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Biomedical subjects

G D'Alessio

Publications and source records attributed to G D'Alessio.

At least 55 records · Page 3Linked to original sources
PubMed1993

Expression of bovine seminal ribonuclease in Escherichia coli.

Bovine seminal RNAase (BS-RNAase), an unusually dimeric member of the pancreatic-like ribonuclease superfamily, is also a multifunctional biological effector, with antitumor, immunosuppressive, and antispermatogenic activities. We report here the cloning of a semi-synthetic cDNA coding for the protein subunit chain, its expression with a T7 expression system in Escherichia coli inclusion bodies, the dimerization of correctly reoxidized monomeric protein, followed by the purification in high yields of the recombinant enzyme, and by its conversion to a protein undistinguishable from BS-RNAase as isolated from seminal vesicles, both in its catalytic activity and in the micro-heterogeneity of its quaternary structure.

Animals↗
PubMed1993

Expression of native dimers of bovine seminal ribonuclease in a eukaryotic cell system.

Bovine seminal ribonuclease, a uniquely dimeric pancreatic-like RNase, with its dimeric structure stabilized by two intersubunit disulfides, and endowed with special, i.e. non-catalytic, actions (antitumor, immunosuppressive, antispermatogenic), was stably expressed in Chinese hamster ovary cells. The recombinant protein, secreted in the culture medium as a correctly folded dimeric enzyme, was purified to homogeneity and found to be fully active both in its catalytic and antitumor activities.

3T3 Cells↗
PubMed1993

Selective deamidation of ribonuclease A. Isolation and characterization of the resulting isoaspartyl and aspartyl derivatives.

Selective deamidation of proteins and peptides is a reaction of great interest, whether it has physiological significance as in protein aging, or occurs as a disturbing event in the preparation of natural or recombinant proteins. Deamidation of bovine pancreatic ribonuclease A, RNase A, a classical model protein, has been reported to occur only after denaturation of the protein, or under harsh conditions. In this paper convenient procedures are described for selective deamidation of Asn67 in native RNase A under mild conditions. Furthermore, for the first time, both products of deamidation were isolated: the aspartyl and the isoaspartyl containing protein derivatives. Replacement of Asn67 with either residue lowers the catalytic activity of the enzyme, on RNA and on model substrates, except when a dinucleotide with a purine on the 5' side is the substrate. In the latter case an intriguing increase in the specificity constant is observed. The Asp67 derivative was found to refold, after full denaturation and reduction, at the same rate as the fully amidated protein, whereas the iso-Asp67 derivative refolded at half that rate. It is hypothesized that this effect is due to a delayed formation of disulfide 65-72 for the presence of the abnormal isopeptide bond between residues 67 and 68.

Amino Acid Sequence↗
PubMed1993

New and cryptic biological messages from RNases.

RISBASEs are RNases with surprising biological actions, or unexpected physiological roles, in which they act as external factors that influence cell behaviour. There are RISBASEs involved in host defence, angiogenesis and the control of pollen fertility, and RISBASEs with antitumour and antispermatogenic actions. Here, Guiseppe D'Alessio describes these unusual roles for RNases, and speculates about the mechanisms underlying their actions.

Journal Article↗
PubMed1992

In vivo and in vitro growth-inhibitory effect of bovine seminal ribonuclease on a system of rat thyroid epithelial transformed cells and tumors.

We investigated the antitumoral effect of bovine seminal RNase (BS-RNase) in vivo and in vitro on a model system of epithelial tumor- and metastasis-derived cells as well as on epithelial tumors derived from the same system. We found that while BS-RNase significantly inhibited the growth in vitro of the epithelial tumor-derived cells, its inhibitory effect was even more dramatic on the growth of metastasis-derived cells. BS-RNase exerted no appreciable growth inhibition on normal thyroid epithelial cells. When administered in vivo to rats bearing solid carcinomas, having the same thyroid origin, BS-RNase induced a drastic reduction in the tumor weight, with no detectable toxic effects on the treated animals. These data show, for the first time on a system of neoplastically transformed epithelial cells, that BS-RNase has a potent specific antitumoral activity.

Animals↗
PubMed1992

The dual-mode quaternary structure of seminal RNase.

Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.

Animals↗
PubMed1991

Seminal RNase: a unique member of the ribonuclease superfamily.

The RNase found in bull semen, although a member of the mammalian superfamily of ribonucleases, possesses some unusual properties. Besides its unique structure and enzymic properties, it displays antispermatogenic, antitumor and immunosuppressive activities. Seminal RNase belongs to an interesting group of RNases, the RISBASES (RIbonucleases with Special, i.e. non catalytic, Biological Actions) other members of which include angiogenin, selectively neurotoxic RNases, a lectin and the self-incompatibility factors from a flowering plant.

Animals↗
PubMed1990

Immunosuppressive activity of bovine seminal RNase on T-cell proliferation.

The interest in the immunosuppressive activity of mammalian seminal plasma depends largely on its putative role in the immunoregulation of both the male and female genital systems. We report here that the immunosuppressive action of bovine seminal plasma is based on the presence in this fluid of copious amounts of an immunosuppressive RNase, bovine seminal RNase. Studies of structure-function relationships have revealed that the immunosuppressive activity of seminal RNase depends on the integrity of the dimeric structure of the enzyme, as well as on the integrity of its catalytic function. While bovine seminal RNase has no effect on the secretion of interleukin-2 by T-cell cultures, the enzyme has been found to decrease drastically the expression of the alpha-chain of the interleukin-2 receptor on the T-cell membrane.

CD3 Complex↗
PubMed1989

Dissociation and reconstitution of bovine seminal RNAase: construction of a hyperactive hybrid dimer.

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.

Amino Acids↗
PubMed1988

Co-operativity in seminal ribonuclease function. Kinetic studies.

Kinetic studies with substrates of the hydrolytic rate-limiting reaction step revealed that the non-hyperbolic kinetics of bovine seminal RNAse may not be ascribed to microheterogeneity of the enzyme or to hysteretic effects. The substrate saturation curves with intermediate plateau and the activating and inhibiting effects of the reaction product, respectively at low and high concentrations, are explained in terms of mixed co-operativity, with binding at subsites that is a prerequisite for full activity of the enzyme. A model is proposed that is supported also by the results of binding studies.

Animals↗
PubMed1988

Repair of isopeptide bonds by protein carboxyl O-methyltransferase: seminal ribonuclease as a model system.

Previous work has shown that in the peptide segment 62-76 of naturally deamidated alpha subunit of bovine seminal ribonuclease (BS-RNase) the alpha-carboxyl group of iso-Asp67 is selectively methylated by S-adenosylmethionine:protein carboxyl O-methyltransferase [Di Donato, A., Galletti, P., & D'Alessio, G. (1986) Biochemistry 25, 8361-8368]. In the present study this reaction has been characterized, by using the tryptic segment 62-76 of the protein chain (peptide alpha 16). The peptide is stoichiometrically methyl esterified with a Km of 6.17 microM and a Vmax of 19.56 nmol min-1 mg-1, and the product of demethylation has been identified as the cyclic succinimidyl derivative of iso-Asp67-Gly68. The cleavage of the succinimidyl ring yields two isomeric peptides containing an aspartyl residue (peptide alpha 17) and an isoaspartyl residue (peptide alpha 16). On the basis of these results conditions were defined in which repeated cycles of methylation-demethylation led to an effective conversion of peptide alpha 16 into peptide alpha 17, a process that can be interpreted as the repair of an altered isopeptide bond. When the methyl esterification reaction was studied on the native dimeric isoenzymes of seminal RNase and on catalytically active monomeric derivatives, including a stabilized alpha-type subunit, the results of these experiments showed that none of the protein forms were substrates for the methyltransferase. Only the unfolded alpha-type subunit was methylated to a stoichiometric extent. These results indicate that the repair of altered isopeptide bonds is chemically feasible in peptides but is hindered in the case of seminal RNase by its three-dimensional structure.

Amino Acid Sequence↗
PubMed1988

Expression of opioid genes in bovine seminal vesicles.

In seminal vesicles, the organ producing most of seminal plasma in the bovine species, the pro-opiomelanocortin and the proenkephalin genes are transcribed and translated, and their translation products processed into opioid peptides, which are secreted into the seminal plasma. By using a micro-organ preparation of seminal vesicles we found that, after 20 h of incubation with labelled methionine, a multiplicity of opioids was produced. Among these, [Met]enkephalin and beta-endorphin were positively identified, whereas in the newly formed secretion only [Met]enkephalin was detected. This may be correlated to the finding that the concentration of beta-endorphin in an extract of seminal plasma was one order of magnitude lower than that of [Leu]enkephalin and [Met]enkephalin. These findings expand the picture of the presence of opioid peptides in the male reproductive tract, indicating that they should have a role(s) in the physiology of reproduction, not only in the hypothalamus-pituitary-gonadal axis, determining the reproductive potential, but also in the so-termed sex accessory glands, determining the actual events leading to reproduction. To our knowledge this is also the first case studied of opioid peptides produced as exocrine hormones.

Animals↗
PubMed1987

Autocrine growth function of human interleukin 1 molecules on ROHA-9, an EBV-transformed human B cell line.

In this study we report that the ROHA-9 cell line, an IL 1-secreting EBV-transformed human B cell line, exhibits an autocrine pathway of growth. In fact, ROHA-9 cells spontaneously secreted an autoregulatory growth factor that co-purified with the constitutively secreted IL 1-like molecules. Accordingly, monocyte-derived human IL 1, free of other known biological activities, also stimulated the growth of ROHA-9 cells in a dose-dependent way. Human recombinant interleukin 2, recombinant IFN-alpha or IFN-gamma and purified IFN-beta were ineffective when used at concentrations up to 1 X 10(3) U/ml. Furthermore, mouse recombinant IL 1, HPLC-purified multi-colony stimulating factor and partially purified preparations of BCGF were ineffective when assayed for growth-promoting activity on ROHA-9 cells. Moreover, a rabbit polyclonal antibody and a mouse monoclonal antibody to human IL 1 molecules blocked the growth of ROHA-9 cells induced by the autologous growth factor and by human IL 1. Lastly, purified human IL 1 increased the clonal efficiency of ROHA-9 cells seeded at a low cell concentration, allowing the isolation of the ROHA-9MC3 subclone, which showed similar growth response specificity and was particularly sensitive to the mitogenic activity of human IL 1.

Animals↗
PubMed1987

Co-operativity in seminal ribonuclease function: binding studies.

Binding of nucleotides to bovine seminal RNAase was studied by differential spectrophotometry and equilibrium dialysis. Cytidine 3'-phosphate, the reaction product of the hydrolytic, rate-limiting step of the reaction, was found to be capable, in contrast to related nucleotides, of discriminating between the two structurally identical active sites of the enzyme. Negative co-operativity, with a 'half-of-sites' reactivity, was found at lower concentrations of ligand, whereas at higher concentrations positive co-operativity was detected. These findings exclude that the non-hyperbolic kinetics previously reported for the hydrolytic step of the reaction are due to hysteretic effect. A model of mixed-type co-operativity is proposed for interpreting the binding data.

Adenosine Monophosphate↗
PubMed1987

Synthesis of seminal ribonuclease in isolated lobules of bull seminal vesicles.

A procedure is described for preparing and maintaining in culture isolated lobules of bovine seminal vesicles, consisting of glandular acini, surrounded by little connective tissue and with free access to the external medium, in which secreted material can be collected. After 48 h in culture, the isolated lobules appeared indistinguishable, by morphological and biochemical criteria, from freshly isolated lobules. After much longer culture times about one third of the glandular cells were still capable of effective protein synthesis. Studying the biosynthesis of seminal ribonuclease with preparations of isolated lobules we found that the enzyme was synthesized and secreted; only the fully amidated isoenzyme was synthesized and secreted, indicating that production of the selectively deamidated isoenzymic forms occurred after secretion, newly synthesized protein was rapidly exported, indicating that the high levels of enzyme previously reported for the seminal vesicle tissue were essentially due to its content of stored secretion.

Animals↗
PubMed1986

Selective deamidation and enzymatic methylation of seminal ribonuclease.

Isoenzymatic forms alpha 2, alpha beta, and beta 2 of bovine seminal ribonuclease are generated by the transformation of beta-type into alpha-type subunit through deamidation of a single amide group [Di Donato, A., & D'Alessio, G. (1981) Biochemistry 20, 7232-7237]. The residue involved in this selective deamidation has been identified as Asn67. Deamidation occurs by formation of a cyclic imide intermediate involving the Gly at position 68. Opening of the cyclic imide may occur on either side of the nitrogen, generating both the normal alpha-aspartyl and an isoaspartyl residue at position 67. The alpha-carboxyl of the isoaspartyl residue is effectively methylated by bovine brain protein carboxylmethyltransferase.

Amino Acid Sequence↗
PubMed1986

Site-directed alkylation and site-site interactions in bovine seminal ribonuclease.

Active-site histidine residues of bovine seminal RNase have been found to react with bromoacetic acid and with 2'(3')-O-bromoacetyluridine (BrAcUrd) at a much faster rate than free histidine. The former reagent reacts preferentially at the pros-N of His119, the latter is specific for the tele-N of His12. Alkylation with bromoacetic acid is mutually exclusive for either His119 or His12 and takes place predominantly at His119, while with BrAcUrd alkylation was found to be selective for His12. These results are very similar to those obtained with the same reagents on RNase A, confirming that seminal and pancreatic ribonucleases have similar geometries at their active sites. On the other hand, the kinetics of reaction of bromoacetyluridine with seminal RNase reveal a 'half-of-the sites' reactivity of the enzyme for this reagent, which is found to discriminate between the two structurally identical active sites of the dimeric enzyme.

Acetates↗