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V Cafaro

Publications and source records attributed to V Cafaro.

11 recordsLinked to original sources
PubMed1998

A recombinant ribosome-inactivating protein from the plant Phytolacca dioica L. produced from a synthetic gene.

Phytolacca dioica L. leaves produce at least two type-I ribosome-inactivating proteins. Each polypeptide chain is subjected to different post-translational modifications giving rise to PD-L1 and PD-L2, and PD-L3 and PD-L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD-L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and preliminarily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome-inactivating activity was generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD-L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.

Base Sequence↗
PubMed1998

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↗
PubMed1998

Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping.

Four residues Pro19. Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins. studied by means of CD measurements. proved reversible and well represented by the two-state N<==>D transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.

Animals↗
PubMed1998

Protein engineering of ribonucleases.

Natural bovine seminal RNase possesses a potent antitumor action. We have mutagenized monomeric bovine pancreatic RNase A, devoid of any cytotoxic action, to insert residues present at corresponding positions in the subunit of dimeric, antitumor, seminal RNase. Like naturally dimeric seminal RNase, the mutant dimeric RNases display selective toxicity for malignant cells, which is absent in the monomeric mutants.

Amino Acid Substitution↗
PubMed1997

From ribonuclease A toward bovine seminal ribonuclease: a step by step thermodynamic analysis.

A proline, a leucine, and two cysteine residues, introduced at positions 19, 28, 31, and 32 of bovine pancreatic RNase A, i.e. the positions occupied by these residues in the subunit of bovine seminal RNase, the only dimeric RNase of the pancreatic-type superfamily, transform monomeric RNase A into a dimeric RNase, endowed with the same ability of BS-RNase of swapping its N-terminal segments. The thermodynamic consequences of the progressive introduction of these four residues into RNase A polypeptide chain have been studied by comparing the temperature- and urea-induced denaturation of three mutants of RNase A with that of a stable monomeric derivative of BS-RNase. The denaturation processes proved reversible for all proteins, and well represented by the two-state N<-->D transition model. The progressive introduction of the four residues into RNase A led to a gradual shift of the protein stability toward that characteristic of monomeric BS-RNase, which, in turn, is markedly less stable than RNase A with respect to both temperature- and urea-induced denaturation. On the other hand, the thermal stability of a dimeric active mutant of RNase A is found to approach that of wild-type seminal RNase.

Animals↗
PubMed1995

Full antitumor action of recombinant seminal ribonuclease depends on the removal of its N-terminal methionine.

Bovine seminal RNase (BS-RNase) is a dimeric member of the pancreatic-like ribonuclease superfamily, with antitumor activity. We report here that recombinant Met(-1) BS-RNase is a less potent cytotoxic factor, while structurally and catalytically indistinguishable from BS-RNase isolated from natural sources. Mature recombinant BS-RNase instead displays full antitumor action. This suggests that the conformation of the N-terminal region of BS-RNase is among the structural determinants of its antitumor action, in addition to its catalytic activity and its quaternary structure.

Amino Acid Sequence↗
PubMed1995

The antitumor action of seminal ribonuclease and its quaternary conformations.

It has been previously shown that the antitumor action of bovine seminal ribonuclease (BS-RNase) is dependent on its dimeric structure. However, two distinct quaternary structures, each in equilibrium with the other, have been described for the enzyme: one in which the two subunits exchange their N-terminal ends, the other with no exchange. Antitumor activity assays, carried out on homogeneous quaternary forms of the enzyme, as well as on dimeric mutants of bovine pancreatic RNase A, reveal that another structural determinant of the antitumor activity of BS-RNase is the exchange of N-terminal ends between subunits.

3T3 Cells↗
PubMed1995

Hints on the evolutionary design of a dimeric RNase with special bioactions.

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.

Animals↗
PubMed1994

Ribonuclease A can be transformed into a dimeric ribonuclease with antitumor activity.

A cDNA coding for bovine pancreatic RNase A was mutagenized to insert a proline, a leucine, and 2 cysteine residues, i.e. the residues present at corresponding positions in the subunit of seminal RNase, the only dimeric RNase of the pancreatic-type superfamily. The mutant, expressed in Escherichia coli, eventually aggregated into catalytically active dimers. Like naturally dimeric seminal RNase, at equilibrium the mutant dimeric RNase A adopted two quaternary structures (one with an exchange of the N-terminal segments between partner subunits, the other with no exchange) and displayed a selective toxicity for malignant cells, absent in the monomeric, parent protein.

3T3 Cells↗
PubMed1981

Abruptio placentae.

Analysis of blood samples from pregnant women has shown that the mean histamine level starts to rise when the plasma ascorbic acid level falls below 1.0 mg/100 ml; it is doubled when the ascorbate level falls to 0.5 mg/100 ml and quadrupled when it falls below 0.2 mg/100 ml. The incidence of abruptio placentae was found to be seven out of 355 (or 2.0%) in women who had plasma ascorbic acid levels above 0.4 mg/100 ml and six out of 31 (19.4%) in women with plasma ascorbate levels below 0.4 mg/100 ml. This difference is highly significant. It is suggested that ascorbic acid deficiency and histamine excess play leading roles in the etiology of abruptio placentae.

Abruptio Placentae↗