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Biomedical subjects

M Rossi

Publications and source records attributed to M Rossi.

At least 271 records · Page 15Linked to original sources
PubMed1998

Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant.

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.

Adjuvants, Immunologic↗
PubMed1998

Structure-function studies on beta-glycosidase from Sulfolobus solfataricus. Molecular bases of thermostability.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a thermostable tetrameric protein with a molecular mass of 240 kDa which is stable in the presence of detergents and has a maximal activity above 95 degrees C. An understanding of the structure-function relationship of the enzyme under different chemical-physical conditions is of fundamental importance for both theoretical and application purposes. In this paper we report the effect of basic pH values on the structural stability of this enzyme. The structure of the enzyme was studied at pH 10 and in the temperature range 25-97.5 degrees C using circular dichroism, Fourier-transform infrared and fluorescence spectroscopy. The spectroscopic data indicated that the enzyme stability was strongly affected by pH 10 suggesting that the destabilization of the protein structure is correlated with the perturbation of ionic interactions present in the native protein at neutral pHs. These experiments give support to the observation derived from the 3D-structure, that large ion pair networks on the surface stabilize Sulfolobus solfataricus beta-glycosidase.

Circular Dichroism↗
PubMed1998

Developmental regulation of NF1 tumor suppressor gene in human peripheral nerve.

Mutations of the NF1 tumor suppressor gene cause type 1 neurofibromatosis, characterized by multiple tumors of the peripheral nerves, as well as other tumor types. The NF1 protein, neurofibromin, is intricately linked to the cell growth regulatory signalling pathways, e.g. by possessing RAS-GTPase activity. The regulation and role of neurofibromin are not known in normal human development. We addressed this issue by studying the regulation of neurofibromin in normal human peripheral nerves, from early fetal development to adulthood. The barely detectable neurofibromin immunosignal in peripheral nerves during the first trimester of gestation contrasted dramatically to its increase in Schwann cells, perineurial cells, and axons during the second and third trimesters. Interestingly, the type I and II isoforms of neurofibromin, differing in their RAS oncoprotein inactivation capacity, displayed clearly different expression profiles throughout these periods. This suggests distinct cellular functions for these neurofibromin isoforms. The results also revealed distinct species-specific differences in neurofibromin expression, potentially bearing relevance to the lack of human neurofibromatosis-like disorders in other species.

Adult↗
PubMed1998

Endometrial ablation with a vaporizing electrode. I. Evaluation of in vivo effects.

BACKGROUND: To evaluate histologically and histochemically the physical and thermal effects of a vaporizing electrode as compared with a standard cutting loop in the performance of endometrial ablation. METHODS: Operative hysteroscopy was performed on 20 menorrhagic patients immediately before hysterectomy. Part of the posterior uterine wall was treated with a cylindrical, grooved, vaporizing electrode and undamped current set at 200 watts, and part with a standard cutting loop and undamped current set at 100 watts. A mucosal strip of about 1 cm width was left intact between the two treatment areas. Specimens underwent histologic examination after hematoxylin and eosin staining and histochemical assessment of thermal injury was based on detection of the respiratory enzyme dihydronicotinamide adenine dinucleotide diaphorase. RESULTS: The mean (standard deviation) endometrial thickness as determined on the untreated area of the posterior uterine wall was 1.08 (0.36) mm. The mean depth of furrows was 3.10 (0.90) mm with the use of the vaporizing electrode and 3.41 (1.11) mm after passage of the cutting loop. Corresponding values when thermal necrosis beneath the ablated area was assessed by the dihydronicotinamide adenine dinucleotide diaphorase technique were, respectively, 1.80 (0.40) mm and 0.41 (0.20) mm (mean difference, 1.39 mm; 95% confidence interval, 1.19 to 1.59: p<0.001, Mann-Whitney U test). CONCLUSIONS: A vaporizing electrode and a standard cutting loop obtained a similar degree of endomyometrial ablation. However, the depth of the thermal effect of the former electrode was significantly greater. Clinical studies are warranted, also considering the potential limitation of fluid absorption and menorrhagia recurrence when a vaporizing electrode is used.

Adult↗
PubMed1998

A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units.

Protein disulfide bond formation is a rate limiting step in protein folding and is catalyzed by enzymes belonging to the protein disulfide oxidoreductase superfamily, including protein disulfide isomerase (PDI) in eucarya and DsbA in bacteria. The first high resolution X-ray crystal structure of a protein disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI. The protein consists of two homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The accessibilities of both active sites are rather different as are, very likely, their redox properties. The protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of disulfide bridges.

Amino Acid Sequence↗
PubMed1998

Lactase haplotype frequencies in Caucasians: association with the lactase persistence/non-persistence polymorphism.

A genetic polymorphism is responsible for determining that some humans express lactase at high levels throughout their lives and are thus lactose tolerant, while others lose lactase expression during childhood and are lactose intolerant. We have previously shown that this polymorphism is controlled by an element or elements which act in cis to the lactase gene. We have also reported that 7 polymorphisms in the lactase gene are highly associated and lead to only 3 common haplotypes (A, B and C) in individuals of European extraction. Here we report the frequencies of these polymorphisms in Caucasians from north and south Europe and also from the Indian sub-continent, and show that the alleles differ in frequency, the B and C haplotypes being much more common in southern Europe and India. Allelic association studies with lactase persistence and non-persistence phenotypes show suggestive evidence of association of lactase persistence with certain alleles. This association was rather more clear in the analysis of small families, where haplotypes could be determined. Furthermore haplotype and RNA transcript analysis of 11 unrelated lactase persistent individuals shows that the persistence (highly expressed) allele is almost always on the A haplotype background. Non-persistence is found on a variety of haplotypes including A. Thus it appears that lactase persistence arose more recently than the DNA marker polymorphisms used here to define the main Caucasian haplotypes, possibly as a single mutation on the A haplotype background. The high frequency of the A haplotype in northern Europeans is consistent with the high frequency of lactase persistence.

Alleles↗
PubMed1998

Improved cloning vectors for Bifidobacterium spp.

The recombinant plasmids pDLI41, pDGA7 and pDCO7 were constructed by cloning in pDG7, a vector based on Bifidobacterium longum replicon pMB1, the following heterologous genes: Pseudomonas fluorescens lipase, Bacillus licheniformis alpha-amylase and Streptomyces sp. cholesterol oxidase. The hybrid plasmids efficiently transformed Bifidobacterium belonging to five different species. A novel Escherichia coli-Bifidobacterium set of shuttle vectors based on the replicon pMB1 (pLF5, pCLJ15, pSPEC1) featuring chloramphenicol, erythromycin and spectinomycin resistance genetic determinants as selection marker for bifidobacteria, was developed. The plasmid pTRE3, a derivative of pLF5, was the smallest (2.8 kb) Bifidobacterium vector, possessed a convenient multicloning site and presented high structural and segregational stability.

Bifidobacterium↗
PubMed1998

Anaesthesia for liver transplantation in cystic fibrosis patients.

INTRODUCTION: Cystic fibrosis (CF) is a disease caused by an inherited genetic defect. While pulmonary and pancreatic abnormalities predominate the clinical spectrum, other organ involvement is common, including liver. The severity of liver disease does not appear to be related to the severity of exocrine pancreatic or lung function. We discuss anaesthesia in four CF patients undergoing liver transplantation. METHODS: We studied haemodynamic and oxygenation modifications during anaesthesia in four patients affected by CF with end-stage liver disease and mild to moderate pulmonary abnormalities. The patients received pancreatic enzyme prior to transplantation and two had insulin-dependent diabetes mellitus. All patients were treated with broad-spectrum antibiotic therapy. After a waiting time ranging one week to three months, all patients were successfully transplanted. General anaesthesia was induced with fentanyl, thiopental and pancuronium, and maintained with isoflurane supplemented by fentanyl in O2:air. Haemodynamic and oxygenation evaluations were made during the main phases of the transplant. After the intubation and at the end of the procedure all patients received a broncho-alveolar toilet through fiberoptic bronchoscopy. RESULTS: During anaesthesia for liver transplantation, PaO2 increased proportionally to the decreasing of Qs/Qt. In postoperative follow-up, Fev1 and FVC improved from preoperative time in all patients. In conclusion, even if cystic fibrosis is a multisystem disease, liver transplantation can be offered to CF patients with endstage liver disease and mild to moderate pulmonary function abnormalities. The four patients are still alive, enjoying good health. The improved respiratory function and quality of life of these children is remarkable.

Adolescent↗
PubMed1998

Computational analysis of the thermal stability in thioredoxins: a molecular dynamics approach.

The knowledge of the relationship between the three-dimensional structure of a protein and its biological and stability is one of the most challenging problem in protein chemistry, since offers the possibility of changing both the specific action of a protein and its stability. In this work, we have approached the problem with studies on a protein family, the thioredoxins, using homology procedures, molecular dynamics simulations in vacuo at 300 K and 500 K and in water solution at 300 K, to determine the relationship between the three-dimensional structure of these proteins and their thermal stability. A comparative analysis, using computational approach, was performed between two thioredoxins with different resistance to temperature. Results obtained using the molecular dynamics techniques and minimization procedures give explanations of the experimental data, underlining that these techniques are able to correlate the increase in protein stabilization with the conformational and structural changes caused by single amino acid replacement. In addition, we report the factors that can be used as a guide in protein engineering and in site-directed mutagenesis to increase or decrease thermal stabilization for this protein family.

Amino Acid Sequence↗
PubMed1998

Temperature effects on the structural and functional properties of GPI-anchored and anchor-less bull seminal plasma ecto-5'-nucleotidase.

The effects of temperature on the three-dimensional organization and on the secondary structure of GPI-anchored 5'-nucleotidase from bull seminal plasma and of its anchor-less form (solubilized ecto-5'-nucleotidase), obtained after GPI anchor removal by phosphatidylinositol-specific phospholipase C were investigated in parallel by circular dichroism and fluorescence spectroscopy. The structural features of the two enzymes were correlated to their functional properties in the temperature range of 25-90 degrees C. The kinetic data indicated that the enzyme activities were temperature dependent, showing the maximal values at 60 degrees C. The relevant Arrhenius plots were linear in the temperature range of 20-60 degrees C and the activation energies were 44.4 and 51.8 kJ/mol for the solubilized and GPI-anchored 5'-nucleotidase, respectively. The time-course measurements of enzyme activity, in the temperature range of 25-55 degrees C, revealed that the two enzymes were of different thermal stability, the solubilized ectoenzyme showing lower thermal deactivation constants and longer half lives. Fluorescence and near UV circular dichroism spectroscopy showed that temperature increases induced remarkable changes in the protein tertiary structure of the two enzymes, whereas far-UV circular dichroism analysis revealed only a small temperature effect on the protein secondary structure content.

5'-Nucleotidase↗
PubMed1998

Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements.

The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity. Two putative structural determinants contributing to the higher stability of ADH-hT had been identified by comparison with the less thermostable ADH (ADH-T) from the less thermophilic B. stearothermophilus NCA 1503. In order to ascertain their role, mutations were designed to eliminate in ADH-hT a salt bridge at the N-terminus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T. Three mutants--Glu11Lys, Pro242Ala, and Glu11Lys/Pro242Ala--were expressed at high level and the proteins purified and characterized. In general, the mutations had little effect on the activity, indicating that they were not disruptive. The thermal resistance was changed displaying quite additive effects.

Alcohol Dehydrogenase↗
PubMed1998

An autonomously replicating transforming vector for Sulfolobus solfataricus.

A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C.

Base Sequence↗
PubMed1998

Microvascular involvement in systemic sclerosis: laser Doppler evaluation of reactivity to acetylcholine and sodium nitroprusside by iontophoresis.

OBJECTIVES: To investigate the skin vasodilatory response to iontophoretically applied acetylcholine (Ach), an endothelium dependent vasodilator, and to sodium nitroprusside (SNP), an endothelium independent vasodilator, in patients with systemic sclerosis (SSc). METHODS: Eleven SSc patients were preliminarily studied (10 females, mean age 40.5; mean disease duration 6.5 years), and 16 age and sex matched control subjects. By means of laser Doppler flowmetry skin blood flow was evaluated at third finger, at baseline, and after postischaemic hyperaemia test and during iontophoretically transcutaneous application of 1% solution of Ach and SNP. RESULTS: No significant differences in basal skin blood flow were detected between SSc patients and controls. Cutaneous vasodilatory response to ischaemia, Ach, and SNP was significantly less pronounced in SSc patients compared with controls (p < 0.001). Moreover, among SSc patients a lower (p < 0.05) vasodilatory response to Ach compared with ischaemia and SNP was recorded. CONCLUSIONS: These data confirm a reduction of skin digital vasodilatory reserve in SSc patients and suggest a defect of both endothelial dependent arteriolar relaxation and wall compliance in the pathogenesis of this dysfunction.

Acetylcholine↗
PubMed1998

A C-terminal fragment of Agouti-related protein increases feeding and antagonizes the effect of alpha-melanocyte stimulating hormone in vivo.

Agouti-related protein (Agrp) is present in rat and human hypothalamus and is structurally related to agouti protein. Overexpression of either of these proteins results in obesity. However the effect of exogenous Agrp and its in vivo interaction with alpha-melanocyte stimulating hormone (alphaMSH), the likely endogenous melanocortin 3 and 4 receptor (MC3-R and MC4-R) agonist, have not been demonstrated. We report that 1 nmol of Agrp(83-132), a C-terminal fragment of Agrp, when administered intracerebroventricularly (ICV) into rats, increased food intake over a 24-h period (23.0+/-1.4 g saline vs 32.9+/-2.3 g Agrp, p<0.05). The hyperphagia was similar to that seen when 1 nmol of the synthetic MC3-R and MC4-R antagonist SHU9119 was given i.c.v. (19.6+/-1.8 g saline vs 32.5+/-1.7 g SHU9119, p<0.001). Both Agrp(83-132) and SHU9119 blocked the reduction in 1-h food intake of i.c.v. alphaMSH at the beginning of the dark phase. This effect occurred independently of whether the antagonists were administered simultaneously, or nine hours prior, to the alphaMSH. We have also shown Agrp(83-132) is an antagonist at the MC3-R and MC4-R, with similar inhibition of cAMP activation to that previously reported for the full length peptide. In conclusion, Agrp(83-132) administered i.c.v. increases feeding with long lasting effects and is able to inhibit the action of alphaMSH. This interaction may be mediated by the MC3-R and/or MC4-R.

Agouti Signaling Protein↗
PubMed1998

Body mass index and mortality in nonsmoking older adults: the Cardiovascular Health Study.

OBJECTIVES: This study assesses the relationship of body mass index to 5-year mortality in a cohort of 4317 nonsmoking men and women aged 65 to 100 years. METHODS: Logistic regression analyses were conducted to predict mortality as a function of baseline body mass index, adjusting for demographic, clinical, and laboratory covariates. RESULTS: There was an inverse relationship between body mass index and mortality; death rates were higher for those who weighed the least. Inclusion of covariates had trivial effects on these results. People who had lost 10% or more of their body weight since age 50 had a relatively high death rate. When that group was excluded, there was no remaining relationship between body mass index and mortality. CONCLUSIONS: The association between higher body mass index and mortality often found in middle-aged populations was not observed in this large cohort of older adults. Over-weight does not seem to be a risk factor for 5-year mortality in this age group. Rather, the risks associated with significant weight loss should be the primary concern.

Age Distribution↗
PubMed1998

Risk and prevention of type 2 diabetes in women with gestational diabetes.

Women with a history of gestational diabetes mellitus (GDM) are at increased risk of future diabetes, predominantly type 2 diabetes, as are their children. The extent of this risk depends both on the diagnostic criteria used to identify GDM and on maternal risk factors, some of which are potentially modifiable whereas others are not. The unmodifiable risk factors are ethnicity, pre-pregnancy weight, age, parity, family history of diabetes, and degree of hyperglycemia in pregnancy and immediately postpartum. The modifiable risk factors are persistent obesity, future weight gain, and subsequent pregnancies. Additional modifiable risk factors in these women are likely to be levels of physical activity, dietary fat, and avoidance of other lifestyle factors that adversely influence insulin resistance, such as smoking and certain drugs. Diabetic prevention strategies need to address the potentially modifiable risk factors using the unmodifiable risk factors to identify women most at risk.

Adult↗