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PubMed2001

Genetic bases of severe junctional epidermolysis bullosa presenting spontaneous amelioration with aging.

Change of the clinical picture with aging is noted in some patients suffering from junctional epidermolysis bullosa (JEB), an inherited blistering disorder caused by extensive disadhesion of the epithelia. We have studied a patient born with severe JEB associated with absent expression of laminin 5. A remarkable reduction of the blistering tendency was observed with aging that correlated with a restored expression of immunoreactive laminin 5 molecules. Genetic analysis of the gene LAMB3 detected compound heterozygosity for the nonsense mutation R635X and a novel 2 bp deletion (1587delAG) resulting in a downstream premature termination codon. RT-PCR amplification of total RNA purified from skin biopsies demonstrated that the mutated beta3 mRNAs underwent rapid decay shortly after birth, and that illegitimate splicing of the mRNA carrying mutation 1587delAG generated a new internally shortened beta3 transcript with advancing age. Our genetic and biochemical data show that (i) the illegitimate splicing of the beta3 pre-mRNA results in synthesis and secretion of a laminin 5 heterotrimer with an internally deleted beta3 polypeptide, (ii) expression of the mutated beta3 polypeptide is up-regulated in the basal keratinocytes with high proliferative potential, (iii) absence of the N-terminal region of the beta3 rod domain II thought to stabilize the tertiary structure of the laminin 5 is not required for the assembly of the protein and (iv) the mutant laminin 5 retains its adhesive potential. Our results demonstrate that mRNA rescue may underlie the evolution of the clinical phenotype in inherited skin conditions.

Age Factors↗
PubMed2003

Mutations in a novel gene Dymeclin (FLJ20071) are responsible for Dyggve-Melchior-Clausen syndrome.

Dyggve-Melchior-Clausen syndrome (DMC) is a rare autosomal-recessive disorder, the gene for which maps to chromosome 18q21.1. DMC is characterized by the association of a spondylo-epi-metaphyseal dysplasia and mental retardation. Electron microscopic study of cutaneous cells of an affected child showed dilated rough endoplasmic reticulum, enlarged and aberrant vacuoles and numerous vesicles. As the etiology of the disorder is unknown, we have used a positional cloning strategy to identify the DMC gene. We detected seven deleterious mutations within a gene predicted from a human transcript (FLJ20071) in 10 DMC families. The mutations were nonsense mutations (R194X, R204X, L219X, Q483X), splice site or frameshift mutations (K626N+92aa to stop). The DMC gene transcript is widely distributed but appears abundant in chondrocytes and fetal brain. The predicted protein product of the DMC gene yields little insight into its likely function, showing no significant homology to any known protein family. However, the carboxy terminal end comprises a cluster of dileucine motifs, highly conserved across species. We conclude that DMC syndrome is consequent upon loss of function of a gene that we propose to name Dymeclin, which may have a role in process of intracellular digestion of proteins.

Abnormalities, Multiple↗
PubMed1983

Decreased microfilarial load and its effect on the calculation of prevalence and the rate of false negatives in the detection of onchocerciasis.

It has been shown in the epidemiological evaluation of onchocerciasis that the method of taking two or more snips per person contains information about the probabilities of false negatives which can be extracted and used to improve the prevalence estimates. Using the method of maximum likelihood, one can obtain expressions for estimating the prevalence, adjusted for the false negatives, and for estimating the proportion of false negatives. The variances of all estimates are also available, making confidence interval estimation possible.

Humans↗
PubMed1991

Age and protein restriction followed by balanced refeeding affect pancreatic digestive enzyme outputs and turnover times in rats.

Outputs and turnover times of trypsinogen 2, chymotrypsinogen 1, lipase and amylase were determined in pancreatic juice of growing male Wistar rats at various times during protein restriction (5% protein) followed by balanced refeeding (20% protein). In control rats fed a 20% protein diet, trypsinogen 2, chymotrypsinogen 1 and amylase outputs increased progressively with age, those of lipase remained constant and the turnover times of the four hydrolases were shortened. With time, protein restriction induced the most rapid decrease in trypsinogen 2 output, followed by that of amylase, then by those of trypsinogen 1 and lipase. Compared with controls, protein restriction enhanced specific radioactivity in total proteins and each hydrolase and diminished the enzyme turnover times at the beginning of the experiment. Refeeding returned each hydrolase output to control values, but the turnover times remained shortened to the end of experiment. Pancreatic lobules were isolated from control and protein-depleted rat pancreata after 23 d of dietary treatment. After a pulse of [3H]leucine, lobules were incubated in Krebs-Ringer-bicarbonate-HEPES. Basal and stimulated (50 pmol cholecystokinin/L) secretions of total proteins, amylase and radioactive proteins were measured. Protein restriction resulted in a lower response of acinar cells to cholecystokinin. The reduced ability of cholecystokinin to stimulate secretion may be attributable to a lower number and/or to the reduced affinity of cholecystokinin receptors in acinar cells. Therefore, the observed output and turnover changes during protein restriction could be partly due to an impairment of cholecystokinin efficiency.

Aging↗
PubMed1992

Changes in serum and lipoprotein fatty acids of growing rats fed protein-deficient diets with low or adequate linolenic acid concentrations.

The effects of a protein-deficient diet associated with sunflower oil [adequate in 18:2(n-6), poor in 18:3(n-3)] or soybean oil [adequate in both 18:2(n-6) and 18:3(n-3)] on lipid serum and lipoprotein compositions were studied in growing rats. Four groups of rats were fed different diets: SFC (20% casein + 5% sunflower oil); SFd (2% casein + 5% sunflower oil); SC (20% casein + 5% soybean oil); Sd (2% casein + 5% soybean oil). After 28 d, both protein-deficient groups exhibited low concentrations of protein, phospholipid, triacylglycerol and total cholesterol in serum and VLDL. Compared with rats fed 20% casein diets, those fed low protein diets had lower 18:2(n-6) and 20:4(n-6) in phospholipids of serum, VLDL and HDL2-3, and the 20:4(n-6)/18:2(n-6) ratio was twofold higher in triacylglycerols of serum and VLDL. In the SFd-fed group, 22:5(n-6) was higher than in the SFC-fed group for both triacylglycerols and phospholipids in overall lipoprotein fractions studied. In addition, the 20:3(n-9)/20:4(n-6) ratio was 0.1 in HDL2-3 phospholipids of the SFd-fed group. Sunflower oil-fed rats compared with soybean oil-fed rats had greater monounsaturated fatty acids and lower total (n-3) fatty acids in both triacylglycerols and phospholipids of serum, VLDL and HDL2-3, as well as lower total (n-6) fatty acids in serum and VLDL triacylglycerols. Apolipoproteins (apo) of VLDL were drastically depressed in rats fed protein-deficient diets, whereas apo-AI of HDL2-3 showed a particular resistance. Likewise, sunflower oil-fed rats had enhanced apo-B48 of VLDL and apo-C, apo-AII and apo-AIV of HDL2-3. The present findings show that some effects of protein malnutrition were enhanced by alpha-linolenic acid deficiency, in particular reduced (n-6) and (n-3) fatty acid bioavailability.

Animals↗
PubMed1995

Rat plasma VLDL composition and concentration and hepatic lipase and lipoprotein lipase activities are impaired during two types of protein malnutrition and unaffected by balanced refeeding.

The relationships between VLDL concentrations and composition and changes in hepatic lipase and lipoprotein lipase activities were determined in rats, during the consumption of two low protein diets (2% casein or 5% gluten) (protein malnutrition) for 28 d, followed by the refeeding of a balanced diet for 14 d (15% casein) (refeeding). A control group was fed 15% casein for 42 d. In the control group, total lipolytic activity increased with age (r = 0.83, P < 0.001), whereas in both depleted groups, this activity remained low and stable throughout the period of protein malnutrition. At d 28 of protein malnutrition, plasma total lipolytic activities were significantly reduced in both depleted groups, (P < 0.05); hepatic lipase values represented 23% of the control value and lipoprotein lipase activity was about 11% of the control value. Moreover, lipid supply was even more dramatically diminished by the strong reduction in plasma VLDL concentration in both depleted groups. At d 14 of refeeding, lipoprotein lipase activities remained low in both depleted groups. Hepatic lipase activity was similar in the control and casein groups, but significantly higher in the gluten group. The VLDL composition varied significantly with each type of protein malnutrition and could be attributable to the different low levels of plasma VLDL-apolipoprotein C of rats fed both depleted protein diets, which involve an inhibiting or activating effect on lipoprotein lipase activity. Therefore, our results indicated that both protein-deficient diets investigated may diminish fatty acid supply in the various tissues involved.

Animals↗
PubMed1998

Digestion and absorption rates of [3H]-oleic acid and [14C]-triolein do not differ in rats fed heated (-) and (+) gossypol cottonseed and soybean flours.

This study was conducted to compare in vivo the acute effects of heated (+) and (-) gossypol cottonseed flours with those of soybean flour on lipid digestion and absorption in growing rats. Rats were fed by gastric intubation mixed [3H]-oleic acid and [14C]-triolein with heated flours or without flour (control). Lipid digestion and absorption were determined for 6 h after meal intubation. Both radioactivities recovered in gastrointestinal tract were significantly higher in rats fed (+) gossypol cottonseed flour than in all other groups. The majority of both recovered radioactivities was found in stomach contents, then in stomach wall and finally in intestinal wall. The distribution of both radioactivities at different gastrointestinal levels was similar. In stomach contents and wall, [14C]-radioactivity was primarily in triacylglycerols, but was also recovered in free fatty acids and diacylglycerols. In intestinal wall (mucosa + tunica) [3H]-radioactivity was found at greatest levels in free fatty acids, then in triacylglycerols and diacylglycerols. Greatest [14C]-radioactivity was found in triacylglycerols, then in free fatty acids, in diacylglycerols and last in phospholipids in rats fed the three flours. Therefore no quantitative differences in lipid digestion and absorption were observed among the rats fed the three flours. In conclusion, both cottonseed flours slowed lipid digestion and absorption compared with soybean flour and this delay was greater in the rats fed (+) gossypol cottonseed flour than in those fed (-) gossypol cottonseed flour. However, this inhibiting effect was probably too low to induce physiologically important effects on lipid digestion or absorption.

Animals↗
PubMed1998

Highly purified soybean protein is not hypocholesterolemic in rats but stimulates cholesterol synthesis and excretion and reduces polyunsaturated fatty acid biosynthesis.

The specific effects of soybean protein on lipid metabolism were determined with highly purified soybean protein. At 5 wk of age, growing rats were fed diets containing 20% highly purified soybean protein or casein supplemented or not with 0.1% cholesterol for 2 mo. Plasma and liver lipid composition, fecal steroid excretion and several hepatic enzyme activities were measured. There were no significant dietary protein-related differences in plasma and liver cholesterol concentrations. When diets were cholesterol free, highly purified soybean protein stimulated fecal neutral and acidic steroid excretion associated with concomitantly higher hydroxy methylglutaryl CoA (HMG-CoA) reductase activity, but lower cholesterol 7alpha-hydroxylase activity. Soybean protein lowered the linoleate desaturation index [20:4(n-6)/18:2(n-6)] in liver microsomal lipids and phospholipids. This may have been due to the reduced microsomal Delta6(n-6) desaturase activity in rats fed soybean protein, whereas Delta5(n-6) desaturase activity did not differ between groups fed the two proteins. Cholesterol supplementation (0.1%) did not affect plasma cholesterol but increased liver cholesterol and triacylglycerol concentrations and reduced HMG-CoA reductase activity; this latter effect was greatest in rats fed soybean protein. Cholesterol 7alpha-hydroxylase activity, however, was diminished only in rats fed casein. Desaturase activities, and particularly Delta5(n-6) activity, were lowered by cholesterol supplementation in rats fed both protein diets, including a significantly lower 20:4(n-6)/18:2(n-6) ratio in liver microsomal lipids and liver phospholipids. Thus although dietary proteins have no effect on serum cholesterol in rats, they affect enzyme activities involved in cholesterol metabolism and fatty acid desaturation.

Animals↗
PubMed2003

VLDL metabolism in rats is affected by the concentration and source of dietary protein.

The present study was designed to determine if changes in dietary protein level and source are related to changes in VLDL lipid concentrations and VLDL binding by hepatic membranes and isolated hepatocytes. Male Wistar rats were fed cholesterol-free diets containing 10, 20 or 30 g/100 g casein or highly purified soybean protein for 4 wk. Hepatic, plasma and VLDL lipids, VLDL apo B-100 and VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane were determined. Increasing casein or soybean protein level (from 10 to 30 g/100 g) in the diet increased VLDL apo B-100, indicating an increase in the number of VLDL particles. VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane increased when the protein level increased from 10 to 20 g/100 g in the diet and decreased with 30 g/100 g protein, regardless of protein type. The dietary protein source did not affect plasma total cholesterol concentrations at any protein level. Feeding 20 g/100 g soybean protein compared with casein lowered plasma triglyceride concentrations and VLDL number as measured by decreased VLDL-protein, -phospholipid, -triglyceride, -cholesterol and -apo B-100. VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane were higher in rats fed soybean protein than those fed casein. The higher VLDL uptake could be responsible for the hypotriglyceridemia in rats fed soybean protein.

Animals↗
PubMed2002

Parental smoking cessation and adolescent smoking.

OBJECTIVE: To examine the relation of parent smoking cessation to adolescent smoking and test its potential mediators. METHOD: Participants were 446 adolescents and their parents who completed a computerized measure of implicit attitudes toward smoking and questionnaires assessing smoking, parenting, and explicit attitudes. RESULTS: Parental smoking cessation was associated with less adolescent smoking, except when the other parent currently smoked. In general, ex-smoking parents showed more antismoking socialization than did smoking parents. However, in children's reports, these effects were negated if the other parent (particularly the mother) smoked. Children's reports of parents' antismoking behavior partially mediated the relation between parental smoking and adolescent smoking. Although children's implicit and explicit attitudes were unrelated to parental smoking, mothers' implicit attitudes were related to both their own smoking and their child's smoking. CONCLUSIONS: Parental smoking cessation may help lower risk for adolescent smoking. However, this benefit may be realized only if the other parent does not currently smoke. Antismoking parenting might be a useful focus in cessation interventions.

Adolescent↗
PubMed1997

DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter.

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.

Acetates↗
PubMed2001

Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.

This study evaluates the utility of Cre-expressing adenovirus for deletion of floxed genes in primary cells using primary murine hepatocytes. Adenovirus infection was very efficient, even at very low MOI (>95% infection at a MOI of 6) and did not reduce viability. High level LacZ expression was cytotoxic to hepatocytes but Cre expression had no effect on viability. Cre-mediated recombination was completed within a timespan that permits experimentation during primary culture (>95% recombination after 24 h), independently of the number of floxed alleles per cell. Recombination did not induce p53 or produce cytological nuclear abnormalities (even in polyploid cells). Contrary to expectation, deletion of DNA ligase 1 did not alter cell cycle progression, although Cre expression hastens entry to S phase from G(1), independently of the presence of floxed sequences. We conclude that adenovirus-mediated deletion of floxed alleles in primary cells is a straightforward and highly efficient tool for conducting preliminary studies of conditional gene targeting. Primary cells have advantages of differentiation, relative purity and ease of experimentation within controlled conditions, while avoiding confounding problems encountered in vivo (i.e. target cell specificity, kinetics and level of recombination, and elicitation of inflammatory and immune responses). This system could help identify important phenotypic effects and design and interpret in vivo studies.

Adenoviridae↗
PubMed1992

Renal venous erythropoietin concentrations in hypertensive patients with unilateral renal disease.

In 24 hypertensive patients with unilateral renal disease, the erythropoietin (Epo) concentration ratio in the renal veins was compared with the renin ratio. Seven patients showed moderately elevated peripheral Epo values. Epo and plasma renin activity were significantly positively correlated both in peripheral and renal veins. This suggests that the reduction of renal blood flow was a common, but not unique, stimulating factor of Epo and renin secretion. Epo ratio appeared insensitive since it was greater than 1.5 in only 30% of patients with a renin ratio > 2. Our results indicate that the Epo concentration ratio in renal veins cannot be proposed as a substitute for the currently used renin ratio.

Adolescent↗
PubMed1995

Specificity of antiperinuclear factor for rheumatoid arthritis in rheumatoid factor-positive sera.

Although antiperinuclear factor (APF) has the same specificity for rheumatoid arthritis (RA) as rheumatoid factor (RF), there is no evidence that this specificity is maintained in patients with positive RF-agglutination tests. Thus, we evaluated the specificity and usefulness of APF for RA diagnosis, regardless of RF titre. APF was tested (1:100 threshold) on 214 sera sent for RF evaluation over a 9-month period. These sera were previously determined to have latex or Rose-Waaler (RW) titres > or = 12 or 4 IU, respectively, but not necessarily above the threshold values of 100 and 32 IU. The APF test was performed blindly, and physicians were not advised of the results. In the patient population (119 RA and 95 non-RA) APF still demonstrated good specificity (0.82) for RA. As expected, APF proved useful for RA diagnosis in 28/33 (85%) RA cases with an RF level below 1:100 for latex and 1:32 for RW, thus reducing the number of 'seronegative' RA from 33/119 to 5/119. However, it also improved the serological positive predictive value for RA, even in cases when RW results were > or = 32 IU. Indeed, the positive predictive value for RA when both tests were positive was 0.94 (68/72), whereas concordant results (either positive or negative) for both APF and RW tests allowed correct classification (RA or non-RA) in 94% of cases.

Antibodies, Antinuclear↗