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Biomedical subjects

M Rossi

Publications and source records attributed to M Rossi.

At least 163 records · Page 9Linked to original sources
PubMed2000

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus.

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.

Archaeoglobus fulgidus↗
PubMed2000

Magnetic resonance findings and ophthalmologic abnormalities are correlated in patients with neurofibromatosis type 1 (NF1).

In subjects with neurofibromatosis type 1 (NF1), we show that a reduction in the visual field is the most sensitive indicator of gliomas along the optic pathway. Therefore, we conclude that a visual field evaluation is the most sensitive clinical test among those evaluated to predict the presence of optic pathway pathology on an MRI examination. These data may contribute to the establishment of more precise guidelines for the evaluation and treatment of children with NF1.

Adolescent↗
PubMed2000

A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor.

Blood glucose is a clinically important analytes for diabetic health care. In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase. The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be used to measure the glucose concentration. Our results suggest that enzymes which use glucose as their substrate can be used as reversible and nonconsuming glucose sensors in the absence of required cofactors. Moreover, the possibility of using inactive apoenzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.

Animals↗
PubMed2000

Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus.

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.

Adenosine Triphosphatases↗
PubMed2000

Identification and molecular characterization of the first alpha -xylosidase from an archaeon.

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.

Archaea↗
PubMed2000

Comparative T cell receptor repertoire selection by antigen after adoptive transfer: a glimpse at an antigen-specific preimmune repertoire.

The low frequency of precursor cells specific for any particular antigen (Ag) makes it difficult to characterize preimmune T cell receptor (TCR) repertoires and to understand repertoire selection during an immune response. We have undertaken a combined adoptive transfer single-cell PCR approach to probe the Ag-specific preimmune repertoires of individual mice. Our strategy was to inject paired irradiated recipient mice with normal spleen cells prepared from individual donors and to compare the TCR repertoires subsequently selected during a CD8 response to a defined model Ag. We found that although some TCRs were shared, the TCR repertoires selected by mice receiving splenocytes from the same donor were not identical in terms of the TCRs selected and their relative frequencies. Our results together with computer simulations imply that individual mice express distinct Ag-specific preimmune TCR repertoires composed of expanded clones and that selection by Ag is a random process.

Adoptive Transfer↗
PubMed2000

A protein biosensor for lactate.

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.

Anilino Naphthalenesulfonates↗
PubMed2000

Can discography cause long-term back symptoms in previously asymptomatic subjects?

STUDY DESIGN: A prospective controlled study of patients without low back pain observed after experimental lumbar discography. OBJECTIVES: To determine whether discography may cause long-term low back symptoms in selected patients. BACKGROUND: Lumbar discography is generally considered safe, although controversial, with few complications. There is little information on long-term follow-up of asymptomatic patients after experimental discography. METHODS: Twenty-six patients without pre-existing low back pain were observed for 1 year after lumbar discography to determine the effects of lumbar discography in three experimental groups: 10 patients who had remote cervical spine surgery with excellent results (pain-free group); 10 patients who had the same surgery with poor results (chronic pain group); and 6 patients who had primary somatization disorders. Two control groups were also observed: a group of patients with somatization disorder who were similarly evaluated but without discography, a group of patients with low back pain observed for 1 year after discography without surgery. RESULTS: In the three experimental groups (no back pain before discography) no subject with normal psychometric testing had persistent pain after discography (0/11), whereas 6 (40%) of 15 with abnormal psychometric test results reported significant new low back pain. None of the pain-free group (0/10), 20% of the chronic neck pain group (2/10), and 66% of the somatization group (4/6) continued to have significant back pain 1 year after injection. Psychological testing and occupational disability strongly predicted continued pain after discography. The control groups had no significant change in low back symptomatology during the year of observation. CONCLUSION: Discography in a subject group without low back pain but with significant emotional and chronic pain problems may result in reports of significant back pain for at least 1 year after injection. Subjects with normal psychometric test results had no reports of significant long-term back pain after discography.

Adult↗
PubMed2000

Discontinuing or withholding primary prophylaxis against Mycobacterium avium in patients on successful antiretroviral combination therapy. The Swiss HIV Cohort Study.

OBJECTIVE: To assess the safety of discontinuing or withholding primary prophylaxis against disseminated Mycobacterium avium infection (MAC) in HIV infected patients on successful antiretroviral combination therapy. SETTING: National prospective multicentre cohort study. DESIGN: HIV-infected patients were eligible for the analysis if: (i) they had a history of at least two CD4 cell counts < 50 x 10(6)/l; (ii) they had never had MAC; (iii) they had discontinued or never begun primary prophylaxis against MAC; (iv) they received antiretroviral therapy and demonstrated an increase in CD4 cell counts to > or = 100 x 10(6)/l that was sustained for at least 12 weeks. From this time point until last follow-up, incidence of disseminated MAC disease was measured, and 99% confidence intervals were calculated assuming a Poisson distribution of events. RESULTS: Two-hundred and fifty-three patients (22.5% female; median age, 37 years, 30% injecting drug users) were eligible for analysis. Sixty-six per cent were in Centers for Disease Control and Prevention (CDC) stage C, and 28% were in CDC stage B. Their median nadir CD4 cell count was 10 x 10(6)/l, the median duration of CD4 cell count < 50 x 10(6)/l was 12 months. During a total follow-up of 364.3 patient-years there was no case of disseminated MAC. The one-sided 99% confidence limit for incidence density of MAC was 1.3 per 100 person-years. CONCLUSION: Discontinuing or withholding primary prophylaxis against MAC is safe in patients who have a sustained increase in their CD4 cell count to > or = 100 x 10(6)/l.

AIDS-Related Opportunistic Infections↗
PubMed2000

Investigation of the melanocyte stimulating hormones on food intake. Lack Of evidence to support a role for the melanocortin-3-receptor.

The melanocortin receptors, melanocortin-3-receptor (MC3-R) and melanocortin-4-receptor (MC4-R), are expressed in many discrete medial hypothalamic nuclei implicated in feeding regulation. The pro-opiomelanocortin product alpha-melanocyte stimulating hormone (alpha-MSH), an MC3/4-R agonist, decreases food intake following intracerebroventricular (ICV) injection in rats. MC4-R's involvement in feeding has been established although a function for the MC3-R is unclear. We investigated endogenous melanocortin ligand binding and activation at the MC3-R and MC4-R and their effects on feeding. We have shown that alpha-MSH, desacetyl-alpha-MSH and beta-MSH bound to the MC3-R and MC4-R with similar affinity and stimulated cAMP with similar potency in HEK 293 cells transfected with MC3-R and MC4-R. In contrast gamma(2)-MSH showed selectivity for the MC3-R over the MC4-R both in binding affinity and cAMP stimulation. alpha-MSH and beta-MSH injected ICV into fasted rats at doses of 1, 3 and 6 nmol resulted in a decrease in food intake, (2 h food intake: alpha-MSH 6 nmol, 1.7+/-0.3 g; beta-MSH 6 nmol, 1.5+/-0.3 g vs. saline 6.0+/-0.5 g, P<0.001). Desacetyl alpha-MSH did not reduce food intake at low doses but was significant at 25 nmol (2 h food intake: desacetyl-alpha-MSH 6.1+/-1.0 g vs. saline 9.5+/-1.4 g, P<0.05). In contrast, gamma(2)-MSH had no effect on food intake when administered ICV to fasted rats. We were unable to establish a role for the MC3-R in feeding regulation. Our evidence, however, strengthens the hypothesis that the melanocortin's effects on food intake are mediated via the MC4-R.

Animals↗
PubMed2000

Synthesis and immunosuppressive activity of novel prodigiosin derivatives.

Prodigiosins (Ps) represent a family of naturally occurring red pigments characterized by a common pyrrolylpyrromethene skeleton. Some members of this family have been shown to possess interesting immunosuppressive properties exerted with a novel mechanism of action, different from that of currently used drugs. In fact, Ps inhibit phosphorylation and activation of JAK-3, a cytoplasmic tyrosine kinase associated with a cell surface receptor component called common gamma-chain, which is exclusive of all IL-2 cytokine family receptors. Blocking common gamma-chain transduction activity results in a potent and specific immunosuppressive activity. With respect to the interesting and unexploited immunomodulating properties of this family of compounds we initiated a medicinal chemistry program aimed at finding novel prodigiosin derivatives with improved immunosuppressive activity and lower toxicity. Utilizing an unprecedented and flexible way of assembling the prodigiosin frame, a number of new derivatives have been prepared and tested leading to the choice of 4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2, 2'-bi-1H-pyrrole (PNU-156804, 16) as a lead immunosuppressant.

Animals↗
PubMed2000

[Anemia in HIV infection].

Anemia is a common finding in HIV-infected patients, especially in late stages of the disease. The grade of anemia is an independent marker for mortality. Beside the classical causes of anemia we find in HIV-infected patients typically cytokine mediated suppression of the bone marrow, infiltration of the latter by opportunistic agents and side effects of drugs. These causes have to be taken into account in the diagnostic approach. The therapeutic intervention should be focused on the aetiology and pathogenesis of anemia in the individual patient. Successful antiretroviral combination therapy is associated with a substantial decrease of morbidity and mortality and lowers the incidence of severe anemia. However, several antiretroviral agents, especially zidovudine, and drugs against opportunistic infections can cause anemia.

Anemia↗
PubMed2000

Cocaine- and amphetamine-regulated transcript, glucagon-like peptide-1 and corticotrophin releasing factor inhibit feeding via agouti-related protein independent pathways in the rat.

The melanocortin-4 receptor (MC4-R) appears to be an important downstream mediator of the action of leptin. We examined to what extent the anorectic effects of cocaine- and amphetamine-regulated transcript (CART), glucagon-like peptide-1 (GLP-1) and corticotrophin releasing factor (CRF) might be mediated via MC4-R. alpha-Melanocyte stimulating hormone (alpha-MSH), the MC4-R agonist, administered intracerebroventricularly (ICV) at a dose of 1 nmol reduced food intake by approximately half. Agouti-related protein (Agrp) (83-132), a biologically active fragment of the endogenous MC4-R antagonist, administered ICV at a dose of 1 nmol completely blocked the anorectic effect of 1 nmol alpha-MSH. CART (55-102) (0.2 nmol), GLP-1 (3 nmol) and CRF (0.3 nmol) produced a reduction in feeding of approximately the same magnitude as 1 nmol alpha-MSH. Agrp (83-132) (1 nmol) administered ICV did not block the anorectic effects of CART (55-102) (1 h food intake, 0.2 nmol CART (55-102), 2.7+/-0.8 g vs. CART (55-102)+Agrp (83-132), 2.6+/-0.6 g, P=0.87; saline control 5.4+/-0.3 g, P<0.001 vs. both groups). Agrp (83-132) also did not block the anorectic effects of GLP-1 or CRF (1 h food intake, 0.3 nmol CRF, 0.7+/-0.3 g vs. CRF+Agrp (83-132), 0.7+/-0.3 g, P=0.91; 3 nmol GLP-1, 1.9+/-0.4 g vs. GLP-1+Agrp (83-132), 1.1+/-0. 5 g, P=0.23; saline control 5.0+/-0.6 g, P<0.001 vs. all four groups). Thus, as previous data suggests, GLP-1 and CRF do not appear to reduce food intake predominantly via MC4-R, we here demonstrate for the first time that CART, in addition to GLP-1 and CRF primarily acts via Agrp independent pathways.

Agouti-Related Protein↗
PubMed2000

The rates of false-positive lumbar discography in select patients without low back symptoms.

STUDY DESIGN: Experimental disc injections in subjects with no history of low back symptoms. OBJECTIVE: To determine in an experimental setting the relative pain response and pain-related behavior in selected subjects without a history of low back pain undergoing lumbar discography. This study aimed to select a study population that more closely represented patients undergoing discography in clinical practice. SUMMARY OF BACKGROUND DATA: Previous work has shown that in young, healthy men with little degenerative disc disease and no history of low back problems, discographic injections usually did not cause significant pain. This group differed from the patients who usually undergo discographic evaluation in clinical practice. Most clinical patients are older, have significant degenerative disc disease, have behavioral changes associated with chronic pain, and often have confounding psychosocial troubles. The authors undertook to study discography in subjects without low back pain but with clinical profiles similar to patients undergoing discography in clinical practice. METHODS: Twenty-six individuals, mean age 43 years, with no history of low back pain had lumber discography according to the strict protocol of Walsh et al. Of these, 10 were pain-free; 10 had chronic neck and arm pain, but no low back symptoms; and 6 had primary somatization disorders without low back symptoms.- RESULTS: Significant positive pain response and pain-related behavior with discography were found in 10% of the pain-free group, in 40% of the chronic cervical pain group, and in 83% of the somatization disorder group completing the injections. Twenty-four subjects had negative control discs. Discs with annular disruption were more likely to be painful on injection, particularly in those individuals with ongoing compensation issues, chronic pain, or abnormal psychological testing.- CONCLUSION: If strict criteria are applied, the rate of false-positive discography may be low in subjects with normal psychometric profiles and without chronic pain. Significantly painful injections were very common in subjects with annular disruption and chronic pain or abnormal psychometric testing.

Adult↗
PubMed2000

Pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius as probe to monitor the sodium concentrations in the blood.

We describe the isolation and characterization of a pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius. This protein appears to be a tetramer composed of four 55-kDa subunits. The intrinsic tryptophan fluorescence of this protein is quenched by approximately 20% upon binding sodium, which occurs with a dissociation constant near 15 mM. Importantly, the intrinsic fluorescence of this pyruvate kinase does not appear to be affected by potassium, magnesium, and calcium at the concentrations found in whole blood. It appears that this pyruvate kinase can provide the basis for a selective protein sensor for sodium with minimal interference from other cations.

Ammonium Sulfate↗
PubMed2000

Cerebrospinal fluid flow dynamics study in Chiari I malformation: implications for syrinx formation.

Cerebrospinal fluid (CSF) flow abnormalities are known to be present in Chiari I malformation and to underlie the origin and progression of associated syringomyelia. The incidence of syrinx formation, however, is variable for unknown reasons. The aim of this study was to investigate whether differences in CSF flow dynamics in patients with Chiari I malformation may account for the different clinical and radiological presentation. Presurgical and postsurgical phase-contrast magnetic resonance imaging investigations were prospectively conducted in 47 adult patients with symptomatic Chiari I malformation. Patients were divided into two groups according to the presence (32 cases) or absence (15 cases) of syrinx. Cerebrospinal fluid flow patterns were evaluated at four regions of interest: prebulbar cistern, foramen magnum, and the ventral and dorsal spinal subarachnoid spaces at the C-5 level. A temporal analysis of CSF flow waveforms was performed with measurement of cranial- and caudal-directed flow durations. All patients underwent a craniocervical decompressive procedure. Preoperatively, a prolonged caudal- directed (systolic) flow pattern was observed in patients with syringomyelia, as compared with normal control values obtained in 15 healthy volunteers. Conversely, a decreased systolic duration was observed in Chiari I patients who had malformation without syrinx. These trends were not statistically significant because of the considerable degree of overlap with the control values recorded in both groups. Additional comparison of the observed preoperative values obtained in patients with and those without syringomyelia indicated that the difference in systolic flow duration was significant at the ventral spinal subarachnoid space level (p = 0.003) and remarkable at the other levels, although not reaching statistical significance. Cerebrospinal fluid flow was minimal or absent at the foramen magnum (dorsal aspect) due to tonsillar herniation, precluding reliable quantitative measurement at this level. There was no evidence of communication between the fourth ventricle and syrinx in any case. Postoperatively, unobstructed CSF flow was recorded across the enlarged foramen magnum and into the artificial cisterna magna in all patients. A gradual restoration of near-normal flow patterns was observed in both groups. Inside the syrinx, fluid motion gradually tapered, no longer being detectable in 12 patients (37.5%) 1 year postsurgery. In patients with Chiari I malformation and associated syringomyelia different CSF flow patterns were demonstrated as compared with patients in whom syrinx was absent. Analysis of this study's findings supports the hypothesis that in Chiari I malformation an elongated systolic flow may prolong the condition of increased spinal subarachnoid pressure caused by the junctional obstruction, thus favoring CSF penetration into the spinal cord. It may be also proposed that a shortened systolic flow may be insufficient to maintain a hypertensive condition for enough time to induce syrinx formation.

Adult↗
PubMed2000

The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature.

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.

Archaeoglobus fulgidus↗