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Biomedical subjects

F David

Publications and source records attributed to F David.

At least 55 records · Page 3Linked to original sources
PubMed1997

Synthesis of [11C]RPR-72840A and its evaluation as a radioligand for the serotonin reuptake site in positron emission tomography.

RPR-72840A, an inhibitor of serotonin reuptake, was labelled with carbon-11. The synthesis of the nonradioactive precursor, which exhibited some unexpected chemistry, and its reaction with [11C]phosgene affording [11C]RPR-72840A are described. Biodistribution studies in rats and PET studies in baboons were conducted to evaluate [11C]RPR-72840A as a tracer for PET imaging of the serotonin reuptake system.

Animals↗
PubMed1997

Diagnosis of infections caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis using polymerase chain reaction in stool specimens.

OBJECTIVE: To study the usefulness of polymerase chain reaction (PCR) for the species identification of microsporidia in stool specimens obtained from HIV-infected patients with Enterocytozoon bieneusi or Encephalitozoon intestinalis infections. SETTING: Infectious disease clinic in a university hospital. PATIENTS: Thirty-seven stool specimens from 29 HIV-infected patients with microsporidiosis were tested. The diagnosis of microsporidian infection was made by light microscopy of stool specimens and species identification was made by transmission electron microscopy of duodenal biopsies. Sixty-one stool specimens from 45 HIV-infected patients without microsporidiosis served as controls. METHODS: PCR was performed using DNA extracted from stools with two primers sets, one specific for E. bieneusi and one specific for E. intestinalis. RESULTS: A 1265 base-pair fragment of the small subunit ribosomal RNA (rrs) gene could be amplified from all 31 stool specimens infected with E. bieneusi. In addition, a 930 base-pair fragment of the rrs gene could be amplified from all six stool specimens infected with E. intestinalis. The 61 control stools were negative with both primers. CONCLUSIONS: These results suggest that a PCR based assay using species-specific primers sets can be used successfully for microsporidian species differentiation from stool specimens, thus obviating the need for invasive biopsy procedures.

AIDS-Related Opportunistic Infections↗
PubMed1997

Selective modulation of protein kinase A and protein kinase C activities in epidermal growth factor (EGF)-stimulated MCF-7 breast cancer cells.

In human MCF-7 breast cancer cells, both protein kinase A (PKA) and different members of the protein kinase C (PKC) family are stimulated upon binding of epidermal growth factor (EGF) to cell surface receptors. Selective stimulation of calcium-dependent PKCs with 10(-6) to 10(-9) M Thymeleatoxin significantly increased the proliferation rate of MCF-7 cells over 5 days in culture. This stimulation was blocked by the PKC antagonist Chelerythrine. In contrast, selective activation of PKA by addition of 1 mM dibutyryl cyclic AMP (dBcAMP) did not affect the proliferation rate of MCF-7 cells. Similarly, activation of the adenylate cyclase by 1 microM Forskolin and inhibition of PKA by the cyclic AMP analogue Rp-cAMPS did not modulate the proliferation rate of these cells. Activation of PKC stimulated the expression of the immediate early gene c-fos but c-myc expression was not significantly enhanced. On the other hand, PKA activation increased both c-myc and c-fos expression in MCF-7 cells. These results suggest that PKA activation and c-myc expression are not obligatory for proliferation of MCF-7 cells.

Alkaloids↗
PubMed1996

Reduced cell surface expression of a mutated dipeptidyl peptidase IV (DPP IV/CD26) correlates with the generation of a beta strand in its C-terminal domain.

Dipeptidyl peptidase IV (DPP IV/CD26) belongs to a non-classical subfamily of serine-proteases. Sequence comparisons have identified Asp599, Ser624, Asp657, Asp702, and His734 as highly conserved residues of mouse DPP IV. We previously reported the identification of Ser624, Asp702 and His734 as the catalytic triad of mouse DPP IV (David, F., Bernard, A. M., Pierres, M., and Marguet, D. (1993) J Biol. Chem. 268, 17247-17252). Using site-directed mutagenesis, we have shown here that substitution of Asp599 for Ala (D599A) specifically decreases the cell-surface expression of DPP IV in stably transfected mouse fibroblasts. The D599A mutant remained as a high mannose immature glycoprotein and was rapidly degraded. This retention/degradation process correlates with the generation of a beta strand in the C-terminal region of DPP IV as shown by three dimensional computer modeling. Our results suggest that conserved residue Asp599 is important for the proper folding, glycosylation and transport of mouse DPP IV.

Amino Acid Sequence↗
PubMed1996

Correction of 13C mass isotopomer distributions for natural stable isotope abundance.

Metabolism of singly or multiply 13C-labeled substrates leads to the production of molecules that contain 13C atoms at various positions. Molecules differing only in the number of isotopic atoms incorporated are referred to as mass isotopomers. The distribution of mass isotopomers of many molecules can be measured by gas chromatography/ mass spectrometry after chemical derivatization. Quantification of metabolite mass isotopomer abundance resulting from biological processes necessitates correction of the measured mass isotopomer distribution of the derivatized metabolite for contributions due to naturally occurring isotopes of its elements. This correction must take into account differences in the relative natural abundance distribution of each mass isotopomer (skewing). An IBM-compatible computer program was developed which (i) calculates the natural abundance mass isotopomer distribution of unlabeled and labeled standards given the molecular formula of the derivatized molecule or fragment ion, and (ii) calculates the natural abundance mass isotopomer distribution of the singly and multiply labeled molecule or fragment via non-linear fitting to the measured mass isotopomer distribution of the unlabeled molecule or fragment. The output of this program is used to correct measured mass isotopomer distributions for contributions from natural isotope abundances and to verify measured values for theoretical consistency. Differences between predicted and measured unlabeled and 13C-labeled isotopomer distributions for hydroxamate di-t-butyl-dimethylsilyl (di-TBDMS) derivatized pyruvate were measured. The program was applied to the mass isotopomer distribution of glucose labeled from [U-13C3]glycerol and of fatty acids labeled from [U-13C6]glucose and either [2-13C2] acetate or [U-13C2]acetate. In some of these cases, the measured mass isotopomer distributions corrected by the program were different from those corrected by the classical technique. Implications of these differences including those on the calculation of glucose production due to gluconeogenesis in isolated perfused rat liver are discussed.

Animals↗
PubMed1996

Assay of the deuterium enrichment of water via acetylene.

A technique is presented for measuring the 2H enrichment of water in biological samples when this enrichment is greater than 0.2%. The sample is reacted with calcium carbide to form acetylene gas, which is determined by gas chromatography electron impact ionization mass spectrometry. Ion-molecule reactions, resulting in proton abstraction, are minimized by lowering the electron ionization energy from the usual 70 eV to 45 eV. This technique is much more rapid and economical than the classical isotope ratio mass spectrometric assay of the enrichment of hydrogen gas derived from reduction of water.

Acetylene↗
PubMed1996

Assay of the 13C and 2H mass isotopomer distribution of phosphoenolpyruvate by gas chromatography/mass spectrometry.

The 13C mass isotopomer distribution of liver phosphoenolpyruvate (PEP) yields important information on the regulation of gluconeogenesis and the citric acid cycle. A convenient technique is presented for measuring the mass isotopomer distribution of PEP in tissue extracts. The procedure involves reduction of extant pyruvate to lactate with NaBH4, enzymatic conversion of PEP to pyruvate, extraction of pyruvate hydroxamate and gas chromatographic/mass spectrometric determination of pyruvate hydroxamate di-tert-butyldimethylsilyl derivative. When PEP is labeled with 2H, the enzymatic conversion of PEP to pyruvate results in the loss of 2H. Therefore, to assay the enrichment of [2H]PEP, the tissue extract is chromatographed on an anion-exchange column. The fraction containing PEP is treated to form PEP tri(trimethylsilyl) derivative. The procedures were applied to liver PEP labeled using [U-13C3]lactate, [U-13C3]glycerol or 2H2O. The results show the compatibility between the mass isotopomer distributions of PEP and glucose in rat livers perfused with [U-13C3]lactate or [U-13C3]glycerol. There is a 78% isotopic equilibration of 2H enrichment between the hydrogens on C-3 of liver PEP and the hydrogens of water in 2 day fasted rats.

Animals↗
PubMed1996

Detection and species identification of intestinal microsporidia by polymerase chain reaction in duodenal biopsies from human immunodeficiency virus-infected patients.

Polymerase chain reaction (PCR) was used for the detection of microsporidian DNA in duodenal biopsies obtained from 28 human immunodeficiency virus (HIV)-infected patients with intestinal microsporidiosis. Duodenal biopsies from 23 HIV-infected patients without microsporidiosis served as controls. A generic primer set for human microsporidia was used at first for the PCR. Amplified products were detected in 26 (93%) of 28 biopsies from patients with intestinal microsporidiosis. All control biopsies were negative. Microsporidia species were identified using Southern blot hybridization with specific probes for Enterocytozoon bieneusi and Encephalitozoon intestinalis. This technique confirmed the transmission electron microscopy-based species identification. Similar results were obtained using PCR with species-specific primer sets for E. bieneusi and E. intestinalis. PCR testing of intestinal biopsy specimens can be used successfully for rapid detection and species differentiation of intestinal microsporidia and thus could be a valuable alternative to transmission electron microscopy.

AIDS-Related Opportunistic Infections↗
PubMed1996

Noninvasive probing of citric acid cycle intermediates in primate liver with phenylacetylglutamine.

In human and primate liver, phenylacetate and glutamine form phenylacetylglutamine, which is excreted in urine. Probing noninvasively the labeling pattern of liver citric acid cycle intermediates with phenylacetylglutamine assumes that the labeling pattern of its glutamine moiety reflects that of liver alpha-ketoglutarate. To validate this probe, we infused monkeys with [U-13C3]lactate, [3-13C]lactate, [1, 2-13C2]acetate, [2-13C]acetate, [U-13C3]glycerol, or 2-[3-13C]ketoisocaproate and compared the labeling patterns of urinary phenylacetyl-glutamine with those of glutamate and glutamine in liver, plasma, muscle, and kidney and liver alpha-ketoglutarate. Only with [U-13C3]lactate or [3-13C]lactate does the labeling pattern of phenylacetylglutamine reflect patterns of liver alpha-ketoglutarate and glutamate. With [13C]acetate, muscle and kidney glutamate are more labeled than liver metabolites. This confirms that with [13C]acetate, the labeling pattern of liver metabolites is influenced by 13CO2 and [13C]glutamine made in peripheral tissues. Our data validate the use of phenylacetylglutamine labeled from [3-13C]lactate or [3-13C]pyruvate to probe noninvasively the pyruvate carboxylase-to-pyruvate dehydrogenase flux ratio in human subjects.

Animals↗
PubMed1996

Contributions of liver and kidneys to glycerol production and utilization in the dog.

The classical concept holds that liver and kidneys are the main sinks of glycerol released by adipose tissue. However, rates of glycerol appearance (Ra) exceed the rate of glycerol delivery to liver and kidneys. We measured the hepatic and renal contributions to glycerol production and utilization in anesthetized dogs that were fasted either overnight or for 24 h after 3 days on a carbohydrate-free diet. Dogs were infused with [2H5]glycerol, and the concentration and 2H enrichment of glycerol were measured across liver and kidney. After a baseline period, either norepinephrine or glucose plus insulin was infused to alter the rate of glycerol production. Our study shows that the production of glycerol by liver and kidneys amounted to 4-9% and 4-7% of the Ra of glycerol, respectively. Uptake of glycerol by liver and kidneys amounted to 26-30 and 10-19% of the Ra of glycerol, respectively. Thus, contrary to the classical concept, the bulk of glycerol utilization occurs in nonhepatic, nonrenal tissues that have very low glycerol kinase activity per gram.

Animals↗
PubMed1996

Haplotype VIII of the Y chromosome is the ancestral haplotype in Jews.

DNA samples from Ashkenazic and Sephardic Jews were studied with the Y-chromosome-specific DNA probes p49f and p49a to screen for restriction fragment length polymorphisms and haplotypes. Two haplotypes (VII and VIII) are the most widespread, representing about 50% of the total number of haplotypes in Jews. The major haplotype in Oriental Jews is haplotype VIII (85.1%); haplotype VIII is also the major haplotype in the Djerban Jews (77.5%) (Djerban Jews represent probably one of the oldest Jewish communities). Together these results confirm that haplotype VIII is the ancestral haplotype in Jews.

Chromosome Mapping↗
PubMed1995

Limitations of the mass isotopomer distribution analysis of glucose to study gluconeogenesis. Substrate cycling between glycerol and triose phosphates in liver.

Mass isotopomer distribution analysis allows studying the synthesis of polymeric biomolecules from 15N, 13C-, or 2H-labeled monomeric units in the presence of unlabeled polymer. The mass isotopomer distribution of the polymer allows calculation of (i) the enrichment of the monomer and (ii) the dilution of the newly synthesized polymer by unlabeled polymer. We tested the conditions of validity of mass isotopomer distribution analysis of glucose labeled from [U-13C3]lactate, [U-13C3]glycerol, and [2-13C]glycerol to calculate the fraction of glucose production derived from gluconeogenesis. Experiments were conducted in perfused rat livers, live rats, and live monkeys. In all cases, [13C]glycerol yielded labeling patterns of glucose that are incompatible with glucose being formed from a single pool of triose phosphates of constant enrichment. We show evidence that variations in the enrichment of triose phosphates result from (i) the large fractional decrease in physiological glycerol concentration in a single pass through the liver and (ii) the release of unlabeled glycerol by the liver, presumably via lipase activity. This zonation of glycerol metabolism in liver results in the calculation of artifactually low contributions of gluconeogenesis to glucose production when the latter is labeled from [13C]glycerol. In contrast, [U-13C3]lactate appears to be a suitable tracer for mass isotopomer distribution analysis of gluconeogenesis in vivo, but not in the perfused liver. In other perfusion experiments with [2H5]glycerol, we showed that the rat liver releases glycerol molecules containing one to four 2H atoms. This indicates the operation of a substrate cycle between extracellular glycerol and liver triose phosphates, where 2H is lost in the reversible reactions catalyzed by alpha-glycerophosphate dehydrogenase, triose-phosphate isomerase, and glycolytic enzymes. This substrate cycle presumably involves alpha-glycerophosphate hydrolysis.

Animals↗
PubMed1995

Isotopomer analysis of citric acid cycle and gluconeogenesis in rat liver. Reversibility of isocitrate dehydrogenase and involvement of ATP-citrate lyase in gluconeogenesis.

We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-13C3]lactate, and 0.2 mM [U-13C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed: (i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002-13004 and Lee, W.-N.P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions inconsistent with Lee's citric acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate data amounted to only 60%, which is unlikely. All these conclusions are independent of the perfusion modes. We provide evidence that the following processes contribute to the observed labeling discrepancy: (i) the reversibility of the isocitrate dehydrogenase reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloacetate carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which we developed to incorporate the above reactions (Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and alpha-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.

ATP Citrate (pro-S)-Lyase↗