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PubMed1991

Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts.

Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts. The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables coincided with bundles of actin microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with the EF-2 antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.

Actin Cytoskeleton↗

PubMedDate not supplied

Postnatal ontogeny of catecholamine and somatostatin neuron systems in the median eminence of the rat as revealed by a colocalization technique.

The ontogenetic development of catecholamine (CA) and somatostatin containing nerve terminals in the rostral, central and caudal median eminence (ME) of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. Somatostatin terminals were detected earlier in development than CA terminals and had already appeared in the lateral part of the external layer of the central ME by the 1st postnatal day. CA nerve terminals were first observed in the same region of the ME on the 7th postnatal day. At about this stage both types of terminals seemed to show early signs of a correlation in their distribution which became progressively closer as maturation proceeded. Their distribution reached a stable condition in density and pattern on the 21st postnatal day when the majority were found in the lateral part of the external layer of the central ME, in the basal part of the brain just dorsal to the tuberoinfundibular sulcus and in the upper and lower labia of the tuberoinfundibular stalk. They subsequently matured to the adult pattern of stabilized distribution and anatomical relationship. It was also established that on the 28th postnatal day the somatostatin immunoreactive terminals reached a greater concentration than at any other stage of development including that of the adult.

Aging↗

PubMed1991

The Sp1 transcription factor gene (SP1) and the 1,25-dihydroxyvitamin D3 receptor gene (VDR) are colocalized on human chromosome arm 12q and rat chromosome 7.

By means of somatic cell hybrids segregating either human or rat chromosomes, the genes encoding the transcription factor Sp1 (SP1) and the 1,25-dihydroxyvitamin D3 receptor (VDR) were both assigned to human chromosome arm 12q and to rat chromosome 7. This result implies that the locus for the clinical disorder vitamin D dependency rickets type II maps on 12q. The phenylalanine hydroxylase (PAH) and the retinoic acid receptor-gamma (RARG) genes also map on human chromosome arm 12q and rat chromosome 7, indicating that a synteny group is conserved on these chromosomes.

Animals↗

PubMed1992

Colocalization of the gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) in the Xq28 region.

The gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) have both been localized in the Xqter region by genetic mapping and functional expression studies, respectively. In this paper genetic evidence that the DIR locus is localized distal to the DXS305 locus and that the functional gene for the V2 receptor is localized between the markers DXS269 and F8 is presented. These further refinements in the localization of both genes strengthen the assumption that both genes are identical and provide a rationale for cloning the gene by reversed genetics strategies.

Animals↗

PubMed1992

Colocalization of the genes for the alpha 3(IV) and alpha 4(IV) chains of type IV collagen to chromosome 2 bands q35-q37.

Each type of basement membrane in man contains between two and five genetically distinct type IV collagens: alpha 1(IV)-alpha 5(IV). Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been isolated. We have recently isolated partial cDNAs for the fifth member of the family, designated alpha 4(IV). On the basis of comparison of the deduced peptide sequences of all five chains, the type IV collagens can be divided into two families: alpha 1-like, comprising alpha 1(IV), alpha 3(IV), and alpha 5(IV); and alpha 2-like, comprising alpha 2(IV) and alpha 4(IV). Genes encoding the alpha 1(IV) and alpha 2(IV) chains (COL4A1 and COL4A2) both map to human chromosome 13q34 and have been shown to be transcribed from opposite DNA strands using a common bidirectional promoter that allows coordinate regulation of the two chains. Indeed, these two chains are commonly found together in basement membrane and form [alpha 1]2.[alpha 2] heterotrimers. Whereas alpha 1(IV) and alpha 2(IV) have been found in all basement membranes studied hitherto, it has been shown that alpha 3(IV) and alpha 4(IV) are found in only a subset of basement membranes. In basement membranes where either of these molecules is present, however, they are found together. In view of this relationship and the structural similarities between alpha 1(IV) and alpha 3(IV) and between alpha 2(IV) and alpha 4(IV), we hypothesized that COL4A3 and COL4A4, the genes encoding alpha 3(IV) and alpha 4(IV), respectively, have a genomic organization similar to that of COL4A1 and COL4A2.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗

PubMed1992

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.

Amino Acid Sequence↗

PubMed1992

Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors.

A complementary DNA clone encoding a transporter for glycine has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The transporter displays high affinity for glycine (KM approximately 100 microM) and is dependent on external Na+ and Cl-. Northern blot analysis indicates that the distribution of the mRNA encoding the glycine transporter is restricted to the nervous system. In situ hybridization data are consistent with roles for the transporter in both glycine neurotransmission and glycine modulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus. The identification of this transporter therefore opens the study of the molecular mechanisms underlying both inhibitory glycinergic transmission and NMDA-mediated excitatory transmission.

Amino Acid Sequence↗

PubMed1993

Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release.

We examined, using physiological and morphological techniques, the distribution of Ca(2+)-gated K+ (gKca) channels relative to the location of Ca2+ channels and transmitter release sites at the frog neuromuscular junction (NM). Charybdotoxin (ChTx) and iberiotoxin, blockers of gKca channels with large conductances, increase transmitter release at the frog NMJ. Intracellular Ca2+ buffers with rapid binding kinetics, dimethyl BAPTA and BAPTA, prevented the effect of ChTx, but EGTA, a Ca2+ buffer with similar affinity for Ca2+ but slower binding kinetics, did not. Dimethyl BAPTA and BAPTA, but not EGTA, caused a temporary increase in transmitter release. Labeling of gKca channels with ChTx-biotin revealed a series of bands located at the sites of Ca2+ channels, but this labeling did not occur in denervated preparations. Cross sections of NMJs revealed that gKca channels are clustered in the presynaptic membrane facing the postsynaptic membrane. We conclude that gKca channels are strategically clustered at the neurotransmitter release sites, where they can be quickly activated by Ca2+ entering the terminal.

Animals↗

PubMed1995

Colocalization of scavenger receptor in CD68 positive foam cells in verruciform xanthoma.

Verruciform xanthoma is a rare variant of xanthoma that appears predominantly on oral mucosa and the anogenital area. The histologic features are characterized by marked accumulation of foam cells in the papillary dermis with verrucous epidermal acanthosis and hyperkeratosis. However, little is known of the nature or origin of foam cells. Recent studies have emphasized the crucial role of macrophage scavenger receptors in the formation of foam cells in atherogenesis. We examined the immunohistologic localization of scavenger receptors in genital verruciform xanthoma. We found that the massively infiltrated foam cells in the papillary dermis were CD68+ monocyte-macrophage lineage cells, and that the majority of CD68+ cells coexpressed scavenger receptors. The in situ staining pattern of scavenger receptor in foam cells was mainly of an intracytoplasmic vacuolar pattern similar to that of dermal resident macrophages found in normal skin. Furthermore, CD1a+ Langerhans cells, completely negative for scavenger receptor, were markedly decreased in number in verruciform xanthoma. These results indicate that scavenger receptor-bearing CD68+ cells are also actively involved in the development of cutaneous verruciform xanthoma, as has been shown to be the case in atherogenesis.

Aged↗

PubMed1991

Colocalization of neurotransmitters analyzed by in situ hybridization.

In situ hybridization and Northern blot analysis has been used to analyse in some detail the localization and regulation of the messenger molecules adrenaline, noradrenaline and neuropeptide tyrosine (NPY) within cells of the sympathetic nervous system and the adrenal medulla. In the rat adrenal gland, a novel NPY containing population of ganglion cells was found. Synthetic oligonucleotide probes complementary to mRNA coding for the catecholamine synthesizing enzymes phenylethanolamine N-methyltransferase (PNMT), tyrosine hydroxylase (TH) and NPY were used to analyse the regulation of these genes following administration of the catecholamine depleting drug reserpine. Twenty-four hours after a single dose of reserpine, a differential regulation of PNMT, TH and NPY was found. Thus, a dramatic decrease in PNMT mRNA was observed in the adrenal medulla. In contrast, mRNA for both TH and NPY exhibited an increase. Different regulatory mechanisms may thus operate for these three compounds coexisting in chromaffin cells of the adrenal medulla. The regulation of enzymes and peptides was also studied in human sympathetic ganglia. After brief electrical preganglionic stimulation of thoracic ganglia in humans, in situ hybridization was performed with synthetic oligonucleotide probes complementary to TH, dopamine beta-hydroxylase (DBH) and NPY mRNA respectively. A several fold increase in all three mRNAs was found in the principal ganglion cells. The results point to a very rapid regulation of genes involved in signal transmission in the sympathetic nervous system of humans. The results also suggest a novel way to define neuronal projections by visualizing increases in mRNA levels following electrical stimulation.

Adrenal Glands↗

PubMed1995

Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development.

Members of the bone morphogenetic protein (BMP) class of transforming growth factor beta (TGF beta)-related molecules have been implicated in a variety of inductive processes throughout vertebrate development. The 60A subclass of BMPs contains at least four vertebrate members, BMPs 5-8. We have shown by library screening and in situ hybridization that of these four genes, BMP 7 is expressed earliest, in gastrulating embryos. Furthermore, BMP 7 transcripts are present at diverse sites throughout development, in a pattern consistent with a role in a variety of inductive interactions. Recent studies have shown that BMP 2/7 heterodimers have unique activities compared to the corresponding homodimers. For this reason, we compared the patterns of expression of BMP 2 and BMP 7 using in situ hybridization. Our results demonstrate that these BMPs are coexpressed in a number of tissues that are known to be the source of inductive signals, including the zone of polarizing activity and apical ectodermal ridge of the developing limb and the notochord, raising the possibility that BMP 2/7 heterodimers may mediate aspects of these tissue interactions. We also show that BMP 2 transcripts are restricted within the developing gut to dorsal endoderm, whereas sonic hedgehog has been localized to ventral and medial regions of the developing gut endoderm. These markers provide the first molecular evidence for dorsal/ventral polarity in the developing gut.

Animals↗

PubMed2005

The neurotrophin receptor TrkB is colocalized to mitochondrial membranes.

The transmembrane tyrosine-specific protein kinase TrkB has been shown to serve as a receptor for the neurotrophic factors BDNF and NT-4. Neurotrophin binding to TrkB isoformes mediates many intracellular signaling pathways, including calcium signalling. Two truncated isoforms of the receptor, lacking the tyrosine kinase activity, signal through a yet unknown pathway. Specific signals modulate the surface expression of TrkB, which is localized in considerable amounts in intracellular pools. These intracellular pools has not been specified so far. We therefore investigated the intracellular distribution of TrkB by colocalisation studies. In contrast to the unspecific neurotrophin receptor NGFRp75, TrkB immunohistochemistry showed a staining pattern very similar to mitochondrial stainings in adult human skeletal muscle fibers. Immunofluorescence techniques revealed in different types of permeabilized cells that TrkB is bound to mitochondrial membranes. This observation was confirmed on isolated astrocyte mitoplasts. Colocalisation of the TrkB ligand NT-4 and the specific mitochondrial marker cytochrome c oxidase was also observed. Western blot analysis of isolated mitochondria from rat brain and skeletal muscle verified that a truncated isoform of TrkB is present in both, brain and muscle mitochondria, and full-length TrkB is additionally present in brain mitochondria. Our results imply that neurotrophins can be stored in mitochondria and possibly act as signalling molecules on mitochondria.

Animals↗

PubMed2005

Distribution of galanin receptor 1-immunoreactive neurons in the ovine hypothalamus: colocalization with GnRH.

Galanin is implicated in numerous physiological functions, including reproduction. Where and how galanin acts in the brain is poorly understood, but recent evidence suggests that it is predominantly through the GAL-R1 receptor. Using an antibody raised against the third intracellular loop of rat GAL-R1, a region that is highly conserved among species, our first objective was to determine the distribution of cells expressing immunoreactive GAL-R1 in the hypothalamus of the sheep. GAL-R1-immunoreactive cells were spread widely in the ovine diencephalon and overlapped with the known distribution of GnRH neurons. Galanin has been shown to enhance GnRH secretion, but it is not known whether this effect is transduced at the level of the GnRH neuron or is indirect. Thus, our second objective was to establish if GnRH neurons throughout the hypothalamus expressed GAL-R1 receptors and, if so, whether GAL-R1 expression in GnRH neurons was influenced by season, gender and/or stage of the estrous cycle. In rams and ewes during the non-breeding season, only a tenth of the GnRH neurons expressed immunocytochemically detectable GAL-R1 receptors. In contrast, a fifth of the GnRH neurons expressed immunocytochemically detectable GAL-R1 in the luteal phase, whereas only a twentieth expressed GAL-R1 in the follicular phase. These data suggest that galanin may affect a subpopulation of GnRH neurons through the GAL-R1 receptor and that this affect may be modulated by steroids.

Analysis of Variance↗

PubMed2006

Expression and colocalization of NADPH-diaphorase and Fos in the subnuclei of the parabrachial nucleus in rats following visceral noxious stimulation.

To investigate whether neural nitric oxide synthase (nNOS) in the parabrachial nucleus (PB) is involved in processing visceral noxious stimulation, we mapped the distribution of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nNOS, and immunohistochemical staining for Fos, a neuronal activity marker, in the subnuclei of the PB following 2% formalin injection into the stomach of rats. NADPH-d and noxious-stimuli induced Fos staining were also examined in tissue containing PB cells labeled by the retrograde transport of fluogold (FG) injected into the central nucleus of the amygdala (CeA). We found that the number of Fos immunoreactive (Fos-IR) neurons was significantly increased in the dorsal lateral (dl), external lateral (el) and Kölliker-Fuse (KF) subnuclei of the PB. We observed that intensely labeled (type 1) NADPH-d positive neurons were mainly located in the rostral part of the PB; they extended long processes adjacent Fos-IR neurons, but no Fos/type 1 NADPH-d double-labeled neurons were seen. In contrast, lightly labeled (type 2) NADPH-d positive neurons were principally localized in the dl of the PB, in which a few Fos/type 2 NADPH-d double-labeled neurons were detected. Additionally, a large number of FG/Fos double-labeled neurons were observed to be surrounded closely by the intensive NADPH-d staining in the el of the PB. These results suggest that neurons in the el of the PB that project to the CeA are activated by visceral noxious stimulation and could be indirectly influenced by nitric oxide in the PB.

Analysis of Variance↗

PubMed2006

Collagen colocalizes with a protein containing a decorin-specific peptide in the tissues of the ascidian Styela plicata.

Decorin is an extracellular matrix dermatan sulfate/chondroitin sulfate proteoglycan found in a variety of vertebrate species. In the extracellular matrix of mammals, decorin interacts with fibrillar collagen and regulates its morphology. We report here the occurrence and distribution of collagen type I and the peptide, CEASGIGPEVPDDRD, which is present in the human decorin proteoglycan, in the extracellular matrix of different tissues of the primitive invertebrate chordate Styela plicata. The content of collagen was estimated by hydroxyproline, and its distribution in the tissues by histochemistry. Collagen was detected biochemically in intestine, heart, pharynx and mantle, occurring in higher amounts in the heart, followed by pharynx, mantle and intestine. Histochemical analysis with Sirius red indicates that collagen is present in the extracellular matrix of intestine and pharynx. Further ultrastructural immuno-gold assays using polyclonal antibodies raised against the decorin-specific peptide CEASGIGPEVPDDRD and collagen type I showed a co-localization of these molecules. These data suggest the occurrence of a protein containing a decorin-like peptide sequence, which may be interacting with fibrillar collagen in this primitive chordate.

Animals↗

PubMed2006

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.

Arabidopsis↗

PubMed2003

CD34+CDw90(Thy-1)+ subset colocated with mesenchymal progenitors in human normal bone marrow hematon units is enriched in colony-forming unit megakaryocytes and long-term culture-initiating cells.

OBJECTIVE: The progress made in the supportive care of allografts and the identification of mesenchymal stem cells in adult human bone marrow (BM) has prompted renewed interest in the use of BM as a form of cell therapy. With the aim of optimizing the collection of BM cells, we evaluated the hematopoietic and mesenchymal immature cell contents of BM hematon units (HUs), which usually are eliminated during graft processing. MATERIALS AND METHODS: Hematopoietic CD34+ progenitors from HU and buffy coat (BC) compartments were characterized in short-term culture. The sorted CD34+CDw90(Thy-1)+ primitive subset was assessed in colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, then further characterized by the expression of additional antigens. In parallel, we evaluated the colony-forming unit fibroblast (CFU-F) number and phenotyped the fresh adherent (D1-3) cells. RESULTS: The plating efficiencies of CD34+ cells derived from HU and BC were identical. However, the HU CD34+CDw90(Thy-1)+ subset was enriched in colony-forming unit megakaryocyte (2.3x), LTC-IC (4.6x), and cells coexpressing CD105 (5x). We found a higher frequency of CFU-F (4.7x), considered to be the mesenchymal stem cell-containing population, correlated with an enrichment in fresh adherent (CD45/GPA)-CD14- cells. CONCLUSIONS: We show for the first time that functional properties of the CD34+CDw90+ subset are related to its in vivo location in HU, which may represent the BM mesenchymal reserve compartment. The location in HU of 35.6%, 59.1%, and 58.7% of CD34+ cells, CD34+CDw90+ LTC-IC, and CFU-F, respectively, justifies the development of a procedure to collect them in order to reduce the therapeutic BM volume.

Antigens, CD34↗

PubMed2006

No effect of nutrient restriction from gestational days 28 to 78 on immunocytochemically detectable growth hormone-releasing hormone (GHRH) neurons and GHRH receptor colocalization in somatotropes of the ovine female fetus.

The maternal environment affects fetal development and may permanently affect the physiology of the adult. Fetal growth hormone (GH) secretion is increased by maternal undernutrition but the physiological mechanisms responsible for this increase are unknown. We have recently found evidence suggesting that the GHRH component of the fetal neuroendocrine GH axis may be perturbed by undernutrition. This study sought to determine the effect of maternal undernutrition on immunocytochemically detectable GHRH neurons and the expression of GHRH receptors by somatotropes in the pituitary gland. Ewes were grouped (n=12 per group) randomly into control (fed 100% of requirements) or nutrient restricted (fed 50% of requirements) from days 28 to 78 of gestation, corresponding to the period from implantation to the end of placentation. At day 78, half the ewes were killed and the fetal brains were perfused. The remaining ewes were re-alimented to 100% of nutritional requirements and killed at day 135. There was no effect of nutrition restriction or age on the number of GHRH neurons. Similarly, the mean density and percentage of somatotropes expressing GHRH receptors was not significantly different between treatment groups at either age. This study found no effect, as determined by immunocytochemistry, of nutrient restriction on the GHRH component of the fetal neuroendocrine GH axis. It remains to be established if the release of GHRH and responsiveness of somatotropes to GHRH in the fetus are affected by undernutrition.

Animals↗