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At least 1,423 records · Page 79Linked to original sources
PubMed1994

Replication and transcription sites are colocalized in human cells.

HeLa cells synchronized at different stages of the cell cycle were permeabilized and incubated with analogues of nucleotide triphosphates; then sites of incorporation were immunolabeled with the appropriate fluorescent probes. Confocal microscopy showed that sites of replication and transcription were not diffusely spread throughout nuclei, reflecting the distribution of euchromatin; rather, they were concentrated in 'foci' where many polymerases act together. Transcription foci aggregated as cells progressed towards the G1/S boundary; later they dispersed and became more diffuse. Replication was initiated only at transcription sites; later, when heterochromatin was replicated in enlarged foci, these remained sites of transcription. This illustrates the dynamic nature of nuclear architecture and suggests that transcription may be required for the initiation of DNA synthesis.

Autoantigens↗
PubMed1996

Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons.

The glucocorticoid receptor and the mineralocorticoid receptor are hormone-dependent transcription factors. They regulate the excitability of rat hippocampus CA1 neurons in a coordinated fashion. We studied the spatial distribution of these transcription factors in nuclei of CA1 neurons by dual labeling immunocytochemistry and confocal microscopy, combined with novel image restoration and image analysis techniques. We found that both receptors are concentrated in about one thousand clusters within the nucleus. Some clusters contain either mineralocorticoid receptors or glucocorticoid receptors, but a significant number of clusters contains both receptors. These results indicate that the two receptor types are targeted to specific compartments in the nucleus. The coordinated action of the glucocorticoid and mineralocorticoid receptor on gene expression may be established in a specific set of nuclear domains that contain both receptors.

Adrenalectomy↗
PubMed2003

Increased chymase in livers with autoimmune disease: colocalization with fibrosis.

Chymase, one of the proteases contained in human mast cells, promotes myocardial and renal interstitial fibrosis by converting angiotensin I to II (AII). We previously established a method for measuring chymase in liver tissue and examined the relationship between chymase and fibrosis in chronic hepatitis. In the present study, chymase was determined in liver specimens affected by autoimmune hepatitis (AIH, n=10) or primary biliary cirrhosis (PBC, n=12). To investigate spatial relationships between hepatic fibrosis and human chymase, mast cell distribution in the specimens was determined immunohistochemically using anti-chymase antibody. The mean amounts of chymase in livers with AIH and PBC were 11.56+/-10.64 and 11.67+/-9.96 ng/mg respectively. Hepatic chymase in AIH and PBC was significantly more abundant than in acute hepatitis (AH, 2.72+/-2.23 ng/mg, n=10; p<0.05). When sections from patients with AIH and PBC were immunostained for chymase, immunoreactive mast cells were detected in portal areas and sinusoidal walls, coinciding with zones of fibrosis. Thus chymase appears to be involved in hepatic fibrosis in AIH and PBC.

Chymases↗
PubMed2001

Colocalization of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephrology syndrome by double-labeling technique.

Simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) was achieved by a double-labeling technique using a combination of immunohistochemistry and in situ hybridization in five pigs with naturally occurring porcine dermatitis and nephropathy syndrome (PDNS). Both PRRSV and PCV2 were isolated from a homogenate of pooled skin and kidney from three pigs. PRRSV RNA was demonstrated by reverse transcription polymerase chain reaction (PCR) in skin, kidney, lymph node, and tonsil homogenates from all pigs. PCV2 DNA was demonstrated by PCR in kidney, lymph node, tonsil, liver, and lung homogenates from all pigs. For double-labeling studies, the tissue samples were processed sequentially, first by immunohistochemistry and then by in situ hybridization. The most consistent and intense staining for PRRSV and PCV2 was in the kidney, lymph node, and tonsil. PRRSV antigen and PCV2 DNA were also detected in the skin. This morphologic study is the first to confirm the presence of both PRRSV and PCV2 in the same tissues in pigs with naturally occurring PDNS.

Animals↗
PubMed2001

Colocalization of noggin and bone morphogenetic protein-4 during fracture healing.

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to bone morphogenetic protein (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, 10, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.

Animals↗
PubMed2005

Colocalization of multiple laminin isoforms predominantly beneath hemidesmosomes in the upper lamina densa of the epidermal basement membrane.

Multiple laminin isoforms including laminins 5 (alpha3 beta3 gamma2), 6 (alpha3 beta1 gamma1), 10 (alpha5 beta1 gamma1), and possibly laminins 7 (alpha3 beta2 gamma1) and 11 (alpha5 beta2 gamma1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (alpha3 beta3 gamma2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the alpha5, beta1, beta2, and gamma1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the alpha5, beta1, and gamma1 chains (present in laminins 10/11) and beta2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the alpha5, beta1, beta2, and gamma1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the alpha5, beta1, beta2, and gamma1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n> or =200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the alpha5beta1/beta2gamma1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane.

Adolescent↗
PubMed2006

The use of combined immunohistochemical labeling and in situ hybridization to colocalize mRNA and protein in tissue sections.

This chapter explores the combination of a nonradioactive in situ hybridization technique to detect mRNA with an imunnohistochemical labeling technique for use in formalin-fixed, paraffin-embedded, or frozen tissue sections. This technique allows the combination of detecting mRNA by in situ hybridization with immunohistochemical detection of a protein product or a cell surface marker without using any radioactive procedures. This technique is ideal for use on tissue sections when the aim is to identify which cells are producing a secreted protein product, such as a cytokine.

Fixatives↗
PubMed2005

Antidepressants and antipsychotic drugs colocalize with 5-HT3 receptors in raft-like domains.

Despite different chemical structure and pharmacodynamic signaling pathways, a variety of antidepressants and antipsychotics inhibit ion fluxes through 5-HT3 receptors in a noncompetitive manner with the exception of the known competitive antagonists mirtazapine and clozapine. To further investigate the mechanisms underlying the noncompetitive inhibition of the serotonin-evoked cation current, we quantified the concentrations of different types of antidepressants and antipsychotics in fractions of sucrose flotation gradients isolated from HEK293 (human embryonic kidney 293) cells stably transfected with the 5-HT3A receptor and of N1E-115 neuroblastoma cells in relation to the localization of the 5-HT3 receptor protein within the cell membrane. Western blots revealed a localization of the 5-HT3 receptor protein exclusively in the low buoyant density (LBD) fractions compatible with a localization within raft-like domains. Also, the antidepressants desipramine, fluoxetine, and reboxetine and the antipsychotics fluphenazine, haloperidol, and clozapine were markedly enriched in LBD fractions, whereas no accumulation occurs for mirtazapine, carbamazepine, moclobemide, and risperidone. The concentrations of psychopharmacological drugs within LBD fractions was strongly associated with their inhibitory potency against serotonin-induced cation currents. The noncompetitive antagonism of antidepressants at the 5-HT3 receptor was not conferred by an enhancement of receptor internalization as shown by immunofluorescence studies, assessment of receptor density in clathrin-coated vesicles, and electrophysiological recordings after coexpression of a dominant-negative mutant of dynamin I, which inhibits receptor internalization. In conclusion, enrichment of antidepressants and antipsychotics in raft-like domains within the cell membrane appears to be crucial for their antagonistic effects at ligand-gated ion channels such as 5-HT3 receptors.

Antidepressive Agents↗
PubMed2006

Compartment-dependent colocalization of Kir3.2-containing K+ channels and GABAB receptors in hippocampal pyramidal cells.

G-protein-coupled inwardly rectifying K+ channels (Kir3 channels) coupled to metabotropic GABAB receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABAB receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABA(B1). Consistent with this observation, a high degree of coclustering of Kir3.2 and GABA(B1) was revealed around excitatory synapses by the highly sensitive SDS-digested freeze-fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K+ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites.

Animals↗
PubMed2004

Consequences of molecular-level Ca2+ channel and synaptic vesicle colocalization for the Ca2+ microdomain and neurotransmitter exocytosis: a monte carlo study.

Morphological and biochemical studies indicate association between voltage-gated Ca2+ channels and the vesicle docking complex at vertebrate presynaptic active zones, which constrain the separation between some Ca2+ channels and vesicles to 20 nm or less. To address the effect of the precise geometrical relationship among the vesicles, the Ca2+ channel, and the proteins of the release machinery on neurotransmitter release, we developed a Monte Carlo simulation of Ca2+ diffusion and buffering with nanometer resolution. We find that the presence of a vesicle as a diffusion barrier alters the shape of the Ca2+ microdomain of a single Ca2+ channel around the vesicle. This effect is maximal in the vicinity of the vesicle and depends critically on the vesicle's distance from the plasmalemma. Ca2+-sensor(s) for release would be exposed to markedly different [Ca2+], varying by up to 13-fold, depending on their position around the vesicle. As a result, the precise position of Ca2+-sensor(s) with respect to the vesicle and the channel can be critical to determining the release probability. Variation in the position of Ca2+-sensor molecule(s) and their accessibility could be an important source of heterogeneity in vesicle release probability.

Calcium↗
PubMed2004

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells.

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Autoantibodies↗
PubMed1993

Electron microscopic immunocytochemistry on the colocalization of ATP synthase and cytochrome P-450 of side chain cleavage enzymes in mitochondria of rat and bovine adrenal cortical cells.

To clarify whether ATP synthase and side chain cleavage enzymes (SCC) can coexist in a mitochondrion of the adrenal cortical cell, and whether the functional heterogeneity of mitochondria can occur in an adrenal cortical cell, the immunostainability of these two enzymes in mitochondria was compared by electron microscope using pre- and post-embedding methods. In the pre-embedding method, the inner membrane of most mitochondria in the adrenal cortical cell was positively stained for SCC, 11 beta-hydroxylase, and ATP synthase, but among these immunopositive mitochondria we often observed negatively immunoreacted ones in the same adrenal cortical cell. In the positively stained mitochondria, immunoreaction products were evenly localized along the inner mitochondrial membrane. We therefore think that these differences in the immunostainability are caused by technical artifacts, namely the inadequate penetration of the antibody. In the post-embedding immunocytochemistry, gold particles showing the presence of the enzymes were observed on all mitochondria. Two different antibodies, anti-ATP synthase antibody and anti-cytochrome P-450scc antibody, which were labelled with gold particles of varying diameter (5 nm, 10 nm each) could be observed on all the mitochondria of the bovine adrenal cortical cells. No heterogeneities as to the stainability for both ATP synthase and SCC were detected in mitochondria of the rat and bovine adrenal cortical cells. These results indicate that in the rat and bovine adrenal cortical cells, both ATP synthase and SCC are evenly and simultaneously present on the inner membrane of all mitochondria. Hence, mitochondria in the adrenal cortical cell seem to be homogeneous in their steroid and ATP synthesizing abilities.

Adrenal Cortex↗
PubMed1986

Immunohistochemical colocalization of GABA and insulin in beta-cells of rat islet.

gamma-Aminobutyric acid (GABA) is found in high concentrations in the pancreatic islet. In addition, enzymes regulating the level of GABA (L-glutamate decarboxylase and GABA-alpha-ketoglutarate transaminase) have been immunohistochemically localized in the medullary cells of the islet. In this study, an immunofluorescence and elution/restaining protocol is used to determine the distribution of GABA and either insulin, glucagon, or somatostatin in a tissue section. GABA was not detected within the islet alpha- or delta-cells but was determined to be localized within the insulin-containing beta-cells.

Animals↗
PubMed1997

The significance of colocalization of plasminogen activator inhibitor-1 and vitronectin in hepatic fibrosis.

BACKGROUND: We examined the relationships among vitronectin (VN), plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor beta 1 (TGF-beta 1) in liver diseases to evaluate the presence of plasmin cascade in human hepatic fibrosis. METHODS: Blood and liver tissues were obtained from 57 patients with liver disease. Plasma VN, PAI-1 antigen, and PAI-1 activity levels were evaluated. Biopsied liver specimens were observed by light and electron microscopy after immunohistochemical staining. Morphometric analysis was performed on these specimens. RESULTS: Plasma VN and PAI-1 activity levels decreased significantly with the progression of hepatic fibrosis and were particularly marked in the liver cirrhosis group. Plasma PAI-1 antigen level increased significantly. The immunolocalization of the active form of TGF-beta became more intense with the progression of hepatic fibrosis, whereas that of the dual-stained positive areas of PAI-1 and VN (PAI-1.VN) decreased. There was a positive correlation between TGF-beta and PAI-1, whereas there was a negative correlation between TGF-beta and PAI-1.VN. Immunoelectron microscopy showed the localization of PAI-1-VN in the extracellular space around the sinusoidal cells or surface of aggregating platelets, TGF-beta mainly in Ito cells, and VN in hepatocytes near the focal necrotic area or fibrous septa. CONCLUSIONS: These findings suggest that VN and PAI-1 are related to the active form of TGF-beta and that it is possible that the plasmin cascade is present in the human liver.

Adult↗
PubMedDate not supplied

Colocalization of immunoglobulin binding factor and prostate specific antigen in human prostate gland.

Immunoglobulin binding factor (IgBF) produced in the prostate is a useful marker for the diagnosis of prostatic tumor. IgBF was localized in the majority of epithelial cells of benign prostatic hypertrophy by an immunohistochemical technique. Prostate specific antigen (PSA), a known marker for prostatic cancer, was localized to all epithelial cells. Double immunolabeling of IgBF and PSA using fluorescent methods revealed that all epithelial cells producing IgBF were also immunopositive for PSA and some cells were positive only for PSA. The present findings suggest that the prostatic glands consist of two types of epithelial cells, one producing both IgBF and PSA and the other producing PSA alone.

Humans↗