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At least 1,153 records · Page 64Linked to original sources
PubMed1989

Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: a beta 3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells.

Human osteoclasts (OCLs) obtained from cell suspensions of surgically excised giant cell bone tumors (osteoclastomas) were attached to glass coverslips and analyzed by immunofluorescence with antibodies to integrins and cytoskeletal proteins. It was found that in OCLs (i) podosomes, identified by their F-actin core and by interference reflection microscopy, were predominantly found in a peripheral belt as described in avian OCLs; (ii) each F-actin core was surrounded by a ring of vinculin and talin; (iii) beta 1 integrin was diffuse in the ventral membrane; (iv) beta 3 integrin was distributed in intensely fluorescent rings surrounding F-actin cores; (v) beta 2 integrin was absent; (vi) beta 4 integrin was absent. The macrophages detected in the same coverslips displayed podosomes containing beta 2 but not beta 3, fibroblasts showed adhesion plaques positive for beta 1 and beta 3 but not for beta 2, and platelets were intensely positive for beta 3. These results indicate that OCLs produce an integrin complex that is absent in the monocyte-macrophage lineage.

Actins↗
PubMed1992

Nuclear colocalization of c-myc protein and hsp70 in cells transfected with human wild-type and mutant c-myc genes.

Using immunofluorescence and electron microscopy we have studied the localization of wild-type and mutant c-myc proteins transiently expressed in CV-1 cells. In agreement with our previous observations, wild-type c-myc protein accumulated in large amorphous globules in the nucleus. All mutant proteins tested accumulated in the nucleus as well, but gave rise to morphologically different inclusion bodies. Many small globules appeared in cells transfected with D145-262 (deletion of amino acids 145-262), while cells transfected with D371-412 or D414-433 generated structures looking like a fine network or like beads on a string. In addition, a particulate cytoplasmic staining appeared in some cells transfected with the wild-type gene and in cells transfected with mutants D145-262 or D414-433. Since the c-myc protein has been reported to stimulate expression of exogenous hsp70 protein, we also examined the intracellular distribution of hsp70 in the transfected cells. Double immunofluorescence microscopy revealed that hsp70 codistributed with the c-myc protein in distinct globules in the nucleus of many but not all myc-positive cells. However, the levels of hsp70 transcripts were not significantly raised compared to nontransfected and vector-transfected cells. Likewise, the levels of hsp70 protein did not vary significantly. These findings indicate that overexpression of c-myc stimulates translocation of preexisting hsp70 from the cytoplasm into the nucleus, rather than influencing hsp70 expression. Conceivably, this may represent one of several mechanisms whereby the cell deals with excessive amounts of c-myc protein.

Animals↗
PubMedDate not supplied

5-HT1A and 5-HT4 receptor colocalization on hippocampal pyramidal cells.

The mixed inhibitory and excitatory effects of 5-HT on hippocampal pyramidal cells were studied on hippocampal slices perfused with a low-Ca2+/high Mg2+ solution that blocked synaptic activity and induced spontaneous pyramidal cell discharge. Extracellular recordings of the spontaneous discharge revealed that, in 65% of the cells, 5-HT (0.5-10 microM) initially inhibited and then, upon washout, facilitated spontaneous discharge. Sometimes the off-stimulation persisted for the duration of the experiment. In 17% of the cells the response to 5-HT was only stimulatory, and in 15% the response was exclusively inhibitory. The 5-HT1 agonists, 8-hydroxy-dipropylamino-tetraline, and 5-carboxamidotryptamine produced inhibition with no excitatory responses upon washout. The inhibition was blocked by spiroxatrine indicating it was mediated by 5-HT1A receptors. The 5-HT3 agonist, 2-methyl 5-HT, had no effect, and the 5-HT2 antagonist, ketanserin, did not alter the excitatory responses to 5-HT. This indicates the excitatory response is not mediated by 5-HT2 or 3 receptors. Cisapride, a 5-HT4 agonist increased pyramidal cell discharge. The 5-HT3 & 4 antagonist, ICS 205-930 antagonized the excitatory responses to 5-HT, alpha-methyl 5-HT, and cisapride, indicating the excitatory response is mediated, in part, by 5-HT4 receptors. The phosphodiesterase inhibitor, isobutyl-methyl-xanthine, stimulated pyramidal cell discharge and potentiated the response to cisapride. This further suggests 5-HT4 receptor involvement since these receptors are positively coupled to adenylyl cyclase.

Animals↗
PubMed1994

Regulatory peptides in gastric endocrine cells of the rainbow trout Oncorhynchus mykiss: general distribution and colocalizations.

The endocrine cells of the rainbow trout (Oncorhynchus mykiss) stomach have been investigated using the immunocytochemical techniques of peroxidase-anti-peroxidase and avidin-biotin-peroxidase complexes on paraffin sections. 33 antisera were tested and eight immunoreactivities were detected: somatostatin-, glucagon- bombesin-, substance P-, serotonin-, met-enkephalin-, CCK/gastrin-, and chromogranin-like containing cells. All of them were present throughout the gastric mucosa except CCK/gastrin-like containing cells that were restricted to the pyloric epithelium. Somatostatin 25 and chromogranin immunoreactive cells are described for the first time in fish stomach. Serotonin immunoreactive cells were also positive for the Grimelius technique and some of them were immunoreactive to anti substance P or anti CCK/gastrin. Immunoreactivities for gastrin 17, gastrin 34 and CCK appeared in the same cells and the absorption controls showed that a molecule containing the carboxi-terminal pentapeptide of this family was present in trout stomach.

APUD Cells↗
PubMed1987

Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor.

Using a combination of a transient expression assay and in vitro mutagenesis, we showed previously that the human glucocorticoid receptor (hGR) is composed of a series of discrete functional domains. Here we report the effects of selective deletion of each of these domains on hGR ability to activate transcription of the MTV-CAT fusion gene. Deletion of the immunogenic domain or the entire amino-terminal half of the protein reduces but does not abolish the ability of the hGR to induce transcriptional activation. Somewhat surprisingly, deletion of the steroid-binding domain engenders a constitutively active receptor, revealing that this domain normally represses receptor function. However, the central, cysteine-rich region contains all the information required for both DNA binding and trans-activation. Taken together, these data delineate a core domain in the hGR spanning 88 amino acids that determines both DNA-binding and transcriptional activation functions. This physical linkage distinguishes the glucocorticoid receptor from other described eukaryotic regulatory proteins, where these two functions have been shown to be separable.

Animals↗
PubMed1990

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor.

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Amino Acid Sequence↗
PubMed1993

Substance P and neurokinin A are colocalized in the central chemosensory pathway of the male golden hamster.

The medial nucleus of the amygdala, bed nucleus of the stria terminalis and medial preoptic area play critical roles in the regulation of mating behavior in the male hamster. Destruction of these nuclei or the pathways that connect them severely disrupt copulation. We have begun identifying the neuropeptides contained in these neurons as a prelude to determining the role of peptide neurotransmitters in the regulation of male copulatory behavior. We have found that substance P is localized within these neurons and is regulated by gonadal steroids. In this study we report 1) that a closely related peptide, NKA (substance K), is also present in the medial nucleus of the amygdala, the bed nucleus of the stria terminalis and the medial preoptic area; 2) that all those neurons which contain SP also contain NKA and 3) testosterone also regulates the production of NKA. Thus, NKA may also play a role in the regulation of male copulatory behavior.

Amygdala↗
PubMed1989

Vasoactive intestinal peptide distribution and colocalization with dopamine-beta-hydroxylase in sympathetic chain ganglia of pig.

The extent to which vasoactive intestinal peptide (VIP) occurs in postganglionic cells of the sympathetic chain and whether these cells are noradrenergic, cholinergic, or neither is unknown. We have (1) established the extent of VIP-containing cell bodies and fibers in all levels of the sympathetic chain in pig and (2) determined the coexistence of VIP and dopamine-beta-hydroxylase (DBH) using immunofluorescence. Weanling pigs were perfusion fixed. Alternate serial frozen sections of cervical, thoracic and lumbar paravertebral ganglia were processed for immunofluorescence with antisera to VIP or DBH. VIP-immunoreactive cells were most evident in thoracic ganglia with a few occurring in stellate and lumbar ganglia. While the cells were distributed throughout individual thoracic ganglia, intensely stained cells consistently occurred just proximal to exiting fiber bundles and sent long processes into these bundles. VIP-immunoreactive fibers occurred in all ganglia. Fiber density varied at each level. Varicose fibers with VIP staining were found to be especially evident from the caudal cervicothoracic ganglion through upper lumbar ganglia, in many cases forming networks of beaded varicosities around principal ganglion cells. Fibers and cells were associated with both interganglionic connectives and rami. A majority of neurons in all ganglia were DBH-immunoreactive but with varying intensities. VIP- and DBH-immunoreactivity coexisted in the same perikarya in some cells in thoracic ganglia, preferentially in cells moderately DBH-immunoreactive. We conclude that VIP-immunoreactive cells are distributed primarily in the thoracic sympathetic chain ganglia and that fibers containing VIP are found at all ganglia levels. Additionally, some VIP-immunoreactive postganglionic neurons in paravertebral ganglia may be noradrenergic.

Animals↗
PubMed1994

The distribution and colocalization of neuropeptides in fish cardiac neurons.

Most if not all intracardiac nerve cell bodies in four species of teleost fish and a primitive air breathing fish contained immunoreactivity (IR) to vasoactive intestinal peptide (VIP). Intracardiac nerve cell bodies contained no other neuropeptide although galanin (GAL)-, substance P (SP)- and calcitonin gene-related peptide (CGRP)-IR were detected in cardiac axons. Varicose VIP-IR axons were observed in close association to the cardiac muscle in the sinus venosus and atrium, but not in the ventricle. Slightly less than half the total number of VIP-IR axons also contained colocalised GAL-IR. A smaller number of varicose axons containing colocalised SP- and CGRP-IR were also present in the sinus venous and atrium. In addition, a subpopulation of CGRP-IR axons present in the sinus venosus and atrium did not contain SP-IR. SP-IR axons lacking CGRP-IR formed boutons around the axon hillock and soma of the majority of VIP-IR nerve cell bodies. Associated with a small number of VIP/-ganglion cells were VIP/- boutons. No neuropeptides were observed in the ventricle of any species of fish studied here. These results suggest that a VIP-like peptide is localised in the cholinergic postganglionic parasympathetic neurons. Associated with some of these neurons are nerve boutons containing either SP alone or VIP alone. In addition, the fish heart is innervated by extrinsic nerve fibres containing: GAL/VIP; CGRP alone; and CGRP/SP.

Animals↗
PubMed1993

Immunocytochemical detection of insulin receptor substrate-1 (IRS-1) in rat brain: colocalization with phosphotyrosine.

In peripheral insulin-sensitive tissues, insulin receptor substrate (IRS-1) undergoes tyrosine phosphorylation immediately after cells are stimulated by insulin or insulin-like growth factor-1 (IGF-1), and may function as a molecular link between insulin/IGF-1 receptor tyrosine kinases and enzymes regulating cell growth and metabolism. A fundamental question pertaining to insulin/IGF-1 action in the brain is whether IRS-1 is expressed by neurons. In this study, the distribution of cells containing immunoreactivity to IRS-1 in the brain was determined by immunocytochemistry with polyclonal IRS-1 antiserum, and compared to the localization of immunostaining for phosphotyrosine using polyclonal phosphotyrosine antiserum. The immunostaining results with ABC-peroxidase method and cryostat sections showed the presence of IRS-1 immunoreactivity in many neuron cell bodies throughout the rat forebrain, particularly in the habenula, cerebral cortex and piriform cortex. In the hypothalamus, IRS-1 immunostaining was present in neurons of the paraventricular nucleus, supraoptic nucleus, and arcuate nucleus. The choroid plexus stained intensely for IRS-1. The populations of cells that stained for IRS-1 also showed strong immunostaining for phosphotyrosine. Studies at the cellular level are needed to verify coexpression of IRS-1 and receptors for insulin or IGF-1 by the same neurons, as well as in cells of the choroid plexus. The present results are the first demonstration of IRS-1 expression by neurons in adult mammalian brain. These findings are consistent with the hypothesis that insulin and IGF-1 actions in the brain involve signal transduction mechanisms common to those found in peripheral tissues.

Animals↗
PubMed1995

Colocalization of vasoactive intestinal polypeptide and nitric oxide in penis-innervating neurons in the major pelvic ganglion of the rat.

By using a combination of fluorescent retrograde labeling and double immunofluorescence histochemistry, the present study revealed in the rat that the vast majority of major pelvic ganglion neurons innervating penile erectile tissue contained both vasoactive intestinal polypeptide (VIP) and nitric oxide synthase. The result suggests that VIP and nitric oxide may exert a cooperative action in penile erection.

Amino Acid Oxidoreductases↗
PubMed1996

Altered expression of Bcl-2, Bcl-X, Bax, and c-Fos colocalizes with DNA fragmentation and ischemic cell damage following middle cerebral artery occlusion in rats.

Permanent occlusion of the middle cerebral artery in rats was used to assess the effects of focal ischemia on the expression of members of the bcl-2 family which have been implicated in the regulation of programmed cell death. Intraluminal occlusion of one middle cerebral artery for 6 h resulted in histologically detectable brain damage within the ipsilateral caudate putamen, basolateral cortex and parts of the thalamus. In the infarcted basolateral cortex and thalamus fragmentation of DNA was detected in many nuclei using in-situ end-labeling of DNA breaks by terminal transferase, whereas only scattered labeled nuclei were visible in the infarcted caudate putamen. Immunohistochemical analysis revealed activation of c-Fos in the infarcted cortex and thalamus and in the non-infarcted cingulate cortex as has been shown by others. A decrease in immunoreactivity for Bcl-2, and Bcl-X and an increase in immunostaining for Bax was observed exclusively in neurons within the ischemic cortex and thalamus. Within the infarcted caudate putamen, however, protein levels of all bcl-2 family members declined and c-Fos remained absent. By reverse transcription and polymerase chain reaction it was demonstrated that levels of bcl-2 mRNA markedly decreased in the ipsilateral hemisphere, whereas the amount of bax mRNA was elevated. These findings suggest that a shift in the ratio of cell death repressor Bcl-2 to cell death effector Bax and a concomitant activation of c-Fos may contribute to neuronal apoptosis in the infarcted thalamus and cortex.

Animals↗
PubMedDate not supplied

Melanin-concentrating hormone (MCH) immunoreactivity in the brain and pituitary of the dogfish Scyliorhinus canicula. Colocalization with alpha-melanocyte-stimulating hormone (alpha-MSH) in hypothalamic neurons.

The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the dogfish Scyliorhinus canicula was determined by indirect immunofluorescence and peroxidase-anti-peroxidase techniques, using an antiserum raised against synthetic salmon MCH. Three groups of MCH-positive cell bodies were localized in the posterior hypothalamus. The most prominent cell group was detected in the nucleus sacci vasculosi. Scattered MCH-immunoreactive cells were observed in the nucleus tuberculi posterioris and in the nucleus lateralis tuberis. At the pituitary level, the caudal part of the median lobe of the pars distalis contained strongly MCH-positive perikarya. Some of these cells were liquor-contacting-type. Immunoreactive fibers originating from the hypothalamic perikarya projected throughout the dorsal wall of the posterior hypothalamus. Positive fibers were also detected within the thalamus and the central gray of the mesencephalon. The distribution of MCH-containing neurons was compared to that of alpha-MSH-immunoreactive elements using consecutive, 5-micron thick sections. Both MCH- and alpha-MSH-immunoreactive peptides were found in the same neurons of the nucleus sacci vasculosi. These data suggest that MCH and alpha-MSH, two neuropeptides which exert antagonistic activities on skin melanophores, may also act in a coordinate manner in the central nervous system of cartilaginous fish.

Animals↗
PubMedDate not supplied

Substance P and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization and effects on uterine contractility.

Immunoreactivity to the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, SP immunoreactivity (SP-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns, with nerves immunoreactive for CGRP being the more numerous. Immunocytochemical double labeling studies revealed SP coexisted with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., SP-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. SP (10(-4) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP(1-37) and CGRP(8-37) had no effect on basal uterine tension. While CGRP(1-37) (10(-7) M) reduced SP-stimulated (10(-5) M) uterine contraction by 56%, CGRP(8-37) had no effect on SP-stimulated uterine contraction. However, CGRP(8-37) (10(-6) M) significantly reduced the ability of CGRP(1-37) (10(-7) M) to inhibit SP-stimulated uterine contraction. These results demonstrate that SP- and CGRP-I are present in, and coexist in some uterine nerves, presumably afferent nerves. The first 7 amino acids are necessary for the inhibitory effect of CGRP(1-37) on stimulated uterine contraction. In addition, CGRP(8-37) acted as an antagonist to this inhibitory action. SP and CGRP could be coreleased from afferent fibers in an "efferent fashion" and influence uterine contractility. SP having a contractile effect and CGRP having a relaxing effect.

Animals↗
PubMedDate not supplied

Galanin and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization, and effects on uterine contractility.

Immunoreactivity to the neuropeptides galanin (GAL) and calcitonin gene-related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, GAL immunoreactivity (GAL-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns and cervix, with nerves immunoreactive for CGRP being more numerous. Immunocytochemical double-labeling studies revealed GAL coexists with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., GAL-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. GAL (10(-5) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP had no effect on basal uterine tension, but CGRP (10(-7) M) reduced GAL-stimulated (10(-7) M) uterine contraction by 92.5%. These results demonstrate that GAL- and CGRP-I are present in, and coexist in, some uterine nerves, presumably afferent nerves. GAL and CGRP could be released from afferent fibers in an "efferent fashion" and influence uterine contractility, GAL having a contractile effect and CGRP having a relaxing effect.

Animals↗
PubMed1996

Neuropeptide Y in hamster limbic nuclei: lack of colocalization with substance P.

The medial nucleus of the amygdala (Me), bed nucleus of the stria terminalis (BNST), and medial preoptic area (MPOA) regulate copulation in the male hamster. The present study identified neuropeptide Y-immunoreactive (NPY-IR) neurons in the BNST and Me with the greatest concentration in the posteromedial and posteriordorsal subdivisions of these nuclei, respectively. NPY-IR filters are found in all three nuclei with dense plexi of NPY-IR varicosities in the most medial subdivisions. Substance P neurons are also densely concentrated in the posterior BNST and Me; however, no neurons contained both peptides. Thus, NPY and substance P neurons comprise two distinct populations within the BNST and Me of the hamster.

Amygdala↗