription efficiency of the G3PDH gene is not altered, and reduction to 50% of the message is due essentially to an approximately twofold decrease in its half-life.

Adipose Tissue↗
PubMed1990

Analysis of the glucocorticoid receptor during differentiation of 3T3-F442A preadipocyte cell line in culture.

A pure glucocorticoid agonist RU 28362 and the potent antagonist RU 38486 were compared with dexamethasone for the evolution and the molecular nature of the GR during insulin-dependent conversion of 3T3-F442A preadipocytes into mature cells. In the whole cell assay system, the affinity for preadipocyte GR was observed in the order RU 38486 greater than RU 28362 greater than dexamethasone. The GR complex was most stable in presence of dexamethasone followed by the antagonist RU 38486 = the agonist RU 28362. Similar results were obtained in mature adipocytes but the binding of RU 38486 was more equivocal. An insulin-dependent differentiation process did not alter any of these parameters but increased the number of GR nearly fivefold over a 2-week period. Ion-exchange analysis of the cytosolic receptor revealed that the differentiation process was not accompanied by the appearance of any novel or new forms of GR, contrary to the situation in the liver, since both RU 38486 and dexamethasone were bound to identical molecular species of GR. These data provide a defined system for further analysis of cellular receptor as a function of steroid, tissue, and species, contrary to the classical dogma where GR is generally thought to be identical as a passive vehicle for the steroid in all circumstances, and affinity for steroid is generally equated with receptor stability.

Adipose Tissue↗
PubMed1990

[Decrease of gene expression of glycerophosphate dehydrogenase by dexamethasone in differentiated 3T3-F442A cells: antagonism with insulin and antiglucocorticoid RU38486].

Preadipocyte subclones derived from mouse 3T3 cells differentiate into adipocytes; this differentiation is characterized by an increased activity of numerous enzymes required for triglyceride synthesis and/or mobilization. Among these enzymes, the role of glycerophosphate dehydrogenase in the differentiation process has been previously reported. In the present work, we studied the hormonal regulation of glycerophosphate dehydrogenase gene expression (G3PDH) in differentiated 3T3-F442A adipocytes. Dexamethasone (DEX) elicited a 50% decrease in both mRNA content and specific activity of G3PDH. This effect was due to a posttranscriptional event since DEX shortened the half life of the mRNA, whereas it did not modify the transcription rate of this gene. The DEX effect is specific to G3PDH, since the expression of another adipose-specific gene, namely adipsin, is not modified by DEX treatment. Insulin counteracts the inhibitory effect of DEX, mainly by stabilizing the mRNA encoding for G3PDH. The antiglucocorticoid RU38486 is able to reverse DEX inhibition. Latter phenomenon suggests that DEX action on G3PDH gene expression could be mediated by glucocorticoid receptors.

Animals↗
PubMed1988

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content.

In vitro senescence of chondrocytes, characterized by a decline in the proliferation rate during late passages, resulted from a rapid growth rate in early subcultures to a complete loss of division after seven to nine passages. One senescent-associated phenotypic change was the apparent increase in the density of cytoplasmic cytoskeletal proteins. We examined the relationship between tubulin content and growth (measured by DNA and total protein contents and cell volume), using flow cytometry, in the assessment of cytoskeleton analysis during in vitro aging. In contrast with previous microscopic observations of tubulin organization, flow cytometry revealed a tubulin content that was modulated as a function of protein content and/or cell volume.

Animals↗