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Biomedical subjects

M F Robert

Publications and source records attributed to M F Robert.

At least 19 recordsLinked to original sources
PubMed2003

In vitro cytotoxicity activity of Diosquinone, a naphthoquinone epoxide.

Diosquinone [1], a naphthoquinone epoxide previously isolated from the root bark of Diospyros mespiliformis (Hostch) and D. tricolor [Ebenaceae] is been assessed for cytotoxicity activity against ten cancer cell lines by standard NIH method. The ethno-pharmacological claim of this plant and the previously observed good antibacterial activity of this compound among the others isolated from this plant suggest its probable cytotoxicity activity. Diosquinone was observed to be very active against most of the cancer cell lines. It shows very good activity against all the cell lines tested with ED50 value ranging between 0.18 microg/ml. against Human Glioblastoma (U373) to 4.5 microg/ml. against Hormone dependent human prostrate cancer( LNCaP).

Antineoplastic Agents↗
PubMed2001

The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice.

OBJECTIVE: To directly ascertain the physiological roles in adipocytes of hormone-sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. RESEARCH METHODS AND PROCEDURES: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. RESULTS: Homozygous HSL-/- mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild-type and deficient littermates. HSL-deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL-/- mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL-/- adipocytes, lipolysis is not significantly increased by beta3-adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL-/- adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48-hour period at 4 degrees C was similar in HSL-/- mice and controls. Overnight fasting was well-tolerated clinically by HSL-/- mice, but after fasting, liver triglyceride content was significantly lower in HSL-/- mice compared with wild-type controls. CONCLUSIONS: In isolated fat cells, the lipolytic rate after beta-adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL-/- adipocytes suggests that HSL-independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.

Adipocytes↗
PubMed2001

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: clinical course and description of causal mutations in two patients.

Hereditary deficiency of mitochondrial HMG-CoA synthase (mHS, OMIM 600234) is a poorly defined, treatable, probably underdiagnosed condition that can cause episodes of severe hypoketotic hypoglycemia. We present clinical follow-up and molecular analysis of the two known mHS-deficient patients. The diagnosis of mHS deficiency is challenging because the symptoms and metabolite pattern are not specific. Moreover, enzyme analysis is technically difficult and requires sampling of an expressing organ such as liver. The patients, now aged 16 and 6 y, have normal development and have had no further decompensations since diagnosis. Patient 1 is homozygous for a phenylalanine-to-leucine substitution at codon 174 (F174L). Interestingly, although the F174 residue is conserved in vertebrate mHS and cytoplasmic HS isozymes, a Leu residue is predicted in the corresponding position of HS-like sequences from Caenorhabditis elegans, Arabidopsis thaliana, and Brassica juncea. Bacterial expression of human F174L-mHS produces a low level of mHS polypeptide with no detectable activity. Similarly, in purified cytoplasmic HS, which in contrast to purified human mHS is stable and can be studied in detail, the corresponding F-->L substitution causes a 10,000-fold decrease in V(max) and a 5-fold reduction in thermal stability. Patient 2 is a genetic compound of a premature termination mutation, R424X, and an as-yet uncharacterized mutant allele that is distinguishable by intragenic single nucleotide polymorphisms that we describe. Molecular studies of mHS are useful in patients with a suggestive clinical presentation.

Adolescent↗
PubMed1998

HMG CoA lyase deficiency: identification of five causal point mutations in codons 41 and 42, including a frequent Saudi Arabian mutation, R41Q.

The hereditary deficiency of 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HL; OMIM 246450 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?246450]) results in episodes of hypoketotic hypoglycemia and coma and is reported to be frequent and clinically severe in Saudi Arabia. We found genetic diversity among nine Saudi HL-deficient probands: six were homozygous for the missense mutation R41Q, and two were homozygous for the frameshift mutation F305fs(-2). In 32 non-Saudi HL-deficient probands, we found three R41Q alleles and also discovered four other deleterious point mutations in codons 41 and 42: R41X, D42E, D42G, and D42H. In purified mutant recombinant HL, all four missense mutations in codons 41 and 42 cause a marked decrease in HL activity. We developed a screening procedure for HL missense mutations that yields residual activity at levels comparable to those obtained using purified HL peptides. Codons 41 and 42 are important for normal HL catalysis and account for a disproportionate 21 (26%) of 82 of mutant alleles in our group of HL-deficient probands.

Base Sequence↗
PubMed1998

3-Hydroxy-3-methylglutaryl-CoA lyase (HL): gene targeting causes prenatal lethality in HL-deficient mice.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL, EC 4.1.3.4) catalyses the last step of ketogenesis from leucine and fatty acids. HL deficiency in humans is one of the many inborn errors of CoA ester metabolism. By gene targeting, we created a strain of HL-deficient mice. Heterozygous HL-deficient mice are clinically normal and fibroblasts from homozygous HL-deficient embryos grow normally despite absence of HL activity. In contrast, homozygous HL-deficient embryos die at approximately 11.5 days post-coitum. Histologically, HL-deficient embryos show marked vacuolization, particularly in liver. Ultrastructural studies of hepatocytes obtained before death from HL-deficient embryos reveal abnormal dilated mitochondria. HL-deficient mice are the first mammalian example of a disease primarily affecting CoA ester metabolism with abnormal prenatal development.

Alleles↗
PubMed1997

Hormone-sensitive lipase (Lipe): sequence analysis of the 129Sv mouse Lipe gene.

Hormone-sensitive lipase (Lipe) catalyzes both the release lease of fatty acids from storage triglycerides in adipocytes and the liberation of cholesterol from cholesterol esters in steroidogenic tissues. Lipe activity is regulated in a tissue-, development- and hormone-specific fashion, the latter in large part by serine phosphorylation. We cloned and sequenced the Lipe gene from the 129Sv strain mouse, including 2.7 kb of the 5' nontranslated region. The primary transcript of the 129Sv Lipe locus spans 9.6 kb and contains 9 exons. We studied the curious hypervariable region immediately 5' to the regulatory serine residues by aligning the peptide and nucleic acid sequences of mouse, human, and rat Lipe. We propose that much of the variability is attributable to differences in the copy number of a 12-nucleotide repeat that shifts the intron 7 acceptor splice site. Introns 1 and 7 contain B1 elements, which in intron 7 are immediately adjacent to a tetranucleotide repeat. The mouse Lipe promoter region contains numerous potential binding motifs for factors implicated in adipose tissue expression and hormone responsiveness including adipocyte determination- and differentiation-dependent factor 1 (ADD1/SREBP1).

Amino Acid Sequence↗
PubMed1996

3-Hydroxy-3-methylglutaryl CoA lyase (HL): mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients.

3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, we found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene.

Animals↗
PubMed1996

Characterization of the hydroxymethylglutaryl-CoA lyase precursor, a protein targeted to peroxisomes and mitochondria.

We previously showed that human liver hydroxymethylglutaryl-CoA (HMG-CoA) lyase (HL; EC 4.1.3.4) is found in both mitochondria and peroxisomes. HL contains a 27-residue N-terminal mitochondrial targeting sequence which in cleaved on mitochondrial entry, as well as a C-terminal Cys-Lys-Leu peroxisomal targeting motif. Because peroxisomal HL has a greater molecular mass and more basic pI value than mitochondrial HL, we predicted that peroxisomal HL retains the mitochondrial leader. To test this hypothesis, we expressed both the precursor (pHL) and mature (mHL) peptides in Escherichia coli and studied their properties. pHL purified by ion-exchange and hydrophobic chromatography had a pI of 7.6 on FPLC chromatofocusing and a molecular mass of 34.5 kDa on SDS/PAGE, similar to our findings for peroxisomal HL. For purified mHL, pI (6.2) and molecular mass (32 kDa) values resemble those of mitochondrial HL. Purified pHL is similar to mHL in K(m) for HMG-CoA (44.8 microM), k(cat) (6.3 min(-1)) and pH optimum (9.0-9.5). However, the quaternary structures of pHL and mHL differ. On Superose 12 FPLC gel filtration and also on ultrafiltration, both in the presence and in the absence of HMG-CoA), pHL behaves as a monomer whereas mHL migrates as a dimer. We conclude that the HL percursor is probably identical to peroxisomal HL, that its catalytic properties resemble those of mature mitochondrial HL, and that the mitochondrial leader peptide prevents dimerization on pHL.

Amino Acid Sequence↗
PubMed1996

Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient.

Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.2.8.3.5) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single approximately 3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes >> fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency.

Amino Acid Sequence↗
PubMed1995

Molecular prenatal diagnosis of 3-hydroxy-3-methylglutaryl CoA lyase deficiency.

We report the first molecular prenatal diagnosis of 3-hydroxy-3-methylglutaryl CoA lyase (HL) deficiency. The proband had a classic but severe presentation with hypoketotic hypoglycaemia and acidosis, secondary mental retardation, and epilepsy, and HL deficiency was documented in cultured fibroblasts. We found him to be homozygous for the frameshift mutation N46fs (+1), which yields a distinct pattern on single-strand conformation polymorphism (SSCP) analysis. In two subsequent pregnancies, molecular prenatal diagnosis was performed using SSCP. In the first, chorionic villus biopsy was normal. In the second pregnancy, amniocentesis revealed an affected fetus. In both pregnancies, the diagnosis was confirmed enzymatically. HL activity was less than 7 per cent of control values in amniocytes and fetal liver of the affected pregnancy. In the second pregnancy, amniotic fluid metabolite measurements by stable isotope dilution-selected ion monitoring mass spectrometry showed greater than 100-fold increases of 3-hydroxy-3-methylglutaric acid and of 3-methylglutaconic acid levels compared with controls.

Amniocentesis↗
PubMed1995

Medical aspects of ketone body metabolism.

Ketone bodies are produced in the liver, mainly from the oxidation of fatty acids, and are exported to peripheral tissues for use as an energy source. They are particularly important for the brain, which has no other substantial non-glucose-derived energy source. The 2 main ketone bodies are 3-hydroxybutyrate (3HB) and acetoacetate (AcAc). Biochemically, abnormalities of ketone body metabolism can present in 3 fashions: ketosis, hypoketotic hypoglycemia, and abnormalities of the 3HB/AcAc ratio. Normally, the presence of ketosis implies 2 things: that lipid energy metabolism has been activated and that the entire pathway of lipid degradation is intact. In rare patients, ketosis reflects an inability to utilize ketone bodies. Ketosis is normal during fasting, after prolonged exercise, and when a high-fat diet is consumed. During the neonatal period, infancy and pregnancy, times at which lipid energy metabolism is particularly active, ketosis develops readily. Pathologic causes of ketosis include diabetes, ketotic hypoglycemia of childhood, corticosteroid or growth hormone deficiency, intoxication with alcohol or salicylates, and several inborn errors of metabolism. The absence of ketosis in a patient with hypoglycemia is abnormal and suggests the diagnosis of either hyperinsulinism or an inborn error of fat energy metabolism. An abnormal elevation of the 3HB/AcAc ratio usually implies a non-oxidized state of the hepatocyte mitochondrial matrix resulting from hypoxia-ischemia or other causes. We summarize the differential diagnosis of abnormalities of ketone body metabolism, as well as pertinent recent advances in research.

3-Hydroxybutyric Acid↗
PubMed1994

Human mitochondrial HMG CoA synthase: liver cDNA and partial genomic cloning, chromosome mapping to 1p12-p13, and possible role in vertebrate evolution.

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholesterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of a HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. The nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistent with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain.

Amino Acid Sequence↗
PubMed1993

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL). Cloning of human and chicken liver HL cDNAs and characterization of a mutation causing human HL deficiency.

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) catalyzes the final step of ketogenesis, an important pathway of mammalian energy metabolism. HL deficiency is an autosomal recessive inborn error in man leading to episodes of hypoglycemia and coma. Using the N-terminal peptide sequence of purified chicken liver HL, we designed degenerate sequence primers and amplified an 89-base pair (bp) chicken liver HL cDNA fragment. Longer cDNA clones for chicken (1384 bp) and human (1575 bp) HL were obtained by library screening. The peptide sequence predicted from the chicken clone contains two peptides from purified chicken HL. Mature human and chicken HL are 298-residue peptides. The sequence of the human clone predicts a 27-residue mitochondrial leader and a 31.6-kDa mature HL peptide. Human fibroblast and liver RNA contain a single 1.7-kilobase HL message. Two Acadian French-Canadian siblings with HL deficiency were homozygous for a 2-base pair deletion within the Ser-69 codon (S69fs(-2)), predicted to result in a truncated nonfunctional HL peptide lacking a complete active site. S69fs(-2) was not present in 12 other HL-deficient patients of 10 other ethnic origins, showing that HL deficiency is genetically heterogeneous.

Amino Acid Sequence↗
PubMed1993

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL): cloning and characterization of a mouse liver HL cDNA and subchromosomal mapping of the human and mouse HL genes.

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) is a homodimeric mitochondrial matrix enzyme that catalyzes the last step of ketogenesis. Using a human HL cDNA as a probe, we isolated a 1.4-kb mouse HL cDNA (HLM) from a mouse liver library and extended the sequence in the 5' direction, using RACE PCR to include the complete coding sequence. The nucleotide sequence of the mouse HL coding region is 85.7% identical to human HL, and 52.6% to Ps. mevalonii HL. Peptide identities of 87.4% and 54.3% respectively were observed. Southern analysis of 29 strains of laboratory mice and of Mus spretus revealed a total of about 25 kb of hybridizing fragments and three polymorphic fragments in both EcoRI and Hin-dIII digestions. The mouse HL locus (Hmgcl) was localized on Chromosome (Chr) 4: Pmv-19-12.6 +/- 3.6 cM-Hmgcl-7.3 +/- 2.3 cM-Xmv-8-1.5 +/- 1.0 cM-Gpd-1. The human HL locus (HMGCL) was mapped to distal Chr 1p by analysis of a human-hamster hybrid cell panel and by in situ hybridization.

Amino Acid Sequence↗
PubMed1992

Ornithine delta-aminotransferase mutations in gyrate atrophy. Allelic heterogeneity and functional consequences.

Ornithine delta-aminotransferase is a nuclear-encoded mitochondrial matrix enzyme which catalyzes the reversible interconversion of ornithine and alpha-ketoglutarate to glutamate semialdehyde and glutamate. Inherited deficiency of ornithine delta-aminotransferase results in ornithine accumulation and a characteristic chorioretinal degeneration, gyrate atrophy of the choroid and retina. We have surveyed the ornithine delta-aminotransferase genes of gyrate atrophy patients for mutations. Using a variety of techniques, we discovered and molecularly characterized 21 newly recognized ornithine delta-aminotransferase alleles. We determined the consequences of these and three previously described mutations on ornithine delta-aminotransferase mRNA, antigen, and enzyme activity in cultured fibroblasts. The majority (20/24) of these alleles produce normal amounts of normally sized ornithine delta-aminotransferase mRNA. By contrast, only 2/24 had normal amounts of ornithine delta-aminotransferase antigen. Reproducing these mutations by site-directed mutagenesis and expressing the mutant ornithine delta-aminotransferase in Chinese hamster ovary cells confirms that several of these mutations inactivate ornithine delta-aminotransferase and cause gyrate atrophy in these patients.

Alleles↗
PubMed1987

Congenital cytomegalovirus infection with osteolytic lesions: use of DNA hybridization in diagnosis.

A case of congenital cytomegalovirus (CMV) infection with long-bone lesions is presented. The bone lesions consisted of broad regions of generalized osteopenia with irregular fragmentation and spiculization at the zone of provisional calcification. Diagnosis of CMV infection was made by DNA spot hybridization of the urine sediment to DNA from the Towne strain of CMV, demonstrating the usefulness of DNA hybridization for identification of CMV in clinical specimens. Bone lesions associated with congenital CMV infection are useful early clues to diagnosis but can be indistinguishable from those of congenital rubella syndrome.

Bone Diseases, Metabolic↗