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Biomedical subjects

M Rossi

Publications and source records attributed to M Rossi.

At least 181 records · Page 10Linked to original sources
PubMed2000

Diurnal variation in orexin A immunoreactivity and prepro-orexin mRNA in the rat central nervous system.

Orexins are a family of neuropeptides originally believed to be important mediators of food intake. The wide distribution of orexins and their receptors, however, has suggested other regulatory functions for these peptides including involvement in sleep and arousal mechanisms. In this study, we have demonstrated diurnal variation in orexin A immunoreactivity in the pons, from where locus coeruleus noradrenergic neurones innervate other brain areas to stimulate arousal, and in the preoptic/anterior hypothalamic region, an area implicated in the regulation of sleep and circadian rhythms. Orexin A immunoreactivity decreased by 50% in the preoptic/anterior hypothalamus from 09:00 to 21:00 h (P < 0.0001), whilst in the pons, it increased by over 30% from 09:00 to 01:00 h (P = 0.02). Prepro-orexin mRNA also displayed diurnal variation. This further suggests that orexins are involved in the regulation of the sleep/wake cycle.

Animals↗
PubMed2000

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR.

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.

Automation↗
PubMed2000

Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus.

A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble and active form at 20 mg/liter of E. coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-kDa monomeric protein, was demonstrated to be a B"-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-hexanoate with K(m) and k(cat) values of 11 +/- 3 microM (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethylsulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [(3)H]DFP, supported our previous suggestion of a catalytic triad made up of Ser(160)-His(285)-Asp(255). The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed.

Amino Acid Sequence↗
PubMed2000

Imaging active lymphocytic infiltration in coeliac disease with iodine-123-interleukin-2 and the response to diet.

Coeliac disease is diagnosed by the presence of specific antibodies and a jejunal biopsy showing mucosal atrophy and mononuclear cell infiltration. Mucosal cell-mediated immune response is considered the central event in the pathogenesis of coeliac disease, and untreated coeliac patients show specific features of T-cell activation in the small intestine. Here we describe the use of iodine-123-interleukin-2 scintigraphy in coeliac patients as a non-invasive tool for detection of lymphocytic infiltration in the small bowel and its use for therapy follow-up, and we demonstrate the specificity of binding of labelled-IL2 to activated lymphocytes by ex-vivo autoradiography of jejunal biopsies. 123I-IL2 was administered i.v. [74 MBq (2 mCi)], and gamma camera images were acquired after 1 h. Ten patients were studied with 123I-IL2 scintigraphy at diagnosis and seven were also investigated after 12-19 months of gluten-free diet. Results were expressed as target-to-background radioactivity ratios in six different bowel regions before and after the diet. At the time of diagnosis all patients showed a significantly higher bowel uptake of 123I-IL2 than normal subjects (P < 0.003 in all regions). A significant correlation was found between jejunal radioactivity and the number of IL2R + ve lymphocytes per millimetre of jejunal mucosa as detected by immunostaining of jejunal biopsy (r2 = 0.66; P = 0.008). Autoradiography of jejunal biopsies confirmed that labelled-IL2 only binds to activated T-lymphocytes infiltrating the gut mucosa. After 1 year of the diet, bowel uptake of 123I-IL2 significantly decreased in five out of six regions (P < 0.03), although two patients still had a positive IL2 scintigraphy in one region. We conclude that 123I-IL2 scintigraphy is a sensitive non-invasive technique for assessing in vivo the presence of activated mononuclear cells in the bowel of patients affected by coeliac disease. Unlike jejunal biopsy, this method provides information from the whole intestine and gives a non-invasive measure of the effectiveness of the gluten-free diet.

Adult↗
PubMed2000

Responses of the skin microcirculation to acetylcholine and to sodium nitroprusside in chronic uremic patients.

The aim of the present study was to assess the endothelial function of the microcirculation in chronic renal failure. We investigated the responses of the cutaneous blood flow to locally delivered acetylcholine and sodium nitroprusside in uremic patients. The study included 60 chronic uremic patients: 40 patients with a creatinine clearance of 4-25 ml/min were on conservative treatment and 20 patients were on maintenance hemodialysis. The changes in skin blood flow following iontophoretic delivery of acetylcholine (an endothelium-dependent vasodilator) and sodium nitroprusside (an endothelium-independent vasodilator) were measured by laser Doppler flowmetry. Acetylcholine induced a progressive increase in blood flow in both groups, reaching approximately 100% of the maximal hyperemic response obtained by sodium nitroprusside delivery. The percent increase in blood flow from baseline was lower in hemodialysis patients than in patients on conservative treatment, after both acetylcholine (550 +/- 44 vs. 718 +/- 61%, P < 0.05) and sodium nitroprusside (553 +/- 46 vs. 735 +/- 69%, P < 0.05) delivery. In the hemodialysis group, the hyperemic responses to acetylcholine and sodium nitroprusside did not improve after the hemodialysis session. Hence, the hyperemic responses of the skin microcirculation are lower in hemodialysis patients than in patients on conservative treatment, and did not ameliorate after hemodialysis. It seems to be independent of endothelial dysfunction, and associated with the severity of uremia and with the maintenance hemodialysis treatment. This microcirculatory abnormality is in keeping with the arterial stiffness and vascular wall damages described in dialysis patients, which contribute to the cardiovascular morbidity of chronic uremia.

Acetylcholine↗
PubMed2000

An archaeal chaperonin-based reactor for renaturation of denatured proteins.

We describe an original chaperonin-based reactor that yields folded and active proteins from denatured materials. We used the 920-kDa chaperonin of the archaeon Sulfolobus solfataricus, which does not require any protein partner for its full activity and assists in vitro folding with low substrate specificity. The reactor consists of an ultrafiltration cell equipped with a membrane that retains the chaperonin in a functional state for folding in solution and permits the flowthrough of the folded substrates. By studying the ATP-dependent functional cycle of the chaperonin, we were able to use the reactor for repeated refolding processes. The scale-up of the reactor is made possible by the overproduction of chaperonin in Sulfolobus solfataricus cells that acquired thermotolerance upon appropriate heat shock.

Adenosine Triphosphate↗
PubMed2000

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal beta-glycosidases.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.

Cellulase↗
PubMed2000

Specific detection of bifidobacterium strains in a pharmaceutical probiotic product and in human feces by polymerase chain reaction.

For PCR specific detection of the strains Bifidobacterium longum Y 10, B. infantis Y 1 and B. breve Y 8 used in a new probiotic product (VSL-3), strains-specific rDNA primers have been developed. Spacer regions between the 16S and 23S rRNA genes (ITS) of the three strains were amplified by PCR with conserved primers and the nucleotide sequence of these ITSs were determined. On the basis of their comparison with the rDNA sequences retrieved from GenBank, we designed new primers which specifically recognize the species B. breve and the two strains B. infantis Y 1 and B. breve Y 8. Specificity of these primers was confirmed through the analysis of 60 bifidobacteria strains belonging to the more representative human species. The feasibility of this PCR method was investigated in commercial VSL-3 product and fecal samples collected from 4 patients affected by inflammatory bowel deseases and two healthy subjects before and after the VSL-3 administration. By PCR analysis of different VSL-3 commercial batches we were successful in differentiating and quantifying the strains B. longum Y 10, B. infantis Y 1 and B. breve Y 8. B. infantis Y 1 and B. breve Y 8 could be detected at high concentration in fecal specimens of both patients and subjects treated with the probiotic preparation, showing a different colonization behaviour. Seven days after the VSL-3 treatment suspension, no patients and subjects harbored B. infantis Y 1 and B. breve Y 8, indicating a transient presence of these exogenous strains.

Bacterial Typing Techniques↗
PubMed2000

Digital radiography using an EBCCD-based imaging device

In the last few years, large efforts were devoted to the development of new intensified imaging systems to be applied mainly in medical radiographic devices for the reduction of the dose irradiated to the patient but also in high-energy physics for particle tracking or in astronomy for low-light imaging. Among a number of different image-intensifier systems, electron-bombarded charge coupled devices (EBCCD) present interesting characteristics providing the opportunity to be used also for special application such as X-ray imaging in industrial field. For this purpose, a prototype EBCCD image-intensifier tube suitable for X-ray digital radiography (DR) and computed tomography (CT) has been set up by the Physics Department of the University of Bologna (Italy) and Geosphaera Research Center of Moscow (Russia). Results concerning tests of EBCCD device and DR analysis of industrial components are presented and discussed.

Journal Article↗
PubMed2000

A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell-bound proteins.

An oxygen-induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose-rich media. This protein was also identified as being associated with the cell-surface, with the amount of the released and cell-bound protein fractions depending on the growth phase of the cells. The steady decrease in cell-associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity. Characterization in comparison with the cytosolic superoxide dismutase revealed identical N-terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes. In order to clarify the physiological role of the cell-associated superoxide dismutase, the prevention of cell-bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme. Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay. Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components.

2,6-Dichloroindophenol↗
PubMed2000

NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius possible determinants of protein stability.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium growing optimally at 333 K, is the first Trx described to date from a moderate thermophilic source. To understand the molecular basis of its thermostability, the three-dimensional structure in the oxidized form was determined by NMR methods. A total of 2276 1H-NMR derived distance constraints along with 23 hydrogen-bonds, 72 phi and 27 chi1 torsion angle restraints, were used in a protocol employing simulated annealing followed by restrained molecular dynamics and restrained energy minimization. BacTrx consists of a well-defined core region of five strands of beta-sheet, surrounded by four exposed alpha-helices, features shared by other members of the thioredoxin family. The BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx) determined by X-ray crystallographic diffraction, and a number of structural differences were observed that may contribute to its thermostabilty. The results of structural analysis indicated that protein stability is due to cumulative effects, the main factor being an increased number of ionic interactions cross-linking different secondary structural elements and clamping the C-terminal alpha-helix to the core of the protein.

Amino Acid Sequence↗
PubMed2000

Combined Surgery for cardiac and carotid disease: management and results of a rational approach.

OBJECTIVES: The aim of the present study was to apply a rational plan for simultaneous cardiac and carotid surgery in high-risk patients. MATERIALS AND METHODS: A consecutive series of 89 patients with coexisting severe cardiac and carotid disease were operated on during a 5-year period with routinary carotid shunting, moderate hypothermia and balanced anaesthesia. The combined surgical procedures were coronary artery by-pass grafts (CABG) + carotid endarterectomy (CEA) in 81 patients, CABG + CEA + aortic valve replacement (AVR) in four patients, and four cases of CEA + AVR. RESSULTS: Two deaths (2%), three acute myocardial infarctions (3%) and one (1%) major stroke occurred in five patients during the perioperative (30 days) period for a combined rate of death and/or disabling stroke of 3%. There were five reversible neurological deficits. Carotid and aortic mean clamping times were 9 and 60 min respectively. Patients were discharged after a mean length of stay in Intensive Care Unit (ICU) of 131 h and 7 days of hospitalisation post-ICU. CONCLUSIONS: Based on our results, combined interventions of CEA and CABG can be performed with an acceptable morbidity and mortality when severe carotid stenosis is associated with advanced, symptomatic cardiac disease. The management of these patients needs careful and appropriate pre-intra and post-operative assessment and timing aimed to reduce the ischaemic injuries, both cardiac and cerebral, especially during CBP time.

Aged↗
PubMed2000

Central neurocytoma: long-term follow-up of a paediatric case.

In order to gain a better understanding of the clinical and histological features of central neurocytoma and the role of radiotherapy in the treatment of these tumours, we present an unusual paediatric case and review the pertinent literature. Most patients present in the second and third decade of life, rarely in the teenage years and exceptionally in children. Our patient was treated by a combined surgical and radiation therapy treatment for a central neurocytoma at the age of 7 years, but the tumour had been diagnosed when she was 3 years old. At a 38-year follow-up, she is well with no evidence of tumour progression. This patient seems to be the youngest histologically proven case of central neurocytoma and with the longest recorded survival.

Brain Neoplasms↗
PubMed2000

Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus.

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.

Affinity Labels↗
PubMed2000

Tissue transglutaminase is the target in both rodent and primate tissues for celiac disease-specific autoantibodies.

BACKGROUND: Endomysial antibodies have recently been shown to react with tissue transglutaminase. This study was undertaken to investigate whether the tissue distribution of transglutaminase is also compatible with reticulin, jejunal, and fibroblast autoantibody binding patterns. METHODS: Sera from patients with and without celiac disease, monoclonal tissue transglutaminase antibodies, and sera from mice parenterally immunized against commercially available tissue transglutaminase, transglutaminase complexed with gliadin, or gliadin were used in indirect immunofluorescence and double-staining studies using both rodent and primate tissues as substrates. Also, antibody competition, affinity chromatography, and potassium thiocyanate extraction studies were undertaken. RESULTS: Tissue transglutaminase antibody binding patterns were identical with the extracellular binding patterns seen with celiac patient sera. Human umbilical cord-derived fibroblasts exhibited both cytoplasmic and extracellular matrix staining. Double staining with patients' sera and tissue transglutaminase antibodies showed complete overlapping. Tissue transglutaminase effectively absorbed reticulin-endomysial antibodies from celiac sera, and patients' sera blocked the staining of the monoclonal tissue transglutaminase antibodies. Potassium thiocyanate extraction abolished the staining patterns, but they were elicited again after readdition of tissue transglutaminase. CONCLUSIONS: Reticulin, endomysial, and jejunal antibodies detect transglutaminase in both rodent and primate tissues, indicating that these tissue autoantibodies are identical.

Animals↗
PubMed2000

Interface dynamics from experimental data

An algorithm is envisaged to extract the coupling parameters of the Kardar-Parisi-Zhang (KPZ) equation from experimental data. The method hinges on the Fokker-Planck equation combined with a classical least-square error procedure. It takes properly into account the fluctuations of surface height through a deterministic equation for space correlations. We apply it to the (1+1)-dimensional KPZ equation and carefully compare its results with those obtained by previous investigations. Unlike previous approaches, our method does not require large sizes and is stable under a modification of sampling time of observations. Shortcomings associated with standard discretizations of the continuous KPZ equation are also pointed out and finally possible future perspectives are analyzed.

Journal Article↗