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R Donato

Publications and source records attributed to R Donato.

At least 109 records · Page 6Linked to original sources
PubMed1983

[Effect of proteins S100 on RNA and protein biosynthesis in neurons and glial cells of the rat brain].

The effects of brain-specific proteins S100 injected into lateral ventricles of rat brain on biosyntheses of RNA, proteins and endogenous proteins S100 in neurons and glial cells were investigated. It was found that exogenous proteins S100 essentially and irreversibly stimulate RNA and total protein syntheses in glial cells, while bovine serum albumin stimulates RNA and protein syntheses in neurons but not in glial cells. The dynamics of changes in the content of newly synthesized endogenous proteins S100 in glial cells and neurons are suggestive of transport of S100 protein molecules synthesized by glial cells, into the neurons. The absence of appreciable changes in the indicated parameters in rats injected with total rabbit sera proteins as well as in animals subjected to "false injections" provide evidence for the specificity of metabolic effects of proteins S100 and serum albumin.

Animals↗
PubMed1982

Immunocytochemical localization of S-100 protein binding sites in synaptosomal fractions and subfractions.

1. Immunocytochemical evidence is presented of the ultrastructural localization of binding sites for S-100 protein in synaptosomal fractions and subfractions. Synaptosomes or isolated synaptosomal subfractions were first incubated with S-100, then centrifuged to remove unbound S-100, and finally fixed before treatment with anti-S-100 antiserum, using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. 2. When intact synaptosomes were used, the immunoreaction product was localized to the postsynaptic density including the postsynaptic membrane. In some reactive synaptosomes, the presynaptic membrane was labeled as well, in the region of synaptic contact. No reaction deposit was found in the synaptic cleft or on intrasynaptosomal structures. Divalent cations (Ca2+ and Mg2+) were essential for S-100 to interact with synaptosomes. Of the synaptosomal subfractions tested, i.e., synaptic vesicles and intraterminal mitochondria, only synaptic vesicles showed immunoreactivity when treated with S-100 and anti-S-100 antiserum as described above. 3. The S-100 immunoreactivity in synaptic structures documented in this report parallels the distribution of the high-affinity binding sites for radiolabeled S-100 in synaptosomal fractions and subfractions.

Animals↗
PubMed1982

Heterogeneity of the S-100 protein specific binding sites in synaptosomal particulate fractions and subfractions.

The specific interaction of S-100 protein with synaptosomal particulate fractions (SYN) was further investigated with special reference to the number of binding components and their localization in synaptosomal subfractions. Binding studies were conducted on SYN from various CNS regions, on synaptosomal subfractions from the cerebral cortex, and on cerebral cortex SYN under various conditions. The results suggest that S-100 binds to two populations of membrane sites: high -affinity sites, which seem to be confined to neuronal membranes (synaptosomal plasma membranes and synaptic vesicles), and low-affinity sites, which are also detected in other membranes. The data are consistent with the view that the biphasic profile of S-100 binding to SYN does not result from heterogeneity of the S-100 molecule, and that the Ca2+ conformation of the protein is as important as the proper conformation of the binding site for full expression of high-affinity binding.

Animals↗
PubMed1982

The specific interaction of S-100 protein with synaptosomal particulate fractions. Modulation of binding by fractional occupancy of sites and by the physical state of membranes.

The nonlinearity of single components of the Scatchard plot of S-100 binding to synaptosomal particulate fractions (SYN) and the observation that dilution of the 125I-labeled S-100 site complex results in a greater extent of dissociation of the tracer in the presence than in the absence of an excess of unlabeled S-100 suggest that sites change their binding behavior depending on fractional occupancy. To study this aspect of the interaction in more detail, 125I-labeled S-100 binding experiments were conducted in the presence of, or after preincubation of SYN with various concentrations of, unlabeled S-100. The results indicate that: (a) S-100 synaptosomal sites do change their binding behavior depending on fractional occupancy; and (b) the nonrapid equilibrium between bound S-100 and the medium, which has been referred to as the formation of a tight complex between S-100 and its binding sites, is related to the activation of high-affinity sites. However, no univocal interpretation of these data in terms of binding model can be offered at present, as the binding models currently employed in the analysis of ligand-site interactions can each account for only part of the results described in this report. In any case, data obtained by studying 125I-labeled S-100 binding to untreated SYN at 2 degrees C and to prefixed SYN at 37 degrees C indicate that the physical state of membranes influences both the extent of the interaction and the binding behavior of the sites.

Animals↗
PubMed1981

Specific binding sites for S-100 protein in isolated brain nuclei.

Isolated brain nuclei possess binding sites for S-100 protein. The interaction of S-100 with these sites is specific and time-, temperature-, and Ca+ -dependent. The profile of the (125)I-labelled S-100 binding inhibition is biphasic, displaying a high-affinity component and a low-affinity component. The S-100 binding to brain nuclei is largely irreversible, probably owing to the formation of a tight complex between the protein and its nuclear binding sites. The S-100 binding to brain nuclei is in most aspects similar to that to synaptosomal membranes. Several lines of evidence indicate, however, that the S-100 binding to nuclei is not due to contamination of these structures with plasma membranes. Isolated liver nuclei do not possess the high-affinity component of S-100 binding.

Animals↗
PubMed1981

Subnuclear distribution of the S-100 protein specific binding sites in rat brain.

Fractionation of isolated brain nuclei previously reacted with (125)I-labelled S-100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of (125)I-labelled S-100 into the nucleus, was observed at 37 percent C, but not at 0-4 percent C. When tested separately for (125)I-labelled S-100 specific binding, both the nuclear membranes and the nucleoli were found to bind (125)I-labelled S-100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.

Animals↗
PubMed1981

The specific interaction of S-100 protein with synaptosomal particulate fractions. Evidence for the formation of a tight complex between S-100 and its binding sites.

The dissociation of the 125I-labelled S-100 specifically bound to synaptosomal particulate fractions (SYN) has been studied under a variety of conditions after different association times. The results indicate that after a critical association time of about 20 min at 37 degrees C, the bound protein becomes progressively less accessible to the dissociating agents of conditions employed. These findings support the view that the partial irreversibility of the 125I-labelled S-100 binding to SYN could be due to the formation of a tight complex between the protein and its synaptosomal sites. These data are discussed mainly in relation to the particulate-bound fraction of native S-100.

Animals↗
PubMed1975

Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor.

Within cerebral cortex synaptosomes, S-100 protein can be recovered in two forms: soluble and membrane-bound. Synaptosomal S-100 is mainly a soluble protein (85 percent). The membrane-bound S-100 is differently distributed in the synaptosomal membranes, intraterminal mitochondria, and synaptic vesicles. S-100 binds to a specific receptor. The binding is time-dependent, reversible and saturable with respect to S-100. The number of receptors is calculated to be about 9 times 10(12)/mg protein, since saturation is achieved at 31 ng [125I]S-100/0.1 mg protein of disrupted synaptosomes. The rate constant for association of S-100 with its receptor at 37 degrees C, k1, is 4.74 times 10(4) M(-1) sec(-1), and the rate constant for dissociation, k-1, 9.24 times 10(-4) sec(-1).

Animals↗
PubMedDate not supplied

Cardiac S-100a0 protein: purification by a simple procedure and related immunocytochemical and immunochemical studies.

A simple procedure is described for the purification of the alpha alpha isoform of S-100 proteins (S-100a0) from porcine heart. Purification steps include the following: i) extraction of the tissue with a hypotonic medium containing EDTA; ii) ammonium sulfate fractionation (0-50%) of the extract; iii) Ca2+-dependent affinity chromatography of the supernatant obtained through the preceding step on phenyl-sepharose and elution of absorbed proteins through a two-chamber gradient of 1.0-0.0 mM CaCl2 and 0.0--1.0 mM EGTA, respectively; and iv) chromatography of the resultant S-100-containing fractions on Sephadex G-200. The yield is 20 mg S-100a0/kg porcine heart. The whole procedure takes five days and is highly reproducible. Data obtained from the phenyl-sepharose step suggest that the affinity of Ca2+ for S-100a0 increases by several orders of magnitude once the protein had interacted with that matrix. This observation is discussed in relation to the role of S-100 proteins in amplification of the Ca2+ signal. Immunocytochemical and immunoblotting analyses indicate that S-100a0 is exclusively found at the level of the sarcolemmal membranes, the membranes of the sarcoplasmic reticulum, the external mitochondrial membranes, and in the adjacent sarcoplasm. No evidence of S-100a0 being associated with the nuclei or with myofibrils has been obtained. Finally, the cardiac tissue does not contain the Triton X-100-extractable fraction of S-100 normally detected in the brain and in adipocytes. Our data suggest that S-100a0 behaves as a peripheral membrane protein in cardiac tissue.

Animals↗
PubMedDate not supplied

[Evaluation of the Yersinia enterocolitica pathogenicity through some phenotypic and genotypic characters: CRMOX agar positivity and presence of the ail gene].

Sixty-nine strains of Y. enterocolitica isolated from environmental and human matrices (waste water, food and faeces) were studied in order to evidence the presence of ail gene, calcium-dependency and Congo Red absorption for pathogenic strains identification. Out of 24 clinical strains, the ail gene was present in 21 (87%), among which 79% were CRMOX-positive as well. On the contrary, none of the 45 environmental strains showed the ail gene although only one (isolated from cooked vegetables) was CRMOX agar positive. Our results confirmed the importance of molecular methods to evidence the Y. enterocolitica pathogenic strains. However, our study pointed also the utility to consider the approach of classic bacteriology, like the subcoltivation on CRMOX agar to show calcium-dependency and Congo Red absorption. In particular, when dealing with environmental isolates, that medium will be useful as a preliminary screening to identify those isolates which need further research to indicate their pathogenic potential by the use of more complex but also more expensive molecular methods.

Agar↗