Guided tissue regeneration of vascular grafts in the peritoneal cavity.
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Animals↗
Biomedical subjects
S Sorrentino
Publications and source records attributed to S Sorrentino.
Degradation of double-stranded RNA by mammalian pancreatic-type ribonucleases.
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Amino Acid Sequence↗
The two dimeric forms of RNase A.
In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868-3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X-ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437-3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A.
Animals↗
Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin.
A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.
Amino Acid Sequence↗
Role and mechanism of action of glial cell deficient/glial cell missing (glide/gcm), the fly glial promoting factor.
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Animals↗
New muteins of RNase A with enhanced antitumor action.
Monomeric bovine pancreatic RNase A has been transformed into a dimeric ribonuclease with antitumor activity (Di Donato, A., Cafaro, V. and D'Alessio, G. (1994) J. Biol. Chem. 269, 17394-17396). This was accomplished by replacing the residues located in the RNase chain at positions 19, 28, 31, and 32, with proline, leucine, and two cysteine residues, respectively, i.e. those present at identical positions in the subunit of bovine seminal RNase, a dimeric RNase of the pancreatic-type superfamily, endowed with a powerful antitumor action. However, as an antitumor agent this mutant dimeric RNase A is not as powerful as seminal RNase. We report here site-directed mutagenesis experiments which have led to the identification of two other amino acid residues, glycine 38 and 111, whose substitution in the polypeptide chain of the first generation dimeric mutant of RNase A, is capable of conferring to the mutein the full cytotoxic activity characteristic of native seminal RNase.
Animals↗
Human extracellular ribonucleases: multiplicity, molecular diversity and catalytic properties of the major RNase types.
Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase A superfamily, can be classified into four different RNase families on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and the information available on their catalytic properties has been analysed and discussed in comparison with those of other animal RNases. On the basis of some results obtained with various single- and double-stranded polyribonucleotides, it has been proposed that while pancreatic-type (pt) RNases could be defined as single-strand/pyrimidine 'preferring' ribonucleases, mammalian nonpancreatic-type (npt) RNases may be referred to as single-strand/pyrimidine 'specific' ribonucleases. In addition, some data concerning human nptRNases may support the suggestion [Cuchillo et al. (1993) FEBS Lett. 333: 207-210] that the enzyme 'ribonuclease' should be reclassified as 'transferase'.
Amino Acid Sequence↗
Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action.
Trimers of bovine pancreatic RNase A were obtained by cross-linking native RNase A with dimethyl suberimidate. They degrade double-stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4-I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross-linked dimers of RNase A appear to be ineffective.
Animals↗
Structure-function relationships in human ribonucleases: main distinctive features of the major RNase types.
Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase superfamily, can be classified into four different enzyme types on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and catalytic differences (action on single- and double-stranded RNAs, dependence of enzyme activity on pH, ionic strength and cations, and hydrolysis of cyclic nucleotides) have been comparatively analyzed and discussed. In addition, some data considered here concerning human nonpancreatic-type RNases may support the suggestion [Chuchillo et al. (1993) FEBS Lett. 333, 207-210] that the enzyme 'ribonuclease', presently classified as 'hydrolase', should be reclassified as 'transferase'.
Amino Acid Sequence↗
Single-strand-preferring RNases degrade double-stranded RNAs by destabilizing its secondary structure.
To establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residues, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U).poly(A), poly(C) and poly(C).poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I).poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U).poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U).poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U).poly(A) are discussed on the basis of their efficient estabilishing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly (U).poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U).poly(A) at physiological ionic strength.
Animals↗
The activity on double-stranded RNA of aggregates of ribonuclease A higher than dimers increases as a function of the size of the aggregates.
Stable bovine RNase A aggregates larger than dimers (identified as trimers, tetramers, pentamers and hexamers) were obtained by lyophilization of RNase A from 40-50% acetic acid solutions. The RNase activity of these aggregates was compared with that of monomeric RNase A on single- and double-stranded polyribonucleotides. Their activity toward poly(U) and yeast RNA slightly decreases as a function of the size of the aggregates. In contrast, their action on poly(A).poly(U) as substrate progressively increases from a relative activity of 1 for the RNase monomer to 10 for the hexamer. These results are discussed in the light of an already advanced hypothesis about a possible mechanism of RNase attack on double-stranded RNA.
Animals↗
Double-stranded RNA: the variables controlling its degradation by RNases.
The kinetics of single-stranded (SS) and double-stranded (ds) polyribonucleotides cleavage by three mammalian pancreatic type ribonucleases have been studied under low and high salt conditions. The values kcat, Km, and kcat/Km for depolymerization of poly(U), poly(A).poly(U), poly(I) and poly(I).poly(C) by bovine RNase A, bovine seminal RNase, and human seminal RNase have been determined and compared to each other. The Km values of bovine RNase A for (ss) or (ds) substrates were of the same order of magnitude under low and high ionic strength conditions, while their kcat values were found to differ considerably. Qualitatively similar results were obtained with bovine and human seminal RNases, i.e., the activity ratios (ssRNA/dsRNA) were mostly determined by the ratio of kcat values. It was shown that the modest levels of activity toward dsRNAs shown by single-strand-preferring RNases may occur by a mechanism consisting in the binding of the RNase to single nucleotides which are wound off the double helix because of thermal fluctuations. A higher activity and its enhancement as a function of number and location of the positive charges present on the RNase surface (human seminal RNase > bovine seminal RNase > bovine RNase A), as well as its increase under low ionic strength conditions, could instead be explained by the increased occurence of the splitting mechanism based on the binding of the RNase to single-stranded RNA sequences transiently exposed from the RNA double-helix.
Animals↗
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↗
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↗
Eosinophil-derived neurotoxin and human liver ribonuclease. Identity of structure and linkage of neurotoxicity to nuclease activity.
Eosinophil-derived neurotoxin (EDN) and human liver RNase were found to be indistinguishable from each other but distinct from the pancreatic ribonucleases in their nucleolytic activity on polynucleotides or small defined substrates. Antibodies to EDN and liver RNase showed identical cross-reactivities in assays of nuclease inhibition and in a radioimmunoassay. In each instance, EDN and liver RNase were easily distinguished from bovine or human pancreatic RNase. When injected intrathecally into rabbits, 5-10 micrograms of EDN or liver RNase each was neurotoxic as judged by induction of the Gordon phenomenon. Human pancreatic RNase was less neurotoxic, and up to 20-fold higher levels of bovine pancreatic RNase showed no effect. Treatment of EDN, liver RNase, and eosinophil cationic protein with iodoacetic acid at pH 5.5 resulted in inactivation of their RNase activity and also destroyed their neurotoxicity. EDN conformation was not greatly affected by iodoacetate treatment since interaction of the modified protein with antibodies was only slightly altered. We conclude that RNase activity is necessary but not sufficient to induce neurotoxic action.
Amino Acid Sequence↗
Left ventricular diastolic function in systemic sclerosis: assessment by radionuclide angiography.
The aim of this study was to assess left ventricular function in subjects with systemic sclerosis. Twenty-four women with systemic sclerosis (mean age 48 +/- 11 yr) and 14 age- and sex-matched normal subjects were studied by radionuclide angiography performed at rest with a temporal resolution of 20 msec/frame. Left ventricular volume curves were generated and indices of systolic and diastolic function were computed. Left ventricular diastolic asynchrony was evaluated by dividing the left ventricle into five regions and then computing the time-to-peak filling rate for each region. After excluding the valvular region, the coefficient of variation of this index was obtained. The isovolumic relaxation period was prolonged in systemic sclerosis patients in comparison to normal subjects (127 +/- 39 msec versus 87 +/- 44 msec, p less than 0.05). Moreover, 38% of the systemic sclerosis patients had a subnormal peak filling rate. Left ventricular diastolic asynchrony was increased in the systemic sclerosis group, as expressed by a higher coefficient of variation of the regional time to peak filling rate (27.9% +/- 11.5% versus 14.5% +/- 8.6%, p less than 0.05). Our results indicate an impaired relaxation and an increased diastolic asynchrony in patients with systemic sclerosis.
Female↗
Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain.
In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined.
Amino Acid Sequence↗
Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP).
The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN') and uridine and cytidine 2',3'-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the 'non-secretory' liver-type enzymes rather than to the 'secretory' pancreatic-type RNases.
Blood Proteins↗