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Biomedical subjects

S Pagani

Publications and source records attributed to S Pagani.

At least 37 records · Page 2Linked to original sources
PubMed2000

Evidence of increased carriage of Corynebacterium spp. in healthy individuals with low antibody titres against diphtheria toxoid.

This study evaluated whether a correlation exists between carriage of corynebacteria and the lack of immunity to diphtheria toxoid. Samples of both nasal and pharyngeal secretions were taken from 500 apparently healthy subjects of both sexes and of all ages and inoculated onto Tinsdale's medium. A serum sample was also taken for ELISA test to determine the titre of diphtheria toxin antibodies. None of the subjects carried Corynebacterium diphtheriae. Ninety-three strains of Corynebacterium spp. were isolated from 93 subjects and 86 of these were classified to species or group level by biochemical tests. C. xerosis was the most common (25.8%) followed by C. pseudodiphthericum (16.1%), C. jeikeium and C. striatum (both 10.8%), and C. urealyticum (9.7%). Three other species accounted for approximately 20% of strains and seven were unclassified as biochemically atypical corynebacteria. Non-protective antibodies to diphtheria toxin were found in 80 of the 93 subjects and a strong statistical association was demonstrated between carriage of corynebacteria and non-protective levels of anti-toxin antibodies. The remaining 13 subjects had protective levels of antitoxin antibodies. In contrast, only 45 of the 407 non-colonized subjects had non-protective antitoxin titres. The prevalence of carriage increased with age among males as did the percentage of non-protected subjects. The prevalence of female carriers of corynebacteria was significantly lower. Serum samples from 12 subjects with different antibody titres to diphtheria toxoid reacted to varying degrees with whole-cell lysates of a number of species of corynebacteria. The results suggest that a causal relationship may exist between nasopharyngeal carriage of corynebacteria and a low anti-diphtheria toxin immune response.

Adolescent↗
PubMed2000

Circulating GH isoforms and GH bioactivity in preterm neonates.

Among the molecular variants of human GH, the monomeric 22-kD is the predominant isoform, whereas the 20-kD is the second most abundant isoform. Because little is known on the pattern of human GH isoforms in the early postnatal period, we evaluated serum levels of 22-kD GH by an immunofluorometric assay and of 20-kD GH by an ELISA using an anti-20-kD antibody, and measured GH bioactivity with the Nb2 cell bioassay in 19 preterm neonates (gestational age, 32 +/- 0.5 wk; mean +/- SEM) on the fourth and 15th days of life. As control subjects, we studied 19 full-term neonates (gestational age, 39 +/- 0.3 wk) on the fourth day of life and 20 healthy adults, aged 20 +/- 0.3 y. Four-day-old preterm neonates showed significantly higher serum values of 20-kD GH (0.99 - 0.14 ng/mL) than full-term neonates (0.33 +/- 0.07 ng/mL; p < 0.001) and adults (0.09 +/- 0.02; p < 0.0001). Likewise, 22-kD GH and GH levels by Nb2 cell bioassay were also significantly higher (p < 0.001) in preterm than in full-term neonates and young adults. A significant decrease (p < 0.01) in 20-kD, 22-kD, and Nb2-determined GH was observed in preterm neonates on the 15th day of life The percentage of the 20-kD isoform was similar in the preterm infants at the fourth and 15th day, in full-term-infants, and in adults (2.7%, 2.7%, 2.8%, and 3.16%, respectively). Our results indicate that 20-kD GH serum levels change throughout life as regards total amount, but not as regards percentage.

Adult↗
PubMed2000

Growth hormone immunoreactivity does not reflect bioactivity.

In childhood, the largest secretory burst of GH occurs during nighttime, and consists of a complex mixture of molecular forms of GH that are thought to have different biologic activity. Standard GH assays cannot distinguish between bioactive and biologically inactive GH isoforms. To examine this relationship, overnight GH secretion was assessed by blood sampling every 30 min in 10 short prepubertal children (7 boys and 3 girls) to evaluate both the serum concentration and the biologic activity of GH. Serum GH concentrations were measured by an immunofluorometric assay and its biologic activity by the Nb2 cell bioassay. The 12-h (2000 h to 0800 h) and 6-h (2000 h to 0200 h and 0200 h to 0800 h) GH profiles were analyzed using the Pulsar program. When GH secretory pattern was measured by immunofluorometric assay, the area under the curve above the 0 line, the mean GH concentration, and the mean height of the secretory peaks were significantly higher during the first than during the second part of the night (29.17+/-5.93 versus 16.29+/-1.87 mIU/L, p<0.05; 7.77+/-1.28 versus 4.83+/-0.33 mIU/L, p<0.05; 4.61+/-0.94 versus 2.68+/-0.27 mIU/L, p<0.05, respectively). In contrast, GH biologic activity was not significantly different during the two parts of the night. In conclusion, a dissociation between GH bioactivity and immunoreactivity is present in physiologic conditions, indicating that standard GH measurements do not provide any information on the biologic activity of the hormone. Although GH secretion is regulated by complex neuroendocrine mechanisms, the biologic activity of the hormone seems to be independent of them and is most probably regulated by peripheral mechanisms acting on its clearance or bioavailability to the target tissues.

Biological Assay↗
PubMed1999

The level of bioavailable growth hormone (GH) after the first GH injection predicts the first year's growth response in GH-deficient children.

Serum GH levels were measured in 9 prepubertal children with growth hormone (GH) deficiency using an immunofluorometric assay (IFMA) and a Nb2 cell bioassay, prior to and 2, 4, 6 and 12 hours after the first hGH subcutaneous injection (sc) (0.1 IU/Kg). After this first acute phase, hGH treatment was continued regularly at a dose of 0.1 IU/kg per day at bedtime. The acute pharmacokinetic profile was similar in the patients irrespectively of the assay used: serum GH usually starts to rise after 2 hours, peaks by 4 or 6 hours and drops back to near baseline levels 12 hours after s.c. GH administration. A great variation in the amplitude of peak levels was observed among the patients. The assays significantly (p<0.0001) correlate with each other in all subjects. An increase in serum GH values as evaluated by the Nb2 bioassay was observed in most children after s.c. GH injection, suggesting that the administered hormone preserves its biological activity. The individual variations in GH bioactivity in our GH-deficient children could be due to different mechanisms of the drug's degradation at the site of injection or in circulation. Pre-treatment height velocity calculated as standard deviation scores (SDS) significantly increased (p<0.001) from -2.77+/-0.42 to 1.22+/-0.87 SDS after a 12-month period of GH treatment in 8 patients who had already completed the first year of therapy, although it varied considerably among subjects. A higher serum GH peak value evaluated by both assays corresponded to a higher growth response during the 1st year, but not in the 2nd year of GH therapy. In conclusion, the degree of GH bioactivity released into circulation after sc GH injection may vary considerably among GHD patients and correlates with the growth response observed during the 1st year of GH therapy.

Adolescent↗
PubMed1999

Crystallization and preliminary crystallographic investigations of rhodanese from Azotobacter vinelandii.

The rhdA gene identified in Azotobacter vinelandii codes for a protein, RhdA, which displays rhodanese (thiosulfate-cyanide sulfurtransferase) activity. RhdA was overexpressed and purified to homogeneity. The protein crystallized in the orthorhombic space group P2(1)2(1)2 with unit-cell parameters a = 44.4, b = 150.8, c = 53.8 A; on a synchrotron source the diffraction patterns could be collected to a resolution limit of 1.8 A. Evaluation of the crystal density indicates that the crystal lattice accommodates one molecule per asymmetric unit and that the solvent content is 59% of the total volume.

Amino Acid Sequence↗
PubMed1998

Evaluation of growth hormone bioactivity using the Nb2 cell bioassay in children with growth disorders.

The Nb2 cell bioassay could be a useful tool for evaluating human growth hormone (hGH) bioactivity, but is not specific for hGH since it relies on the ability of the hormone to produce effects by cross-reacting with the lactogenic receptor on Nb2 cells. We studied the biological activity of both endogenous and exogenous hGH in short patients using the Nb2 bioassay after inhibiting the mitogenic effect of the other lactogenic hormone, that is human prolactin (hPRL), by adding a specific antibody against hPRL to each assay. The in vitro study showed a significant (p<0.0001) increase in Nb2 cell proliferation when increasing concentrations of highly purified hGH were added to the cell culture. A complete inhibition of Nb2 cell replication was observed after adding a specific antibody against hGH. The in vivo study showed a significantly (p<0.0001) lower hGH bioactivity (4.90+/-0.28 ng/ml) evaluated during stimulation tests in 9 children with total idiopathic GH deficiency, mean age 9.25+/-1.99 years, in comparison with that found in 11 short children with normal growth velocity, mean age 8.22+/-1.41 years (12.25+/-1.19 ng/ml). Likewise, serum GH levels measured by immunofluorometric assay IFMA in the same serum samples were significantly (p<0.001) lower in the 9 GH-deficient (1.97+/-0.37 ng/ml) than in the 11 short children (21.85+/-2.71 ng/ml). Moreover, we evaluated GH concentrations using both IFMA and the Nb2 cell bioassay in serum samples collected from another 11 idiopathic GH-deficient children, mean age 10.71+/-1.18 years, before and then, 6 and 24 hours following the 1 st injection of r-hGH (0.1 IU/kg sc). Serum GH values measured by both IFMA and Nb2 bioassay significantly (p<0.0001) increased 6 hours after r-hGH administration and decreased to reach basal levels after 24 hours. In conclusion, the Nb2 cell bioassay with minor modifications seems to provide a specific and sensitive assessment of hGH bioactivity.

Animals↗
PubMed1996

Cloning, sequence analysis and overexpression of the rhodanese gene of Azotobacter vinelandii.

A gene encoding rhodanese (rhdA) was cloned from Azotobacter vinelandii on a 2.3-kb SphI fragment. This fragment was identified by its hybridization to a PCR product obtained by amplification of genomic DNA using degenerate primers encoding the N-terminal sequence of rhodanese purified from A. vinelandii. The sequence of a 1.2-kb region revealed an 813-bp open reading frame that encoded a polypeptide of 271 amino acids, the N-terminal sequence of which was identical to that of A. vinelandii rhodanese. In a search of database entries, eukaryotic rhodaneses and rhodanese-like proteins from bacteria gave the highest scores of identity (27-30%) with the predicted product of the 813-bp open reading frame. A. vinelandii RhdA shows less sequence similarity to vertebrate rhodaneses than it does to prokaryotic rhodanese-like proteins which did not show typical rhodanese activity. Basic residues thought to be catalytically important in bovine rhodanese are not conserved in A. vinelandii rhodanese. The sequence similarity between the two structurally similar domains of rhodanese is more pronounced for the A. vinelandii enzyme than the bovine enzyme, and supports the hypothesis that the complete structure was originally generated by gene duplication. When rhdA was overexpressed in Escherichia coli, rhodanese represented 30% of total cell protein and thiosulfate:cyanide sulfurtransferase activity increased >600 fold in cell-free extracts. A. vinelandii rhdA insertion/deletion mutants had no discernible phenotype distinct from the wild-type strain with respect to growth on various sulfur sources or nitrogenase activity. Mutants retained 20% of wild-type rhodanese thiosulfate:cyanide sulfurtransferase activity suggesting the presence of redundant sulfurtransferase enzymes in A. vinelandii.

Amino Acid Sequence↗
PubMed1996

Biochemical analysis of muscle biopsy in overnight fasting patients with severe chronic heart failure.

To better characterize the role of skeletal muscle in chronic heart failure we studied energetic charge, metabolites and enzyme activity in the energy production pathway. We selected 15 males with severe chronic heart failure (NYHA class III, stable clinical conditions and in normal nutritional status) and seven controls. Controls and patients were submitted to biopsy of the vastus lateralis muscle in resting and fasting conditions. Hormone profiles were also evaluated. Our results showed near normal ATP, ADP and AMP concentrations, but there were substantially more reductions in glycogen (46 +/- 5 vs 77 +/- 6 mumoles glycosidic units.g-1 fresh tissue) and creatine phosphate (5 +/- 1 vs 13 +/- 1 mumoles.g-1 fresh tissue) in patients than in controls. We also found a reduction in glycolytic activity (pyruvate kinase 1009 +/- 79 vs 1625 +/- 26 nmoles. min-1.mg protein-1), despite normal tricarboxylic acid cycle velocity, an increase in alanine amino-transferase (964 +/- 79 vs 425 +/- 34 nmoles. min-1.mg protein-1) and in aspartate aminotransferase (515 +/- 44 vs 291 +/- 56 nmoles.min-1.mg protein-1). An increase was also observed in total NADH cytochrome c reductase (128 +/- 14 vs 68 +/- 5 nmoles.min-1.mg protein-1), while cytochrome oxidase activity was normal. The cortisol/insulin ratio was slightly elevated (77 +/- 4 vs 32 +/- 12). In conclusion, normonutritive patients with severe heart failure show an imbalance in the energy production/utilization ratio. The impairment is probably due both to a decrease in production and an increase in consumption of energy owing to greater cellular workload and/or a hypercatabolic state.

Adenine Nucleotides↗
PubMed1995

Purification and characterization of an iron superoxide dismutase from the nitrogen-fixing Azotobacter vinelandii.

Two electrophoretically distinct forms of superoxide dismutase (SOD; EC 1.15.1.1) which show different inhibition patterns to hydrogen peroxide have been identified in Azotobacter vinelandii. The SOD inhibited by hydrogen peroxide was purified to homogeneity, and turned out to be an iron superoxide dismutase. The enzyme is present in only one molecular form with an isoelectric point of 4.1, and it is composed of two identical subunits with an apparent molecular weight of 21,000 Da. Spectroscopic analyses indicated that this enzyme contains ferric iron (1.4-1.6 mol/mol protein) in the typical high-spin form present in other prokaryotic Fe-SODs. N-Terminal sequence alignments (up to the 49th residue) showed that A. vinelandii Fe-SOD has high similarity with other prokaryotic Fe-SODs.

Amino Acid Sequence↗
PubMed1994

Some structural features of cluster-coordinating cysteines of Clostridium pasteurianum ferredoxin are revealed by 2D TOCSY 1H NMR on the oxidized protein.

Different sets of geminal J coupling constants for the eight beta-CH2 protons in the iron-coordinating cysteines in Clostridium pasteurianum ferredoxin were detected by 2D TOCSY 1H NMR experiments on the oxidized protein. Four resonances were characterized by quite similar high values of J, two more resonances had a J value about half of the former ones, while the last two had extremely low J values. These findings suggest that the cysteines required for cubane symmetry around the iron atoms are constrained into different geometries. The simplified model used for fine tuning of tau m in these TOCSY experiments is also presented and discussed.

Clostridium↗
PubMed1994

Reversible and non-denaturing replacement of iron by cadmium in Clostridium pasteurianum ferredoxin.

Incubation of native, reduced Clostridium pasteurianum ferredoxin with different metals gave a range of modifications in the electronic and EPR spectrum of the protein, or made the signals disappear. The reduced protein, isolated after incubation with different metals under identical conditions (50 microM protein, 1 mM metal, 1 h incubation) was found to contain amounts of foreign metals increasing with their thiophylicity, i.e. Cd2+ >> Zn2+ > Co2+. Little, if any, incorporation was observed for Ni2+, Cu2+, Mn2+ or in the absence of reductant. The activity of substituted ferredoxins in a hydrogenase-coupled assay was proportional to the amount of residual iron, suggesting that the residual iron is present in a population of intact active molecules rather than in partially substituted clusters distributed among individual molecules. The cadmium-substituted ferredoxin did not contain iron, but contained eight cadmium atoms and six labile sulfide atoms/mol. Folding of the isolated, substituted proteins was investigated by CD and 1H-NMR. Both techniques showed retention of the main structural features of the protein upon metal substitution. The rate and extent of the substitution of iron by cadmium were essentially independent of pH, but were found to decrease with increasing ionic strength and to increase with the cadmium concentration. In the cadmium-substituted protein, cadmium was replaced by iron upon incubation with iron and mercaptoethanol in the absence of dithionite. In the presence of dithionite, cadmium was not replaced by iron upon incubation of the cadmium-substituted protein with excess iron and mercaptoethanol. In competition experiments, incubation of iron-containing ferredoxin with stoichiometric amounts of cadmium in the presence of dithionite and excess iron and mercaptoethanol resulted in quantifiable replacement of iron by cadmium. Therefore, substitution of iron by cadmium was only achieved under reducing conditions, and was only reversible in the absence of strong reductants.

Cadmium↗
PubMed1993

Properties of Azotobacter vinelandii rhodanese.

Rhodanese (thiosulfate: cyanide sulfurtransferase, E.C. 2.8.1.1) was purified from the nitrogen fixing organism Azotobacter vinelandii, and its amino acid composition was determined. The enzyme is a single polypeptide chain of M(r) 29,000 which showed an apparent pI of 4.7 and no presence of isoenzymes. Steady-state initial velocity measurements indicate that the enzyme catalyzes the transfer of sulfane sulfur of thiosulfate either to cyanide or to dihydrolipoate by way of a double displacement mechanism.

Amino Acids↗
PubMed1992

1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum.

The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.

Clostridium↗
PubMed1991

Uptake of iron by apoferritin from a ferric dihydrolipoate complex.

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.

Animals↗
PubMed1991

Identification of sulfurtransferase enzymes in Azotobacter vinelandii.

Rhodanese and 3-mercaptopyruvate sulphurtransferase have been identified in A. vinelandii. Two distinct active fractions of the two sulphur transferases were obtained after FPLC ion-exchange chromatography of material partially purified from crude extracts. Rhodanese has been purified to homogeneity, and it consists of one polypeptide chain of Mr ca 25,000. A partial purification of 3-mercaptopyruvate sulphurtransferase was obtained.

Azotobacter↗
PubMed1989

Molecular aspects of the removal of ferritin-bound iron by DL-dihydrolipoate.

The removal of ferritin-bound iron by the physiologic dithiol DL-dihydrolipoate was studied over the pH range 5.5-9.0. A novel method was devised for the determination of iron removal, making it possible to study the actual release of iron from ferritin, regardless of the oxidation state or complexation form. The overall iron-removal process appears to depend upon a balance between the deprotonation of the dithiol and the protolytic dissolution of the iron core inside the ferritin molecule. The amount of iron removed at equilibrium increases with the pH, at any of the dihydrolipoate/ferritin iron ratios tested. The formation of the binuclear iron-dithiol complex [Fe2(dihydrolipoate)3]-3 is not strictly required for iron mobilization, but it seems to affect the efficiency of the dithiol in iron mobilization by providing a stable complexation form for the released iron outside the ferritin protein shell. Comparison of the release of ferritin-bound iron by free and immobilized dihydrolipoate indicates that mobility of the dithiol is mandatory for the removal process to take place.

Chemical Phenomena↗
PubMed1989

Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size.

We have separated von Willebrand factor (vWF) multimers of different size into several fractions which were characterized by SDS-agarose gel electrophoresis and by measuring the ratio between ristocetin cofactor activity (Ricof) and von Willebrand antigen (vWF:Ag) content. The pooled fractions contained vWF with multimeric structures and Ricof similar to those in plasma. The pool was labelled with 125I and used for inhibition binding studies with individual fractions to calculate the dissociation constants (Kd values expressed in mol/l) of the individual fractions for ristocetin-dependent binding to GP Ib and thrombin-induced binding to GP IIb/IIIa. Direct binding studies of the 125I-vWF pool gave mean Kd values of 2.02 +/- 0.05 x 10(-8) for GP Ib and 1.15 +/- 0.02 x 10(-8) for the GP IIb/IIIa complex. Inhibition binding studies gave Kd mean values one third to one tenth as high for larger multimers and 3-10 times higher for smaller multimers, for both GP Ib and IIb/IIIa complex. Similar results were observed when binding studies were carried out in the presence of platelets from a patient with afibrinogenaemia. These data on binding correlated very well with ristocetin- and thrombin-induced aggregation of afibrinogenaemic platelets, since equal concentrations of the higher molecular weight forms gave significantly higher aggregation rates. Based on these results, we conclude that the affinity of the vWF molecule for its two platelet receptors is greater for the largest multimers.

Afibrinogenemia↗