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PubMed1995

Evidence for apoptosis in advanced human atheroma. Colocalization with interleukin-1 beta-converting enzyme.

This study sought evidence for apoptosis, a form of programmed cell death, in human atheromatous coronary and carotid arteries. Markers for apoptotic cells included in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), genomic DNA electrophoresis, and morphological analysis. Intimal lesions contained more TUNEL+ cells (34 +/- 6%, n = 8) than non-atherosclerotic arterial intima (8 +/- 3%, n = 5, P < 0.05). The tunica media of the diseased arteries had a percentage of TUNEL+ cells (5 +/- 1%) similar to that in the normal vessels (3 +/- 1%, N.S.). Oligonucleosomal DNA fragments were visualized in extracts from 12 atheromatous plaques but in none of 5 non-atherosclerotic vessels. Both smooth muscle cells (SMC) and macrophages, two major cell types in the atherosclerotic intima, bore markers of apoptosis, but with different patterns, as determined by double histochemical labeling for cell types and TUNEL. The TUNEL+ SMC localized mainly in the fibrotic portion of the atheroma, whereas TUNEL+ macrophages clustered near or within the lipid-rich core of the lesion. Atheromatous lesions expressed mRNA encoding interleukin-1 beta-converting enzyme (ICE), a mammalian cell death gene, as demonstrated by reverse transcriptase polymerase chain reaction. Immunohistochemistry revealed that ICE localized in regions of TUNEL+ SMC and macrophages. TUNEL- cells showed little or no immunoreactive ICE. These data point to a role for apoptosis in regulation of cell accumulation during atherogenesis and suggest involvement of ICE in SMC death in fibrous regions of complex atheroma, and in macrophage death in the lipid-rich core of the lesion. Apoptosis of vascular cells in fibrous cap may impede maintenance or repair of the matrix in this region and affect stability of the plaques.

Apoptosis↗
PubMed1994

Immunohistochemical colocalization of Met5-enkephalin-Arg6-Gly7-Leu8 with tyrosine hydroxylase in neurons of the porcine inferior mesenteric and coeliac-superior mesenteric ganglia.

The localization and distribution of the proenkephalin A-derived octapeptide Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was studied in the porcine sympathetic prevertebral ganglia by using the indirect immunofluorescence technique. In the inferior mesenteric ganglion, a small population of principal neurons and SIF cells contained intense MEAGL immunoreactivity. A very dense plexus of MEAGL-immunoreactive nerve fibers was distributed in different parts of the ganglion. A low number of MEAGL- immunoreactive principal ganglion cells and SIF cells, as well as numerous nerve fibers were also present in the coeliac-superior mesenteric ganglion. Double staining experiments indicated that the MEAGL-immunoreactive principal neurons in the inferior mesenteric and coeliac-superior mesenteric ganglia also contained tyrosine hydroxylase immunoreactivity.

Animals↗
PubMed1994

Colocalized alterations in connexin32 and cytochrome P450IIB1/2 by phenobarbital and related liver tumor promoters.

Direct intercellular signal transduction is achieved by the passage of small molecules through gap junctions (GJ). Previous studies in our laboratory showed that the liver tumor promoter phenobarbital (PB) reversibly decreases the abundance of the GJ protein connexin32 (Cx32) in both preneoplastic-altered hepatic foci and centrolobular hepatocytes (M. J. Neveu et al., Cancer Commun., 2: 21-31, 1990). Because the inhibitory effects of PB on GJ intercellular communication are prevented by the nonspecific cytochrome P-450 inhibitor SKF-525A (J. E. Klauning, et al., Toxicol. Appl. Pharmacol., 102: 533-563, 1990), we investigated whether alterations in Cx32 are coincident with changes in the major PB-inducible cytochrome P-450, termed b/e or IIB1/2. Immunostaining of liver cryosections from rats fed dietary PB demonstrated that centrolobular hepatocytes that exhibit reduced Cx32 express enhanced cytochrome P450IIB1/2 protein. In contrast, no change in the periportal distribution of connexin26 immunoreactivity was found in PB-treated rats. In addition, rats were treated with the structurally related barbiturates pentobarbital, amobarbital, barbital, and barbituric acid. We found that the extent of the hepatic lobule occupied by coincident centrolobular alterations in Cx32 and P-450 staining correlates with the ability of the compounds to promote liver oncogenesis. To determine the molecular mechanisms responsible for the modification in Cx32 staining, we examined the mRNA and protein levels of Cx32 and P450IIB1/2 in total-tissue homogenates from PB-treated rats. Northern blotting demonstrated thatdietary PB dramatically induced P-450IIB1 mRNA, but the same RNA samples failed to show alterations in Cx32 steady-state transcripts. Consistent with these findings, the level of Cx32 protein in total liver homogenates did not change in rats chronically fed PB. Examination of Cx32 solubility in 20 mM NaOH demonstrated that PB treatment results in the generation of a NaOH-soluble form of Cx32 (i.e., 47 kDa). In addition, trypsinized paraffin-embedded liver sections from PB-treated rats exhibited diffuse cytoplasmic Cx32 staining that was restricted to centrolobular cells. Our results show that PB and related barbiturate tumor promoters reversibly down-regulate punctate Cx32 staining in centrolobular hepatocytes posttranslationally, possibly through modification(s) in the transport, assembly, and/or turnover of GJs.

Animals↗
PubMed1996

Colocalization of GABA, glycine, and their receptors at synapses in the rat spinal cord.

To determine whether GABA and glycine can act as cotransmitters at synapses in the rat spinal cord, we have compared the ultrastructural distribution of GABAA-receptor beta 3 subunit with that of the glycine receptor-associated protein gephyrin and combined this with postembedding detection of GABA and glycine. We also used a dual-immunofluorescence method to confirm that gephyrin was associated with the glycine-receptor alpha 1 subunit throughout the cord. GABAA beta 3-subunit immunoreactivity was restricted primarily to synapses, and at a majority of these synapses the presynaptic axon was GABA-immunoreactive. Many synapses showed both GABAA beta 3 and gephyrin immunoreactivity, and at most of these synapses GABA and glycine were enriched in the presynaptic axon. These results strongly support the idea that cotransmission by GABA and glycine occurs in the spinal cord.

Animals↗
PubMed1996

Nitric oxide synthase and tyrosine hydroxylase are colocalized in nerves supplying the postnatal human male genitourinary organs.

PURPOSE: The objective of this study was to examine the distribution of nitric oxide synthase (NOS) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in nerve fibers supplying the human neonatal male genitourinary organs. MATERIALS AND METHODS: An indirect double label immunofluorescence technique was employed on specimens obtained from infants and children at postmortem examination. RESULTS: Many nerve fibers immunoreactive for both NOS and TH were observed in the muscle coat of the vas deferens and the seminal vesicle, within the fibromuscular stroma of the prostate gland and at the bladder neck, and also formed perivascular plexuses in each of these organs. Double-labeled nerves occurred less frequently in the intramural ureters and superficial trigone while similar nerves in the bladder body were relatively sparse. Numerous nerves immunoreactive for NOS but not TH were observed at the base of the epithelium of each organ examined. Four types of autonomic ganglion cell were observed in nearby pelvic ganglia: those which contained NOS and TH, those which contained NOS alone, those which contained TH alone and those which contained neither NOS nor TH. CONCLUSION: The results indicate that many of the noradrenergic nerves as well as non-noradrenergic nerves supplying the male genitourinary organs have the capacity to synthesize nitric oxide (NO) and that NO may play a significant role in the autonomic control of both the urinary and genital organs in the postnatal human male.

Cadaver↗
PubMed1995

Nitric oxide synthase and vasoactive intestinal polypeptide colocalization in neurons innervating the human penile circulation.

The question which neurotransmitters mediate penile erection in humans is still unsettled. Although functional studies have established a role of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) in human penile smooth muscle relaxation, they did not allow a conclusion as to whether they are released by neurons or other cells. This article outlines the identification of numerous nitric oxide synthase (NOS) and VIP-containing axons in the human penis. More than 50% of the perivascular nerve fibres and more than 90% of the trabecular nerve fibres within the corpus cavernosum stained positive for both NOS and VIP. In contrast, endothelial cells stained only faintly for NOS-immunoreactivity, NOS/VIP-immunoreactivity was reduced (diabetes) or absent (lesion of the cavernous nerve) in penile tissue taken from patients with neurogenic impotence. These findings support the concept that NO and VIP act as neural comediators of penile erection in humans.

Humans↗
PubMed1996

Association of p120 ras GAP with endocytic components and colocalization with epidermal growth factor (EGF) receptor in response to EGF stimulation.

Epidermal growth factor (EGF) stimulation of cells induces down-regulation of EGF receptors through receptor-mediated endocytosis. Simultaneously, activated autophosphorylated EGF receptors become associated with the pTyr-binding domains of various intracellular signaling proteins, including those implicated in the regulation of the plasma membrane-associated small G protein p21 Ras. Included among these EGF receptor-binding proteins is the M(r) 120,000 Ras GTPase-activating protein (GAP). Herein we show by subcellular fractionation, immunoprecipitation and Western blotting, indirect immunofluorescence, and double immunogold labeling and electron microscopy that, in response to EGF stimulation of MDCK cells, GAP becomes first plasma membrane localized and subsequently internalized with EGF receptors. In various subcellular membrane fractions examined, EGF receptor and GAP were physically associated. EGF stimulation also induced association of GAP with p190 Rho-GAP and the GAP-p190 complex coimmunoprecipitated from membrane fractions. Recruitment of SH2 domains to activated receptor tyrosine kinases at the plasma membrane is undoubtedly an important mechanism in controlling the subcellular localization and substrate accessibility of signaling proteins. Our results illustrate that another level of signaling protein regulation may occur by virtue of their association with endosomes.

Animals↗
PubMed1996

The Synapse-Associated Protein Rapsyn Regulates Tyrosine Phosphorylation of Proteins Colocalized at Nicotinic Acetylcholine Receptor Clusters

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.

Journal Article↗
PubMed1997

1.25-Dihydroxyvitamin D3 receptor is partly colocalized with oxytocin immunoreactivity in neurons of the male rat hypothalamus.

With receptor immunocytochemistry, neurons receptive for the steroidhormone 1.25-dihydroxyvitamin D3 have been observed in hypothalamic nuclei. In the present paper we report that a fraction of 1.25-dihydroxyvitamin D3 receptor (VDR) immunoreactive neurons in the hypothalamus of male rats are immunoreactive for oxytocin (OT), suggesting a direct genomic action of this steroid on OT expression. While only 10% of neurons with OT immunofluorescence in the periventricular nucleus contained nuclear VDR immunostaining, up to 50% of the OT neurons in the supraoptic nucleus and 30% in the magnocellular portion of the paraventricular nucleus were VDR positive. VDR immunostaining in the magnocellular nuclei was in many cases confined to the perinuclear cytoplasm. We assume that 1.25-dihydroxyvitamin D3 has effects on hypothalamic peptidergic systems similar to other steroid hormones.

Animals↗
PubMed1997

Distribution and colocalization of calcitonin gene-related peptide, tachykinins, and vasoactive intestinal peptide in normal and idiopathic unstable human urinary bladder.

The distribution of nerves containing calcitonin gene-related peptide (CGRP), substance P (SP), neurokinin A (NKA), and vasoactive intestinal peptide (VIP) was examined in the human urinary bladder, using both single- and double-label immunohistochemistry. Nerves containing CGRP and tachykinins were typically present within the subepithelial region, encircling intramural ganglia and around blood vessels. These nerves were sparsely distributed, and only very rarely projected to the smooth muscle bundles of the detrusor. In contrast, VIP-containing nerves formed a dense subepithelial plexus and also projected to the detrusor muscle bundles. The double-label studies revealed that approximately 26% +/- 10% of CGRP-immunoreactive nerves also contained SP and NKA, but that no CGRP fibers coexpressed VIP. Conversely, all SP-immunoreactive fibers also contained CGRP, and many coexpressed NKA. At least three neurochemically distinct populations of nerves can therefore be discerned in the human bladder: VIP/-, CGRP/-, and CGRP/SP/ +/- NKA. The density of CGRP and SP-immunoreactive nerves within the subepithelium of 14 women with urodynamically-proven idiopathic detrusor instability was increased by 82% (p = 0.003) and 94% (p = 0.036), respectively, relative to that of 14 control women with no symptoms of frequency or urgency. This effect was not due to an increase in overall nerve density, because immunoreactivity for the general nerve marker, protein gene product, was not significantly different between the two groups (p = 0.21). The results indicate that at least some patients with detrusor instability demonstrate increases in a specific class of nerve fibers containing CGRP and SP. These peptides are characteristic of sensory neurons in a number of species, suggesting that abnormalities in the afferent arm of the micturition reflex may be associated with detrusor instability.

Adult↗
PubMed1998

DR/CLIP (class II-associated invariant chain peptides) and DR/peptide complexes colocalize in prelysosomes in human B lymphoblastoid cells.

In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1998

Expression of nerve growth factor and neurotrophin-3 mRNAs in hippocampal interneurons: morphological characterization, levels of expression, and colocalization of nerve growth factor and neurotrophin-3.

We have investigated the distribution and morphology of hippocampal interneurons that express the neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the rat. For this study, we combined in situ hybridization for the detection of NGF and NT-3 mRNAs and immunocytochemistry against the calcium-binding proteins parvalbumin (PARV), calretinin (CALR), and calbindin (CALB). Whereas the majority of PARV+ interneurons expressed NGF mRNA, only subsets of CALR- and CALB-immunoreactive interneurons (23% and 24%, respectively) displayed NGF hybridization. Most CALB/NGF+ cells were located in the stratum oriens/alveus of the CA3-CA1 regions, suggesting that they may include the population of CALB+, hippocamposeptal, nonpyramidal neurons. Most of the nonspiny CALR/NGF+ neurons were located within or in the vicinity of the pyramidal layer and had faint CALR immunostaining and stellate, thin dendrites. Regarding the spiny CALR-immunoreactive cells, we found that most of these neurons in the hilus were NGF+, whereas only 59% of displayed NGF hybridization in the stratum lucidum of the CA3 region. A small subset of PARV- and CALR-immunoreactive cells expressed NT-3 mRNA (16% and 13%, respectively). NT-3 message was not found in the large basket cells of the dentate gyrus, whereas the distribution and morphology of CALR/NT-3+ cells were similar to those of nonspiny CALR/NGF+ cells. In fact, double in situ hybridization analysis confirmed that most NT-3+ neurons also expressed NGF mRNA, indicating coexpression of both neurotrophins in subpopulations of PARV+ and CALR+ neurons. Moreover, the level of NGF mRNA expression was higher in PARV+ neurons than in CALR- and CALB-immunoreactive interneurons, whereas NT-3 message was expressed similarly in PARV+ and CALR+ neurons. The present findings show a differential expression of NGF and NT-3 mRNAs in subsets of hippocampal interneurons and suggest that the expression of these transcripts depends on factors intrinsic to particular cell types.

Animals↗
PubMed1998

Colocalization of 11-cis retinyl esters and retinyl ester hydrolase activity in retinal pigment epithelium plasma membrane.

PURPOSE: To identify the subcellular locale of 11-cis retinyl esters in bovine retinal pigment epithelium (RPE) and to characterize the enzymic mechanism responsible for liberation of 11-cis retinoids in this compartment. METHODS: Endoplasmic reticulum (ER)- enriched and plasma membrane (PM)-enriched protein fractions were prepared from bovine RPE microsomes using sequential discontinuous sucrose and Percoll gradient fractionation. Enzyme markers for ER (such as carboxylesterase), and PM (such as 5'-nucleotidase [5'-ND]; alkaline phosphatase [AP]; and ouabain-sensitive Na+,K+-ATPase [ATPase]) were used to identify the subfractions. Membrane-associated retinoids were quantified by high-performance liquid chromatography (HPLC) and retinyl ester hydrolase (REH) activities were determined by radiometric and chromatographic (HPLC) means. RESULTS: Chromatographic analyses of membrane-associated retinoids showed that 11-cis retinyl esters are localized mainly in PM-enriched fractions, whereas all-trans retinyl esters are associated predominantly with ER-enriched membranes; profiles of the distribution of 11-cis- and all-trans REH activities were consistent with the retinyl ester distribution. Further purification of the crude PM fraction yielded a fraction (P2) that was significantly enriched with 5'-ND (fivefold), ATPase (15-fold), AP (10-fold), and 11-cis retinyl ester hydrolase (11-cis REH; threefold) activities, but was relatively devoid of carboxylesterase and all-trans REH activities. Apparent kinetic constants (Km(app) and Vm(app)) for 11-cis REH activity in P2 were 18 microM and 1800 picomoles/min per mg, respectively. CONCLUSIONS: This is the first identification of an 11-cis-specific REH activity in RPE plasma membrane. Results from these studies demonstrate the capacity of RPE plasma membranes to accommodate and hydrolyze 11-cis retinyl esters. Plasma membrane storage and mobilization of 11-cis retinyl esters represents a novel compartmentalization of retinoid metabolism that is distinct from the sites where 11-cis retinoids are produced. The implication of these findings for present theories of visual chromophore biosynthesis are discussed.

5'-Nucleotidase↗
PubMed1998

An in vivo topoisomerase II cleavage site and a DNase I hypersensitive site colocalize near exon 9 in the MLL breakpoint cluster region.

The human myeloid-lymphoid leukemia gene, MLL (also called ALL-1, Htrx, or HRX ), maps to chromosomal band 11q23. MLL is involved in translocations that result in de novo acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), mixed lineage leukemia, and also in therapy AML (t-AML) and therapy ALL (t-ALL) resulting from treatment with DNA topoisomerase II (topo II) targeting drugs. MLL can recombine with more than 30 other chromosomal bands, of which 16 of the partner genes have been cloned. Breaks in MLL occur in an 8. 3-kb breakpoint cluster region (BCR) encompassing exons 5 through 11. We recently demonstrated that 75% of de novo patient breakpoints in MLL mapped in the centromeric half of the BCR between two scaffold-associated regions (SAR), whereas 75% of the t-AML patient breakpoints mapped to the telomeric half of the BCR within a strong SAR. We have mapped additional structural elements in the BCR. An in vivo DNA topo II cleavage site (induced with several different drugs that target topo II) mapped near exon 9 in three leukemia cell lines. A strong DNase I hypersensitive site (HS) also mapped near exon 9 in four leukemia cell lines, including two in which MLL was rearranged [a t(6;11) and a t(9;11)], and in two lymphoblastoid cell lines with normal MLL. Two of the leukemia cell lines also showed an in vivo topo II cleavage site. Our results suggest that the chromatin structure of the MLL BCR may influence the location of DNA breaks in both de novo and therapy-related leukemias. We propose that topo II is enriched in the MLL telomeric SAR and that it cleaves the DNase I HS site after treatment with topo II inhibitors. These events may be involved in recombination associated with t-AML/t-ALL breakpoints mapping in the MLL SAR.

Cell Transformation, Neoplastic↗
PubMed1999

High level expression of the Xlr nuclear protein in immature thymocytes and colocalization with the matrix-associated region-binding SATB1 protein.

The X-linked lymphocyte-regulated (Xlr) protein is a 30,000 Mr nuclear protein bearing homology with meiosis-specific proteins and expressed in late stage B lymphoid cell lines. In the present study we investigated its expression in the T lymphoid lineage. In adults, a high level of expression was detected in CD4-CD8- thymocytes. Most remarkably, the peak of Xlr expression occurred early during thymus cell ontogeny, precisely on days 14-15 of gestation, and was associated with the first wave of pre-T cell differentiation. Its onset preceded the rearrangement of TCR genes, as Xlr expression was conserved in thymus cells from RAG1(0/0) mice. The lower expression of Xlr on day 13 of fetal development, the bright Thy1+ phenotype of Xlr-positive cells, their large size, and their absence from subcapsular areas suggest that Xlr expression must be turned on within the thymus and not in prethymic precursors. From day 16 of gestation, Xlr expression decreased markedly. At birth and later, Xlr(high) cells were mostly large cells scattered throughout the cortical area. As shown by confocal microscopy, expression of Xlr closely overlapped that of SATB1, which binds special AT-rich DNA sequences associated with the nuclear matrix and plays an important regulatory role for many genes. The remarkably regulated expression of Xlr in the lymphoid cell lineage and of its homologue Xmr in the germ cell lineage suggests that they might play an important role in chromatin metabolism at critical stages of differentiation during which the genome undergoes irreversible rearrangements.

Animals↗
PubMed1999

T cell epitopes of a lipocalin allergen colocalize with the conserved regions of the molecule.

In this study we characterized the human T cell-reactive sites of the major cow dander allergen, Bos d 2, a member of the lipocalin protein family. We showed that Bos d 2 contains only a limited number of epitopes. This is in contrast to many other allergens, which usually contain multiple T cell epitopes throughout the molecule. The epitopes of Bos d 2 were primarily concentrated in the conserved regions of the molecule. One of the epitopes was recognized by all the cow-asthmatic individuals regardless of their HLA phenotype. Computer-predicted T cell epitopes on Bos d 2, other lipocalin allergens, and human endogenous lipocalins were situated in similar locations on these molecules and corresponded to experimentally identified epitopes on Bos d 2. The results suggest that human endogenous lipocalins could be involved in the modulation of immune responses against exogenous lipocalin allergens. In addition, our findings are likely to facilitate the development of new forms of immunotherapy against allergies induced by the important group of lipocalin allergens.

Allergens↗