Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “colocalization analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45Linked to original sources
PubMed2006

Quantitative analysis of immunofluorescent punctate staining of synaptically localized proteins using confocal microscopy and stereology.

We established a protocol for the immunofluorescent detection of glutamate receptor subunits at synaptic sites using laser scanning confocal microscopy and stereological procedures. An in vitro model of eyeblink classical conditioning from turtles was used for this study. Triple-labeling of the presynaptic marker synaptophysin, the NR1 subunit of NMDA receptors, and the GluR4 subunit of AMPA receptors was performed on pseudoconditioned (control) and conditioned in vitro brain stem preparations in which punctate staining for each individual protein, as well as for the colocalization of GluR4 and NR1 with synaptophysin, was analyzed. For every tissue section analyzed, images of two consecutive optical planes were taken using confocal microscopy. Protein puncta were counted in one optical section (sample section) if they were not present in the optical section immediately above the sample section (look-up section). We found a significant increase in the colocalization of GluR4-containing AMPA receptors with synaptophysin after conditioning compared with the control group. Colocalization of NR1 subunits with synaptophysin was unchanged after conditioning. The described protocol, therefore, can be used for the quantitative analysis of changes in synaptic localization of different types of proteins. The protocol is designed to provide a more accurate and uniform approach in studying receptor trafficking during various forms of synaptic plasticity.

Animals↗
PubMed2000

Tid1/Rdh54 promotes colocalization of rad51 and dmc1 during meiotic recombination.

Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1/RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54 mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also contain a tid1/rdh54 mutation. The role of Tid1/Rdh54 in coordinating RecA homolog assembly may be very direct, because Tid1/Rdh54 is known to physically bind both Dmc1 and Rad51. Also, Dmc1 foci appear early in a tid1/rdh54 mutant. Thus, Tid1 may normally act with Rad51 to promote ordered RecA homolog assembly by blocking Dmc1 until Rad51 is present. Finally, whereas double-staining foci predominate in WT nuclei, a subset of nuclei with expanded chromatin exhibit individual Rad51 and Dmc1 foci side-by-side, suggesting that a Rad51 homo-oligomer and a Dmc1 homo-oligomer assemble next to one another at the site of a single double-strand break (DSB) recombination intermediate.

Animals↗
PubMed1995

Cell type differences in Golgi retention signals for transmembrane proteins.

The transmembrane domain of Golgi resident proteins such as beta-galactoside alpha 2,6-sialyltransferase (ST) and N-acetylglucosaminyltransferase 1 (NT) contain a Golgi retention signal which confers Golgi retention to reporter proteins appended to them in the appropriate context. Thus, chimeras of the cell surface protein dipeptidyl peptidase IV containing the transmembrane domain of ST and NT are retained in the Golgi apparatus in MDCK and COS cells, as assessed by indirect immunofluorescence microscopy. Transfection of these chimeric constructs into CHO cells, however, results in their transport to vesicular structures which do not colocalize with that of an endogenous Golgi marker, mannosidase II. Furthermore, the staining pattern of these structures are not affected by brefeldin A. Biochemical analysis of the transgene products in pulse-chase experiments revealed that the chimeric proteins eventually become resistant to endoglycosidase H, suggesting that they are transported beyond the medial Golgi and therefore the vesicular structures are likely to be post-Golgi. The vesicular structures colocalized well with a lysosomal marker, cathepsin D, and also with internalized FITC-dextran chased into the lysosomal compartment. Monitoring the cell surface appearance of the chimeric protein suggests that the majority is transported directly to the lysosomal compartment. Golgi retention can be completely restored for ST and improved for NT by the inclusion of sequences flanking the transmembrane domain. Our results reflect cell type differences in the interpretation of the transmembrane domain Golgi retention signal, established that general Golgi retention of type II glycosyltransferases requires the hydrophilic flanking sequence as well as the transmembrane domain, and demonstrate that proteins which escape Golgi retention may be channeled to the lysosomal pathway.

Animals↗
PubMed2006

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Genome-wide associations between hybrid sterility QTL and marker transmission ratio distortion.

Marker transmission ratio distortion (TRD) in genetic mapping populations is frequently ascribed to selection against allelic combinations that cause hybrid incompatibility. Accordingly, genomic regions of TRD should be nonrandomly associated (colocated) with loci that underlie hybrid incompatibility. To directly test this hypothesis, we evaluated the genome-wide qualitative and quantitative agreement between chromosomal regions exhibiting marker TRD and those known to contain hybrid incompatibility quantitative trait locus (QTL). Incompatibility data came from a near-isogenic line (NIL) analysis of pollen and seed sterility in a cross between two Solanum (formerly Lycopersicon) species. We assessed (1) whether these incompatibility loci are colocated with markers that show significant TRD in two earlier generations preceding these introgression lines and (2) whether the magnitude of marker distortion quantitatively matches the estimated strength of selection against each incompatibility locus. We found evidence that TRD regions are chromosomally colocated with hybrid incompatibility loci more frequently than is expected by chance: pollen sterility QTLs were most closely associated with distorted heterozygote frequencies in later-generation backcrosses. Nonetheless, there was no evidence for an association between TRD and seed sterility and little evidence of a quantitative association between the magnitude of marker TRD and the fitness effects of heterospecific alleles at each chromosomal location. We propose and test a model (the "dance partner" model) to explain several cases where regions of TRD are not associated with hybrid incompatibility loci. Under this model, some NILs containing greater than one heterospecific introgression may not express hybrid incompatibility phenotypes because they carry both appropriate genetic dance partners required for a fully functional interaction. Accordingly, negative interactions expressed in earlier backcross generations are masked in these double-introgression NILs. Based on this model, we identify the location of several new putative pairwise interactors underlying hybrid incompatibility in this species cross.

Chromosome Mapping↗
PubMed2006

A rat homologue of CED-6 is expressed in neurons and interacts with clathrin.

We isolated from a brain library a cDNA encoding an isoform of rat CED-6 that has not been previously described. This transcript results from alternative splicing of the ced-6 gene present on chromosome 9. We expressed this isoform as his-tagged protein in E. coli and used the purified protein to raise antibodies to investigate the expression of CED-6 in rat brain. Immunoblot analysis showed the presence of CED-6 as a doublet of approximately 34 and 33 kDa in cortex, hippocampus and cerebellum, indicating that the protein was present in different regions of the brain. Subcellular fractionation experiments showed that CED-6 immunoreactivity did not concentrate in GFAP-containing glial vesicles, whereas it showed a distribution similar to the synaptotagmin in synaptosomes-enriched fractions, suggesting that CED-6 is present in neurons. CED-6 immunoreactivity was also investigated using immunohistochemistry analysis and it was found in several brain regions, being particularly strong in the cell body of some groups of neurons such as Purkinje cell layer of cerebellum, and pyramidal cells of the hippocampal formation and also in epithelial cells from the choroid plexus. Importantly, CED-6 immunoreactivity colocalized with a neuronal marker but not with a glial marker. Considering that several PTB-containing proteins bind clathrin, we investigated whether rat CED-6 would also have this property. Yeast two-hybrid and GST pull-down analysis indicated that ratCED-6 interacts with clathrin and in cultured cells we detected colocalization between CED-6 and clathrin-coated vesicles. The present findings suggest that CED-6 may have a role in endocytic trafficking or signaling in neurons.

Amino Acid Sequence↗
PubMed1993

Analysis of Candida albicans adhesion to salivary mucin.

Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.

Animals↗
PubMed1989

A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity.

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.

Animals↗
PubMed2000

Herpes simplex virus UL17 protein is associated with B capsids and colocalizes with ICP35 and VP5 in infected cells.

A previous study using a mutant lacking the UL17 gene has suggested that the UL17 protein of herpes simplex virus type 1 (HSV-1) is required for the cleavage/packaging of viral DNA. In this study, we have raised a rabbit polyclonal antiserum which specifically reacted with the UL17 protein which has an apparent molecular mass of 78-kDa in the lysates of HSV types 1- and 2-infected Vero cells. Western blot analysis of intracellular capsids demonstrates that the UL17 protein was associated with B and C capsids. Indirect immunofluorescence studies reveal that it colocalized with the major capsid protein VP5 and the scaffolding protein ICP35 within the nucleus. These results suggest that the association of the UL17 protein with immature B-type capsids is important for its role in cleavage/packaging.

Animals↗
PubMed2001

Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus.

We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.

Animals↗
PubMed2006

Involvement of integrin-linked kinase in carbon tetrachloride-induced hepatic fibrosis in rats.

Integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transduction between integrins and growth factor receptors. Although its expression is upregulated in pulmonary and renal fibrosis, its role in the development of hepatic fibrosis remains to be determined. Therefore, we considered it important to investigate whether ILK is involved in activation of hepatic stellate cells and thus plays a role in the development of hepatic fibrosis. Immunohistochemical analysis of liver sections obtained from rats with CCl4-induced cirrhosis revealed increased expression and colocalization of ILK and alpha-smooth muscle actin in hepatic stellate cells in perisinusoidal areas. In addition, hepatic stellate cells isolated from fibrotic livers expressed high levels of ILK and alpha-smooth muscle actin, and their expression was sustained in culture. In contrast, hepatic stellate cells (HSCs) isolated from normal rat liver did not express ILK, but its expression was increased when the cells were activated in culture. Our studies also showed that ILK is involved in the phosphorylation of ERK 1/2, p38 MAPK, JNK, and PKB and that selective inhibition of ILK expression by siRNA results in a significant decrease in their phosphorylation. These changes were accompanied by significant inhibition of cell spreading and migration without affecting cell proliferation. In conclusion, ILK plays a key role in HSC activation and could be a possible target for antifibrogenic therapy.

Animals↗
PubMed2001

Expression and cellular distribution of alpha(v)integrins in beta(1)integrin-deficient embryonic stem cell-derived cardiac cells.

beta(1)integrin-deficient (beta(1)-/-) ES cells showed increased differentiation of cardiac cells characterized by reduced adhesion and high beating frequency. Whereas in whole embryoid body outgrowths of beta(1)-/- cells maximum levels of alpha(v), beta(3)and beta(5)integrin mRNA were delayed and transiently upregulated, in cardiac clusters isolated from beta(1)-/- cells, only beta(3)integrin mRNA levels were enhanced in comparison to wild-type (wt) cells. To answer the question, whether alpha(v)and beta(3)integrins may compensate, at least partially, the loss of beta(1)integrin function during cardiac differentiation, the distribution of alpha(v)and beta(3)integrins in beta(1)-/- and wt pacemaker-like cardiac cells was analyzed. A different distribution of alpha(v)and beta(3)integrins in beta(1)-/- v wt cardiac cells was found. In wt cardiac cells, beta(1)integrin was localized in specialized subsarcolemmal regions, in particular, at focal contacts and costameres, but alpha(v)integrin was diffusely distributed. In contrast, in beta(1)-/- cardiac cells, alpha(v)integrin was preponderantly localized at cell membranes, focal contacts and costameres. beta(3)integrin displayed a diffuse pattern both in wt and in beta(1)-/- pacemaker-like cells at early differentiation stages, whereas at terminal stages, beta(3)was colocalized with sarcomeres in wt, but not in beta(1)-/- pacemaker-like cells. Quantitative immunofluorescence analysis revealed increased alpha(v)and beta(3)integrin levels in beta(1)-/- pacemaker-like cardiac cells. Our results led us to conclude that altered cellular distribution of alpha(v)integrin and upregulation of beta(3)integrin correlate with growth and survival of beta(1)-/- cardiac pacemaker-like cells at an early developmental state. However, alpha(v)and beta(3)integrins cannot functionally compensate the loss of beta(1)integrin during terminal differentiation of cardiac cells implicating that cardiomyocytes require specific beta(1)integrin functions for cardiac specialization.

Animals↗
PubMed2004

Rubella virus P90 associates with the cytokinesis regulatory protein Citron-K kinase and the viral infection and constitutive expression of P90 protein both induce cell cycle arrest following S phase in cell culture.

In utero infection of developing fetus by Rubella virus (RV) causes cell division inhibition of critical precursor cells in organogenesis, CNS-associated birth defects and induction of apoptosis in cell culture. The underlying mechanisms of RV-induced congenital abnormalities are not known. Here, we identified a novel interaction between RV replicase P90 protein and a cytokinesis-regulatory protein, the Citron-K kinase (CK), in a yeast two-hybrid cDNA library screen. Aberrations in cytokinesis and subsequent apoptosis do occur in specific cell types when the CK gene is knocked out or, its regulatory function is perturbed. Our analysis found that full-length P90 binds CK and in RV-infected cells P90 colocalizes with CK in the cytoplasm. Furthermore, during RV infection as well as cellular expression of P90 alone, we identified a discrete subpopulation of cells containing 4N DNA content, indicating that these cells are arrested in the cell cycle following S phase, suggesting that cellular expression of viral P90 during RV infection perturbs cytokinesis. Previous reports by others established that RV infection leads to apoptosis in cell culture. These observations together taken to the fetal organogenesis level, favor the idea that RV P90, by binding to cellular CK, invokes cell cycle aberrations resulting in the cell- and organ-specific growth inhibition and programmed cell death during RV infection in utero, which commonly is referred to as RV-induced teratogenesis.

Amino Acid Sequence↗
PubMed2006

Novel localization of CD38 in perivascular sympathetic nerve terminals.

Using high performance liquid chromatography fraction analysis we have recently established that numerous smooth muscle preparations, including the canine mesenteric artery and vein, release beta-nicotinamide adenine dinucleotide upon short-pulse electrical field stimulation in tetrodotoxin- and omega-conotoxin GVIA-sensitive manners [ Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder. J Biol Chem 279:48893-48903.]. The beta-nicotinamide adenine dinucleotide metabolites ADP-ribose and cyclic ADP-ribose are also present in the tissue superfusates. CD38 is a multifunctional enzyme involved in the degradation of beta-nicotinamide adenine dinucleotide to ADP-ribose and cyclic ADP-ribose. Western immunoblot analysis revealed that CD38 is expressed in both artery and vein. Confocal laser scanning microscopy established colocalization of CD38 with tyrosine hydroxylase, synaptotagmin and synaptic vesicle protein in both blood vessels. High performance liquid chromatography with fluorescence detection demonstrated that whole tissue segments metabolize 1,N(6)-etheno-nicotinamide adenine dinucleotide to 1,N(6)-etheno-ADP-ribose and nicotinamide-guanine dinucleotide to cyclic GDP-ribose, suggesting the presence of both nicotinamide adenine dinucleotide-glycohydrolase and ADP-ribosyl cyclase activities in these blood vessels. Both enzymes appear to be associated with the membrane fraction, and therefore might be attributed to CD38. These data demonstrate a previously uncharacterized localization of CD38 in perivascular autonomic nerve terminals. Therefore, the beta-nicotinamide adenine dinucleotide/CD38 system may provide new mechanisms in autonomic neurovascular control.

ADP-ribosyl Cyclase 1↗
PubMed2002

Survival motor neuron protein in the nucleolus of mammalian neurons.

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by mutations in the survival motor neuron gene (SMN1). While it has been shown that the SMN protein is involved in spliceosome biogenesis and pre-mRNA splicing, there is increasing evidence indicating that SMN may also perform important functions in the nucleolus. We demonstrate here through the use of a previously characterized polyclonal anti-SMN antibody, abSMN, that the SMN protein shows a striking colocalization with the nucleolar protein, fibrillarin, in both nucleoli and Cajal bodies/gems of primary neurons. Immunoblot analysis with antifibrillarin and two different anti-SMN antibodies reveals that SMN and fibrillarin also cofractionate in the insoluble protein fraction of cultured cell lysates. Immunoprecipitation experiments using whole cell extracts of HeLa cells and cultured neurons revealed that abSMN coprecipitated small amounts of the U3 small nucleolar RNA (snoRNA) previously shown to be associated with fibrillarin in vivo. These studies raise the possibility that SMN may serve a function in rRNA maturation/ribosome synthesis similar to its role in spliceosome biogenesis.

Animals↗
PubMed1997

Immunohistochemical and genetic evidence of myeloperoxidase involvement in multiple sclerosis.

The myeloperoxidase enzyme (MPO) is expressed specifically in myeloid cells and catalyzes the formation of hypochlorous acid and other cytotoxic oxidants. We previously reported that two alleles of MPO exist which differ in promoter strength due to a base difference in an Alu-encoded hormone response element. The present study shows that the higher expressing MPO genotype is overrepresented in early onset multiple sclerosis in females, implicating MPO in this demyelinating disease. Contrary to the general conception that macrophages lack MPO, immunohistochemical analysis shows that MPO is present in microglia/macrophages in and around MS lesions as shown by colocalization with major histocompatibility antigens HLA-DR and phagocytized myelin. Also, MPO mRNA sequences are detected in cDNA derived from isolated human adult microglia. This is the first evidence that MPO is present in microglia/macrophages at MS lesions, that MPO gene expression occurs in microglia and that MPO plays a role in MS pathogenesis as shown by the allelic disequilibrium in early onset disease.

Adult↗
PubMed2002

Measles virus matrix protein is not cotransported with the viral glycoproteins but requires virus infection for efficient surface targeting.

As we have shown earlier, the measles virus (MV) glycoproteins H and F are expressed on both, the apical and the basolateral membrane of polarized Madin-Darby canine kidney cells. In contrast to the glycoproteins, we found the viral matrix protein (M) to accumulate selectively at the apical plasma membrane of MV-infected cells. M did not colocalize with the glycoproteins at basolateral membranes of polarized cells indicating an independent surface transport mechanism. Analysis of infected cells treated with monensin supported this view. When H and F were retained in the medial Golgi by monensin treatment, M did not accumulate in this cellular compartment. To elucidate the subcellular transport mechanism of the cytosolic M protein, M was expressed in the absence of other viral proteins. Flotation analysis demonstrated that most of the M protein coflotated in infected or in M-transfected cells with cellular membranes. Thus, the M protein possesses the intrinsic ability to bind to lipid membranes. Unexpectedly, plasmid-encoded M protein was rarely found to accumulate at surface membranes. Although cotransport with the viral glycoproteins was not needed, M transport to the plasma membrane required a component only provided in MV-infected cells.

Animals↗
PubMed2002

Interactions among adenosine deaminase, adenosine A(1) receptors and dopamine D(1) receptors in stably cotransfected fibroblast cells and neurons.

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding. The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed2001

Fine structure and neurotransmitter cytochemistry of neurons in the rat ventral cochlear nucleus projecting to the ipsilateral dorsal cochlear nucleus.

The neural tracer wheat germ agglutinin conjugated to horse radish peroxidase was injected into the rat dorsal cochlear nucleus and acoustic stria. Some labelled neurons in the ipsilateral ventral cochlear nucleus were found as a result. These neurons were studied at the ultrastructural level, and their axo-somatic synaptic profile and glycine immunoreactivity were determined. Most neurons were glycine negative and classified as type I multipolar neurons. The latter showed a different synaptic profile from that of neurons projecting to the contralateral inferior colliculus or cochlear nucleus. This suggests the presence of differing populations of multipolar cells based on their synaptic profile. Few labelled multipolar neurons of type II were found, which appeared glycine negative and, rarely, glycine positive. The latter show an ultrastructure and axo-somatic profile similar to that of glycinergic commissural neurons in the dorsal and ventral cochlear nucleus. In particular, about one-third of boutons contained round synaptic vesicles, which are believed to contain an excitatory neurotransmitter. The ultrastructural analysis of the synaptic boutons in the cochlear nucleus confirms the presence of numerous cases of colocalization of glycine and GABA where flat and pleomorphic synaptic vesicles are mixed. The present study is in accordance with previous tract-tracing light microscopic studies which have indicated that large glycinergic neurons in the ventral cochlear nucleus act as broad-band inhibitory neurons in microcircuits of the dorsal cochlear nucleus and contralateral cochlear nucleus.

Animals↗