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Biomedical subjects

G Siest

Publications and source records attributed to G Siest.

At least 91 records · Page 5Linked to original sources
PubMed1998

Biological variations and genetic reference values for apolipoprotein E serum concentrations: results from the STANISLAS cohort study.

Serum apolipoprotein (apo) E concentrations were determined by immunoturbidimetry in 4284 subjects from 4 to 71 years of age and belonging to 1003 nuclear families recruited for the STANISLAS cohort study between January 1994 and August 1995. Values for apo E ranged from 16 to 169 mg/L, with a geometric mean +/- SD values of 46.6 +/- 13.8 mg/L in the overall sample. The interindividual variability varied from 24.6% to 32.0% among family members. Females exhibited higher apo E values than males until the age of 17-26 years. Conversely, after the age of 26 years, serum apo E concentrations were higher in men than in women. Biological factors affecting serum apo E concentrations were described in fathers, mothers, sons, and daughters and explained up to 32.0% of the apo E variability in daughters and 19.0% in fathers. The main biological factors affecting apo E concentrations were the following: apo E polymorphism, waist-to-hip ratio, oral contraceptive intake, puberty, body mass index, age, and gender. Given the importance of apo E polymorphism in the regulation of apo E concentrations, we recommend the use of genetic-based reference values for the clinical interpretation of serum apo E concentrations.

Adolescent↗
PubMed1997

Low and very low density lipoprotein composition and resistance to copper-induced oxidation are not notably modified in smokers.

To study whether tobacco use was associated with oxidative phenomena affecting lipoproteins, we estimated susceptibility of LDL and VLDL to an in vitro copper-mediated oxidation, and measured serum autoantibody titers against oxidized LDL in 45 middle-age healthy nonsmokers, 35 smokers and 37 ex-smokers of both sexes, taking into account the detailed lipid composition of the lipoproteins. VLDL from female smokers had higher triglyceride, phospholipid, apolipoprotein E and alpha-tocopherol content and showed a higher rate of copper-induced oxidation in comparison with those from nonsmokers (P < or = 0.05) whereas the relative composition of these particles in saturated, mono- or poly-unsaturated fatty acids was not modified by tobacco consumption. After adjustment for triglyceride content, no statistically significant difference in oxidation rate was observed. Lipid, alpha-tocopherol and protein composition of LDL did not appear to be influenced by smoking; in accordance with these observations, no difference in indices of in vitro oxidizability of LDL was noticed between the different groups. Autoantibody titers against oxLDL were similar in smokers and nonsmokers. We conclude that, in supposed healthy individuals, smoking does not seem to be associated with notable variations in composition of VLDL and LDL or with an increase of oxidizability of these atherogenic lipoproteins.

Adolescent↗
PubMed1997

Gene transfer technologies for the production of enzyme and protein reference materials.

To maintain the success of recommended methods and to allow comparison among various methods of enzyme analysis, enzyme reference materials are required, having catalytic properties as close as possible to those of the corresponding human enzymes. Though human sources are preferable, ethical reasons require the extraction and purification from animal tissues. By providing theoretically unlimited amounts of material, gene transfer technologies and mass culture can overcome the need of human or mammalian tissues. We have used these technologies to produce human gamma-glutamyltransferase (GGT) and pancreatic lipase (PL) in various types of host cells. Different strategies were tested, especially for GGT, depending on the inherent properties and requirements of the human enzyme. Expression and purification protocols were optimized, yielding good amounts of recombinant enzymes which share many physico-chemical and catalytic features with their natural counterparts. Kinetic constants and catalytic behavior were very similar, demonstrating the usefulness of these products as reference materials. We assume recombinant DNA technologies could be successfully applied to most enzymes or proteins assayed in clinical chemistry laboratories.

Animals↗
PubMed1997

Cerebrospinal fluid apolipoprotein E level is increased in late-onset Alzheimer's disease.

Worldwide evidence has recently shown that the allele epsilon4 of apolipoprotein E (ApoE) is a genetic risk factor for Alzheimer's disease (AD) underlining the possible role of apoE in the physiopathology of AD. To evaluate the usefulness of apoE concentration in pathogenesis of AD, we measured the cerebrospinal fluid (CSF) levels of apoE. CSF apoE level was significantly higher in 38 patients with late-onset AD than in 31 control patients and 47 patients suffering from other neurological and related diseases. Higher levels of CSF apoE were also present in a subset of patients with meningoencephalitis, motor neuron disease, and low back pain. The increase of CSF-apoE in AD is in agreement with results from studies that find an increase of mRNA apoE in the brains of AD patients. Compared to other works, these results underline the importance and the difficulties of the selection of the controls. The CSF apoE level seems to be a reflection of neuronal damage and/or an inflammatory reaction that may be common to AD and other neurological and related diseases.

Age of Onset↗
PubMed1997

Monometoxytrityl derivatives of uridine as inhibitors of a human recombinant UDP-glucuronosyltransferase: UGT1*6.

A series of inhibitors of the human liver recombinant UDP-glucuronosyltransferase 1*6 derived from uridine were synthetized as probes of the binding site of the cosubstrate, UDP-glucuronic acid. If triphenylmethanol or uridine alone failed to inhibit the glucuronidation of 4-methylumbelliferone, the trityl derivatives of uridine were found to be very effective inhibitors of the enzyme (Ki 4.4 to 73 microM). The type of inhibition (competitive or mixed) varied with the substitutions on the uracile or on the triphenylmethyl moiety by halogen atoms or methyl groups. Structural features for the binding of the cofactor are postulated.

Animals↗
PubMed1997

Characterization of three human apolipoprotein E isoforms (E2, E3 and E4) expressed in Escherichia coli.

Apolipoprotein E is one of the apolipoproteins involved in cholesterol metabolism. Three major isoforms are present in men: E2, E3, E4 corresponding to the products of three alleles. They have different affinities for receptors and the epsilon 4 allele is a risk factor for cardiovascular diseases and more recently for Alzheimer's disease. We describe here the production, by heterologous expression in Escherichia coli, of the three apolipoprotein E isoforms for use in both research and clinical laboratories. By Surface Plasmon Resonance, the purified recombinant apolipoprotein E isoforms were able to recognize three monoclonal anti-human apolipoprotein E antibodies with affinity constants close to those of purified human apolipoprotein E. For receptor binding studies, the recombinant apolipoprotein E isoforms were associated with VLDL isolated from apolipoprotein E knockout mice. Although the association of the recombinant apolipoproteins E with the mouse VLDL was less efficient than that of human plasma apolipoprotein E3, the recombinant apolipoprotein E3 and apolipoprotein E4 complexes competed efficiently with 125I-labelled LDL for binding to the LDL receptor in J774 macrophages, whereas the recombinant apolipoprotein E2-VLDL complexes did not. These results suggest that the recombinant apolipoprotein E isoforms have biological properties similar to the human apolipoprotein E isoforms.

Animals↗
PubMed1997

High sensitivity of laser-induced fluorescence detection in capillary gel electrophoresis for accurate apolipoprotein E genotyping.

Human apolipoprotein E (apoE) is a product of a polymorphic gene. In the general population, it shows two major mutations, which lead to the appearance of three common alleles encoding for three protein isoforms. This polymorphism is important in the regulation of lipid metabolism. Accurate apoE phenotyping or genotyping has become essential in clinical laboratories, since the epsilon 4 allele has been associated with cardiovascular and Alzheimer's diseases. Endonuclease restriction isotyping, followed by slab gel electrophoresis, is a rapid and convenient method for the investigation of common apoE genotypes. However, during the large-scale apoE genotyping of the STANISLAS cohort, we were confronted with a partial lack of sensitivity and resolution power of this traditional method, which sometimes leads to the misclassification of the genotypes epsilon 2/2 and epsilon 3/2. We have overcome this difficulty by separating the restriction fragments with capillary gel electrophoresis linked to laser-induced fluorescence detection. The baseline resolution was 2 bp, and the sensitivity limit attainable was similar to that by radioactive detection. The distinction between the epsilon 3/2 and the epsilon 2/2 genotypes became unequivocal, even when only low amounts of DNA were available for amplification.

Alleles↗
PubMed1997

Combined effects of lipoprotein lipase and apolipoprotein E polymorphisms on lipid and lipoprotein levels in the Stanislas cohort.

We have genotyped 1101 supposedly healthy subjects from the Stanislas cohort for the lipoprotein lipase (LPL) gene Ser417(C)-->stop (G) polymorphism and/or for the apolipoprotein (apo)E common polymorphism. Genotypic effects of the two polymorphisms on fasting serum triglycerides (TG), total cholesterol (Tchol), high density lipoprotein-cholesterol (HDLc), low density lipoprotein-cholesterol (LDLc), apoB, apoA-I, and apoE levels were studied separately for each polymorphism and in conjunction. epsilon 4 allele and high apoE levels were associated with high levels of LDLc, Tchol, apoB, and TG. The G allele of LPL was significantly associated with low TG levels. We found a clear interaction between the LPL/apoE polymorphisms and apoE levels on serum TG variation. Total variability of TG levels in women and men of 42.31% and 53.62% respectively, were mainly explained by apoE concentration and these two polymorphisms. ApoE and LPL genes simultaneously modulated TG levels.

Adult↗
PubMed1996

Cloning and expression of a novel type (III) of human gamma-glutamyltransferase truncated mRNA.

We report the characterization of a novel human gamma-glutamyltransferase mRNA type. This type III mRNA differs from type I and type II mRNAs previously described by several point mutations and the presence of an unspliced 81 bp intron in the open reading frame. Further, type III mRNAs are truncated ones and are tissue and pathology specifically expressed. In fact, type III mRNAs are present in human placenta, sigmoid, lung and in 50% of acute lymphoblastic leukemia blood cells but they are never found in healthy lymphocytes.

Adult↗
PubMed1996

Effects of apo B and apo E gene polymorphisms on lipid and apolipoprotein concentrations after a test meal.

The role of apo B signal peptide and apo E polymorphisms, and individual factors (age, sex, etc.) have been investigated on the interindividual variability of the postprandial response of 274 subjects ingesting a 1.260-KJ milkshake. The mean postprandial response, observed during 4 h, is significantly positive for total cholesterol (P < 0.005), LDL-cholesterol (P < 0.0001), triglycerides (P < 0.001), apo E (P < 0.0001) and glucose (P < 0.0001), whereas HDL-cholesterol, apo A-I and apo B do not present mean postprandial variation. Independently of the mean response, some parameters present a large interindividual variability of response, which is significantly influenced by cofactors, such as weight or BMI, for total and LDL-cholesterol, apo B and apo E or tobacco use for HDL-cholesterol. Sex has no effect on any lipid levels. Total, LDL-cholesterol and apo E responses are correlated with their corresponding fasting values. ApoB signal peptide polymorphism is not involved in the postprandial responses, whereas apo E polymorphism explains a significant part of the variability of HDL-cholesterol and apo A-I responses.

Adult↗
PubMed1996

Apolipoprotein E is highly susceptible to oxidation by myeloperoxidase, an enzyme present in the brain.

Apolipoprotein E, the most common apolipoprotein found in the brain, is linked to several pathologies like Alzheimer's disease. Apolipoprotein E directly binds to beta-amyloid with a strong affinity. Myeloperoxidase, a protein secreted by neutrophils and involved in the inflammatory process, is also present in the brain. In vitro myeloperoxidase oxidation of recombinant human apolipoprotein E leads to fragmentation of the protein with low concentrations of hydrogen peroxide and polymerization with higher concentrations. Comparison with bovine serum albumin shows a higher susceptibility of apolipoprotein E to myeloperoxidase oxidation, which may have importance in the Alzheimer's disease process.

Alzheimer Disease↗
PubMed1996

Interaction between human amphipathic apolipoproteins and amyloid beta-peptide: surface plasmon resonance studies.

Several apolipoproteins including apoE and apoA-I are known to be associated with amyloid beta-peptide, a major component of senile plaques in Alzheimer's disease. In the present study the interaction between three human amphipathic apolipoproteins apoE3, apoA-I and apoA-II and immobilized amyloid beta-peptide (1-40) was quantified by plasmon resonance. The interactions were saturable and reversible. The results demonstrated a high affinity of the binding of amphipathic apolipoproteins to amyloid beta-peptide. On the other hand, only a small population of synthetic amyloid beta-peptide participated in the interaction. The apparent equilibrium dissociation constants K(D) were 10 nM for apoE3, 25 nM for apoA-I and 80 nM for apoA-II under physiological conditions. The affinity of the apoE3-amyloid beta-peptide binding was not affected by pH in the range 6.0-8.0 but was significantly increased by high salt concentration. ApoA-I mainly followed similar patterns. A major participation of hydrophobic forces in the binding of apoE3 and apoA-I to amyloid beta-peptide was suggested.

Amyloid beta-Peptides↗
PubMed1996

Family study of lipoprotein lipase gene polymorphisms and plasma triglyceride levels.

To better characterize the role of the lipoprotein lipase (LPL) gene in the determination of triglyceride levels in healthy subjects, a study was performed in 193 nuclear families (384 parents, means age = 42.0 +/- 5.2 years; 399 offspring, mean age = 14.6 +/- 4.3 years) volunteering to have a free health checkup examination. The pattern of familial resemblance was compatible with a zero correlation between spouses, a weak father-offspring correlation (0.099 +/- 0.054; P < 0.07), and significant mother-offspring (0.235 +/- 0.053; P < 10(-4)) and sib-sib (0.294 +/- 0.064; P < 10(-4)) correlations. Associations of triglyceride levels with the LPL HindIII and PvuII polymorphisms were investigated by a familial measured genotype analysis, specifying sex- and age-dependent polymorphism effects. The effects associated with both polymorphisms were significant only in fathers, the H+ and P+ alleles being associated with raised triglyceride levels. The HindIII and PvuII polymorphisms explained 3.5% and 3%, respectively, of the variability of triglycerides in fathers. The relationship was weakened after prior adjustment on body mass index, but remained significant for PvuII. Because of the lack of effect in mothers and offspring, the polymorphisms did not contribute to the covariance of triglyceride levels in relatives. In conclusion, this family study showed a weak relationship of the HindIII and PvuII polymorphisms to plasma triglyceride levels in young healthy male subjects. The effects detectable only in fathers suggest a possible modulation of the LPL expression by hormonal or lifestyle factors.

Adolescent↗
PubMed1996

Single-step purification of two functional human apolipoprotein E variants hyperexpressed in Escherichia coli.

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH2-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into the Escherichia coli strain BL21(DE3). Recombinant strains were grown at 37 degrees C in a Luria and Bertani medium and the addition of isopropyl beta-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.

Antibodies, Monoclonal↗
PubMed1996

The rat, a useful animal model for pharmacological studies on apolipoprotein E.

Apolipoprotein E is a major protein component of lipoproteins and plays an important role in cholesterol transport. The structure of the gene and the polymorphism of apolipoprotein E have been studied in human and rat, which show similar structures of apolipoprotein E. The wide tissue distribution of this apolipoprotein suggests diverse functions like cholesterol distribution between cells, intracellular cholesterol trafficking and tissue reparation. Nevertheless, the presence of apolipoprotein E in atherosclerotic plaques and amyloid deposits in brains of Alzheimer's disease patients also indicate pathologic functions staying misunderstood. The aim of this paper is to review the present knowledge on the distribution of apolipoprotein E between the different organs with the related functions and to make an overview of the implications of this apolipoprotein is physiological events and pathological states in the rat. The rat is widely used for drug metabolism studies. Its serum levels are 5-10 times higher than in human and thus this animal provides an useful pharmacological model to elucidate the functions of apo E.

Animals↗
PubMed1996

Evaluation of urinary D-glucaric acid excretion in workers exposed to butyl glycol.

The biological follow-up of subjects exposed to butyl glycol (BG) is generally accomplished using a standard blood count that is not sensitive enough to reveal early intoxication by this molecule. For this reason we have used an indirect test for evaluating the induction of hepatic enzymes, the measurement in urine of D-glucaric acid (DGA), which reflects the activity of the glucuronic acid enzyme pathway. This study was performed on 17 foundry workers exposed to BG emissions coming from paints used in cataphoresis. The airborne concentration of BG was less than 0.3 times the average limit exposure value. This study shows that BG emissions at low concentrations are able to increase the activity of the enzymes of the glucuronic acid pathway. DGA urinary excretion increased by 165% in winter (p < .01) and by 85% (p < .05) in summer when the doors are open and the BG concentration lower. DGA urinary excretion is significantly higher in smoking than in nonsmoking exposed workers. None of these workers had a perturbed blood count. This study shows that the urinary level of DGA provides a good test for the follow-up of exposure to BG in the electrophoresis painting plant, and that the exposed smoking workers seem to be more sensitive to BG exposure than do the nonsmokers. In conclusion, the measurement of urinary DGA might be considered as a useful test for the surveillance of subjects exposed to vapors containing BG.

Adult↗
PubMed1996

Frequencies of five genetic polymorphisms in coronarographed patients and effects on lipid levels in a supposedly healthy population.

Allele frequencies of genetic polymorphisms were compared between supposedly healthy subjects and angiographically proven coronary artery disease patients. The polymorphic candidate loci investigated were the apolipoprotein (apo) B signal peptide and XbaI polymorphism, the apo E polymorphism and two polymorphism of lipoprotein lipase (LPL) gene: Hind/III and PvuII. Apo B signal peptide and HindIII/LPL polymorphisms showed significant differences in allele partition between cases and controls; the rare alleles of both polymorphisms were less frequent (p < 0.05) in cases. We looked for associations between the polymorphisms and lipid concentration variability in a supposedly healthy population (145 men and 144 women). Apo B signal peptide, apo E and PvuII/LPL polymorphisms seem to influence some lipid metabolism parameters significantly. Apo AI and LpCIII levels were significantly different among apo B signal peptide genotypes: Del homozygotes had the highest concentrations of both variables. The epsilon 4 allele of apo E polymorphism was associated with increased concentrations of total cholesterol, LDL cholesterol and apo B. Increased LpAI:AII levels observed in E3 homozygotes (p < 0.01) have not previously been reported. LpAI:AII concentration was also influenced by PvuII/LPL polymorphisms.

Adult↗