Maria Grazia Rambotti

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves.

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.

S100B and S100A1 proteins in bovine retina: their calcium-dependent stimulation of a membrane-bound guanylate cyclase activity as investigated by ultracytochemistry

The Ca2(+)-binding proteins of the EF-hand type, S100B and S100A1, were detected in the outer segment of bovine retina photoreceptors where they are localized to disc membranes, as investigated by immunofluorescence and immunogold cytochemistry. S100B and S100A1 stimulate a membrane-bound guanylate cyclase activity associated with photoreceptor disc membranes in dark-adapted retina in a Ca2(+)-dependent manner, although with different Ca2+ requirements, as investigated by an ultracytochemical approach. Other retinal cell types express S100B and S100A1 as well. S100B is detected in the outer limiting membrane, fine cell processes in the outer nuclear layer and the outer plexiform layer, cell bodies in the inner nuclear layer and the ganglion cell layer, and the inner limiting membrane, whereas S100A1 has a more discrete distribution. S100B and S100A1 also stimulate a membrane-bound guanylate cyclase activity in photoreceptor cell bodies and Muller cells, but their effect appears independent of the light- or dark-adapted state of the retina and is observed at relatively high Ca2+ concentrations. These data represent the ultrastructural counterpart of recent biochemical observations implicating S100B and, possibly, S100A1 in the Ca2(+)-dependent stimulation of a photoreceptor membrane-bound guanylate cyclase activity [T. Duda, R. M. Goraczniak and R. K. Sharma (1996) Molecular characterization of S100A1-S1000B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclast. Biochemistry 35, 6263-6266; A. Margulis, N. Pozdnyakov and A. Sitaramayya (1996) Activation of bovine photoreceptor guanylate cyclast by S100 proteins. Biochem. Biophys. Res. Commun. 218, 243-247]. Our data suggest that at least S100B may take part in the regulation of a membrane-bound guanylate cyclase-based signalling pathway in both photoreceptors and Muller cells.

Detection of guanylate cyclases A and B stimulated by natriuretic peptides in gastrointestinal tract of rat

SummaryThe ultracytochemical localization of membrane-bound guanylate cyclases A and B has been studied after stimulation with atrial natriuretic peptide, C-type natriuretic peptide and brain natriuretic peptide in the gastrointestinal tract of rat. The two isoforms are stimulated differently by the three peptides. The results showed that the atrial and C-type natriuretic peptides stimulated guanylate cyclase activity, whereas the brain peptide seemed not to activate enough of the enzyme to detect. The guanylate cyclase activity had a wider distribution in stomach and small intestine than in large intestine; nevertheless, the reaction product of guanylate cyclase A activity had a wider localization in the stomach, whereas the reaction product of guanylate cyclase B activity had a wider distribution in the small intestine. In the small and large intestine, we detected mostly similar localizations of guanylate cyclase activity irrespective of the peptide used; in the stomach the reaction products of guanylate cyclase A and B were detected in different cell types or in different sites of the same cell. In all the gastrointestinal tract, guanylate cyclase activity was detected mainly in three types of cells: exocrine and endocrine cells; undifferentiated and mature epithelial cells; and smooth muscle cells. These localizations of guanylate cyclase activity suggest its role in regulating glandular secretion, cellular proliferation and muscular activity.

Immunocytochemical localization of S-100 beta beta protein in olfactory and supporting cells of lamb olfactory epithelium.

By immunocytochemistry, we have identified two novel cell types, olfactory and supporting cells of lamb olfactory epithelium, expressing S-100 beta beta protein. S-100 immune reaction product was observed on ciliary and plasma membranes, on axonemes and in the cytoplasm adjacent to plasma membranes and to basal bodies of olfactory vesicles. A brief treatment of olfactory mucosae with Triton X-100 before fixation is necessary for detection of S-100 beta beta protein within olfactory vesicles. In the absence of such a treatment, the immune reaction product is restricted to ciliary and plasma membranes. On the other hand, irrespective of pre-treatment of olfactory mucosae, S-100 beta immune reaction product in supporting cells is restricted to microvillar and plasma membranes. The anti-S-100 beta antiserum used in these studies does not bind to basal cells of the olfactory epithelium or to cells of the olfactory glands, whereas it binds to Schwann cells of the olfactory nerve. An anti-S-100 alpha antiserum does not bind to cellular elements of the olfactory mucosa, Schwann cells, or axons of the olfactory nerve. The present data provide, for the first time, evidence for the presence of S-100 beta beta protein in mammalian neurons (olfactory cells).

Differential involvement of reactive oxygen species and nucleoside transporters in cytotoxicity induced by two adenosine analogues in human prostate cancer cells.

Elevated levels of cellular oxidative stress represent a specific vulnerability of malignant cells and exposure to cytotoxic drugs is known to induce oxidative stress in cancer cells. The effects of two adenosine analogues, 2‐chloroadenosine and 2‐chlorodeoxyadenosine, were investigated to assess their mechanism of action in prostate cancer cells.

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells.

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.

Novel Localization of Low Affinity NGF Receptor (p75) in the Stroma of Prostate Cancer and Possible Implication in Neoplastic Invasion: An Immunohistochemical and Ultracytochemical Study

The localization of low affinity nerve growth factor receptor (p75) in prostate carcinogenesis is still unclear. Our aim was to reinvestigate the localization of p75 in normal and pathological prostate and to check a possible correlation to neoplastic grading.

Atrial Natriuretic Peptide and Guanylin-activated Guanylate Cyclase Isoforms in Human Sweat Glands

The ultracytochemical localization of membrane-bound guanylate cyclases A and C, stimulated by atrial natriuretic peptide and guanylin respectively, has been studied in human sweat glands. The results showed that the peptides stimulated guanylate cyclases A and C in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was present on the plasma membranes and on intracellular membranes involved in the secretory mechanism. In eccrine glands, the cells of the excretory duct also presented enzymatic activity on the plasma membranes. In both glands, myoepithelial cells, surrounding the secretory cells, exhibited only guanylate cyclase A activity. These localizations of enzymatic activity suggest a role for both atrial natriuretic peptide and guanylin in regulating glandular secretion.

A Novel Role for Tm7sf2 Gene in Regulating TNFα Expression

We have explored the role of Tm7sf2 gene, which codifies for 3β-hydroxysterol Δ14-reductase, an endoplasmic reticulum resident protein, in the sensitivity to endoplasmic reticulum stress and in the resulting inflammatory response. We used mouse embryonic fibroblasts, derived from Tm7sf2+/+ and Tm7sf2−/− mice, to determine the in vitro effects of thapsigargin on NF-κB activation. Our results show that the Tm7sf2 gene controls the launch of the unfolded protein response and presides an anti-inflammatory loop thus its absence correlates with NF-κB activation and TNFα up-regulation. Our data also show that Tm7sf2 gene regulates liver X receptor activation and its absence inhibits LXR signalling. By expressing the hTm7sf2 gene in KO MEFs and observing a reduced NF-κB activation, we have confirmed that Tm7sf2 gene is linked to NF-κB activation. Finally we used genetically modified mice in an in vivo model of ER stress and of inflammation. Our results show a significant increase in renal TNFα expression after tunicamycin exposure and in the oedematogenic response in Tm7sf2−/− mice. In conclusion, we have shown that the Tm7sf2 gene, to date involved only in cholesterol biosynthesis, also controls an anti-inflammatory loop thereby confirming the existence of cross talk between metabolic pathways and inflammatory response.

Detection of particulate guanylate cyclase in rat neurohypophysis after stimulation with ANF and BNP : an ultracytochemical study

We investigated the ultracytochemical localization of particulate guanylate cyclase (GC) in the rat neurohypophysis after activation with rat atrial natriuretic factor (rANF) or porcine brain natriuretic peptide (pBNP). Under our experimental conditions, the presence of GC reaction product indicated that rANF and pBNP were strong activators of particulate GC since samples incubated in basal conditions without rANF or pBNP did not reveal any GC reaction product. The rANF-stimulated GC was localized both to pituicytes and to nerve fibers and endings whereas the pBNP-stimulated GC was present exclusively in nerve fibers and endings. Recently, two subtypes of receptors for natriuretic peptides have been identified as two isoforms of particulate GC [24,50]. Our data indicate that the receptors of the two hormones have a partially distinct distribution in the rat neurohypophysis. In pituicytes, GC reaction product was found on plasma membrane of finger-like processes and on the membranes surrounding the lipid droplets. In nerve fibers and endings, GC reaction product was associated with intracellular membranes. This finding suggests that the enzyme could mediate an internal inhibitory action of these hormones on the release of vasopressin and oxytocin.