Biomedical subjects
Publications and source records attributed to M Signorini.
Two patients presented with multicystic lymphatic malformations of the penis. These were congenital and progressive, and both were successfully treated by excision after injection of methylene blue.
We developed a procedure for purification to homogeneity of cytosolic transglutaminase from bovine testes, by means of anion exchange, size exclusion and hydrophobic chromatography. The crucial step is chromatography on Phenyl sepharose, with specific elution by the inhibitor GTP. The enzyme displays the properties of type 2 transglutaminases.
1. The involvement of cysteine residues in the catalytic mechanism of UDP-glucose pyrophosphorylase was suggested by the rapid inactivation of the enzyme by N-ethylmaleimide, even at 1:1 reagent/enzyme stoichiometric ratios. 2. The inactivation is largely prevented by uridine substrates (UDP-glucose and UTP) in agreement with the assumption that the reactive cysteine is located at the active site.
The purpose of this research is to evaluate the changes of cutaneous pain threshold induced by vibratory stimuli applied to the skin. The pain threshold was tested by means of electrical stimuli using the method of limits. Both vibratory stimuli (conditioning stimuli) and electrical stimuli (test stimuli) were driven by a computer. The experiments were carried out on the dorsal surface of the second phalanx of the index finger in the dominant hand of 34 healthy subjects (18 males and 16 females). The conditioning stimuli were sinusoidal waves (250 microm peak to peak) at a rate of 30 and 300 Hz. During the experiment (6 min of vibration and 9 min after) the cutaneous temperature of the stimulated area was recorded. Both vibratory rates induced significant increases of the pain threshold but the 30 Hz vibrations were the more effective. After stimulation the pain threshold lowered to a steady value which was higher than before the start of vibrations. Such threshold changes showed no correlation throughout the experiment with changes in skin temperature. We therefore advance the hypothesis that the main factors which modulate the pain threshold are neuronal connections between tactile and pain pathways.
Glycogen phosphorylase is progressively degraded during irradiation with near UV light in the presence of vanadate. The pattern of protein cleavage by monovanadate is characterised by fewer peptides than that by decavanadate, which leads to fragmentation in a ligand dependent way. In both instances, the initial cleavage releases a peptide of 82,000 daltons which accounts for the N-terminal portion of the subunit, including the regulatory phosphorylation site.
A controlled prospective study of 2165 outpatients undergoing skin surgery was performed to evaluate the utility and the effects of several antibiotic schedules for prophylaxis of wound infections. The patients were divided into four groups. Twenty-three of the 541 group A patients, given no antibiotics, had wound infections. Eight of the 542 group B patients, given systemic antibiotics from immediately after surgery until the third day, had wound infections. Four of the 540 group C patients, treated only with local sterile antibiotic powder sprinkled into the wound during surgery, had wound infections develop, and only one infection occurred in the 542 group D patients given systemic antibiotics from 2 days before surgery until the second day after surgery. This last schedule was the best for prophylaxis of wound infections in contamination-prone regions. Local antibiotic administration is a simple method for prevention of infections in routine skin surgery.
During irradiation in the presence of decavanadate, the subunits of phosphofructokinase underwent progressive degradation to a fragment of about 78,000 daltons. This cleavage pattern was altered when the photoirradiation was performed in the presence of monomeric vanadate with formation of several smaller peptides. The specificity of the decavanadate induced cleavage was proved by the resistance of other enzymes to the treatment and by the effects of phosphofructokinase ligands. During irradiation, the activity of the enzyme declined. Differences between the rate of inactivation and of cleavage of enzyme subunits suggest the occurrence of multiple processes.
The activity of liver and brain transglutaminase is rapidly lost following i.p. injection of acrylamide (50-200 mg/kg). Other enzymes investigated were not modified by the treatment, with the exception of brain enolase.
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A case of congenital infiltrating lipomatosis of the face is presented, associated with a congenital cytomegalovirus infection.
Skeletal-muscle UDP-glucose pyrophosphorylase is inactivated by reaction with 2-ethoxy-N-(ethoxy-carbonyl)-1,2-dihydroquinoline (EEDQ) and 1-(3-dimethylaminopropyl-3-ethylcarbodi-imide (EDAC), two reagents specific for carboxylate groups. The former reagent is a more effective inactivator than EDAC. Although no evidence of reversible enzyme-reagent complexes of the affinity-labelling type was obtained by kinetic analysis of the inactivation, the selective protection of UDP-glucose pyrophosphorylase activity against inactivation by EEDQ in the presence of uridine substrates is indicative of an active-site-directed effect. The results are consistent with the hypothesis that EEDQ modifies a single carboxylate group located in a hydrophobic domain close to the substrate-binding site, leading to enzyme inactivation. In contrast, the reaction between UDP-glucose pyrophosphorylase and EDAC appears to involve a different region of the enzyme.
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The report describes a patient with 45 per cent BSA burns who developed Legionnaire's disease 3 days after the acute injury. The diagnosis of this life-threatening complication was late because most of its signs and symptoms can be encountered in the burned patient. This delay could have been fatal to the patient and required the evacuation of the burn centre for disinfection.
We have investigated the interaction between calcium ions and erythrocyte transglutaminase and the enzyme activation. The binding involves both high and low affinity sites, but only the former ones are relevant for activation. The binding of calcium and the activation are modified by treatment with NBD-Cl and with PLP suggesting the presence of cysteine and lysine residues at the high affinity binding sites. The interaction of the enzyme with calcium is not calmodulin dependent and is easily detected as a shift in electrophoretic mobility in the presence of SDS.
Addition of low concentrations of acrylamide to the assay mixture of erythrocyte transglutaminase leads to a strong inhibition of the enzyme with a mechanism consistent with a non-competitive inhibition against both substrates. For the quenching of the intrinsic fluorescence of the protein, much higher concentrations of acrylamide are required so that both phenomena appear independent of each other. In this particular case, therefore, acrylamide can be employed to obtain information on ligand-triggered conformational changes.
Erythrocyte transglutaminase was purified by anion-exchange chromatography, size exclusion and affinity chromatography. Homogeneity was achieved by an additional step of HPLC size-exclusion chromatography. The molecular mass of the purified enzyme was calculated to be 65,000 Da by size-exclusion chromatography and sucrose-gradient centrifugation, and 92,000 Da by SDS-PAGE, thus suggesting a high degree of asymmetry. The amino-acid composition of erythrocyte transglutaminase differed substantially from that of the guinea-pig liver enzyme, notably with respect to the number of histidine, cysteine and acidic amino-acid residues. The enzyme has an absolute requirement for divalent cations for activity: calcium, manganese, and the lanthanides terbium and gadolinium activate the enzyme in decreasing order of efficacy, while no activity is displayed in the presence of magnesium. In the presence but not in the absence of calcium ions, the enzyme is rapidly inactivated by N-ethylmaleimide and by diethylpyrocarbonate suggesting that the cation influences the reactivity of amino acids essential for catalysis. When erythrocyte proteins are employed as amine acceptors in the presence of calcium, the erythrocyte transglutaminase appears to preferentially modify membrane-associated proteins, although, in the absence of calcium ions and exogenous amines, it displays a pH-dependent interaction with soluble proteins.
When human endometrial transglutaminase was measured a 10-fold higher activity was detected during the secretive phase. This change was not related to either differences in solubility of the enzyme or to selective contamination by plasma factor XIII and rather appears to depend on the expression of the tissue form of transglutaminase, suggesting that this enzyme is regulated in vivo by progesterone.
Purified transglutaminase from human erythrocytes was shown to undergo time dependent inactivation by acrylamide added at millimolar concentration: this effect was wholly dependent on calcium ions and some protection was produced by glutamine substrates and GTP. The enzyme activity was recovered by addition of thiol based reducing agents, while ascorbic acid and sodium borohydrate were ineffective. These data are suggestive of an active site directed action of acrylamide.