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Biomedical subjects

L Rossetti

Publications and source records attributed to L Rossetti.

At least 19 recordsLinked to original sources
PubMed2003

Molecular typing of Lactobacillus delbrueckii of dairy origin by PCR-RFLP of protein-coding genes.

Thirty-five strains of Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus isolated from dairy products were typed by restriction fragment length polymorphism (RFLP) of protein-coding genes. The strains were analysed by RFLP of PCR amplified, infragenic fragments of the following housekeeping genes: beta-galactosidase, lactose permease, and proline dipeptidase. Sequencing of the variable regions of the 16S rDNA was then performed on a reduced number of strains. PCR-RFLP analysis evidenced wide strain heterogeneity. Strains were grouped into genotypes according to both subspecies assignment and infra-species genetic polymorphism. This polymorphism was related to the presence of microbial groups within subspecies populations. The low infra-species sequence polymorphism detected in the variable region of the 16S rRNA gene did not enable to group the strains with the same sensitivity reached by PCR-RFLP of protein-coding genes. PCR-RFLP of protein-coding genes applied to L. delbrueckii seems a promising tool to evaluate microbial diversity within bacterial subpopulations. Differences among bacterial subpopulations based upon molecular heterogeneity in protein-coding genes would enable to better understand the role of strains from different ecological niches.

Bacterial Typing Techniques↗
PubMed2003

Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice.

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic alpha cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr(-/-)). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr(-/-) mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to alpha cell, and to a lesser extent, delta cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr(-/-) mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and alpha and delta cell numbers. Furthermore, the lean phenotype of Gcgr(-/-) mice suggests glucagon action may be involved in the regulation of whole body composition.

Animals↗
PubMed2003

Biodiversity in Lactobacillus helveticus strains present in natural whey starter used for Parmigiano Reggiano cheese.

AIMS: Lactobacillus helveticus is the dominant microflora of the natural whey starters used for Parmigiano Reggiano cheese making. The aim of this work was to study the biodiversity of different strains of Lact. helveticus present in six cultures and to compare them with strains of the same species previously isolated from natural whey cultures used for Grana Padano and Provolone cheeses. METHODS AND RESULTS: Twenty different biotypes of Lact. helveticus strains were identified combining the results deriving from SDS-PAGE of cell surface proteins and PCR fingerprinting using M13 as a primer. The biotypes were present in varying amounts in the six natural whey starters and the biodiversity was demonstrated not only within the whey cultures, but also between the whey cultures. CONCLUSIONS: Lact. helveticus strains isolated from Parmigiano Reggiano whey cultures analysed by PCR M13, SDS-PAGE and RFLP were distinguishable from Lact. helveticus strains of different dairy origin, namely Grana Padano and Provolone natural whey starters. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of different Lact. helveticus biotypes seems to be related to the specific ecosystem of cheese making and may be considered as one of the elements contributing to the typicality of Parmigiano Reggiano cheese.

Bacterial Typing Techniques↗
PubMed2001

Specific interactions of the telomeric protein Rap1p with nucleosomal binding sites.

The telomeres of Saccharomyces cerevisiae are structurally and functionally well characterized. Their telomeric DNA is packaged by the protein Rap1p (repressor activator protein 1). Rap1p is a multifunctional, sequence-specific, DNA-binding protein which, besides participating in the regulation of telomeres structure and length, is also involved in transcriptional regulation of genes essential for cell growth and in silencing. Whereas the long tracts of telomeric DNA repeats of higher eukaryotes are mostly organized in closely spaced canonical nucleosomal arrays, it has been proposed that the 300 base-pairs of S. cerevisiae telomeric DNA are organized in a large non-nucleosomal structure that has been called the telosome. Recently, nucleosomes have been found also in Tetrahymena thermophila telomeres, suggesting that, in general, telomere structural differences between lower and higher eukaryotes could be quantitative, rather than qualitative. Using an in vitro model system, we have addressed the question of whether Rap1p can form a stable ternary complex with nucleosomes containing telomeric binding sites, or competes with nucleosome core formation. The approach we have taken is to place a single Rap1p-binding site at different positions within a nucleosome core and then test the binding of Rap1p and its DNA-binding domain (Rap1p-DBD). We show here that both proteins are able to specifically recognize their nucleosomal binding site, but that binding is dependent on the location of the site within the nucleosome core structure. These results show that a ternary complex between a nucleosome and Rap1p is stable and could be a possible intermediate between telomeric nucleosomes and telosomes in the dynamics of S. cerevisiae telomere organization.

Binding Sites↗
PubMed2001

Prevalence of retinopathy in patients with anemia or thrombocytopenia.

BACKGROUND AND OBJECTIVES: Fundus lesions can be the accompanying symptom in many hematological diseases. In cases of anemia or thrombocytopenia, the exact mechanism leading to fundus abnormalities is not completely understood. The aim of this study was to calculate the prevalence of fundus lesions in anemic and thrombocytopenic patients, and define the risk factors for retinopathy. DESIGN AND METHODS: This cross-sectional study involved 226 patients with anemia and/or thrombocytopenia and a control group of 47 age-matched subjects. The studied variables were age, gender, hemoglobin levels, mean cell volume (MCV), red cell distribution width coefficient variation (RDW-CV), the type of onset of anemia, platelet counts, mean platelet volume (MPV), platelet distribution width (PDW), the platelet-large-cell ratio (P-LCR), prothrombin time (PT) and partial thromboplastin time (PTT). RESULTS: Retinopathy was observed in 28.3% of the patients as a whole, the presence of fundus lesions being closely associated with severe anemia (Hb < 8 g/dL) and severe thrombocytopenia (PLT < 50 x 109/L). Among the patients with concomitant anemia and thrombocytopenia, the incidence of retinopathy was 38%. Age, low hemoglobin levels, platelet counts, RDW-CV, and increased MCV, MPV and P-LCR were all significantly associated with the presence of fundus lesions at univariate analysis. INTERPRETATION AND CONCLUSIONS: Retinopathy is a frequent finding in anemic and thrombocytopenic patients, although it is often not significant clinically. As retinal hemorrhages were found in all of the patients with concomitant severe anemia and thrombocytopenia, it is recommended that all such patients undergo routine fundus examinations.

Aged↗
PubMed2001

Rapid identification of Lactobacillus brevis using the polymerase chain reaction.

AIMS: Species-specific PCR was applied to identify Lactobacillus brevis and the sensitivity and the specificity of the protocol were determined. METHODS AND RESULTS: Strains of Lact. brevis obtained from foods, particularly dairy products, and various strain collections, were identified by PCR using primers which amplified a 1340 bp fragment within the 16S rRNA gene. The PCR product was obtained after amplification of all the Lact. brevis strains tested; the size of the amplicon was as expected. No PCR products were observed after amplification from DNA of several lactic acid bacteria (LAB) species. CONCLUSIONS: A PCR method was optimized to identify Lact. brevis. The protocol was highly efficient and sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Conventional phenotypic methods often lead to ambiguous identification of LAB species belonging to Lact. brevis. The proposed protocol is sensitive, specific, and can be applied to total DNA extracted by use of chelating matrix with loss of neither sensitivity nor specificity.

Bacterial Typing Techniques↗
PubMed2001

Impaired biliary lipid secretion in obese Zucker rats: leptin promotes hepatic cholesterol clearance.

Human obesity is associated with elevated plasma leptin levels. Obesity is also an important risk factor for cholesterol gallstones, which form as a result of cholesterol hypersecretion into bile. Because leptin levels are correlated with gallstone prevalence, we explored the effects of acute leptin administration on biliary cholesterol secretion using lean (FA/-) and obese (fa/fa) Zucker rats. Zucker (fa/fa) rats become obese and hyperleptinemic due to homozygosity for a missense mutation in the leptin receptor, which diminishes but does not completely eliminate responsiveness to leptin. Rats were infused intravenously for 12 h with saline or pharmacological doses of recombinant murine leptin (5 microg x kg(-1) x min(-1)) sufficient to elevate plasma leptin concentrations to 500 ng/ml compared with basal levels of 3 and 70 ng/ml in lean and obese rats, respectively. Obesity was associated with a marked impairment in biliary cholesterol secretion. In biles of obese compared with lean rats, bile salt hydrophobicity was decreased whereas phosphatidylcholine hydrophobicity was increased. High-dose leptin partially normalized cholesterol secretion in obese rats without altering lipid compositions, implying that both chronic effects of obesity and relative resistance to leptin contributed to impaired biliary cholesterol elimination. In lean rats, acute leptin administration increased biliary cholesterol secretion rates. Without affecting hepatic cholesterol contents, leptin downregulated hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, upregulated activities of both sterol 27-hydroxylase and cholesterol 7alpha-hydroxylase, and lowered plasma very low-density lipoprotein cholesterol concentrations. Increased biliary cholesterol secretion in the setting of decreased cholesterol biosynthesis and increased catabolism to bile salts suggests that leptin promotes elimination of plasma cholesterol.

Animals↗
PubMed2001

Hyperglycemia modulates angiotensinogen gene expression.

Elevated plasma angiotensinogen (AGT) levels have been demonstrated in insulin-resistant states such as obesity and type 2 diabetes mellitus (DM2), conditions that are directly correlated to hypertension. We examined whether hyperinsulinemia or hyperglycemia may modulate fat and liver AGT gene expression and whether obesity and insulin resistance are associated with abnormal AGT regulation. In addition, because the hexosamine biosynthetic pathway is considered to function as a biochemical sensor of intracellular nutrient availability, we hypothesized that activation of this pathway would acutely mediate in vivo the induction of AGT gene expression in fat and liver. We studied chronically catheterized lean (approximately 300 g) and obese (approximately 450 g) Sprague-Dawley rats in four clamp studies (n = 3/group), creating physiological hyperinsulinemia (approximately 60 microU/ml, by an insulin clamp), hyperglycemia (approximately 18 mM, by a pancreatic clamp using somatostatin to prevent endogenous insulin secretion), or euglycemia with glucosamine infusion (GlcN; 30 micromol. kg(-1). min(-1)) and equivalent saline infusions (as a control). Although insulin infusion suppressed AGT gene expression in fat and liver of lean rats, the obese rats demonstrated resistance to this effect of insulin. In contrast, hyperglycemia at basal insulin levels activated AGT gene expression in fat and liver by approximately threefold in both lean and obese rats (P < 0.001). Finally, GlcN infusion simulated the effects of hyperglycemia on fat and liver AGT gene expression (2-fold increase, P < 0.001). Our results support the hypothesis that physiological nutrient "pulses" may acutely induce AGT gene expression in both adipose tissue and liver through the activation of the hexosamine biosynthetic pathway. Resistance to the suppressive effect of insulin on AGT expression in obese rats may potentiate the effect of nutrients on AGT gene expression. We propose that increased AGT gene expression and possibly its production may provide another link between obesity/insulin resistance and hypertension.

Adipose Tissue↗
PubMed2001

Central melanocortin receptors regulate insulin action.

Energy balance and insulin action are tightly coregulated. Leptin regulates energy intake and expenditure partly by modulation of the melanocortin pathway in the hypothalamus. Here we demonstrate potent effects of the melanocortin pathway on insulin action and body distribution of adiposity. Conscious rats received week-long infusions of either a melanocortin receptor agonist, alpha-melanocyte-stimulating hormone (alpha-MSH), or antagonist, SHU9119, in the third cerebral ventricle while food intake was maintained constant in each group. alpha-MSH decreased intra-abdominal fat and markedly enhanced the actions of insulin on both glucose uptake and production, while SHU9119 exerted opposite effects. Our findings elucidate a neuroendocrine network that is likely to play a central role in the coupling of energy intake and insulin action.

Animals↗
PubMed2001

Endogenous glucose production is inhibited by the adipose-derived protein Acrp30.

Intraperitoneal injection of purified recombinant Acrp30 lowers glucose levels in mice. To gain insight into the mechanism(s) of this hypoglycemic effect, purified recombinant Acrp30 was infused in conscious mice during a pancreatic euglycemic clamp. In the presence of physiological hyperinsulinemia, this treatment increased circulating Acrp30 levels by approximately twofold and stimulated glucose metabolism. The effect of Acrp30 on in vivo insulin action was completely accounted for by a 65% reduction in the rate of glucose production. Similarly, glucose flux through glucose-6-phosphatase (G6Pase) decreased with Acrp30, whereas the activity of the direct pathway of glucose-6-phosphate biosynthesis, an index of hepatic glucose phosphorylation, increased significantly. Acrp30 did not affect the rates of glucose uptake, glycolysis, or glycogen synthesis. These results indicate that an acute increase in circulating Acrp30 levels lowers hepatic glucose production without affecting peripheral glucose uptake. Hepatic expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and G6Pase mRNAs was reduced by more than 50% following Acrp30 infusion compared with vehicle infusion. Thus, a moderate rise in circulating levels of the adipose-derived protein Acrp30 inhibits both the expression of hepatic gluconeogenic enzymes and the rate of endogenous glucose production.

Adiponectin↗
PubMed2001

Overfeeding rapidly induces leptin and insulin resistance.

In common forms of obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia coexist. Here, we demonstrate rapid induction of insulin and leptin resistance by short-term overfeeding. After 3 and 7 days on the assigned diet regimen, rats were tested for their biological responses to acute elevations in plasma insulin and leptin concentrations. Severe resistance to the metabolic effects of both leptin and insulin ensued after just 3 days of overfeeding. During the insulin clamp studies, glucose production was decreased by approximately 70% in control rats and 28-53% in overfed rats. Similarly, leptin infusion doubled the contribution of gluconeogenesis to glucose output in control rats but failed to modify gluconeogenesis in overfed animals. These findings demonstrate a paradoxical and rapid collapse of the leptin system in response to nutrient excess. This partial failure is tightly coupled with the onset of insulin resistance.

Animals↗
PubMed2001

Physiological increase in plasma leptin markedly inhibits insulin secretion in vivo.

The demonstration of leptin receptors on the pancreatic beta-cells suggests the possibility of direct actions of leptin on insulin secretion. In vitro studies on islets or perfused pancreas and beta-cell lines produced inconsistent results. We performed an in vivo study to distinctly examine whether leptin has an effect on glucose-stimulated insulin secretion. Young chronically catheterized Sprague-Dawley rats (n = 28) were subjected to a 4-h hyperglycemic clamp study (approximately 11 mmol/l). At minute 120 to 240, rats were assigned to receive either saline or leptin (0.1, 0.5, and 5 microg x kg(-1) x min) infusion. Leptin decreased plasma insulin levels abruptly, and an approximately twofold decrease in plasma insulin levels compared with saline control was sustained over the 2 h of the study (14.8 +/- 5.8 vs. 34.8 +/- 2.6 ng/ml with leptin and saline infusion, respectively, P < 0.001). Moreover, a dose-dependent decrease in plasma insulin levels was noted (r = -0.731, P < 0.01). Since milrinone, an inhibitor of cAMP phosphodiesterase (PDE) 3, did not reverse the effect of leptin on glucose-induced insulin secretion, its action may be independent of PDE3. These findings suggest that acute physiological increase in plasma leptin levels acutely and significantly inhibits glucose-stimulated insulin secretion in vivo. The site of leptin effects on insulin secretion remains to be determined.

Animals↗
PubMed2001

Small increases in insulin inhibit hepatic glucose production solely caused by an effect on glycogen metabolism.

Based on our earlier work, a 2.5-fold increase in insulin secretion should completely inhibit hepatic glucose production through the hormone's direct effect on hepatic glycogen metabolism. The aim of the present study was to test the accuracy of this prediction and to confirm that gluconeogenic flux, as measured by three independent techniques, was unaffected by the increase in insulin. A 40-min basal period was followed by a 180-min experimental period in which an increase in insulin was induced, with euglycemia maintained by peripheral glucose infusion. Arterial and hepatic sinusoidal insulin levels increased from 10 +/- 2 to 19 +/- 3 and 20 +/- 4 to 45 +/- 5 microU/ml, respectively. Net hepatic glucose output decreased rapidly from 1.90 +/- 0.13 to 0.23 +/- 0.16 mg. kg(-1). min(-1). Three methods of measuring gluconeogenesis and glycogenolysis were used: 1) the hepatic arteriovenous difference technique (n = 8), 2) the [(14)C]phosphoenolpyruvate technique (n = 4), and 3) the (2)H(2)O technique (n = 4). The net hepatic glycogenolytic rate decreased from 1.72 +/- 0.20 to -0.28 +/- 0.15 mg. kg(-1). min(-1) (P < 0.05), whereas none of the above methods showed a significant change in hepatic gluconeogenic flux (rate of conversion of phosphoenolpyruvate to glucose-6-phosphate). These results indicate that liver glycogenolysis is acutely sensitive to small changes in plasma insulin, whereas gluconeogenic flux is not.

Animals↗
PubMed2000

Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation.

The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Sp1 followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta(1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.

Acetylglucosamine↗