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Biomedical subjects

M Libonati

Publications and source records attributed to M Libonati.

At least 19 recordsLinked to original sources
PubMed1994

Human pancreatic-type and nonpancreatic-type ribonucleases: a direct side-by-side comparison of their catalytic properties.

The catalytic properties and substrate preference of several highly purified human ribonucleases from different organs and body fluids have been examined in detail using various low-molecular-weight compounds and single- or double-stranded polyribonucleotides as substrates. All single-stranded polyribonucleotides were degraded by nonpancreatic-type (npt) RNases at a slower rate than by pancreatic-type (pt) enzymes: ptRNases were 20 times more active on RNA and poly(U) substrates and more than 6000 times more active on poly(C). Pancreatic-type RNases degraded poly(C) faster than RNA, showing a strong preference for poly(C) over poly(U) with the following activity ratios: RNA/poly(C), 0.44; RNA/poly(U), 12; poly(C)/poly(U), 27. In contrast, nptRNases cleaved RNA more rapidly than synthetic homopolymers, preferring poly(U) over poly(C) with the following ratios: RNA/poly(C), 130; RNA/poly(U), 10; poly(C)/poly(U), 0.08. Human ptRNases degraded poly(A) and double-stranded polyribonucleotides about 100 and 400 times faster, respectively, than bovine RNase A. However, no measurable activity could be detected on these substrates with nptRNases. The activities of ptRNases on dinucleoside phosphates (CpN and UpN) or uridine and cytidine 2',3'-cyclic phosphates were similar to those of bovine RNase A; nptRNases, instead, cleaved only CpA and UpA at an appreciable rate. The effects of pH, ionic strength, and divalent cations on the activity of these ribonucleases were also investigated using yeast RNA as a substrate.

Catalysis↗
PubMed1993

Differential effects of tyrosine kinase inhibition in CD69 antigen expression and lytic activity induced by rIL-2, rIL-12, and rIFN-alpha in human NK cells.

The effect of rIL-12 on induction of CD69 antigen expression and cytolytic activity in purified human NK cells was evaluated in comparison to the effects of rIL-2 and rIFN-alpha. It was found that rIL-12 directly induced CD69 antigen expression in NK cells, although the period of incubation required by rIL-12 was longer than the period required by rIL-2 or by rIFN-alpha. Similarly, the cytolytic activity induced by rIL-12 in NK cells against the NK-resistant target cell line Raji was consistently lower than the cytolytic activity induced by rIL-2 or rIFN-alpha when measured after 6 hr of incubation, and increased during the following 18 hr of incubation. To compare the involvement of tyrosin kinases in activation of NK cells induced by rIL-2, rIL-12, and rIFN-alpha, the effect of the specific inhibitor of tyrosin kinases, genistein, was evaluated on induction of CD69 antigen expression and lytic function mediated by the three cytokines. It was found that genistein inhibited CD69 antigen expression induced by rIL-2 and by rIL-12, but not that induced by rIFN-alpha. Unlike the effect on CD69 antigen expression, the cytolytic activity induced by all three cytokines was inhibited by genistein. These results, together with the finding that CD69 antigen expression induced by rIL-2 but not by rIL-12 or rIFN-alpha was inhibited by addition of rIL-4, strongly suggest that IL-2, IL-12, and IFN-alpha mediate their effects, leading to induction of CD69 antigen expression through different activation pathways.

Antigens, CD↗
PubMed1992

Revisiting the action of bovine ribonuclease A and pancreatic-type ribonucleases on double-stranded RNA.

Single-strand-preferring ribonucleases of the pancreatic type, structurally and/or catalytically similar to bovine RNase A but endowed with a higher protein basicity, are able to degrade double-stranded RNA (dsRNA) or DNA:RNA hybrids under standard assay conditions (0.15 M NaCl, 0.015 M sodium citrate, pH 7), where RNase A is inactive. This enzyme too, however, becomes quite active if assay conditions are slightly modified or its basicity is increased (polyspermine-RNase). In the attempt to review these facts, we have analyzed and discussed the role that in the process have the secondary structure of dsRNA as well as other variables whose influence has come to light in addition to that of the basicity of the enzyme protein, i.e., the ionic strength, the presence of carbohydrates on the RNase molecule, and the structure (monomeric or dimeric) of the enzyme. A possible mechanism by which dsRNAs are attacked by pancreatic-type RNases has been proposed.

Amino Acids↗
PubMed1992

Involvement of DNA polymerase beta in proliferation of rat liver induced by lead nitrate or partial hepatectomy.

We have studied the expression pattern of DNA polymerase beta in two different models of in vivo cell proliferation. Both mRNA levels and enzyme activity of DNA polymerase beta markedly increased before and/or during DNA synthesis in proliferating hepatocytes in mitogen-treated and partially hepatectomized rats. The time-courses of the expression of the gene coding for DNA polymerase beta were significantly different in the two cell systems. A 5-fold increase in DNA polymerase beta mRNA was observed 8 h after lead nitrate administration, i.e. well before the onset of DNA synthesis. In the regenerative liver cells a 3-fold increase in the amount of mRNA was observed 24-48 h after partial hepatectomy, the event being coincident with extensive DNA synthesis. In both systems, the increase of mRNA levels was always paralleled by an increase in enzyme activity, suggesting that DNA polymerase beta activity may be regulated at a pre-translational level.

Animals↗
PubMed1992

Regulation of poly(ADP-ribose) polymerase gene expression in mitogen-stimulated human peripheral blood mononuclear cells.

The level of mRNA for poly(ADP-ribose) polymerase in human PBMC increased 8 h after addition of PHA, reaching its maximum (9-fold over the basal level) 3-4 days after the stimulation and decreasing thereafter. mRNA maximum slightly preceded in time the maximal value of DNA synthesis. The half-life of poly(ADP-ribose) polymerase mRNA, which is 1.2 h in quiescent PBMC, increased up to 3.4 h in stimulated PBMC. This PHA-induced stabilization of the mRNA for poly(ADP-ribose) polymerase could account for the accumulation of the transcript in mitogen-treated PBMC.

Blotting, Northern↗
PubMed1991

Induction of DNA polymerase beta during proliferation of mitogen-stimulated human lymphocytes.

On induction of proliferation of human peripheral blood mononuclear cells by phytohemagglutinin treatment, DNA polymerase beta activity increases markedly before and during DNA replication. The increase of enzymatic activity seems to be well correlated with the increase of DNA polymerase beta mRNA, which is induced by enhanced expression of the DNA polymerase beta gene. These data suggest that DNA polymerase beta is involved in DNA repair, which is linked to replicative DNA synthesis, or directly in replicative DNA synthesis in normal proliferating cells.

Blotting, Northern↗
PubMed1989

Human seminal ribonuclease. Immunological quantitation of cross-reactive enzymes in serum, urine and seminal plasma.

The distribution of secretory-type ribonuclease in human serum, urine and seminal plasma has been studied by immunological measurements. Inhibition of enzyme activity by antibodies against pure human seminal RNAase shows that a cross-reactive enzyme is predominant (90%) in seminal plasma and is a significant component (70-80%) in urine and serum. A competitive binding radioimmunoassay has been developed by using specific antibodies and 125I-labelled RNAase as radioligand. The procedure, very sensitive, reproducible and specific, has been used to determine seminal RNAase levels in seminal plasma samples from 48 healthy individuals (age range, 20-58 years). The mean concentration of the enzyme was found to be 6.6 micrograms/ml (S.D. +/- 1.9).

Adult↗
PubMed1988

Differences in glycosylation pattern of human secretory ribonucleases.

The major secretory ribonuclease (RNase) of human urine (RNase HUA) was isolated and sequenced by automatic Edman degradation and analysis of peptides and glycopeptides. The isolated enzyme was shown to be free of other urine RNase activities by SDS/polyacrylamide-gel electrophoresis and activity staining. It is a glycoprotein 128 amino acids long, differing from human pancreatic RNase in the presence of an additional threonine residue at the C-terminus. It differs from the pancreatic enzyme in its glycosylation pattern as well, and contains about 45 sugar residues. Each of the three Asn-Xaa-Ser/Thr sequences (Asn-34, Asn-76, Asn-88) is glycosylated with a complex-type oligosaccharide chain. Glycosylation at Asn-88 has not been observed previously in mammalian secretory RNases. Preliminary sequence data on the major RNase of human seminal plasma have revealed no difference between it and the major urinary enzyme; their similarities include the presence of threonine at the C-terminus. The glycosylation pattern of human seminal RNase is very similar to that of the pancreatic enzyme. The structural differences between the secretory RNases from human pancreas, urine and seminal plasma must originate from organ-specific post-translational modifications of the one primary gene product. Detailed characterization of peptides and the results of gel filtration of tryptic and tryptic/chymotryptic digests of performic acid-oxidized RNase have been deposited as Supplementary Publication SUP 50146 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.

Amino Acid Sequence↗
PubMed1988

Structure of the bovine pancreatic ribonuclease gene: the unique intervening sequence in the 5' untranslated region contains a promoter-like element.

Although pancreatic ribonucleases are extensively studied proteins, little information is available on nucleic acids coding for these enzymes. Here, for the first time, the structure of a gene coding for such an enzyme, the well known bovine pancreatic ribonuclease, is reported. The coding region of this gene is devoid of introns, whereas the 5' untranslated sequence of the pancreatic transcript contains an intron of 735 nucleotides. This intervening sequence is endowed with signals (CAAT and TATA boxes) which might act as regulatory elements. The structural organization of this gene suggests that the sequence coding for the bovine pancreatic ribonuclease might be expressed under the control of two different promoters.

Amino Acid Sequence↗
PubMed1986

Cardiac dysrhythmias associated with ophthalmic atropine.

Atropine sulfate, a mydriatic and cycloplegic agent, is frequently used in patients undergoing glaucoma surgery. Trabeculectomy with peripheral iridectomy is the most common glaucoma surgery performed to decrease intraocular pressure and preserve vision. Systemic absorption of ophthalmic atropine does occur and may result in toxic and adverse side effects. Cardiac dysrhythmias are one of the major adverse reactions. This case study reviews three patients who had a trabeculectomy for glaucoma and received ophthalmic atropine. One patient received both systemic and ocular atropine. Two patients developed atrial fibrillation and one a supraventricular tachycardia. Two patients required admission to a cardiac intensive care unit for management of the dysrhythmia and a third reverted to normal sinus rhythm spontaneously. The cardiac effects of ophthalmic atropine should be considered in the preoperative and postoperative assessment of patients with dysrhythmias.

Absorption↗
PubMed1985

Sequence analysis of a cloned cDNA coding for bovine seminal ribonuclease.

The sequence of a cloned cDNA coding for bovine seminal ribonuclease, an enzyme secreted in the bull seminal vesicles, was determined. The cDNA starts at the amino acid residue 47 and terminates 12 nucleotides beyond the consensus sequence AAUAAA in the 3' non-coding region of the mRNA. Northern blotting analysis shows that the mRNA for bovine seminal ribonuclease consists of about 950 nucleotides, a value that is similar to that of other mRNAs coding for ribonucleases of the pancreatic type.

Amino Acid Sequence↗
PubMed1985

Human seminal ribonuclease. A tool to check the role of basic charges and glycosylation of a ribonuclease in the action of the enzyme on double-stranded RNA.

Human seminal ribonuclease (a basic protein occurring in a glycosylated and in a non-glycosylated form) is very active against double-stranded RNAs (De Prisco, R., Sorrentino, S., Leone, E. and Libonati, M. (1984) Biochim. Biophys. Acta 788, 356-363). The action of the two enzyme forms on single-stranded and double-stranded substrates was studied as a function of pH and ionic strength. Results indicate (1) that glycosylation of the RNAase molecule does not affect enzyme action on single-stranded RNAs, while (2) degradation of double-stranded RNAs is moderately increased by the presence of carbohydrates in the enzyme molecule. Human seminal RNAase shows a marked helix-destabilizing activity on poly(dA-dT) X poly(dA-dT). Under various conditions, this action (1) is definitely stronger than that of bovine RNAase A, and (2) seems to be less dependent on the glycosylation than on the basicity of the enzyme protein. The remarkable activity of human seminal RNAase on double-stranded RNA may, at least partly, be related to the enzyme properties mentioned above.

Glycoproteins↗
PubMed1984

A ribonuclease from human seminal plasma active on double-stranded RNA.

A ribonuclease, active on single- and double-stranded RNAs, has been isolated from human seminal plasma 3-5 micrograms of enzyme were recovered per ml of seminal plasma, equivalent to 71% of total activity and a 2500-fold purification (measured with poly(A) X poly(U) as substrate) from the initial dialyzed material. Similar amounts of RNAase were found per g (wet weight) of human prostate, where the enzyme appears to be produced. Human seminal RNAase degrades poly(U) 3-times faster than poly(A) X poly(U), and poly(C) or viral single-stranded RNA about 10-times faster than poly(U). Degradation of poly(A) X poly(U), viral double-stranded RNA, and poly(A) by human seminal RNAase is 500-, 380- and 140-times more efficient, respectively, than by bovine RNAase A. The enzyme, a basic protein with maximum absorbance at 276 nm, occurs in two almost equivalent forms, one of which is glycosylated. Mr values of the glycosylated and non-glycosylated form are 21000 and 16000, respectively. The amino-acid composition of the RNAase is very similar to that of human pancreatic RNAase. The same is true for the carbohydrate content of its glycosylated form.

Amino Acids↗
PubMed1983

Bovine seminal ribonuclease precursor synthesized in vitro.

Native bovine seminal ribonuclease is a dimeric protein, whose identical subunits (Mr 14500), linked through two disulfide bridges, can be dissociated by a selective reduction procedure. Evidence is presented that the synthesis in vitro, under reducing conditions, of bovine seminal RNAase, directed by polyadenylated RNA isolated from bull seminal vesicles (where the enzyme is synthesized in vivo), occurs in the form of a precursor, 18000-Da polypeptide. The precursor nature of this translation product was deduced by two criteria: (1) its specific immunoprecipitation with anti-bovine seminal RNAase antibodies; (2) its processing by dog pancreas microsomal membranes to produce a protein with a molecular weight similar to that of the subunit(s) of bovine seminal RNAase. Moreover, evidence is offered that the precursor polypeptide is able to form in vitro a dimeric molecule under conditions where no exogenous reducing agents were added.

Animals↗
PubMed1983

Influence of protein net charge on the nucleic acid helix-destabilizing activity of various pancreatic ribonucleases.

Helix-destabilization of double-stranded poly[d(A-T)]induced by various homologous pancreatic ribonucleases which differ in their net charges has been studied under different ionic strength conditions. The response of the destabilizing activity of the various proteins to ionic strength is represented by bell-shaped curves, whose maxima are shifted to higher ionic strength values the higher the number of positive charges of the RNAase involved in the nucleic acid-protein complex. This observation is discussed, and a model proposed, that could explain the experimental results presented.

Animals↗
PubMed1982

Dimerization of deoxyribonuclease I, lysozyme and papain. Effects of ionic strength on enzymic activity.

Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A., Furia, A., Doskocil, J., and Libonati, M. (1980) Biochim. Biophys. Acta. 609, 40-52]. To see whether this phenomenon could be common to other enzyme-substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease, hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg-white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength. Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross-linkage with dimethyl suberimidate. The main results of the investigation, similar to those obtained with ribonuclease A, are the following. 1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at given pH values: the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one. 2. The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme. A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non-specific interactions with polyelectrolytic substrates.

Animals↗
PubMed1981

Nucleic acid-protein interactions. Degradation of double-stranded RNA by glycosylated ribonucleases.

1. Extensively glycosylated ribonucleases, like the enzymes from pig and horse pancreas, show a much higher activity on double-stranded RNAs than similarly charged, carbohydrate-free RNAases under stranded assay conditions (relatively high salt concentrations). Glycosylated pig and horse pancreas RNAases also show a larger destabilizing effect on double-stranded poly[d(A-T)] X poly[d(A-T)], than that displayed by bovine RNAase A under these conditions. Both activities show a similar dependence on the ionic strength of the medium. 2. A partial enzymic removal of the heterosaccharide side chains from pig and horse RNAases reduces but their degradative activity on double-stranded RNA and their destabilizing action on poly[d(A-T)] X poly[d(A-T)]. 3. These results are tentatively correlated with a modification of the microenvironment of the enzyme protein caused by its extensive glycosylation.

Animals↗