Immunocytochemical localization of endothelial nitric oxide synthase (e-NOS) and inducible nitric oxide synthase (i-NOS) in rat neurohypophysis after transient cerebral ischemia.

The expression pattern of endothelial and inducible forms of nitric oxide synthase (e-NOS, i-NOS) in rat neurohypophysis after transient ischemia was investigated using post-embedding immunogold method. We demonstrate that ischemia induced an early (10 min) expression of e-NOS not only in endothelium but also in the mast cells. Expression of i-NOS was almost exclusively confined to glial cells (pituicytes) and perivascular macrophages of experimental animals, and peaked at 24 h after ischemia. This evidence indicates that NO plays a significant role in mechanisms of cerebral ischemia. Taking into account the known beneficial role of e-NOS in ischemia it is likely that mast cells protect against post-ischemic brain damage by producing vasodilatation via nitric oxide. In contrast, cerebral macrophages and pituicytes may mediate neuronal and endothelial damage in late period after ischemia in rat neurohypophysis.

Pilot ultrastructural evaluation of human preauricular skin before and after high-energy pulsed carbon dioxide laser treatment.

BACKGROUND: Carbon dioxide laser resurfacing has recently come into favor for the treatment of photodamaged skin. While the clinical and histologic effects of high-energy short-pulse carbon dioxide lasers on human skin have been investigated, the ultrastructural effects of these lasers have not been documented. Our objective was to study the ultrastructural effects of a high-energy pulsed carbon dioxide laser on photodamaged human skin. OBSERVATIONS: Before laser surgery, the ultrastructural changes characteristic of photodamaged skin were evident. Immediately after treatment, there was extensive coagulation necrosis of the epidermis and papillary dermis. Thirty days after treatment, there was no evidence of intercellular or intracellular edema, and ordered differentiation of the epidermal keratinocytes, with a loss of keratinocyte dysplasia, was seen. Increased numbers of desmosomes and tonofibrils were noted. New deposition of collagen was present in the papillary dermis. The ultrastructural findings seen at 90 days after treatment were similar to those seen at 30 days, apart from increased organization of collagen fibers in the papillary dermis. CONCLUSIONS: Treatment with the high-energy pulsed carbon dioxide laser appears to reverse the epidermal and dermal changes of photoaging on an ultrastructural level. These changes appear morphologically to be consistent with previously described clinical and histologic changes following laser resurfacing.

Fluorescent and radiolabeling of polysaccharides: binding and internalization experiments on vascular cells.

Glycosaminoglycans (GAGs) such as heparan sulfates are complex carbohydrate polymers. These structural components of the extracellular matrix are essential for the adhesion, migration, and regulation of cellular growth. To understand the physiological role of GAGs and GAG analogues, a practical approach consists of labeling and detecting them in cell extracts, or analyzing binding domains and their distributions into the cells. We propose a convenient and reliable method for preparing and labeling amino-enriched, polysaccharides with the fluorescent derivative 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein (DTAF). Radioiodination is then performed on the DTAF moiety. This method was applied to polysaccharides known to inhibit vascular smooth-muscle cell (SMC) proliferation such as functionalized dextrans derived from poly(alpha 1-6 glucose) and fucan, poly(L-fucose 4-sulfate) extracted from brown seaweed. Using autoradiography and confocal microscopy, we observed the fixation and internalization of labeled antiproliferative products in SMCs from rat aorta. These probes can be useful for the understanding of polysaccharide-cell interactions. In addition, the method presented here can be applied to various synthetic or natural biomedical materials.

Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase.

An aminopeptidase B (Ap-B) was previously purified to homogeneity from rat testis extracts and characterized. In the present work, by using oligonucleotides selected on the basis of partial amino acid microsequences of pure Ap-B and PCR techniques, the nucleotide sequence of a 2.2-kb cDNA was obtained. The deduced amino acid sequence corresponds to a 648-residue protein (72.3 kDa) containing the canonical "HEXXHX18E" signature, which allowed its classification as a member of the M1 family of metallopeptidases. It exhibits 33% identity and 48% similarity with leukotriene-A4 hydrolase, a relation further supported by the capacity of Ap-B to hydrolyze leukotriene A4. Both enzymes also were closely related to a partially sequenced protein from Dictyostelium discoideum, which might constitute the putative common ancestor of either aminopeptidase or epoxide hydrolase, or both. Ap-B and its mRNA were detected in the germ line and in the Sertoli and peritubular cells of the seminiferous tubules. Because the enzyme was found in the medium conditioned by spermatocytes and spermatids and in the acrosome during spermatozoa formation, together these observations suggested an involvement of this exometallopeptidase in the secretory pathway. It is concluded that this ubiquitous enzyme may be involved in multiple processing mechanisms.

Protracted elevation of endothelin immunoreactivity in hypothalamo-neurohypophysial system after ischemia.

In the present study we investigated the effects of brain ischemia on endothelin expression in the hypothalamo-neurohypophysial system of the rat using the post-embedding immunogold technique for electron microscopy. From 24 hours to six months after ischemia, a relatively high endothelin immunoreactivity was observed in some neurosecretory cells of the supraoptic and paraventricular nuclei as well as in endothelial cells of some microvessels of the hypothalamus and neurohypophysis. The results indicate that ischemia is a potent stimulus for increased production of endothelin. The distribution of endothelin-immunoreactive neurons in the hypothalamus was similar to that of vasopressin and oxytocin, so it seems possible that endothelin participates directly in controlling hormonal synthesis and release from vasopressinergic and oxytocinergic neurons in the hypothalamo-neurohypophysial system.

Intracellular distribution of ampicillin in murine macrophages infected with Salmonella typhimurium and treated with (3H)ampicillin-loaded nanoparticles.

The intracellular distribution of (3H)ampicillin-loaded polyisohexylcyanoacrylate nanoparticles was studied in murine macrophages (peritoneal cells and the J774 cell line) infected by Salmonella typhimurium C5, using ultrastructural autoradiography. Ampicillin penetration and retention into the cells obviously increased by means of nanoparticles. After short-term (2-4 h) treatment with the nanoparticle formulation, numerous intracellular bacteria were seen to be in the process of destruction. The tritium labelling was located in the cell cytoplasm and inside vacuoles in which bacteria undergoing degradation were often present. After long-term (12 h) treatment, numerous spherical bodies (d: 100 nm to 500 nm) and larger forms (2 microns) were seen in the vacuoles. Radioactivity was mainly found to be localized on the spherical bodies, indicating marked damaging action of the ampicillin on the bacterial walls. The targeting of ampicillin therefore allowed its penetration into the macrophages and vacuoles infected with S. typhimurium.

Localization of endothelin-like immunoreactivity in the hypothalamo-neurohypophysial system of the rat after ischemia.

Endothelin is a very powerful endogenous vasoconstrictor substance produced by endothelial cells. To examine the potential role of endothelin as a neuropeptide, we studied its distribution in the neurosecretory system. Endothelin-like immunoreactivity (EN-IR) has been demonstrated by an immunogold method in the paraventricular and supraoptic nuclear neurons and their terminals in the posterior pituitary of the rat. ET-IR was enhanced after ischemia suggesting its modulatory role in neurosecretory functions.

The PML growth-suppressor has an altered expression in human oncogenesis.

Altered sub-nuclear localisation of the nuclear body-associated PML protein in acute promyelocytic leukaemia, has been proposed to contribute to leukaemogenesis. We have recently shown that PML is a primary target gene of interferons. Here, it is shown that PML has growth suppressive properties and displays an altered expression pattern during human oncogenesis. PML is widely expressed in cell-lines and is cell-cycle regulated. Overexpression of the protein induces a sharp reduction in growth rates in vitro and in vivo. In contrast with cell-lines, in normal tissues (including those that rapidly proliferate) only a few cells have detectable PML levels. However, these can be upregulated by soluble factors (e.g. IFN, estrogens). Human epithelial tumors show a gradual increase of PML levels as the lesion progresses from benign dysplasia to carcinoma. A similar induction is found in the surrounding stroma and vessels, which likely results from paracrine interactions. Strikingly, when malignant cells turn invasive, they loose PML expression, while expression is conserved in the stromal compartment. These observations point to the existence of a consistent deregulation in the expression of the PML growth-suppressor during human oncogenesis.

Induction of apoptosis by transiently transfected metabolically stable wt p53 in transformed cell lines.

Biochemical and functional properties of wild-type (wt) and mutant p53 were studied under the same cellular environment by transient transfection. Exogenous wt p53 expressed in transformed cell lines was found to be as metabolically stable as mutant p53. Yet only mutant p53 bound to hsp70 whereas wt p53 did not, suggesting that the metabolic stability of p53 does not depend on its ability to form complexes with hsp70. The wt protein was expressed essentially in the nucleus, while mutant p53 showed both nuclear and cytoplasmic expression, as determined by immunofluorescence staining with PAb122. In addition, staining with PAb1801 revealed a number of strongly fluorescent cell fragments in cultures transfected by wt p53. Morphological features of apoptosis were observed in these cultures. Quantitative analysis by flow cytometry confirmed that only the cell population expressing wt p53 had a significant amount of cell debris. Thus, transient expression of a metabolically stable wt, but not mutant, p53 induces cell death by apoptosis. The present study demonstrates a model system to investigate the functional domains of p53 in the induction of apoptosis.

The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion.

Nuclear bodies (NBs) are ultrastructurally defined granules predominantly found in dividing cells. Here we show that PML, a protein involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), is specifically bound to a NB. PML and several NB-associated proteins, found as auto-antigens in primary biliary cirrhosis (PBC), are co-localized and co-regulated. The APL-derived PML-RAR alpha fusion protein is shown to be predominantly localized in the cytoplasm, whereas a fraction is nuclear and delocalizes the NB antigens to multiple smaller nuclear clusters devoid of ultrastructural organization. RA administration (which in APL patients induces blast differentiation and consequently complete remissions) causes the re-aggregation of PML and PBC auto-antigens onto the NB, while PML-RAR alpha remains mainly cytoplasmic. Thus, PML-RAR alpha expression leads to a RA-reversible alteration of a nuclear domain. These results shed a new light on the pathogenesis of APL and provide a molecular link between NBs and oncogenesis.

Protein kinase C-like immunoreactivity in gerbil hippocampus after a transient cerebral ischemia.

This study describes immunocytochemical distribution of the protein kinase C (PKC) subspecies: alpha, beta and gamma in the CAI sector of gerbil hippocampus. Immunolabelling was performed with 10 nm gold-antibody complexes against each of the PKC subspecies. The subspecies of PKC were expressed specifically in different populations of hippocampal cells. An enhanced PKC immunoreactivity was noted in the animals after ischemia. We propose that this phenomenon reflects an activation of PKC in the early phase of brain ischemia.

Nuclear localization of the Epstein-Barr virus/C3d receptor (CR2) in the human Burkitt B lymphoma cell, Raji.

Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the cell surface interacted with soluble and particle-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells.

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.

SV40 large T-antigen and transformation related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells.

The localization of SV40 large T-antigen (T-Ag) and the cellular protein p53 in the nuclei of mouse and human SV40-transformed cells and of a methylcholanthrene-transformed mouse cell line, was studied. Their detection by ultrastructural immunocytochemistry with specific monoclonal antibodies employed two complementary methods used in parallel. These consisted of indirect immunoperoxidase labelling carried out before embedment on Triton-permeabilized cells, or indirect immunogold labelling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that in SV40-transformed cells both proteins are chiefly localized on peri- and interchromatin RNP fibrils. This shows that they occur in structures involved in the synthesis and processing of hnRNA. The nucleoli and chromatin did not appear to be labelled. In methylcholanthrene-transformed cells the protein p53 (in the absence of large T-Ag) was also detected on peri- and interchomatin fibrils. Taken together with recent results which demonstrated that, during lytic infection, T-Ag was associated chiefly with cellular chromatin (Harper, F, Florentin, Y & Puvion, E, Exp cell res 161 (1985) 434) [33], our experiments provide evidence that the transforming function of SV40 large T-Ag is dissociable from its function in SV40 lytic infection in terms of its subnuclear distribution.

High resolution autoradiographical detection of RNA in the interchromatin granules of DRB-treated cells.

Isolated rat liver cells were pulse-labelled with tritiated uridine and post-incubated in the presence of an excess of unlabelled uridine and of adenosine analog DRB (5-6-dichloro-1-beta-D-ribofuranosyl benzimidazole). Nuclear radioactivity was detected with high resolution autoradiography. A significant labelling of the interchromatin granules was revealed in these conditions. Pretreatments of cells with low doses of actinomycin D in order to preferentially inhibit ribosomal RNA (rRNA) synthesis prevented the labelling of the interchromatin granules during subsequent DRB treatments. These observations indicate that in DRB-treated cells, the interchromatin granules are sites of transfer or of accumulation of nucleolar RNA. Our results are discussed in connection with our knowledge of the action of DRB on RNA metabolism in mammalian cells and with recent data concerning the still enigmatic interchromatin granules which are present in the nuclei of most cells.

Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells.

Small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 small nuclear RNAs were detected by ultrastructural immunocytochemistry in the nuclei of isolated rat hepatocytes using Fab fragments of anti-Sm and anti-RNP autoantibodies. Their localization was carried out in normal cells and in cells treated with two drugs, the adenosine analog DRB and CdCl2, which alter the number and distribution of nuclear RNP components. It was found that more precise determination of the distribution of these small RNAs could be obtained by using two complementary procedures in parallel rather than either one alone. They consisted of an indirect immunoperoxidase labeling carried out before embedment and an indirect immunogold labeling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that snRNPs are associated with all extranucleolar perichromatin fibrils and granules and interchromatin fibrils, which confirms that they occur in structures involved in the synthesis and processing of hnRNA. The snRNPs are not associated with nucleolar perichromatin granules induced by DRB, which confirms that there may be two kinds of perichromatin granules. The snRNPs are also associated with the still enigmatic interchromatin granules which apparently do not contain hnRNA but at least in DRB-treated cells, also contain ribosomal RNA.

Ultrastructural cytochemistry of intranuclear dense granules in nasopharyngeal angiofibroma.

Electron-dense round granules (ING) were found in the nuclei of fibroblastic cells of all the tumors examined by us (6 cases) and diagnosed clinically and histologically as nasopharyngeal angiofibroma (NPAF). The ING were consistently associated with this tumor and can be regarded as pathognomonic of NPAF. We studied at the ultrastructural level the composition of these particles using various cytochemical techniques. Enzymatic digestions carried out on thin sections as well as staining methods preferential or specific for nucleoproteins have revealed that ING represent tightly bound RNA-protein complexes and do not contain DNA. There are no data available that the ING takes part in the metabolism of NPAF fibroblastic cells.

Structural organization of rat hepatocyte chromatin as visualized in thin frozen sections selectively stained for DNA.

We studied the structure of rat hepatocyte chromatin in situ using thin frozen sections selectively stained for DNA after aldehyde fixation. Our results indicate that intranucleolar chromatin is arranged into three different organization levels, confirming the observations on Epon-embedded chromatin. These are: completely extended DNA filaments, with a thickness of approximately 3 nm, clustered in loose, roundish agglomerates, very long fibers with a thickness ranging from 15 to 35 nm and compact chromatin clumps. Both the fibers and the chromatin clumps frequently appeared to be composed of nucleosome-like particles. In the extranucleolar chromatin, agglomerates of extended DNA filaments and long fibers were never visualized. In contrast to data from Epon-embedded chromatin, we noticed that in frozen sections neither the nucleolar nor the extranucleolar compact chromatin appear to be organized into discrete, 20 to 30 nm superordered fibers.

Preferential nucleolar localization of Cis-DDP in human fibroblasts.

The use of complex platinum salts (Cis-DDP) in antitumoral therapy is well known. However the mode of action of this salt remains unknown. The present study was performed with human transformed cells in culture (WI-98-VAD), grown in the presence of Cis-DDP. The intracellular localization of platinum was studied by associating the technique of microanalysis by electronic probe (analysis by wavelength dispersion: W. D. S.) and that of fine tissue sections at low temperature: ultracryotomy. The platinum was found to be concentrated mainly in the nucleolar region.