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In vivo colocalization of 2-nitroimidazole EF5 fluorescence intensity and electron paramagnetic resonance oximetry in mouse tumors.

BACKGROUND AND PURPOSE: The primary objective of this study was to establish in vivo the relationship between 2-2-nitro-1H-imidazol-1yl-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) adduct formation and intratumoral oxygen concentrations measured by electron paramagnetic resonance (EPR) in a tumor model mimicking a clinical situation. The secondary objective was an attempt to calibrate in situ the immunofluorescence (IF) signal with EPR oximetry. MATERIALS AND METHODS: IM syngeneic fibrosarcoma (NFSA) bearing C3H mice were used. Three days after injection of a paramagnetic charcoal into the tumor, the mice were anesthetized, injected with the hypoxic marker EF5, and monitored every 20 min for 3 h with a low-frequency EPR spectrometer. Animals were allowed to breath either under 21 or 100% O(2). Tumors were then harvested, frozen, cut into sections including the charcoal and processed for EF5 adducts detection using monoclonal antibodies. Slices were viewed with a fluorescence microscope and 190x140 micrometer areas surrounding the charcoal were digitized and analyzed with the NIH-Image and Adobe Photoshop software. The fluorescence intensity (FI) was measured in the whole pictures and in strips of 10 micrometer around the charcoal. RESULTS: EF5 binding increased with decreasing pO(2), most substantially at pO(2) below 5 mm Hg. Baseline (ambient air) pO(2) reached 3.2+/-2.1 mm Hg in NFSA tumors. It increased to 9.8+/-3.2 mm Hg under 100% O(2). A statistically significant correlation was observed on an individual tumor basis between the FI in the first 10 micrometer strip around the charcoal and the pO(2) determined by EPR oximetry (Wilcoxon signed rank test: P<0.001). CONCLUSIONS: The present study confirms the intrinsic relationship between EF5 adduct binding and intratumoral pO(2) in an in vivo environment under biologically-relevant pO(2) values of less than 10 mm Hg.

Algorithms↗

Colocalization of neuronal nitric oxide synthase and androgen receptor immunoreactivity in the premammillary nucleus in rats.

To analyze the importance of premammillary region in male behavior such as aggression and mating, distribution of androgen- and estrogen-receptors (AR and ER), aromatase (ARO) and neuronal nitric oxide synthase (nNOS) was studied in the dorsal and ventral premammillary nuclei (PMd and PMv) of the rat by immunohistochemistry. The nNOS-immunoreactivities (-IR) were present both in the PMd and the PMv, while AR-IR were detected only in the PMv. AR-IR became undetectable after orchidectomy but they recovered by an injection of 5 alpha-dihydrotestosterone (DHT). In both nuclei, no clear signals of ER-IR were encountered. On the other hand, ARO-IR were found in the PMv but only very few. In the PMv, although DHT did not increase nNOS-IR significantly in castrated males, all the nNOS-IR cells contained AR-IR at least in intact and castrated-DHT injected males. Thus, involvement of nNOS-nitric oxide system in the PMv in the androgenic action on male behaviors was suggested.

Animals↗

Colocalization of estrogen receptor alpha and NMDA-2D mRNAs in amygdaloid and hypothalamic nuclei of the mouse brain.

Interactions between gonadal steroid hormones and glutamatergic neurons participate in limbic and hypothalamic functions. Glutamate receptors are divided into metabotropic and ionotropic receptors. Among ionotropic receptors, N-methyl-D-aspartate (NMDA) is involved in a variety of neurophysiological processes. In turn, NMDA receptors are composed of subunits from two families: NR1 and NR2. Recently, molecular studies have shown that the expression of NMDA-2D receptor is regulated by estrogen. Although the expression patterns of NMDA-2D and ERalpha in the rodent brain appear to overlap, it remained to be determined whether or not these two receptors co-exist, in vivo, at the level of single neurons. To test the hypothesis that NMDA-2D and ERalpha messenger ribonucleic acid (mRNA) are co-expressed in the same neurons of the adult mouse brain, we used a dual-label in situ hybridization technique. Neuronal populations were identified with digoxigenin-tagged complementary RNA probes for NMDA-2D and 35S-labeled cRNA probes for ERalpha. Our results demonstrate that a majority of the ERalpha-positive neurons also express NMDA-2D mRNA. Quantitative examination of the cellular expression in the ventromedial and arcuate nuclei of the hypothalamus (VMH and Arc) showed that 52.5% and 61.5%, respectively, of the neurons endowed with ERalpha mRNA also contain NMDA-2D mRNA. In the amygdala, 51% of ERalpha-positive cells also contain NMDA-2D mRNA. These findings provide the first anatomical evidence that ER and NMDA-2D receptors can be found in the same hypothalamic and amygdaloid neurons. Co-expression of ERalpha and NMDA-2D receptors supports the hypothesis of the interactions between glutamate receptors and estrogens in brain regions where estrogens control female reproductive behaviors and neuroendocrine functions.

Amygdala↗

Cloning of rat ARHGAP4/C1, a RhoGAP family member expressed in the nervous system that colocalizes with the Golgi complex and microtubules.

The Rho GTPase family of intracellular molecular switches control multiple cellular functions via the regulation of the actin cytoskeleton. Increasing evidence implicates a critical involvement of these molecules in the nervous system, particularly during neuronal migration and polarity, axon and growth cone guidance, dendritic arborization and synaptic formation. However, the molecules regulating Rho GTPase activities in the nervous system are less known. Here, we present the cloning of rat ARHGAP4, a member of the Rho GTPase activating protein family, and also demonstrate its close linkage to the vasopressin 2 receptor gene. In vitro, recombinant ARHGAP4 stimulated the GTPase activity of three members of Rho GTPases, Rac1, Cdc42 and RhoA. ARHGAP4 mRNA expression was observed in multiple tissues with marked expression throughout the developing and adult nervous systems. On closer analysis of protein levels, ARHGAP4 was significantly restricted to specific regions in the nervous system. These included the stratum lucidem in the CA3 area of the hippocampus, neuronal fibers in the ventral region of the brainstem and striatum, and in the cerebellar granule cells. Subcellularly, endogenous ARHGAP4 expression localized to the Golgi complex and could redistribute to the microtubules, for example during mitosis. In addition, distinct protein expression was observed in the tips of differentiating neurites of PC12 cells. Collectively, these results demonstrate that ARHGAP4 is more widely expressed than previously thought but potentially possesses specialized activity in regulating members of the Rho GTPase family in specific cellular compartments of the nervous system.

Amino Acid Sequence↗

Anion exchanger 2 (AE2) binds to erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells.

The hydrochloric acid secreting parietal cells of the human stomach mucosa have been shown to express anion exchanger 2 (AE2). AE2 is restricted to the basolateral membrane domain and is responsible for the basolateral uptake of Cl- and release of HCO3-. It is unknown which mechanism is responsible for the basolateral positioning of AE2 in parietal cells. We raised the question whether AE2 might be immobilized at the cell surface by linkage via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we communicate two observations that support this hypothesis, namely that in parietal cells ankyrin is localized with AE2 along the basolateral cell surface and, secondly, that purified erythrocyte ankyrin binds to the in vitro-translated cytoplasmic domain of AE2. We conclude from these observations that AE2 in parietal cells might be linked via ankyrin to the basolateral membrane cytoskeleton and that this type of linkage might play a role in immobilizing AE2 in a non-random fashion along the basolateral membrane domain.

Anion Transport Proteins↗

Evidence for colocalization of GABA and glycine in afferents to retrogradely labelled rat abducens motoneurones.

The coexistence of gamma-aminobutyric acid (GABA) and glycine in axon terminals impinging on rat abducens motoneurones was investigated using a double staining procedure combining retrograde labelling of the motoneurones with HRP and post-embedding immunocytochemical staining of axon terminals. Adjacent ultrathin sections of cell bodies of identified motoneurones were individually treated with GABA or glycine antibodies. The terminals single labelled for GABA represented 11.4% of the terminals analyzed, while 8% of them were glycine immunoreactive and 9% were both GABA and glycine immunoreactive. All the labelled terminals contained pleomorphic vesicles. The mean length of apposition of the double labelled terminals was statistically larger (2.20 +/- 0.97 microns) than the GABA (1.65 +/- 0.57 microns) or glycine immunoreactive ones (1.37 +/- 0.35 microns).

Abducens Nerve↗

Colocalization of acetylcholinesterase and vasoactive intestinal peptide (VIP) in nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) positive neurons in the intralingual ganglia and perivascular nerve fibers around lingual arteries in the porcine, monkey and canine tongue.

Distribution of nitric oxide synthase in the intrinsic ganglia in the porcine, monkey and canine tongue was histologically investigated using the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) method, acetylcholinesterase histochemistry and vasoactive intestinal peptide (VIP) immunohistochemistry. The majority of intralingual ganglionic cells showed intense NADPH-d reactivity with positive acetylcholinesterase reaction or positive VIP immunohistochemistry. The NADPH-d positive, acetylcholinesterase-rich and the NADPH-d positive, VIP immunoreactive nerve fibers are particularly conspicuous around intralingual blood vessels. These fibers around the arteries in the tongue may be partly derived from the intralingual ganglion cells, because some bundles associated with these nerve cells were easily traced on the wall of blood vessels. The present study suggests the view that the three markers coexist in the axons and nerve terminals of these intralingual neurons.

Acetylcholinesterase↗

Amyloid beta protein and transthyretin, sequestrating protein colocalize in normal human kidney.

The localization of amyloid beta protein (A beta), A beta 40, A beta 42, and transthyretin (TTR) was investigated immunohistochemically in the autopsied human kidney, using polyclonal antibodies against TTR, A beta and C-terminal end-specific antibodies against A beta 40 and 42. Immunoreactivities of A beta and A beta 40 were found both in the proximal and distal tubular epithelial cells. But the immunolocalization of A beta 40 was observed predominantly in the distal tubules whereas that of A beta 42 was predominantly recognized in the proximal tubules. TTR, sequestrating protein for A beta, was present in the proximal tubules. The mechanism by which A beta does not form amyloid in Alzheimer's disease outside the brain remains unknown. The tubular epithelial cells in the kidney may provide a useful system to shed light on this issue.

Amyloid beta-Peptides↗

Examination of colocalization of calcitonin gene-related peptide- and substance P-like immunoreactivity in the knee joint of the dog.

It is generally assumed that the majority of substance P (SP)-containing afferents are also immunoreactive for calcitonin gene-related peptide (CGRP). In order to determine whether this is also the case in articular afferents where the contents of these peptides are low, we carried out a double labeling study using Fast Blue (FB) as a retrograde tracer injected into the center of the knee joint cavity of the dog together with immunohistochemistry for SP and CGRP. After 7-36 days of survival, dorsal root ganglia (DRGs, L4-S1) were removed. Labeled cells were found mainly (94%) in L5 - 6 DRGs, and SP- and CGRP-like immunoreactivity was found in about 17 and 29% of FB-labeled cells, respectively. The coexistence of SP and CGRP was observed in 10.4% of articular afferents and only 62.7% of SP-positive articular neurons contained CGRP, a much lower ratio than in other afferents of the dog such as testicular afferents. Our data suggest that these peptides are not always released together and that they do not always work together in the joint under normal conditions.

Animals↗

Colocalization of orexin a and glutamate immunoreactivity in axon terminals in the tuberomammillary nucleus in rats.

The orexins (also known as hypocretins) are peptide neurotransmitters made by hypothalamic neurons that are thought to play an important role in regulating wake-sleep states. One terminal area for orexin neurons is the tuberomammillary nucleus, a histaminergic cell group that is wake-active, but the relationship of the orexinergic terminals to the tuberomammillary neurons has not been examined in detail. We studied the ultrastructure of orexin A-immunoreactive axons and terminals in the tuberomammillary nucleus using pre- and post-embedding electron microscopic protocols. We confirmed an abundant projection of orexin-immunoreactive boutons to both dorsal and ventral divisions of the tuberomammillary nucleus. These terminals made asymmetric synaptic contacts with proximal and intermediate dendrites of tuberomammillary neurons. They contained small, clear synaptic vesicles and up to 30-40 dense core vesicles were seen per terminal in a single section. Both pre- and post-embedding immunostaining revealed that orexin immunoreactivity was localized to the dense core vesicles, which were always at a distance from the synaptic specialization. We also found glutamate immunoreactivity in the small synaptic vesicles which were at the active zone of the synapses of many of the same terminals. Orexinergic afferents to the tuberomammillary neurons contain separate populations of orexinergic and glutamatergic vesicles, suggesting that the release of these neurotransmitters may be differentially regulated.

Adenosine Deaminase↗

Identification of neuronal plasma membrane microdomains that colocalize beta-amyloid and presenilin: implications for beta-amyloid precursor protein processing.

Alzheimer's disease (AD) is associated with the accumulation of extracellular deposits of the beta-amyloid protein (Abeta). Abeta is a result of misprocessing of the beta-amyloid precursor protein (APP). Gamma-secretase is involved in APP misprocessing and one hypothesis holds that this secretase is identical to PS1. We tested this hypothesis by determining whether PS is co-localised with Abeta in situ. Using confocal analyses and a sensitive immunogold procedure we show that PS and Abeta are co-localised within discrete microdomains of neuronal plasma membranes in AD patients and in aged dogs, an established model of human brain aging. Our data indicate that APP misprocessing occurs in discrete plasma membrane domains of neurons and provide evidence that PS1 is critically involved in Abeta formation.

Aged↗

Evidence for colocalization of calcineurin and calcium channels in dorsal root ganglion neurons.

Calcineurin is a serine/threonine protein phosphatase 2B widely distributed in the brain. However, its role in brain function remains unknown. Recent data indicate that calcineurin can participate in long-term depression or long-term potentiation in rat hippocampus. Obviously, calcineurin can also be involved in numerous brain diseases, such as ischaemic hippocampal damage when the protein dephosphorylation system is markedly altered and hyperphosphorylation of the microtubule system in Alzheimer's disease. Besides, abnormal phosphorylation of the cytoskeletal proteins affecting the synaptic signalling can lead to different pathological disorders in the brain. In this study we analysed in more detail the localization of calcineurin in neuronal elements by using confocal microscopy and immunocytochemical approaches to record the enzyme expression in cultured rat dorsal root ganglion neurons. This is the first report showing that calcineurin immunoreactivity is highly expressed in dorsal root ganglion neurons and it is localized mainly near the inner surface of the plasma membrane. Immunostaining of these cells by anti-beta subunits of voltage-operated Ca2+ channels showed that distribution of calcium channel beta-subunit and calcineurin is very similar. Our findings confirm that the function of calcineurin can be directly connected with the activity of voltage-operated calcium channels.

Animals↗

B-cell epitopes in HIV-1 Tat and Rev proteins colocalize with T-cell epitopes and with functional domains.

We describe the characterization of the B-cell epitopes of HIV-1 regulatory proteins Tat and Rev. The prevalence of antibodies to these proteins among human immunodeficiency virus (HIV)-1-infected individuals was examined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The Tat and Rev antibody-positive sera were selected for epitope mapping performed with partially overlapping synthetic peptides bound to polyethylene pins. Eighteen and twelve percent of HIV-infected individuals had antibodies against Tat or Rev, respectively. In Tat, four epitopic regions were identified, situated within amino acids 6-10 (PRLEP), 21-37 (ACTNCYCKKCCFHCQVC), 39-58 (ITKALGISYGRKKRRQRRRA) and 74-82 (TSQSRGDPT). The most frequently recognized epitopic regions were located in the middle of the protein. In Rev, the two most frequently recognized epitopic regions were near the amino terminus of the protein within amino acids 12-20 (LIRTVRLIK) and 38-49 (RRNRRRRWRERQ). A third epitope was mapped around amino acids 55-62 (ISERILGT) and a fourth around amino acids 78-83 (LERLTU). To analyze the specificity of Tat and Rev epitopes, soluble synthetic peptides representing the identified epitopes were used in an ELISA assay, and the recognition of most epitopes was shown to be specific for HIV-1-infected individuals. In addition, many of the Tat and Rev epitopes were shown to overlap with regions having functional activity or with regions previously identified as T-cell epitopes.

Amino Acid Sequence↗

No colocalization of immunoreactivities for VIP and neuronal NOS, and a differential relation to cGMP-immunoreactivity in bovine penile smooth muscle.

The distribution of immunoreactivity (IR) for the neuropeptide vasoactive intestinal polypeptide (VIP) and neuronal nitric oxide synthase (nNOS) in the bovine retractor penis muscle (RP) and penile artery (PA) was studied by using two different methods. The distribution of these immunoreactivities was also compared with that of the immunoreactivity for cyclic guanosine monophosphate (cGMP). In both tissues the nerve fibers and terminals immunoreactive for VIP had a distribution that was completely different from that of the nerve fibers and terminals immunoreactive for nNOS. This contrasts with the previous observations in penile smooth muscle of other species. In the RP, as well as in the PA, many of the VIP-IR fibers were also immunoreactive for neurofilaments (NF), whereas the nNOS-IR fibers were consistently devoid of NF-IR. Stimulation with sodium nitroprusside, a nitric oxide donor, considerably increased cGMP-IR in the smooth muscle cells in both RP and PA, and in several nerve fibers in PA. Many of these cGMP-IR nerve fibers exhibited nNOS-IR, whereas none of them was immunoreactive for VIP. Our results suggest that the degree of coexistence of VIP-IR and nNOS-IR in the nerve fibers and terminals innervating penile smooth muscle show wide species differences. They also suggest that the mechanisms by which VIP could be involved in neurogenic penile erection may vary between species.

Animals↗

Colocalization of parvalbumin, calretinin and calbindin D-28k in human cortical and subcortical visual structures.

Several studies have demonstrated that three calcium-binding proteins parvalbumin (PV), calbindin D-28k (CB) and calretinin (CR) mark distinct subsets of cortical interneurons. This study demonstrates, in cortical and subcortical visual structures, the coexistence of two calcium-binding proteins in some neuronal subpopulations. The human visual cortex (VC), lateral geniculate nucleus (LGN). lateral inferior pulvinar (LIP) and superior colliculus (SC) were examined by a double-labelling immunocytochemical technique. The VC showed mostly separate populations of PV, CB and CR immunoreactive (-ir) interneurons, but also small populations of double-stained PV + CR and CR + CB neurons, while PV + CB neurons were less frequent. An average of 2.5% of the immunoreactive neurons were double-stained for PV + CR and 7.1% for CR + CB in area 17, while this percentage was slightly higher in association area 18 (3.3 and 7.4%, respectively). In the LGN and LIP, double-stained neurons were scarce, but in the fibre capsule of these nuclei, as well as in the optic radiation (OR) and white matter underlying area 17, both double-stained PV + CR or CR + CB and separate populations of PV-ir, CB-ir and CR-ir neurons and fibres were observed. Unlike the thalamic regions, the SC showed some double-stained PV + CR and CR + CB neurons, scattered both in the superficial and deep layers. These findings are discussed in the light of similar observations recently reported from other regions of the human brain.

Adult↗

CD157 undergoes ligand-independent dimerization and colocalizes with caveolin in CHO and MCA102 fibroblasts.

CD157, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, has recently been shown to induce protein tyrosine phosphorylation in monocytes differentiated from HL-60 cells (mHL-60) in a ligand-dependent manner, but in a ligand-independent manner in stable CD157-transfected CHO (CHO/CD157) and MCA102 (MCA/CD157) fibroblasts [Cell Signal. 11 (1999) 891-897.]. Many GPI-anchored proteins need to be clustered by their ligands or antibodies to induce redistribution to caveolae and a concomitant activation of the associated signal-transducing proteins [Nature 387 (1997) 569-572.]. Here, we demonstrate that CD157, independent of antibody crosslinking, undergoes dimerization with disulfide bond formation and localization in caveolae in CHO/CD157 and MCA/CD157 fibroblasts. However, the native CD157 induced in mHL-60 cells remains a monomer form. The structural integrity of caveolae is required for the association of CD157 with caveolin and CD157-mediated tyrosine kinase signalling in the fibroblasts. We propose that an overexpression of CD157 could lead to its dimerization and relocation to caveolae and to further result in the initiation of signalling processes.

ADP-ribosyl Cyclase↗

Characterization of Drosophila Presenilin and its colocalization with Notch during development.

Mutant Presenilin proteins cause early-onset familial Alzheimer's disease in humans and Caenorhabditis elegans Presenilins may facilitate Notch receptor signaling. We have isolated a Drosophila Presenilin homologue and determined the spatial and temporal distribution of the encoded protein as well as its localization relative to the fly Notch protein. In contrast to previous mRNA in situ studies, we find that Presenilin is widely expressed throughout oogenesis, embryogenesis, and imaginal development, and generally accumulates at comparable levels in neuronal and nonneuronal tissues. Double immunolabeling with Notch antibodies revealed that Presenilin and Notch are coexpressed in many tissues throughout Drosophila development and display partially overlapping subcellular localizations, supporting a possible functional link between Presenilin and Notch.

Amino Acid Sequence↗