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To assess and compare sensory-motor status and clinical features of type I and type II acute acquired concomitant esotropia (group A) and decompensated monofixation syndrome (group B).In a retrospective, comparative study, 26 patients with a confirmed postoperative diagnosis of type I and II acute acquired concomitant esotropia and monofixation syndrome were enrolled. The 2-tailed unpaired t test and 2-tailed χ(2) test were performed to compare angle of deviation and sensory-motor status under the environmental conditions and after prismatic adaptation test and progressive prism test of 2 groups.All of the patients of group A and 4 patients (33%) of group B experienced diplopia under viewing conditions in the Worth's 4 dot and Bagolini striated glass tests. The TNO stereo test showed the total absence of stereopsis in 6 patients in group B and a significantly lower stereoacuity in group A in the remaining 6 patients (p0.0001). The prismatic adaptation test was positive in all of the patients in group B and in 10 patients (71%) in group A (p = 0.39). The value of the angle of deviation after the progressive prism test was significantly higher in group B than in group A (p = 0.02). At the end of the progressive prism test, all of the patients in group A and only 2 patients in group B were orthotropic (p = 0.01).Bagolini striated glass and Worth's 4 dot tests under viewing conditions and responses under prisms allow for the differentiation of the 2 forms and lead to an accurate aesthetic and functional prognosis.

Cardiovascular magnetic resonance (CMR) is a useful noninvasive technique for determining the presence of microvascular obstruction (MVO) and intramyocardial hemorrhage (IMH), frequently occurring in patients after reperfused myocardial infarction (MI). MVO, or the so-called no-reflow phenomenon, is associated with adverse ventricular remodeling and a poor prognosis during follow-up. Similarly, IMH is considered a severe damage after revascularization by percutaneous primary coronary intervention (PPCI) or fibrinolysis, which represents a worse prognosis. However, the pathophysiology of IMH is not fully understood and imaging modalities might help to better understand that phenomenon. While, during the past decade, several studies examined the distribution patterns of late gadolinium enhancement with different CMR sequences, the standardized CMR protocol for assessment of IMH is not yet well established. The aim of this review is to evaluate the available literature on this issue, with particular regard to CMR sequences. New techniques, such as positron emission tomography/magnetic resonance imaging (PET/MRI), could be useful tools to explore molecular mechanisms of the myocardial infarction healing process.

Dendritic cells (DC) sense saprophytic yeast and pathogenic, filamentous forms of Candida albicans in a specific way, resulting in disparate patterns of DC and T(h) cell activation. Using human and murine DC, such disparate patterns could be traced to the exploitation of distinct recognition receptors. Although usage of mannose receptors led to protective type 1 responses in mice, entry through Fcgamma receptors was responsible for suppression of mannose receptor-dependent reactivity, onset of type 2 responses and associated pathology. As the usage of distinct receptors selectively occurred with yeast or hyphal forms of the fungus, these findings suggest that the responsibility for pathogenicity of C. albicans is shared by the organism and DC, with implications for fungal virulence, immunity and vaccine development.

To find out whether polymorphonuclear neutrophils (PMN), abundantly recruited in disseminated Candida albicans infection, could directly affect the activation of Th cells we addressed the issues as to whether murine PMN, like their human counterparts, express costimulatory molecules and the functional consequence of this expression in terms of antifungal immune resistance. To this purpose, we assessed 1) the expression of CD80 (B7-1) and CD86 (B7-2) molecules on peripheral, splenic, and inflammatory murine Gr-1+ PMN; 2) its modulation upon interaction with C. albicans in vitro, in vivo, and in human PMN; 3) the effect of Candida exposure on the ability of murine PMN to affect CD4+ Th1 cell proliferation and cytokine production; and 4) the mechanism responsible for this effect. Murine PMN constitutively expressed CD80 molecules on both the surface and intracellularly; however, in both murine and human PMN, CD80 expression was differentially modulated upon interaction with Candida yeasts or hyphae in vitro as well as in infected mice. The expression of the CD86 molecule was neither constitutive nor inducible upon exposure to the fungus. In vitro, Gr-1+ PMN were found to inhibit the activation of IFN-γ-producing CD4+ T cells and to induce apoptosis through a CD80/CD28-dependent mechanism. A population of CD80+Gr-1+ myeloid cells was found to be expanded in conventional as well as in bone marrow-transplanted mice with disseminated candidiasis, but its depletion increased the IFN-γ-mediated antifungal resistance. These data indicate that alternatively activated PMN expressing CD80 may adversely affect Th1-dependent resistance in fungal infections.

Aspergilli are respiratory pathogens and pulmonary infections are usually acquired through the inhalation of conidia, able to reach small airways and the alveolar space where the impaired host defense mechanisms allow hyphal germination and subsequent tissue invasion. The invasive pulmonary aspergillosis is the most common manifestation of Aspergillus fumigatus infection in immunocompromised patients and is characterized by hyphal invasion and destruction of pulmonary tissue. A Th1/Th2 dysregulation and a switch to a Th2 immune response may contribute to the development and unfavorable outcome of invasive pulmonary aspergillosis. Dendritic cells (DC) have a primary role in surveillance for pathogens at the mucosal surfaces and are recognized as the initiators of immune responses to them. In the present study, we assessed the functional activity of pulmonary DC in response to A. fumigatus conidia and hyphae, both in vitro and in vivo. We analyzed mechanisms and receptors for phagocytosis by DC as well as DC migration, maturation, and Th priming in vivo upon exposure to either form of the fungus. We found a remarkable functional plasticity of DC in response to the different forms of the fungus, as pulmonary DC were able to: 1) internalize conidia and hyphae of A. fumigatus through distinct phagocytic mechanisms and recognition receptors; 2) discriminate between the different forms in terms of cytokine production; 3) undergo functional maturation upon migration to the draining lymph nodes and spleens; and 4) instruct local and peripheral Th cell reactivity to the fungus.

Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg2+ and Mn2+, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg2+ or Mn2+, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg2+ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum–ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn2+ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg2+ and ATP atbreak pH 8 and 7.4.

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.

The ultracytochemical localization of guanylate cyclases A and B activity has been studied after stimulation with atrial natriuretic peptide and C-type natriuretic peptide in light- and dark-adapted retinas and pigmented epithelium. The results showed that both peptides stimulated guanylate cyclases A and B activity in light-adapted retinas only. Guanylate cyclases A and B activity was detected on plasmamembrane of body of photoreceptors, bipolar, horizontal and ganglion cells, on plasma membranes of interneuronal connections at plexiform layers and on the plasma membrane of fibres at the nerve fibres layer. Independently of the light- or dark-adapted state, the pigmented epithelium also presented guanylate cyclases A and B activity on basal and lateral plasma membranes.

The 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.

We studied the activity and the ultracytochemical localization of membrane-bound guanylate cyclase (GC) after stimulation with rat atrial natriuretic peptide (rANP), porcine brain natriuretic peptide (pBNP), rat brain natriuretic peptide (rBNP), or porcine C-type natriuretic peptide (CNP) in rat C6 glioma cells during proliferation or following exposure of confluent cells to dibutyryl cyclic AMP (db-cAMP) or retinoic acid (RA). Under our experimental conditions all peptides were activators of GC as demonstrated by the accumulation of cGMP within cells. During proliferation of C6 cells, the amounts of cGMP remained approximately constant. However, at subconfluency, confluency and postconfluency, the GC reaction product was located at different sites in C6 cells. At subconfluency, GC reaction product was on membranes of protoplasmic extensions, at postconfluency, GC reaction product was in association with membranes of cell bodies, and at confluency, both localizations of GC reaction product were detected. Incubation of confluent cells in culture medium containing db-cAMP or RA induced the appearance of long and slender protoplasmic extensions. Under these conditions, the GC reaction product was localized exclusively to these processes. These data suggest that GC is differently located depending on the state of growth of glial cells, and that in differentiating glial cells GC is preferentially located in cell processes.

S-100 protein and annexin II 2 -p11 2 were reported to inhibit and to stimulate the assembly of glial fibrillary acidic protein (GFAP), respectively, in a Ca 2+ -dependent manner. Here we show by a number of experimental approaches that S-100 protein contrasts all the effects of annexin II 2 -p11 2 on GFAP assembly and, conversely, that annexin II 2 -p11 2 contrasts the inhibitory effects of S-100 protein on GFAP assembly, in a dose-dependent manner in both cases. Altogether, these data suggest that two specific Ca 2+ effecters, i.e., annexin II 2 -p11 2 and S-100 protein, might regulate the state of assembly of glial filaments in a concerted manner.

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.

The cytochemical localization of particulate guanylate cyclase and adenylate cyclase activities in rabbit platelets were studied after stimulation with various agents, at the electron microscope level. In the presence of platelet aggregating agents such as thrombin and ADP, the particulate reaction product of guanylate cyclase activity was detectable on plasma membrane and on membranes of the open canalicular system. In contrast, samples incubated with platelet-activating factor showed no activation of the cyclase activity. Atrial natriuretic factor stimulated the particulate guanylate cyclase. The ultracytochemical localization of this activated cyclase was the same as that of thrombin- or ADP-stimulated guanylate cyclase. Adenylate cyclase activity was studied in platelets incubated with prostaglandin E1 plus or minus insulin. The enzyme reaction product was found at the same sites where guanylate cyclase was detected. Therefore guanylate and adenylate cyclase activities do not seem to be preferentially localised in platelet membranes.

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.

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. The discovery that inhibition of IDO activity reduces the survival of MHC-mismatched fetuses in mice and that the risk of fetal allograft rejection correlates with the degree of parental tissue incompatibility has led to the hypothesis that IDO activity protects fetal allografts from maternal T cell-mediated immunity. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We have found that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions and was associated with the activation of casapase-8 and the release of cytochrome c from mitochondria. In vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data may represent the first experimental evidence for the involvement of tryptophan catabolism in the regulation of T cell apoptosis and maintenance of peripheral T cell tolerance.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We show here that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and of Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions, and was associated with the activation of caspase-8 and the release of cytochrome c from mitochondria. When administered in vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data suggest that the selective deletion of T lymphocytes may be a major mechanism whereby tryptophan metabolism affects immunity under physiopathologic conditions.

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca2+ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca2+-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.

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.

Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg2+ and Mn2+, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg2+ or Mn2+, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg2+ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum-ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn2+ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg2+ and ATP at pH 8 and 7.4.

The ultracytochemical localization of guanylate cyclases A and B activity has been studied after stimulation with atrial natriuretic peptide and C-type natriuretic peptide in light- and dark-adapted retinas and pigmented epithelium. The results showed that both peptides stimulated guanylate cyclases A and B activity in light-adapted retinas only. Guanylate cyclases A and B activity was detected on plasma membrane of body of photoreceptors, bipolar, horizontal and ganglion cells, on plasma membranes of interneuronal connections at plexiform layers and on the plasma membrane of fibres at the nerve fibres layer. Independently of the light-or dark-adapted state, the pigmented epithelium also presented guanylate cyclases A and B activity on basal and lateral plasma membranes.

The 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.

A minireview is presented on the ultracytochemical localization of membrane-bound guanylate cyclase (GC) in various tissues and in cultured cells after activation with three natriuretic peptides, the atrial natriuretic factor (ANF), the brain natriuretic peptide (BNP), and the C-type natriuretic peptide (CNP). GC, two subtypes of which have been recently identified, is the receptor for these peptides. The GC isoforms are differently stimulated by ANF, BNP and CNP. Under our experimental conditions, the natriuretic peptides were strong activators of GC since samples incubated without natriuretic peptides do not reveal any cyclase activity. The natriuretic peptide-stimulated GC activity was studied in rat kidney, lung, adrenal gland and neurohypophysis, in rabbit platelets, in lamb olfactory mucosa, and in rat C6 glioma cells. On the basis of the subcellular GC localization some additional functions of peptides are hypothesized.

The ultracytochemical localization of particulate guanylate cyclase has been studied in lamb olfactory mucosa after activation with rat atrial natriuretic factor (rANF), porcine brain natriuretic peptide (pBNP), porcine C-type natriuretic peptide (pCNP) or rat brain natriuretic peptide (rBNP). Particulate guanylate cyclase is the receptor for these peptides and recently two subtypes of the cyclase have been identified. These isoforms are stimulated differently by ANF, BNP and CNP. Under our experimental conditions, rANF, pCNP and pBNP were strong activators of particulate guanylate cyclase in lamb olfactory mucosa, as demonstrated by the presence of reaction product. Samples incubated in basal conditions without rANF, pCNP or pBNP, or samples incubated in presence of rBNP did not reveal any cyclase activity. The rANF-stimulated cyclase activity was localized in the apical portion of olfactory epithelium. pCNP-stimulated guanylate cyclase was detected to the lamina propria in association with secretory cells of Bowman's glands and with cells in close relation with Bowman's glands (elongated cells and myoepithelial cells). The cyclase activity stimulated by pBNP was limited to cells of Bowman's glands. The present data indicate that ANF and CNP are recognized by different receptors and that BNP and CNP bind to the same receptor.

Glucocorticoid hormones (GCH) induce apoptotic cell death in immature thymocytes through an active mechanism, characterized by extensive DNA fragmentation into oligonucleosomal subunits. This requires macromolecular synthesis and is inhibited by protein kinase C (PKC) inhibitors, interleukin-4 (IL-4) and heat shock (hs). We performed experiments to analyse the possible effect of GCH on more differentiated lymphocytes, i.e. mouse natural killer (NK) cells and CD8+ alloreactive cytotoxic T lymphocytes (CTL). The results show that dexamethasone (DEX) induces DNA fragmentation and cell death in NK cells and CTL in vitro. In both NK cells and CTL, DEX-induced apoptosis is inhibited by IL-2 and IL-4 but, unlike that induced in thymocytes, is augmented by mRNA and protein synthesis inhibitors, PKC inhibitors and HS.

We studied the cytochemical localization of particulate guanylate cyclase (GC) in rat adrenal gland after stimulation with porcine brain natriuretic peptide (pBNP) by electron microscopy. In the adrenal cortex, GC activity, as demonstrated by the presence of reaction product, was prevalently localized to the zona glomerulosa and zona fasciculata, while the zona reticularis showed little GC reaction product. In the adrenal medulla, GC reaction product was present only in adrenalin-containing cells. All GC positivity was associated with intracellular membranes. No GC reaction product was detected in specimens incubated in media devoid of pBNP. In parallel samples incubated in the presence of rat atrial natriuretic factor (rANF), the distribution of rANF-stimulated GC activity was similar to that of pBNP-stimulated GC activity.

The cellular and subcellular localization of annexins V and VI, two members of a superfamily of Ca(2+)-dependent phospholipid- and membrane-binding proteins, was investigated in chorionic villi of human placentae of different gestational ages by postembedding immunocytochemistry at the electron microscope level. All cell types of placental villi, i.e., the syncytiotrophoblast, Langhans cells, Hofbauer cells, fibroblasts, and capillary endothelial cells, appeared to express the two proteins, irrespective of the gestational age. By immunogold particle counts, annexin V was observed to be 2-3 times as much abundant as annexin VI. Syncytiotrophoblast cells appeared to contain the largest amounts and Langhans cells appeared to contain the least amounts of annexins V and VI, as judged by immunocytochemistry. The two proteins were found associated with plasma, Golgi, and vacuolar membranes, and with membranes of the endoplasmic reticulum, as well as diffusely in the cytoplasm. Annexin V appeared to be distributed in nearly equal proportions between cell membranes and the cytoplasm in stromal cells and to be about 30% associated with cell membranes in trophoblast cells, whereas annexin VI appeared almost equally distributed between cell membranes and the cytoplasm in trophoblast and stromal cells. Also, annexins V and VI appeared to be more abundant in trophoblast cells than in stromal cells. The present data strongly support the idea that placenta is a preferential site of annexin-regulated activities, and suggest that annexins V and VI are actively involved in the Ca(2+)-dependent regulation of membrane processes in trophoblast cells.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.

We studied the cytochemical localization of particulate guanylate cyclase (GC) activity after stimulation with atrial natriuretic factor (ANF) in rat lung, at the electron microscope level. Samples incubated in the absence of ANF did not reveal any GC reaction product. These results indicate that ANF is a strong activator of the enzyme in this organ. In intrapulmonary bronchi, the ANF-activated GC reaction product was localized on mucus secreting goblet cells. GC was seen in bronchioles, alveoli and capillaries. All of the GC reaction product was associated with plasma membranes of Clara cells, of great alveolar cells and of endothelial cells in alveolar capillaries. Our data suggest that, by activation of particulate GC, ANF acts directly on cells where Na+ reabsorption occurs.

Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na+ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.

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.

Guanylate cyclase activity has been studied biochemically and cytochemically in guinea pig testis. The results of the biochemical assays indicate an equal distribution of this enzyme between the soluble and particulate fractions, which have a different sensitivity to adenosine triphosphate. The cytochemical results demonstrate that the reaction product of guanylate cyclase is detectable in the interstitial capillary endothelial cells and, in the seminiferous epithelium, mainly at the level of the adjacent surfaces of Sertoli and germ cells of the intermediate and adluminal compartments. Guanylate cyclase activity appears at the level of pachytene spermatocytes and persists throughout subsequent stages of development. The distribution in the seminiferous epithelium seems to indicate that guanylate cyclase is involved in the interrelationships between Sertoli and germ cells during gamete differentiation.

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).

The behaviour of rat mast cells under conditions of high and low vitamin A The authors have studied the mast cells in normal rats and in rats under hypo- and hyper-vitaminosis A. The main results of their studies are: (a) in the skin (dermis), in the case of A hypervitaminosis, there is a significant rise in the number of the mastocytes with swelling and dcgranulation; (b) in lung and bone marrow, the number of mastocytes is increased under hypovitaminosis A and there is also blood congestion and lipomatosis of bone marrow. The authors give an interpretation of their results.

Previous biochemical and cytochemical studies have indicated that in human term placenta the enzyme guanylate cyclase (GC) is associated mostly with the cytosolic fraction of homogenates and localized on the syncytiotrophoblast microvillous border. In the present study we have shown cytochemically the GC particulate form in early human placenta using guanylyl-imidodiphosphate [Gpp(NH)p] as substrate and NaN3 as activator. In samples of placental villi taken from the 6th to 12th week of pregnancy, the GC reaction product was always found on the apposing Langhans cytotrophoblast and syncytiotrophoblast plasma membranes. Furthermore, GC was present on cells in mitosis of the Langhans cytotrophoblast. From the 11th week GC was also visible on basal plasma membranes of Langhans cytotrophoblast and on endothelial cells of fetal capillaries. In samples of human term placenta GC was detectable on the syncytiotrophoblast microvillous border. This suggests a shift of enzyme localization during pregnancy.

Guanylate cyclase activity has been studied biochemically and cytochemically in guinea pig testis. The results of the biochemical assays indicate an equal distribution of this enzyme between the soluble and particulate fractions, which have a different sensitivity to adenosine triphosphate. The cytochemical results demonstrate that the reaction product of guanylate cyclase is detectable in the interstitial capillary endothelial cells and, in the seminiferous epithelium, mainly at the level of the adjacent surfaces of Sertoli and germ cells of the intermediate and adluminal compartments. Guanylate cyclase activity appears at the level of pachytene spermatocytes and persists throughout subsequent stages of development. The distribution in the seminiferous epithelium seems to indicate that guanylate cyclase is involved in the interrelationships between Sertoli and germ cells during gamete differentiation.

Two kinds of secretions, both merocrine and apocrine, have been detected in the serous adenomeres of the human olfactory glands. While the low-density vesicles secrete their products into the glandular lumen by a merocrine way (exocytosis), the electron-dense granules are produced by an apocrine mechanism.

Biopsies taken from the anterior two-thirds of the inferior turbinate of 3 female patients affected by unilateral complete choanal atresia were studied at the light, transmission and scanning electron microscope. Several changes, such as the uneveness of the mucosal surface, the thickening of the basal membrane, and the presence of ciliary abnormalities in the respiratory mucosa of the affected cavity, were observed. Ciliary abnormalities consisted of the presence of several 'adhesive' and 'bulging' compound cilia, of cilia with excessive cytoplasm and cytoplasmatic processes, and of ciliary structures with loss of the peripheral membrane. At SEM, cilia of the affected cavity appeared bundled, irregularly oriented, flattened and covered by an increased and/or accumulated secretion. Choanal atresia represents, according to the authors, a chronic suffering condition of the nasal mucous membranes. It is characterized by peculiar aspects which are probably related, on one hand, to the increased and/or accumulated mucous secretions and, on the other, to the congenital and long-standing airflow deprivation.