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Biomedical subjects

F Paoletti

Publications and source records attributed to F Paoletti.

At least 73 records · Page 4Linked to original sources
PubMed1990

Superoxide-driven NAD(P)H oxidation induced by EDTA-manganese complex and mercaptoethanol.

A purely chemical system for NAD(P)H oxidation to biologically active NAD(P)+ has been developed and characterized. Suitable amounts of EDTA, manganous ions and mercaptoethanol, combined at physiological pH, induce nucleotide oxidation through a chain length also involving molecular oxygen, which eventually undergoes quantitative reduction to hydrogen peroxide. Mn2+ is specifically required for activity, while both EDTA and mercaptoethanol can be replaced by analogs. Optimal molar ratios of chelator/metal ion (2:1) yield an active coordination compound which catalyzes thiol autoxidation to thiyl radical. The latter is further oxidized to disulfide by molecular oxygen whose one-electron reduction generates superoxide radical. Superoxide dismutase (SOD) inhibits both thiol oxidation and oxygen consumption as well as oxidation of NAD(P)H if present in the mixture. A tentative scheme for the chain length occurring in the system is proposed according to stoichiometry of reactions involved. Two steps appear of special importance in nucleotide oxidation: (a) the supposed transient formation of NAD(P). from the reaction between NAD(P)H and thiyl radicals; (b) the oxidation of the reduced complex by superoxide to keep thiol oxidation cycling.

Catalysis↗
PubMed1989

Analysis of transketolase and identification of an enzyme variant in human leukocytes.

Human leukocyte transketolase of fresh cell extracts has been analyzed by isoelectrofocusing on agarose gels (pH 3-10). The enzyme was then transblotted on nitrocellulose and detected with specific anti-transketolase IgG coupled to an avidin/biotin-immunoperoxidase system. Each sample yielded multiple enzyme forms, within a pI range of about 7.4-8.4. Transketolase profile, however, was not identical in all extracts. There are two mainly distinct patterns, showing qualitative and quantitative differences: a standard profile, which is predominant, and a variant, found in three unrelated subjects out of the two hundred and twenty. Standard and variant enzyme have similar Km values for ribose 5-P and xylulose 5-P and the same mobility on SDS-PAGE.

Cytosol↗
PubMed1989

Transketolase from human leukocytes. Isolation, properties and induction of polyclonal antibodies.

Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM-cellulose by specific elution with a D-xylulose-5-phosphate/D-ribose-5-phosphate mixture and the isolated product exhibited a specific activity of about 10 units/mg protein at 37 degrees C. Transketolase preparations are contamination-free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D-xylulose 5-phosphate and D-ribose 5-phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDS/PAGE as a single band, with a molecular mass of about 66 kDa. The isoelectrophoretic heterogeneity of transketolase was assessed either by activity staining or immunovisualization with anti-transketolase antisera, previously induced in rabbits. These techniques yielded two practically overlapping patterns consisting of 6-8 distinct bands within a pI range of 6.5-8.5. Both pure and crude transketolase preparations showed a similar heterogeneous profile, thus confirming the stability of the enzyme throughout purification. The occurrence of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.

Antigen-Antibody Reactions↗
PubMed1989

[Atracurium, vecuronium and pancuronium. Results of a polygraphic study].

The Authors have studied three nondepolarizing muscle relaxants widely used: pancuronium v/s atracurium and vecuronium. The Train of Four was used to detect the magnitude of the neuromuscular blockade. The intubation follows 90 minutes after administration of the drugs and for each patient the Authors valued: 1) the T1 and TR values at intubation; 2) the onset-time; 3) the duration of neuromuscular blockade; 4) the recovery time. Furthermore, the degree of neuromuscular blockade was clinically checked both at the time of induction and at recovery. The results of the present study show, in accordance with the literature, that all three drugs tested are capable of obtaining complete muscle relaxation. Atracurium and vecuronium particularly allow an adequate intubation at the adopted doses (0.6 mg/kg and 0.1 mg/kg respectively) with a relatively short onset-time (medium values 136" in the group of atracurium and 146" in the group of vecuronium) and a restoration time faster than pancuronium.

Atracurium↗
PubMed1988

Changes in CuZn-superoxide dismutase during induced differentiation of murine erythroleukemia cells.

Superoxide dismutase (SOD) activity in murine erythroleukemia cells (MELC) was determined during differentiation induced by hexamethylene bisacetamide. SOD levels in hexamethylene bisacetamide-treated MELC were about twice as high as those of controls. Dose response and kinetic experiments have shown that SOD activity variations are closely related to the amount of inducer and the duration of treatment in culture. Moreover, phorbol 12-myristate 13-acetate, which inhibits hexamethylene bisacetamide-induced MELC maturation, was also effective in reducing the extent of the SOD increase. SOD changes mainly involved the CuZn form of the enzyme, having a molecular weight of about 32,000 and a striking sensitivity to cyanide inhibition. In addition, the isoelectrophoretic analysis of CuZn-SOD from both treated and untreated cells yielded an identical pattern. This suggests that quantitative rather than qualitative enzyme changes occurred during MELC terminal division. SOD levels are directly related to the degree of differentiation and particularly to the amount of cytosolic hemoglobin, whose synthesis in committed cells is paralleled by a rise in enzyme activity. The SOD increase represents a distinctive marker of erythroleukemia maturation and might tentatively be interpreted as a cellular response to oxidative stress from hemoglobin autoxidation.

Acetamides↗
PubMed1987

A spectroscopic investigation of cobalt(II) substituted alkaline phosphatase.

The electronic and 1H NMR spectra are reported for the cobalt(II) alkaline phosphatase (EC 3.1.3.1.) system at pH around 6 in the range 0-2 mol of cobalt per mol protein. It is shown that under the present experimental conditions cobalt(II) selectively populates the A sites. Three isotropically shifted NH signals have been detected in the A site that indicate the presence of three histidines in the coordination sphere of cobalt(II). The electronic spectra and the nuclear relaxation properties are consistent with pentacoordination of cobalt(II) in the A site. The finding of reproducible preparation routes for the derivatives, and of appropriate experimental conditions for the observation of their 1H NMR spectra, open new possibilities for the spectroscopic investigation of alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1987

Buprenorphine vs. morphine via the epidural route: a controlled comparative clinical study of respiratory effects and analgesic activity.

Twelve patients with intense or very intense pain of the non-incident type, secondary to neoplasia, were divided at random into two groups and treated with an epidural dose of 3 mg of morphine in 10 ml of glucose solution (6 patients = group M) or with 0.3 mg of buprenorphine in the same vehicle (6 patients = group B). None of the patients had previously been treated with opioids by any route. After first determining basal values, the following assessments were carried out: (1) evaluation of the analgesic effect of the drugs with checks at 30 min and at 1, 2, 3, 4, 6 and 18 h after administration, using a visual analogue scale, a numerical rating scale and a simple descriptive scale; and (2) evaluation of effects on respiration by means of checks at 30 and 90 min and at 6 and 18 h, on control of breathing indices (P0.1; VE; VA; Ti/Ttot; VT/Ti; RR), gas exchange indices (delta(A-a)O2; VD/VT; pAO2; R) and blood gas and acid-base indices (paO2; paCO2; pH; HCO3-). The data obtained were analyzed statistically using analysis of variance and Student's t test. The study results showed very similar analgesic efficacy for both treatments at a single dosage level of morphine (3 mg) compared to buprenorphine (0.3 mg), which was approximately 3 times greater than an equivalent parenteral dose of morphine (10 mg). Analysis of the results revealed statistically, though not clinically, significant changes in respiratory function indices, only in the buprenorphine-treated group. The effects of buprenorphine on respiratory function, when administered epidurally at the above dosage, are less favourable than those of morphine in the early measurements, probably because of its greater systemic absorption; nevertheless, the risk of delayed respiratory depression appears to be less after buprenorphine than after morphine.

Aged↗
PubMed1987

Immunoblot analysis of Salmonella typhi lipopolysaccharide (LPS) using typhoid sera.

Lipopolysaccharide (LPS) of Salmonella typhi has been analyzed by immunoblotting with pooled sera from typhoid patients. Pooled typhoid sera have recognized all the antigenic determinants of S. typhi LPS, giving a strong reaction with the repeating units on the O-side chains as well as with the core region. Cross-reacting antigens have been observed with the LPS of S. typhimurium and S. enteritidis, while no heterologous reactions were seen with the LPS of E. coli strains.

Cross Reactions↗
PubMed1986

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts.

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.

Animals↗
PubMed1986

Immunoaffinity purification of rat liver transketolase: evidence for multiple forms of the enzyme.

Rat liver transketolase (TK) has been purified, in a single step, by immunoaffinity chromatography on specific TK antibodies covalently linked to Sepharose 4B. The procedure described also involves the raising and isolation of rabbit TK antibodies to the conventionally purified enzyme [F. Paoletti (1983) Arch. Biochem. Biophys. 222, 489-496]. Affinity chromatography allows a 100-fold purification of TK from the cell cytosol and a recovery of about 70% of the original activity. The TK isolated has a specific activity of 2.7-3.2 at 25 degrees C and migrates as a single band on polyacrylamide gel electrophoresis at pH 9.1. Multiple forms of the enzyme, with distinct pI values in the range 7-8, have been detected in purified preparations by means of analytical isoelectric focusing and staining for TK. No addition of either Mg2+ or thiamine pyrophosphate is required for the activity of the enzyme which, in the native form, exhibits a molecular weight of about 139,000. Two moles of thiamine pyrophosphate can be resolved for each mole of enzyme. Affinity TK preparations are virtually free of glyceraldehyde-3-phosphate dehydrogenase, pentose-phosphate epimerase, and isomerase, although slight contamination by phosphohexose isomerase may occur.

Animals↗
PubMed1985

Immune mechanisms for hepatic fibrogenesis. T-lymphocyte-mediated stimulation of fibroblast collagen production in chronic active hepatitis.

Lymphocytes can produce soluble factors capable of enhancing fibroblast proliferation and collagen synthesis. T-lymphocytes, adherent cells and undifferentiated peripheral blood mononuclear cells from patients with HBsAg-positive chronic active hepatitis and from HBsAg healthy carriers were triggered with purified HBsAg and tested for their ability to enhance collagen production by human dermal fibroblast cultures. Purified HBsAg did not induce any proliferative response in the mononuclear cell cultures. Addition of patient T-lymphocyte supernates to monolayers of fibroblast consistently resulted in a significant enhancement of collagen production. On the contrary, supernates harvested from adherent cell cultures did not demonstrate any stimulatory activity. We therefore assumed that the observed enhancement of collagen production was probably the result of lymphokine(s) produced by T-lymphocytes. This fibrogenic factor (or factors), which is released by T-cells independently of the presence in vitro of HBsAg, is stable at -80 degrees C, not dialyzable and, by Sephadex G-100 gel filtration, is present in a fraction which collects substances of a molecular weight between 50 000 and 100 000. Mononuclear cell supernates from HBsAg healthy carriers did not influence fibroblast collagen accumulation. These data emphasize the possible role that lymphokines may play in the pathogenesis of fibrosis during the natural history of chronic liver disease.

Adult↗
PubMed1983

Purification and properties of transketolase from fresh rat liver.

Transketolase has been purified from rat liver. The final product is judged to be homogeneous by the criteria of gel filtration on Sephadex G-200 and Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The estimated Mr's are 139,000 for the native protein and 69,000 for the dissociated subunits. The purified enzyme does not require either Mg2+ or thiamine pyrophosphate for optimum activity. The best C2-donor substrate is D-xylulose 5-phosphate, with Km = 25 microM. D-Fructose 6-phosphate is an active C2-donor, but beta-hydroxypyruvate was found to be inactive, even at high concentration. The product of the C2-transfer from D-xylulose 5-phosphate or D-fructose 6-phosphate to D-ribose 5-phosphate has been identified as D-sedoheptulose 7-phosphate. Neither D-glucose 6-phosphate nor D-arabinose 5-phosphate showed C2-acceptor activity. This enzyme does not appear to be responsible for the formation of octulose 8-phosphates which have been detected in liver extracts.

Animals↗
PubMed1981

The role of respiration in tumor cell transition from the noncycling to the cycling state.

Resting Yoshida AH130 hepatoma cells, harvested at the plateau of tumor development in vivo, were recruited into the cycling state following transfer to an in vitro system whereby these cells were incubated in the autologous ascites plasma diluted with buffered saline and enriched with glucose. In this system, cell recruitment into the phase of DNA synthesis (S phase) strictly depends on the activity of the respiratory chain and is abolished by anaerobiosis as well as by antimycin A, although the intracellular levels of ATP and the rate of protein synthesis are practically unaffected by these treatments. Furthermore, 2,4-dinitrophenol, at concentrations which uncouple the respiratory phosphorylation and hence enhance both glycolysis and oxygen consumption, does not hinder cell promotion into S phase. Thus, the absolute respiration dependence of cycling resumption by resting ascites cells does not seem to rely on respiratory ATP supply, but rather is linked to the electron flow through the respiratory chain.

Adenosine Triphosphate↗