[Proteome and transcriptome].
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Genome, Human↗
Biomedical subjects
D Porquet
Publications and source records attributed to D Porquet.
Expression of pregnancy-associated plasma protein-A (PAPP-A) during human villous trophoblast differentiation in vitro.
Pregnancy-associated placental protein-A (PAPP-A), first isolated from maternal serum, has been identified as a metalloprotease cleaving insulin-like growth factor binding protein-4 (IGFBP-4). The source of PAPP-A during pregnancy is unclear. We therefore investigated PAPP-A expression during in vitro human villous cytotrophoblast cell (CT) differentiation into syncytiotrophoblast (ST). CT were isolated from normal first trimester, second trimester and term placentae (n=10) and cultured to form ST. PAPP-A mRNA was quantified by real-time PCR, and PAPP-A protein expression was studied by immunocytochemistry and TRACE technology with specific monoclonal antibodies. PAPP-A mRNA expression in total placental extracts increased during the course of pregnancy. PAPP-A protein was detected in the cytoplasm of both CT and ST. ST formation in vitro was associated with a 19-fold increase in PAPP-A mRNA expression and an 8-fold increase in PAPP-A secretion into the culture medium. No significant difference in PAPP-A production was observed between cultured cells isolated from early and term placentae. In conclusion, PAPP-A production in vitro, is associated to the differentiation of villous cytotrophoblast cells into syncytiotrophoblast, independently of the age of gestation.
Adult↗
Molecular and structural analysis of two novel mutations in a patient with mut(-) methylmalonyl-CoA deficiency.
Inherited defects in the gene encoding the methylmalonyl-CoA mutase (MCM) result in the mut forms of methylmalonic aciduria (MMA). Twelve mutations have been identified associated with the mut(-) phenotype. We report two novel mutations (K621N and D156N) in a compound heterozygote mut(-) patient. These two mutations and three previously published ones (H627N, A191E, Y231N) were mapped onto a three-dimensional homology model of the human MCM constructed from the crystal structure of the Propionibacterium shermanii enzyme.
Acyl Coenzyme A↗
Establishment of reference values of five amniotic fluid enzymes. Analytical performances of the Hitachi 911. Application to complicated pregnancies.
OBJECTIVES: to evaluate the analytical performances of the Hitachi 911 analyzer (Roche Diagnostics, F) for gamma-glutamyl-transferase, 5'nucleotidase, amylase, aspartate-amino-transferase and total-alkaline-phosphatase assay in amniotic fluid. To establish reference intervals for these five enzymes throughout gestation. To determine their antenatal diagnostic value. DESIGN AND METHODS: amniotic fluid samples were collected between 14 and 35 weeks of gestation. Weekly numbers ranged from 31 to 92. Techniques developed for serum enzyme assays were applied to amniotic fluid. Two pools of amniotic fluid containing low and high marker levels were used. Within-day and between-day variations were calculated, together with the limits of detection and linearity. Reference ranges were established on 508 amniotic fluid samples, including 23 samples from pregnancies with chromosomal aberrations, 14 with gastrointestinal tract defects and 5 with gastroschisis. RESULTS: the assay technique for total-alkaline-phosphatase assay had to be adapted to amniotic fluid, but no adaptation was necessary for the other markers. The within-day CV ranged between 2.2 and 11.2% for low-level samples and from 1.1 to 3.4% for high-level samples. The between-day CV ranged from 6.3 to 13.3% for low-level samples and 1.2 to 4.7% for high-level samples. Total-alkaline-phosphatase activity fluctuated throughout gestation. Amylase levels and aspartate-amino-transferase levels increased whereas gamma-glutamyl-transferase and 5'nucleotidase levels fell until delivery. All trisomy 18 and trisomy 13 pregnancies, 65% of Down's syndrome pregnancies and all pregnancies with digestive tract defects were associated with marked changes in the level of at least one enzyme. CONCLUSION: The Hitachi 911 is suited to rapid, reliable quantification of amniotic fluid enzymes with only minor adaptation. Useful reference intervals can be obtained throughout gestation. Gamma-glutamyl-transferase, 5'nucleotidase, total-alkaline-phosphatase and amylase assay can help to confirm echographic evidence of bowel disorders and thereby improve patient management especially in case of gastroschisis.
Alkaline Phosphatase↗
N219Y, a new frequent mutation among mut(degree) forms of methylmalonic acidemia in Caucasian patients.
Mutations in the MUT locus encoding for the methylmalonyl-CoA mutase (MCM) apoenzyme are responsible for the mut forms of methylmalonic acidemia (MMA). To date, 49 different mutations have been identified in mut MMA. Only two frequent mutations have been reported in the Japanese population and in African-Americans. Here we report a new missense mutation N219Y (731 A-->T) which we found in five unrelated families of French and Turkish descent. All the patients exhibited a severe mut(degree) phenotype and three of them were homozygotes for N219Y. Direct involvement of the mutation in the loss of enzyme activity was demonstrated by mutagenesis and transient expression study. Mapping of the mutation onto a three-dimensional model of human MCM constructed by homology with the Propionibacterium shermanii enzyme shows that it lies in a highly conserved secondary structure motif and might suggest impaired folding and/or poor stability compatible with the mut(degree) phenotype. Finally, a 1% N219Y carrier frequency was observed in a French anonymous control population. Thus, N219Y is the first frequent mut mutation to be reported in the Caucasian population.
Amino Acid Sequence↗
Utility of insulin-like growth factor-I and its binding protein assays.
Insulin-like growth factor-I circulates in serum either in free form or bound to insulin-like growth factor-binding proteins that modulate its bioavailability. Insulin-like growth factor-binding proteins interfere with insulin-like growth factor-I assay, which remains technically difficult. Many assays have been developed, but their results are somewhat discordant. The choice of separation method, standard and tracer considerably influences the results. The circulating concentration of insulin-like growth factor-I, however, is clearly dependent on nutritional status, and total levels are a valuable marker of nutritional status. The clinical utility of free insulin-like growth factor-I assay and simultaneous assay, in the same sample, of total insulin-like growth factor-I and its binding proteins (reflecting the bioavailable insulin-like growth factor-I fraction), remains to be evaluated.
Biomarkers↗
Prenatal diagnosis and follow up of biliary atresia.
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Adult↗
Biochemical investigation of foetal and neonatal thyroid function using the ACS-180SE analyser: clinical application.
Despite sonographic detection of foetal goitre, uncertainty persists in the initial diagnosis of thyrotoxicosis and hypothyroidism. The aim of this study was to establish foetal and neonatal iodothyronine and thyrotrophin reference values for the ACS-180SE analyser. From 22 to 36 weeks of gestation, median foetal serum free thyroxine (FT4) levels increased from 6.0 pmol/L to 143 pmol/L, while free tri-iodothyronine (FT3) levels increased from 0.7 pmol/L to 1.9 pmol/L and mean thyrotrophin (TSH) levels remained stable (10.2 +/- 3.8mU/L; n = 33). At birth, concentrations were independent of gender and gestational age. Among the 10 cases of sonographically detected foetal goitre, serum TSH and FT4 were measured in five, showing hypothyroidism (3/5) or hyperthyroidism (2/5). Cord blood TSH levels reflected the efficacy of prenatal therapy. Measurement of foetal FT4 and TSH can be used to confirm foetal thyroid dysfunction, whereas treatment efficacy can be assessed sonographically and confirmed by measurement of TSH assay at birth.
Female↗
Alpha-1-acid glycoprotein.
Alpha-1-acid glycoprotein (AGP) or orosomucoid (ORM) is a 41-43-kDa glycoprotein with a pI of 2.8-3.8. The peptide moiety is a single chain of 183 amino acids (human) or 187 amino acids (rat) with two and one disulfide bridges in humans and rats,respectively. The carbohydrate content represents 45% of the molecular weight attached in the form of five to six highly sialylated complex-type-N-linked glycans. AGP is one of the major acute phase proteins in humans, rats, mice and other species. As most acute phase proteins, its serum concentration increases in response to systemic tissue injury, inflammation or infection, and these changes in serum protein concentrations have been correlated with increases in hepatic synthesis. Expression of the AGP gene is controlled by a combination of the major regulatory mediators, i.e. glucocorticoids and a cytokine network involving mainly interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF alpha), interleukin-6 and IL-6 related cytokines. It is now well established that the acute phase response may take place in extra-hepatic cell types, and may be regulated by inflammatory mediators as observed in hepatocytes. The biological function of AGP remains unknown; however,a number of activities of possible physiological significance, such as various immunomodulating effects, have been described. AGP also has the ability to bind and to carry numerous basic and neutral lipophilic drugs from endogenous (steroid hormones) and exogenous origin; one to seven binding sites have been described. AGP can also bind acidic drugs such as phenobarbital. The immunomodulatory as well as the binding activities of AGP have been shown to be mostly dependent on carbohydrate composition. Finally, the use of AGP transgenic animals enabled to address in vivo, functionality of responsive elements and tissue specificity, as well as the effects of drugs that bind to AGP and will be an useful tool to determine the physiological role of AGP.
Adjuvants, Immunologic↗
Influence of the software algorithm on risk calculation in Down's syndrome screening.
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Algorithms↗
Evolution of GH secretion in urine during an in-patient slimming course in obese children.
OBJECTIVE: To estimate the change in GH excretion in urine (GH-U) during a slimming course, and if increased, to assess the components of the course related to the increase in obese children. DESIGN: Observational follow-up study of patients admitted for primary obesity to an in-patient slimming course lasting at least 10 weeks. SUBJECTS: 48 complete observations out of 54 consecutive pre-pubertal patients admitted to a paediatric centre for treatment of primary obesity (BMI greater than the 90th percentile of the national reference curves). MEASUREMENTS: GH excretion in urine by immunoradiometric assay, at entry and after 10 weeks, various anthropometric measurements, nutritional intake and departure from the prescribed diet, time spent in physical activity, sleep duration. RESULTS: A mean decrease of 0.90 standard deviations for BMI was accompanied by a 34% increase of GH-U. Time spent in physical activity was the only component of the course found to be related to the magnitude of GH-U increase. CONCLUSION: The results of this observational study confirm that GH-U is increased after a slimming course in children, and suggest that physical activity is a major contributor to the restoration of normal GH-U levels.
Body Constitution↗
Serum levels of insulin-like growth factor-1 in the three months following surgery for a hip fracture in elderly: relationship with nutritional status and inflammatory reaction.
BACKGROUND AND AIMS: Serum IGF-1 level declines with advancing age and is a reliable index of protein-energy undernutrition in elderly patients in a metabolically stable condition. We have examined the value of IGF-1 as an indicator of malnutrition in elderly patients during recovery within 1 month after surgery for a hip fracture and we have studied the relationship between changes in IGF-1 levels, nutritional status and inflammatory reaction during 2 month follow up. STUDY DESIGN: Forty elderly patients (mean age 84.0+/-1.9) were nutritionally assessed upon admission to our rehabilitation unit (D0) then monthly (D30-D60) by measurement of anthropometric and biological parameters. Two groups of patients were defined on the basis of mid-arm circumference (MAC) and tricipital skinfold thickness (TST) measurement at D0: group 1, undernourished (n=13) and group 2, normally nourished (n=27). Changes in anthropometric and biological parameters during the study were compared between these two groups of patients. RESULTS: IGF-1 level was significantly lower in group 1 (79.5+/-9.1 vs 108+/-8.6 ng/ml, P<0.05) while usual serum protein levels were in the same range in the two groups. Orosomucoid level significantly decreased during 2 month follow-up in group 2 (P<0.01), CRP level decreased but not significantly. TST and MAC decreased in group 2 (respectively P<0.02 and P<0.05) while anthropometric parameters tended to slightly increase in group 1 in relation with an increase in caloric intake (P<0.05). IGF-1 level increased significantly during the study in group 2 (P<0.05) and was significantly lower in patients with complications occurring between D0 and D30 in comparison with patients without complications (respectively P<0.05 at D0, P<0.02 at D30). IGF-1 level correlated with body mass index, MAC, TST, muscle midarm circumference, albumin and transthyretin levels. Changes in IGF-1 levels positively correlated with those in transthyretin levels and negatively with those in orosomucoid levels. CONCLUSION: IGF-1 level seems a reliable index of protein-energy under-nutrition in elderly patients in the recovery period after surgery for a hip fracture. This marker is influenced by the inflammatory reaction. Decline of inflammatory reaction, rather than change in nutritional status, is responsible for the variation in IGF-1 level during a 2 month follow-up.
Aged↗
Defect of villous cytotrophoblast differentiation into syncytiotrophoblast in Down's syndrome.
The syncytiotrophoblast (ST) is one of the major components of the human placenta, as it is involved in feto-maternal exchanges and the secretion of pregnancy-specific hormones. The aim of this study was to elucidate the formation and function of the ST in trisomy 21 (Down's syndrome). We first used the in vitro model of cytotrophoblast differentiation into ST. Cytotrophoblasts were isolated from 15 trisomy 21-affected placentas (12-35 weeks gestation) and 10 gestational age-matched control placentas. In vitro cytotrophoblasts isolated from normal placenta fused to form the ST. This was associated with an increase in transcript levels and in the secretion of hCG, human placental lactogen, placental GH, and leptin. In trisomy 21-affected placentas, we observed a defect (or a delay) in ST formation and a dramatic decrease in the synthesis and secretion of these hormones compared to those in cultured cells isolated from control age-matched placentas. These results were confirmed by a significant (P < 0.001) decrease in gene expression in total homogenates of trisomy 21-affected placentas compared to controls. These results will be of help in understanding the maternal hormonal markers of fetal trisomy 21 and the consequences of placental defects for fetal development.
Adult↗
Effects on bone metabolism of one year recombinant human growth hormone administration to children with juvenile chronic arthritis undergoing chronic steroid therapy.
OBJECTIVE: To study the effects on bone metabolism of treatment with recombinant human growth hormone (rhGH) in children with juvenile chronic arthritis (JCA) who are undergoing treatment with glucocorticoids (GC) and have severe bone lesions. METHODS: We assessed the effects of rhGH treatment (1.4 U/kg/week) on bone metabolism markers and bone density measured during a one year treatment course in 14 patients with systemic forms of JCA undergoing longterm GC treatment. RESULT: All patients at inclusion showed severe bone demineralization (mean bone density: -3.7 standard deviation score for chronological age). Compared to pretreatment values, bone formation markers (blood levels of osteocalcin and C-terminal propeptide of type 1 procollagen) and bone resorption markers (urinary hydroxyproline, pyridinoline, and deoxypyridinoline levels) increased significantly during treatment and returned to pretreatment values after discontinuation of rhGH. We observed that plasma level of osteocalcin was the best predictive variable of growth response to rhGH treatment in these patients. CONCLUSION: The results reflect an increase in bone turnover in these patients. Despite these biochemical changes no improvement of bone density was observed during the one year treatment. Treatment of longer duration is necessary to evaluate the curative effects of GH.
Adolescent↗
[Failure of differentiation of the trophoblast in trisomy 21].
The syncytiotrophoblast is the major component of the human placenta as it is involved in the feto-maternal exchanges and the secretion of pregnancy-specific hormones. In normal placenta, the multinucleated syncytiotrophoblast is formed by fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placenta, we show that it exists a defect (or a delay) in the syncytiotrophoblast formation and a decrease of the production of pregnancy-specific hormones. We show that it is related to the over expression of the SOD-1 located on chromosome 21. These results will be of help for the understanding of maternal hormonal markers of fetal trisomy 21 and the consequences of the placental defects on the fetal development.
Biomarkers↗
NF-kappaB is involved in the induction of the rat hepatic alpha1-acid glycoprotein gene by phenobarbital.
Phenobarbital, a classical inducer of the drug-metabolizing cytochrome P450 genes, induces alpha1-acid glycoprotein gene expression through a PB-responsive element (PBRE) located at position -142 to -126 from the transcriptional start site. The aim of this study was to investigate nuclear protein binding to the PBRE sequence after PB treatment. Cycloheximide treatment showed that de novo protein synthesis was not required for PB to induce AGP gene expression, pointing to post-translational modifications. Studies of the DNA-protein complex with the PBRE showed that phosphorylation status is a key regulator of the binding capacity of transactivating proteins involved in PB transcriptional activation. This DNA-protein complex, analyzed by southwestern blotting and UV cross-linking, involves three nuclear factors with molecular weights of 43, 52, and 65 kDa. Supershift and competition experiments showed that the 43-kDa factor can be related to C/EBPalpha and the 52- and 65-kDa factors to the two subunits of NF-kappaB.
Animals↗
Growth hormone inhibits rat liver alpha-1-acid glycoprotein gene expression in vivo and in vitro.
The gene encoding alpha-1-acid glycoprotein (AGP), one of the major acute-phase proteins, is positively controlled at the transcriptional level by cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) and glucocorticoids. Here, we show that growth hormone (GH) treatment of isolated rat hepatocytes in vitro reduces AGP messenger RNA (mRNA) expression. AGP gene expression remained inducible by IL-1, IL-6, and phenobarbital (PB) in GH-treated hepatocytes. Interestingly, the repressive effect of GH on AGP gene expression was also observed in vivo: liver AGP mRNA content was strongly increased in hypophysectomized rats, and GH treatment of these animals led to a decrease in mRNA to levels lower than those in untreated control animals. Moreover, the inhibitory effect of GH mainly occurs at the transcriptional level and can be observed as little as 0.5 hours after GH adding in vitro to isolated hepatocytes. These results show negative regulation of AGP gene expression and strongly suggest that GH is a major endogenous regulator of constitutive AGP gene expression. Moreover, transfection assays showed that the region of the AGP promoter located at position -147 to -123 is involved in AGP gene regulation by GH. Furthermore, GH deeply modifies the pattern of nuclear protein binding to this region. GH treatment of hypophysectomized rats led to the release of proteins of 42 to 45 and 80 kd and to the binding of proteins of 48 to 50 and 90 kd.
Animals↗
Rapid detection of insulin-like growth factor-binding protein-1 and foetal fibronectin in cervico-vaginal secretions to diagnose premature membrane rupture.
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Cervix Uteri↗