A new case for a presynaptic role of dendrites: An immunocytochemical study of the N. Raphé Dorsalis

The distribution of serotonin (5-HT) was determined by the application of the prembedding peroxidase-anti-peroxidase (PAP) technique in vibratome and ultrathin sections of the brain stem. The antiserum stained the neuronal groups B₁ to B₉. Somata, dendrites and axons of multipolar and bipolar neurons were recognized in the usual locations. The most commonly found profiles in the area of the n.raphe dorsalis were dendrites. The search for axon terminals was unsuccesful. The labeled dendrites appear in synaptic contact with unlabeled endings containing round or pleomorphic vesicles, and occasionally some large dense core vesicles. Contacts between two labeled dendrites or processes were not found. Occasionally a dendrodendritic junction between a 5-HT labeled dendrite and an unlabeled dendrite has been found. There are areas of the dendritic membrane free of synaptic junctions and free of glial insulation. Results are discussed in relation with the previously proposed presynaptic role of the dendrites in the neuronal circuitry of then. raphé dorsalis.Special Issue dedicated to Prof. Eduardo De Robertis.Research supported by grants from the CONICET and SECYT, Argentina.     

Antioxidant Properties of Sulfinates: Protective Effect of Hypotaurine on Peroxynitrite-Dependent Damage

It has been proposed that hypotaurine may function as an antioxidant in vivo. We investigated whether this compound can act as protective agent able to prevent damage from peroxynitrite, a strong oxidizing and nitrating agent that reacts with several biomolecules. The results showed that the compound efficiently protects tyrosine against nitration, alpha1-antiproteinase against inactivation, and human low-density lipoprotein against modification by peroxynitrite. Hypotaurine is also highly effective in inhibiting peroxynitrite-mediated nitration of tyrosine in the presence of added bicarbonate. This result suggests that hypotaurine could play an important role as protective agent under physiological conditions. Moreover, it was found that cysteine sulfinic acid, but not taurine, possesses protective properties against peroxynitrite-dependent damage similar to hypotaurine. These findings indicate that the protective effects exerted by these compounds may be attributable to the presence of the sulfinic group oxidizable into sulfonate by scavenging peroxynitrite and/or its derived species.     

Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine

In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1O2) we found that azide, usually used as 1O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661-665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed.     

Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes

The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3' end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.     

Reversible cyclization ofS-(2-oxo-2-carboxyethyl)-L-homocysteine to cystathionine ketimine

Summary S-(2-oxo-2-carboxyethyl)homocysteine (OCEHC), produced by the enzymatic monodeamination of cystathionine, is known to cyclize producing the seven membered ring of cystathionine ketimine (CK) which has been recognized as a cystathionine metabolite in mammals. Studies have been undertaken in order to find the best conditions of cyclization of synthetic OCEHC to CK and for the preparation of solid CK salt product. It has been found that ring closure takes place at alkaline pH and is highly accelerated in 0.5 M phosphate buffer. The sodium salt of CK has been prepared by controlled additions of NaOH to water-ethanol solution of OCEHC under N₂ atmosphere. A solid product is obtained which, dissolved in water, shows the spectral features of CK. Solutions of the sodium salt of CK show the presence of a pH depending reversible equilibrium with the open OCEHC form. Plot of the absorbance at 296 nm in function of pH indicates that at pH 9 the compound is completely cyclized while at pH 6 is totally in the open OCEHC form. At intermediate pHs variable ratios between the two forms occur. According to the results obtained by the spectral analysis, HPLC assays of the sodium salt of CK show different patterns depending on the pH of the elution buffer.Abbreviations CK cystathionine ketimine - OCEHC S-(2-oxo-2-carboxyethyl) homocysteine - HPLC high performance liquid chromatography     

The oxidation of aminoethylcysteine ketimine dimer by oxygen reactive species

Summary The prominent spontaneous reaction of aminoethylcysteine ketimine in the neutral pH range is the concentration-dependent dimerization (Hermann, 1961). The carboxylated dimer first produced loses the free carboxyl yielding the more stable decarboxylated dimer (named simply the dimer in this note). In the search for a possible biochemical activity of this uncommon tricyclic compound we have assayed whether it could interact with oxygen reactive species (H₂O₂, O₂ ^–,OH) thus exhibiting a scavenging effect of possible biomedical interest. The dimer interacts with H₂O₂ producing compounds detectable by chromatographic procedures. The presence of Fe²⁺ stimulates the oxidative reaction by yielding the hydroxyl radical (the Fenton reaction). Using the system xanthine oxidase-xanthine as superoxide producer, the dimer oxidation by O₂ ^– has also been documented. Among the oxidation products the presence of taurine and cysteic acid has been established. Identification of remaining oxidation products and investigation of the possible function of the dimer as a biological scavenger of oxygen reactive species are now oncoming.Abbreviations HPLC high performance liquid chromatography - AAÅ amino acid analyzer - SOD superoxide dismutase - EDTA ethylenediaminetetraacetic acid     

Retinal alterations induced by continuous light in immature rats

Summary Immature albino rats were subjected to (a) continuous illumination for 5–9 days, or (b) continuous illumination followed by prolonged darkness. Their electroretinographic responses and the ultrastructural characteristics of the rod outer segments, as revealed by a mixture of zinc iodine-osmium tetroxide (ZIO) at different temperatures, were studied and compared with those of a control group maintained in a cyclic rhythm of light and darkness.Noteworthy differences in the distribution of ZIO reactive sites were observed in the rats exposed for 5–9 days to continuous illumination (no electroretinographic responses) as compared with normal controls. At 4°C, ZIO staining was negative in the rods of illuminated rats whereas at 20 and 60°C it was positive inside the tubular and vesicular structures.After prolonged darkness, in rats with a partial electroretinographic recovery and ultrastructural restoration, the ZIO reaction showed a similar pattern to that observed in the control group, ZIO deposits being found both in the intra and extradiscal spaces.Supported by Grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and the National Institutes of Health (51 NS 06953-09 NEUA), U.S.A., and by the Universidad Nacional de Buenos Aires and Fight for Sight Inc., N.Y., USA.We are indebted to Miss Margarita López for her skilful technical assistance and to Mr. Alberto Saenz for the electron micrographs.