Hepatocellular heterogeneity in ammonia metabolism: demonstration of limited colocalization of carbamoylphosphate synthetase and glutamine synthetase.
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Ammonia↗
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Results for “Colocalization”
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Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells.
Calcium ions serve as intracellular messengers in 2 activities of hair cells: in conjunction with Ca2(+)-activated K+ channels, they produce the electrical resonance that tunes each cell to a specific frequency of stimulation, and they trigger the release of a chemical synaptic transmitter. Our experiments indicate that both of these functions are conducted within a region that extends a few hundred nanometers around each presynaptic active zone. In focal electrical recordings from the plasma membranes of isolated anuran hair cells, we found nearly all of a cell's Ca2+ channels and Ca2(+)-activated K+ channels clumped at a fixed ratio in an average of 20 clusters on the basolateral membrane surface. Because serial-section electron microscopy indicated that each hair cell has approximately 19 afferent synaptic contacts with a similar distribution upon its basolateral surface, we conclude that the channel clusters coincide with synaptic active zones. Ensemble-variance analysis of current fluctuations indicated that each cell has a total of approximately 1800 Ca2+ channels and approximately 700 Ca2(+)-activated K+ channels; if these are uniformly divided, we estimate that each channel cluster contains approximately 90 Ca2+ and approximately 40 Ca2(+)-activated K+ channels. Freeze-fracture electron microscopy demonstrated an average of 133 large intramembrane particles in the presynaptic membrane at each active zone, an observation that suggests that the particles are the clustered channels. We used the K+ channel's sensitivity to intracellular Ca2+ to assay the concentration of free Ca2+ in the presynaptic cytoplasm, which we found to vary between 10 microM and 1 mM over the physiological range of membrane potentials. The inferred concentrations agreed with the values predicted for free diffusion of Ca2+ away from Ca2+ channels scattered randomly within a 300-nm-diameter synaptic active zone. The close association among Ca2+ channels, Ca2(+)-activated K+ channels, and synaptic active zones is necessary both for the rapid activation of K+ currents required in electrical resonance and for the transmission at afferent synapses of information about the phases of high-frequency stimuli.
Animals↗
Colocalization of messenger substances with special reference to the hypothalamic arcuate and paraventricular nuclei.
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Animals↗
Alcoholic pancreatitis. Possible role of digestive enzyme and lysosomal hydrolase colocalization and abnormal trafficking of proteins after exit site at trans Golgi network (TGN).
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Alcoholism↗
Immunocytochemical localization of glucocorticoid receptor in human gingival fibroblasts and evidence for a colocalization of glucocorticoid receptor with cytoplasmic microtubules.
The cellular distribution of the glucocorticoid receptor (GR) in relation to various intracellular and plasma membrane structures in human fibroblasts was studied using indirect immunofluorescence techniques with monoclonal and polyclonal antibodies. During interphase, GR was located predominantly in the cytoplasm, showing a similar pattern as tubulin. In mitotic cells, GR and tubulin were localized in mitotic spindles and in telophase midbodies. Colchicine and vinblastine induced a similar redistribution of GR and tubulin to the cell periphery. This redistribution was reversible for colchicine but not for vinblastine. Vinblastine also induced paracrystals containing GR and tubulin. These results support the hypothesis that GR interacts in vivo with cytoplasmic microtubules.
Blotting, Western↗
Colocalization of neurotensin receptors and of the neurotensin-degrading enzyme endopeptidase 24-16 in primary cultures of neurons.
This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with 125I-Tyr3-neurotensin, whereas endopeptidase 24-16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24-16 is present in 80-85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35-40% after 4-8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific 125I-Tyr3-neurotensin labeling is maximal after 4 d of culture and is located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24-16, supporting the hypothesis that one of the functions of endopeptidase 24-16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24-16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides.
Animals↗
Cerebellar localization and colocalization of GABA and calcium binding protein-D28K.
Immunocytochemical studies using antibodies raised against the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) and against the 28 Kd vitamin D dependent calcium binding protein (calbindin) in the cerebellum, are reviewed. The GABA immunoreactive neurones found in the cerebellar cortex were the Purkinje cell (PC), the three classes of intrinsic inhibitory interneurones, stellate, basket and Golgi cells and the cells of Lugaro. Some of the neurons of the cerebellar nuclei were also found to be GABA immunoreactive. A part of these could be identified as extrinsic neurones projecting either back to the cerebellar cortex, or to the inferior olive, both these pathways being topographically highly organized but arising from independent parent neurons. The presumed inhibitory function of these two pathways are discussed. Calbindin immunoreactivity in the cerebellum was confined to the PCs, staining concerned the whole cell including soma, branching dendrites, axons and axons terminals. The antibody, which appears to be tightly bound to the PC in vivo, failed to stain some of the PC when cerebellar slices maintained in vitro were studied. The stability of the antigen-antibody binding and the use of calbindin as a marker specific for the PC in the cerebellum, is discussed. Co-localization of GABA with calbindin as well as with other calcium binding proteins are reported to be found in the PCs. While these co-localizations have led to much speculation, conclusive functional roles for them have not been identified at present.
Animals↗
Colocalization of vinculin-containing adhesion plaques, microfilaments and cytokeratin filaments in cytochalasin-treated PtK1 cells.
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Actin Cytoskeleton↗
Colocalization of elastase and myeloperoxidase in human blood and bone marrow neutrophils using a monoclonal antibody and immunogold.
The authors have localized elastase in human blood and bone marrow neutrophils by immunoelectron microscopy using a monoclonal anti-human elastase antibody (NP 57) and compared its distribution with myeloperoxidase (MPO) and lactoferrin (LF), which mark primary and secondary neutrophil granule, respectively. Human bone marrow and blood polymorphonuclear leukocytes (PMN), either unstimulated or after phagocytosis of latex microbeads, were fixed in 4% paraformaldehyde. Ultrathin frozen sections were immunolabeled with NP 57, followed by an immunogold probe. In bone marrow granulocyte precursors elastase appeared simultaneously in the immature first granules of myeloblasts with MPO. As these granules became denser with maturation, labeling for both enzymes became weaker and sometimes negative (possibly due to masking of immunoreactivity). The ellipsoidal primary granules were strongly labeled by NP57. LF positive granules appeared later, at the myelocyte stage, and contained neither MPO nor elastase. In mature neutrophils, immunolabeling for elastase was found together with MPO in the large electron-dense primary granules and in a different granule population from the LF-positive secondary granules. Double labeling with two different-sized gold particles was used to compare the kinetics of degranulation of secondary and primary granules. The observation and the analysis of single phagosome content was made possible by this new technique. In conclusion, immunoelectron microscopy was used to show elastase in the primary granules of neutrophils, where it appears simultaneously with MPO. This technique has also allowed comparison of the kinetics of degranulation of both types of granules, and could be applied to different experimental and pathologic conditions.
Antibodies, Monoclonal↗
Polyamines, molecules necessary for cell division, colocalize with peptide growth factors.
The naturally occurring polyamines spermidine and spermine are necessary for cell division and growth. By restaining experiments, using three independent polyamine cytochemical methods, together with peptide immunocytochemistry, we show that substantial amounts of polyamines occur in a number of peptide growth factor-producing cell types. These include submandibular granular convoluted duct cells producing epidermal growth factor (EGF), pancreatic islet cells producing insulin and anterior pituitary cells producing growth hormone (GH). Other cell types in these tissues display only weak or no polyamine reactivity. Also blood platelets, known to contain platelet-derived growth factor (PDGF), are strongly stained for polyamines. Moreover, in EGF cells, insulin cells and blood platelets, polyamines are clearly localized in secretory granules. The possibility that polyamines may be coreleased and act in concert with peptide growth factors is discussed.
Animals↗
Colocalization of acetylcholine receptors and basal lamina proteoglycan.
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Animals↗
Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain.
The generation of antiserums against a peptide that has met the criteria predicted for corticotropin-releasing factor (CRF) has allowed the immunohistochemical localization of CRF immunoreactive neurons in the rat brain. Although CRF-stained cells have been found to be widely distributed in the central nervous system, attention has focused on neurons in the paraventricular nucleus of the hypothalamus (PVH), which is now acknowledged to be the principal source for delivery of CRF to the hypophyseal portal system. Some 2000 CRF-stained neurons can be counted in the PVH of the colchicine-treated rat, and there is evidence that enkephalin, PHI, and neurotensin coexist with CRF in subsets of parvocellular neurons. Consistent with the established negative feedback effects of adrenal steroids on CRF production and release, adrenalectomy enhances CRF immunoreactivity in parvocellular neurosecretory neurons in the PVH. In addition, immunoreactive vasopressin can be demonstrated in a majority of CRF-stained parvocellular neurons after adrenalectomy, which suggests a form of plasticity that allows for synergy of the two peptides in stimulating adrenocorticotropin secretion. The effects of adrenalectomy appear to be glucocorticoid-dependent, and specific to these peptides and this cell type. A survey of neural inputs to the hypophyseotropic zone of the PVH suggests potential substrates for the control of CRF release and/or synthesis by interoceptive stimuli, by the limbic region, and by a number of cell groups in the basal forebrain. Finally, CRF may also participate in other (nonadenohypophyseal) modes of regulation that are represented in the PVH. Thus, CRF immunoreactivity has been demonstrated in a discrete subset of oxytocinergic magnocellular neurosecretory neurons that project to the posterior pituitary, and in a small fraction of cells in the parvocellular division that project to cell groups in the brain stem and spinal cord that are associated with the control of autonomic functions.
Adrenalectomy↗
Regulation of neuroendocrine gene expression by alternative RNA processing. Colocalization of calcitonin and calcitonin gene-related peptide in thyroid C-cells.
The generation of two discrete mRNAs encoding different polypeptide products in expression of the rat calcitonin, CGRP gene provides an example of developmentally determined alternative RNA processing events in the neuroendocrine system. Both mature mRNA transcripts and their peptide products, encoding either calcitonin or calcitonin gene-related peptide (CGRP) are co-produced in identical thyroid C-cells, in a ratio of 95:1, respectively. These data suggest that the generation of the two mRNAs reflect the actions of a factor(s) dictating alternative RNA processing events.
Animals↗
[Colocalization of peptides and classic neurotransmitters in neurons of the central nervous system].
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Animals↗
Localization of pregnancy-associated plasma protein-A and colocalization of pregnancy-associated plasma protein-A messenger ribonucleic acid and eosinophil granule major basic protein messenger ribonucleic acid in placenta.
BACKGROUND: The human eosinophil granule major basic protein (MBP), a 13.8 kilodalton cationic polypeptide constituting the core of the eosinophil granule, is cytotoxic to parasites and numerous mammalian cells. Concentrations of a molecule immunochemically similar to eosinophil granule MBP are present in maternal plasma, and MBP mRNA has been localized to placental X cells by in situ hybridization. Eosinophil granule MBP is initially translated as a nontoxic precursor (proMBP), containing a 9.9 kilodalton acidic pro-portion that is believed to neutralize MBP toxicity. Recent analyses of sera from pregnant women have revealed that pregnancy-associated plasma protein-A (PAPP-A), previously thought to be a homotetramer of PAPP-A subunits, is actually composed of PAPP-A subunits bound by disulfide bonds to equimolar amounts of proMBP molecules to form a complex, PAPP-A/proMBP. In addition, the PAPP-A subunit nucleotide and deduced amino acid sequence have been determined from cloned cDNA. The PAPP-A monomer found in plasma contains 1547 amino acid residues. EXPERIMENTAL DESIGN: Because of the new evidence that PAPP-A is complexed with proMBP, previous studies on the localization of PAPP-A using antibodies to PAPP-A must be questioned. To determine the localization of the PAPP-A subunit, immunofluorescence was performed on normal placental tissues using proMBP absorbed anti-PAPP-A antibody. Furthermore, the expression of PAPP-A mRNA was investigated by in situ hybridization. RESULTS: Immunofluorescence staining with proMBP absorbed anti-PAPP-A antibody showed that PAPP-A is localized to placental septa, anchoring villi, and the syncytia of chorionic villi, whereas MBP is localized only to septa and anchoring villi. By in situ hybridization, PAPP-A mRNA is detected in placental X cells and syncytiotrophoblasts, but MBP mRNA is localized only to placental X cells. CONCLUSIONS: The presence of PAPP-A mRNA and PAPP-A subunit protein in placental X cells and syncytiotrophoblasts indicates that both X cells and syncytiotrophoblasts synthesize the PAPP-A subunit, whereas only X cells synthesize proMBP.
Blood Proteins↗
Colocalization of glutamate and glycine in giant fiber synapses of the lamprey spinal cord.
Using electron microscope immunocytochemistry in serial and semi-serial sections, glutamate and glycine immunoreactivity was analyzed in various synapses of the lamprey spinal cord. Immunoreactivity to glutamate was observed over the synaptic vesicle clusters in the giant reticulospinal axons and dorsal/dorsolateral column fibers, both of which established "en passant" asymmetrical contacts. Most of giant axons (70%) were also observed to express an immunoreactivity to glycine. Glutamate or glycine immunopositive axon terminals were present throughout the spinal cord regions. Glutamate positive boutons made asymmetrical contacts and contained rounded synaptic vesicles. Glycine reactive axon terminals with rounded synaptic vesicles established asymmetrical contacts and those with flattened synaptic vesicles established symmetrical contacts. The possibility that these two amino acids could be released from the same reticulospinal synapse in the lamprey, with a glycine modulatory effect on NMDA receptors, is suggested.
Animals↗
Peripheral benzodiazepine receptors are colocalized with activated microglia following transient global forebrain ischemia in the rat.
In mammalian brain the expression of peripheral benzodiazepine receptors (PBRs) can be markedly induced following different types of neuronal injury. PBRs are believed to be expressed on non-neuronal cells in the brain, yet the specific cell type that expresses these receptors following CNS insult has not been defined. In the present study, we investigated the effects of transient global forebrain ischemia on PBRs by autoradiographic localization of 3H-PK11195 binding. The distribution of PBRs was compared to glial fibrillary acidic protein (GFAP) as a marker for astrocytes and OX42 as a marker for microglia. Five to 6 d following four-vessel occlusion (4-VO), an increase in PBRs was seen in the CA1 region of all 15 brains examined. In brains from rats subjected to 4-VO, microglia were selectively activated in stratum pyramidale of the CA1 layer. In contrast, astrocytes appeared to be activated in multiple hippocampal cell layers including stratum radiatum and stratum oriens. Activated astrocytes were also found in regions that did not exhibit increased 3H-PK11195 binding. In some brains, selected regions of secondary lesion, specifically necrotic thalamic nuclei and the isocortex were found to be strongly immunoreactive for OX42 but lacked GFAP immunoreactive cells. In adjacent sections, these same regions displayed high densities of 3H-PK1195 binding. These observations lend further support to the application of 3H-PK11195 binding as a marker of neuronal injury in the brain. Furthermore, the data strongly suggest that activated microglia rather than astrocytes express PBRs following ischemic insults.
Animals↗
Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain.
Recent cloning of auxiliary subunits associated with voltage-gated ion channels and their subsequent coexpression with the channel forming alpha-subunits has revealed that the expression level, gating and conductance properties of the expressed channels can be profoundly affected by the presence of an auxiliary subunit polypeptide. In the present study, we raised antibodies against the beta-subunit associated with the bovine dendrotoxin sensitive K(+)-channel complex and used these antibodies to characterize the related beta-subunit polypeptides in rat brain. The anti-beta-subunit antibodies displayed a specific reaction on immunoblots of rat brain membranes with a major 38 kDa polypeptide, and a minor 41 kDa polypeptide, which correspond closely to the predicted sizes of the Kv beta 2 and Kv beta 1 beta-subunit polypeptides, respectively, recently cloned from rat brain. Reciprocal coimmunoprecipitation experiments revealed that the beta-subunit polypeptides are associated with Kv1.2 and Kv1.4, but not Kv2.1, alpha-subunits. Immunohistochemical staining revealed that the beta-subunit polypeptides were widely distributed in adult rat brain. Moreover, the cellular distribution of beta-subunit immunoreactivity corresponded closely with immunoreactivity for Kv1.2, and to a lesser extent Kv1.4, but not with Kv2.1. These results suggest that neuronal mechanisms may exist to direct the selective interaction of K+ channel alpha- and beta-subunit polypeptides, and that the properties of K+ channels in specific subcellular domains may be regulated by the formation of heteromultimeric K+ channel complexes containing specific combinations of alpha- and beta-subunits.
Amino Acid Sequence↗