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PubMed1997

Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney.

NG-monomethylarginine (L-NMA) and asymmetric NG, NG-dimethylarginines (ADMA) are endogenous inhibitors of cellular L-arginine uptake and/or nitric oxide (NO) synthesis that are implicated in renal parenchymal and Dahl salt-sensitive hypertension. Since the L-arginine:(L-NMA + ADMA) ratio determines NO synthase (NOS) activity, we compared the immunohistochemical distribution of NOS with NG, NG-dimethylarginine dimethylaminohydrolase (DDAH), which inactivates dimethylarginines (DMA) and L-NMA by hydrolysis to L-citrulline. Neuronal NOS (nNOS) was expressed predominantly in tubular epithelial cells of macula densa (MD), endothelial NOS (eNOS) in vascular endothelial cells (EC), and inducible NOS (iNOS) quite widely in tubular epithelium, including proximal tubules (PT), thick ascending limbs of Henle (TAL), distal convoluted tubule and intercalated cells (IC) of the collecting duct. Immunostaining for DDAH was present in PT, TAL, MD, and IC, and was also present in the glomerulus, Bowman's capsule, and endothelium of blood vessels. DDAH was detected in small vesicles of TAL and PT by electron microscopic (EM) immunocytochemistry. To study the effects of methylarginines on tubuloglomerular feedback (TGF) response, vehicle or methylarginines (10(-3) M) were added to artificial tubular fluid (ATF) perfused orthogradely from the late PT at 40 nl. min-1 while assessing changes in glomerular capillary pressure from proximal stop flow pressure (PSF). Whereas the maximal TGF responses were unchanged by vehicle (delta TGF 0 +/- 0%) or symmetric DMA (SDMA; +1 +/- 2%, NS), they were enhanced by L-NMA (+22 +/- 4%, P < 0.001) and asymmetric DMA (ADMA; +28 +/- 3%, P < 0.001). Since L-arginine transport can regulate renal epithelial NO generation, methylarginines (10(-3) M) or vehicle were co-perfused orthogradely with [3H]-L-arginine from the late PT and collected at the early distal tubule to study arginine uptake from the perfused loop of Henle. All methylarginines reduced fractional loop [3H] absorption significantly (P < 0.001; vehicle, 84 +/- 6; ADMA, 49 +/- 6; SDMA, 56 +/- 6; L-NMA, 41 +/- 6%). In conclusion, sites of DDAH expression in the vasculature or nephron are all sites of expression of an isoform of NOS. L-NMA, ADMA, and SDMA all inhibit renal tubular L-arginine uptake, whereas L-NMA and ADMA, but not SDMA, enhance TGF responses. Therefore, DDAH may regulate the cellular L-arginine: methylarginine levels in specific renal cells, thereby governing cell-specific L-arginine uptake and NO generation in renal tubular epithelium.

Amidohydrolases↗
PubMed1997

Immunohistochemical colocalization of the alpha-subunit of neutrophil NADPH oxidase and ecto-5'-nucleotidase in kidney and liver.

In kidney and liver, fibroblasts and fibroblast-like cells, respectively, are sources of erythropoietin (Epo) formation, and these cells also bear a number of other similarities. Renal Epo expression is localized in peritubular type 1 fibroblasts of the cortical labyrinth, and in the liver, apart from parenchymal cells, transcription is found in Ito cells. Both the renal peritubular cells and Ito cells contain ecto-5'-nucleotidase (5'NT). It had been suggested that 5'NT is involved in the oxygen sensing mechanism via a hydrolysis of AMP to adenosine, which in turn may stimulate EPO synthesis. However, the molecular mechanism of the cellular response to hypoxia is currently not well understood. Based on the notion that a heme protein probably acts as the oxygen sensor, it has recently been proposed that a b-type cytochrome as part of the neutrophil NADPH oxidase may influence intracellular superoxide levels depending on local oxygen tension. Superoxide levels were otherwise shown to determine the EPO production in hepatoma cell lines. By double immunofluorescence labeling the alpha-subunit of cytochrome b558 (alpha-SU) and 5'NT were simultaneously localized in rat kidney and liver, and in the kidney Epo mRNA and alpha-SU were double-labeled. Positive signal for alpha-SU was found in the majority of renal peritubular fibroblasts in the cortex and outer medulla, and in Ito cells. In both organs, the cells that coexpress 5'NT and Epo mRNA also contain an immunoreactivity for alpha-SU. In these cells, cytochrome b558 as part of an NADPH oxidase may be involved in a presumptive oxygen sensing mechanism using H2O2 as a possible second messenger for EPO gene regulation.

5'-Nucleotidase↗
PubMed2000

Distribution of Tsc1 protein detected by immunohistochemistry in various normal rat tissues and the renal carcinomas of Eker rat: detection of limited colocalization with Tsc1 and Tsc2 gene products in vivo.

We and others previously demonstrated that hereditary mutation and a subsequent second hit in the rat homolog of tuberous sclerosis gene (Tsc2) are responsible for Eker renal carcinomas (RC). In humans, alteration in the TSC2 gene is known to cause the tuberous sclerosis complex (TSC) that results in hamartomatous lesions in multiple organs, but the function of TSC2 is not fully understood. In recent years, a second gene (TSC1) responsible for human TSC has been cloned, and binding between TSC1 and TSC2 proteins was reported. In this study, to clarify associations between Tsc proteins in vivo, the expression of Tsc1 protein was detected by immunohistochemistry, and compared with Tsc2 expression. Tsc1 protein was expressed in the nervous system and in many endocrine tissues, including pancreatic islets, the parathyroids, testis, and ovary. Tsc1 was also detected in the many epithelial tissues of organs, such as kidney, uterus, small and large intestine, and liver. Our results indicate overlapping, but not identical, organ distributions of Tsc1 and Tsc2 proteins. At the intracellular distribution, double fluorescent immunolabeling allowed the determination that only a partial portion of Tsc1 signals overlapped with Tsc2 in some organs. These results suggest the existence of co-localizing and independent forms of Tsc proteins in endogenous expressions. Additionally, relatively high expression of Tsc1 protein was detected in RC in the Tsc2 mutant (Eker) rat.

Animals↗
PubMed2004

Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides.

We have previously shown that apolipoprotein E (Apoe) promotes the formation of amyloid in brain and that astrocyte-specific expression of APOE markedly affects the deposition of amyloid-beta peptides (Abeta) in a mouse model of Alzheimer disease. Given the capacity of astrocytes to degrade Abeta, we investigated the potential role of Apoe in this astrocyte-mediated degradation. In contrast to cultured adult wild-type mouse astrocytes, adult Apoe(-/-) astrocytes do not degrade Abeta present in Abeta plaque-bearing brain sections in vitro. Coincubation with antibodies to either Apoe or Abeta, or with RAP, an antagonist of the low-density lipoprotein receptor family, effectively blocks Abeta degradation by astrocytes. Phase-contrast and confocal microscopy show that Apoe(-/-) astrocytes do not respond to or internalize Abeta deposits to the same extent as do wild-type astrocytes. Thus, Apoe seems to be important in the degradation and clearance of deposited Abeta species by astrocytes, a process that may be impaired in Alzheimer disease.

Amyloid beta-Peptides↗
PubMed1997

An autoradiographic study of dextromethorphan high-affinity binding sites in rat brain: sodium-dependency and colocalization with paroxetine.

1. The distribution and some pharmacological properties of centrally located dextromethorphan high-affinity binding sites were investigated by in vitro autoradiography. 2. Sodium chloride (50 mM) induced a 7 to 12 fold increase in dextromethorphan binding to rat brain in all areas tested. The effect of sodium was concentration-dependent with a higher dose (120 mM) exerting a smaller effect on binding. 3. [3H]-dextromethorphan binding in the presence of sodium was inhibited in the presence of the anticonvulsant phenytoin at a concentration of 100 microM, while the sigma ligand (+)-3-(-3-hydroxyphenyl)-N-(1-propyl)pipendine ((+)-PPP) had no effect on the binding, suggesting an interaction with the DM2 site. 4. The distribution of the sodium-dependent binding identified in this study correlated significantly with the distribution of the selective 5-HT uptake inhibitor [3H]-paroxetine, and paroxetine and dextromethorphan mutually displaced their binding at concentrations in the low nanomolar range. 5. These data show that dextromethorphan and paroxetine share a sodium-dependent high affinity binding site in rat brain, and suggest that dextromethorphan might interact, in the presence of sodium, with the 5-HT uptake mechanism in rat brain.

Animals↗
PubMed2002

Interaction and colocalization of PGP9.5 with JAB1 and p27(Kip1).

PGP9.5 (UCH-L1) is a member of the ubiquitin C-terminal hydrolase (UCH) family of proteins that is expressed in neuronal tissues. Our previous studies have shown that PGP9.5 was highly expressed in primary lung cancers and lung cancer cell lines. Additionally, the frequency of PGP9.5 over expression increases with tumor stage, indicating that PGP9.5 may play a role in lung cancer tumorigenesis. We used the yeast two-hybrid system to identify proteins that interact with PGP9.5. We show that PGP9.5 interacts with at least three proteins, one of which is JAB1, a Jun activation domain binding protein that can bind to p27(Kip1) and is involved in the cytoplasmic transportation of p27(Kip1) for its degradation. We also show that PGP9.5 is associated with JAB1 in vitro and in vivo; and that both proteins can be a part of a heteromeric complex containing p27(Kip1) in the nucleus in lung cancer cells. Furthermore, under serum-restimulation, nuclear translocation of both PGP9.5 and JAB1 coincides with a reduced level of p27(Kip1) in the nucleus. In contrast, when cells are contact inhibited, both PGP9.5 and JAB1 became more perinuclear and cytoplasmic in localization while p27(Kip1) was present only in the nucleus. Therefore, PGP9.5 may contribute to p27(Kip1) degradation via its interaction and nuclear translocation with JAB1.

Active Transport, Cell Nucleus↗
PubMed2001

Colocalization of CGRP with 5-HT1B/1D receptors and substance P in trigeminal ganglion neurons in rats.

Vasodilatation in the dura mater has been implicated in migraine pathogenesis. Anti-migraine triptan drugs block vasodilatation by binding to 5-HT1B/1D receptors localized on the peripheral sensory terminals and dural blood vessel smooth muscles. Previous studies suggest that calcitonin gene-related peptide (CGRP) released from Adelta-fibres plays a more important role than substance P (SP) released from C-fibres in inducing dural vasodilatation and that one of the antimigraine mechanisms of triptan drugs is inhibiting CGRP release. In the present study, the relationship between CGRP and 5-HT1B/1D receptors, and between CGRP and SP in the trigeminal ganglion neurons in rats was examined by double immunohistochemical staining. CGRP, 5-HT1B, 5-HT1D and SP-positive trigeminal ganglion neurons were all predominantly small and medium-sized. In the trigeminal ganglia, approximately 50% of CGRP-positive neurons were 5-HT1B positive. Similarly, approximately 55% of CGRP-positive neurons were 5-HT1D immunoreactive. Approximately 50% of CGRP-positive neurons were SP-positive, while 93% of SP-positive neurons were CGRP-positive, suggesting that nearly all SP-positive neurons also contain CGRP. The fibre types of the 5-HT1B- and 5-HT1D-positive neurons were further investigated with an antibody against the A-fibre marker 200-kDa neurofilaments (NF200). Approximately 46% of the 5-HT1B-positive and 43% of the 5-HT1D-positive trigeminal ganglion neurons were also NF200 positive, indicating that many A-fibre trigeminal neurons express 5-HT1B or 5-HT1D receptors. These results support the hypothesis that one important action of antimigraine drugs is the inhibition of CGRP release and that Adelta-fibres may play an important role in migraine pathogenesis.

Animals↗
PubMed2002

Persistent increase in myofibroblasts in Helicobacter heilmannii-infected mice but not in Helicobacter pylori-infected Mongolian gerbils: colocalization of COX-2 and bFGF immunoreactivity.

BACKGROUND: The clinical significance of Helicobacter heilmannii infection remains uncertain, owing to the lack of a specific detection method. Recently, we reported a marked increase in myofibroblasts in the early stage of Helicobacter pylori infection in Monglian gerbils. AIM: The present study was designed to clarify changes in myofibroblasts, and in the immunoeactivities of basic fibroblast growth factor, cyclooxygenase-2 and inducible nitric oxide synthase after H. pylori infection in Monglian gerbils and H. heilmannii infection in mice. METHODS: After oral inoculation, changes in the location of bacteria and the immunoreactivity of myofibroblasts, basic fibroblast growth factor, cyclooxygenase-2 and inducible nitric oxide synthase were stained with the indirect immunofluorescent method and observed by confocal laser microscopy. RESULTS: In H. heilmannii-infected mice, the increases in myofibroblasts and in immunoreactivities of these three markers were sustained 12 months after infection. In H. pylori-infected Monglian gerbils, however, these increases were significant at 3 months but had returned to control levels at 12 months. CONCLUSIONS: Two types of Helicobacter infection showed different patterns of myofibroblast proliferation, coinciding with the extent of inflammation. These findings suggest that this difference may be related to the consequences of the infection.

Animals↗
PubMed2001

Damaged myocytes as detected by the colocalization of DNA fragmentation and tissue transglutaminase and their prognostic significance in enterovirus-associated dilated cardiomyopathy.

BACKGROUND: Apoptotic cardiac myocytes have been described in chronic heart failure, but no data exist on the relationship between these 'damaged' myocytes and myocardial detection of enterovirus RNA often associated with dilated cardiomyopathy (DCM). DESIGN: In patients with idiopathic DCM, endomyocardial biopsy samples were studied for enteroviral RNA by one step reverse transcription-polymerase chain reaction (PCR) and a subsequent hybridization of the PCR product using a Southern blot technique. The endomyocardial biopsies were further investigated for markers of cell damage and apoptosis: DNA fragmentation and expression of tissue-transglutaminase (TTG) in the myocytes using the in-situ endlabelling method or an anti-TTG-staining, respectively. To assess the prognostic significance of these two markers the correlation between the percentage of myocytes positive both for DNA fragmentation and TTG (the index of damaged myocytes) and the hemodynamic course of the patients during a mean follow-up period of 15.9 +/- 6.2 months was investigated prospectively by echocardiography. RESULTS: In 14 (45%) of the 31 patients with idiopathic DCM, enteroviral RNA was found in the endomyocardial biopsy samples, while 17 patients (55%) were enterovirus-negative. In enterovirus-positive patients, the index of 'damaged' myocytes was significantly lower (10.7 +/- 4.9% vs. 19.2 +/- 8.8%, P = 0.002) and the left ventricular ejection fraction (LVEF) improved significantly (P = 0.00017 vs. P = 0.13) during long-term follow-up. In addition, a weak negative correlation was seen between the index of damaged myocytes and the changes in LVEF in all patients during long-term follow-up (r = - 0.48, P = 0.004). CONCLUSION: Our results favour the view that enterovirus-positive patients with DCM have less damaged myocytes and a better haemodynamic course than enterovirus-negative patients.

Adult↗
PubMed2000

Expression of the Fusarium resistance gene I-2 colocalizes with the site of fungal containment.

The tomato resistance gene I-2 is one of at least six members of a gene family that are expressed at low levels in the roots, stems and leaves of young tomato plants. Plants transformed with constructs containing a functional I-2 promoter fused to the beta-glucuronidase (GUS) reporter gene were used in detailed expression studies. Highest GUS activity was found in stems of young tomato plants. Histochemical analysis revealed that the I-2 promoter drives expression of the reporter gene in vascular tissue of fruits, leaves, stems and mature roots. In younger roots, expression was most abundant at the base of lateral root primordia. Microscopical analysis of young tomato plants revealed expression in tissue surrounding the xylem vessels. We show that in resistant plants, fungal growth into this region of the vascular tissue is prevented, suggesting a correlation with the I-2-mediated resistance response.

Base Sequence↗
PubMed1998

Preprodynorphin mRNA-expressing neurones in the rat parabrachial nucleus: subnuclear localization, hypothalamic projections and colocalization with noxious-evoked fos-like immunoreactivity.

The dorsal lateral subnucleus of the rat pontine parabrachial nucleus is a major target for ascending nociceptive information from the spinal cord. With in situ hybridization histochemistry, using a radiolabelled cRNA probe, we demonstrate that neurones in and near the dorsal lateral subnucleus express preprodynorphin mRNA. The cRNA probe was constructed from a PCR product amplified from rat genomic DNA. Sequencing of the PCR product revealed that it corresponded to the sequence 466-1101 of the rat preprodynorphin gene exon 4. Tract tracing experiments, using injection of cholera toxin subunit B into the hypothalamic median preoptic nucleus, showed a retrograde labelling pattern of neurones in the parabrachial nucleus that was almost identical to that of the preprodynorphin mRNA expressing neurones. Double-labelling, combining immunohistochemical detection of tracer and in situ hybridization, revealed that the retrogradely labelled neurones expressed preprodynorphin mRNA. A similar double-labelling, combining in situ hybridization with immunohistochemical detection of noxious-evoked fos following formalin injection into one hindpaw of awake animals, showed that almost all fos-immunoreactive neurones in the dorsal lateral parabrachial subnucleus also expressed preprodynorphin mRNA. Quantitative analysis suggested that the evoked fos immunoreactivity was accompanied by an increased preprodynorphin mRNA expression. The findings provide evidence that neurones in the dorsal lateral subnucleus produce dynorphin and project to the median preoptic nucleus, and that noxious stimulation in awake animals synaptically activates the dynorphinergic neurones in this subnucleus. These observations are consistent with the idea of a functional and chemical heterogeneity among different parabrachial subnuclei that serves to produce specific homeostatic responses to stimuli that changes the physiological status of the organism, including tissue damage.

Animals↗
PubMed1999

Distribution, regulation and colocalization of the genes encoding the EP2- and EP4-PGE2 receptors in the rat brain and neuronal responses to systemic inflammation.

It is currently believed that prostaglandin (PG) of E2 type plays a crucial role in transferring the information received from circulating immune factors to brain parenchymal cells. Although PGE2 is synthesized quite essentially by cells of the blood-brain barrier, the organization and regulation of its receptor subtypes within neuronal elements remain unknown. In this study, intravenous (i.v.) injection of the endotoxin lipopolysaccharide (LPS) or recombinant rat interleukin-1beta (IL-1beta), and intramuscular (i.m.) injection of turpentine were used as different models of systemic immune stimuli. Rats were perfused at various times after the insults (30 min to 24 h), their brains cut and hybridized with full-length rat cRNA probes. Double-labelling procedures were accomplished to determine the cellular phenotype and activity. A very distinct distribution of both EP2 and EP4 receptors was found across the brain under basal conditions; the hybridization signal for the type 2 was detected in the bed nucleus of the stria terminalis (BNST), lateral septum, subfornical organ (SFO), ventromedial hypothalamic nucleus (VMH), central nucleus of the amygdala (CeA), locus coeruleus (LC) and the area postrema (AP), whereas the ventral septal/anterior preoptic area, the magnocellular paraventricular nucleus (PVN), supraoptic nucleus, parabrachial nucleus, LC, the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM) exhibited moderate to strong levels for the EP4 mRNA under basal conditions. Upregulation of the genes encoding EP2 and EP4 receptors was detected in selective regions and neuronal populations during systemic inflammatory challenges. The most dramatic one being the robust transcriptional activation of the EP4 subtype within corticotropin-releasing factor (CRF) neurons of the parvocellular PVN following i.v. LPS and IL-1beta injection, and the localized i.m. aggression. These neurons of the endocrine hypothalamus as well as those of numerous autonomic-related nuclei were activated by the proinflammatory cytokine, as they were immunoreactive (ir) to Fos nuclear protein. The EP4 transcript was also present in activated catecholaminergic neurons of the LC, NTS and VLM, although only the A1 cell group exhibited an increase in EP4 transcription in response to circulating IL-1beta. Moreover, the systemic immunogenic insults caused a significant increase in the EP2 mRNA levels in the CeA, SFO, AP and the leptomeninges. These data provide a distinct pattern of EP2 and EP4 expression throughout the rat brain under both basal and immune-challenged conditions, and underlie the possible role of the EP4 subtype in mediating the effects of PGE2 on different autonomic and neuroendocrine functions. The presence of Fos-ir nuclei in various populations of EP4 neurons of IL-1beta-treated animals clearly supports this concept and suggests that the selectivity of the neuronal response during systemic inflammation may depend on the expression of specific PGE2 receptors in key structures of the brain.

Animals↗
PubMed1994

Evidence for the colocalization of parvalbumin and glutamate, but not GABA, in the perforant path of the gerbil hippocampal formation: a combined immunocytochemical and microquantitative analysis.

Gerbils (Meriones unguiculatus) are known for their seizure sensitivity, which is dependent on an intact perforant path from the entorhinal cortex to the hippocampus. In contrast with other species, the perforant path in gerbils contains parvalbumin, a cytosolic high-affinity calcium-binding protein. Parvalbumin is known to be present in a subpopulation of GABA-containing neurons and is thought to be responsible for their physiological characteristics of fast spiking activity and lack of spike adaptation. Therefore, the question arose of whether this projection in gerbils is GABAergic or glutamatergic as in other species. In a first approach to this question, the effect of lesioning the origin of the perforant path, the entorhinal cortex, on levels of GABA and glutamate was determined by enzymatic-luminometric assay in single layers of the dentate gyrus of lyophilized brain sections. Parallel sections were cryofixed using an acidified acetone-formaldehyde mixture at -20 degrees C for 48 h, and subsequently stained for parvalbumin immunocytochemistry. Seven days after ablation of the entorhinal cortex, parvalbumin staining was undetectable in the termination zone of the perforant path, the outer two-thirds of the stratum moleculare. In parallel, glutamate content was reduced to 80% of controls (and of the unoperated contralateral side) but unchanged in the inner third of the stratum moleculare and in stratum granulare. GABA content was not significantly altered by the lesion. From these results, we conclude that in the gerbil as in other species, the perforant path contains glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Visualization of D1 dopamine receptors on living nucleus accumbens neurons and their colocalization with D2 receptors.

To examine the substrate for dopamine (DA) synaptic action in the nucleus accumbens (nAcc), we visualized the cellular and subcellular distribution of DA receptors on postnatal nAcc neurons in culture using fluoroprobe derivatives of DA receptor ligands. Previously, we have shown that rhodamine-N-(p-aminophenethyl)-spiperone (NAPS) (10 nM), a derivative of the D2 antagonist spiperone, labels D2-like receptors on living nAcc neurons. We now show that rhodamine-Sch-23390 (30 nM), a derivative of the D1 antagonist, labels D1-like receptors. Putative specific membrane labeling reached a plateau after about 20 min. Labeling was stereospecific, as it was unaffected by competition with (-)-butaclamol, but blocked with (+)-butaclamol. We found that 52 +/- 7% of nAcc medium-sized neurons showed D1 labeling, which extended onto the dendrites. Labeling was also seen on presynaptic terminals, often abutting D1-positive and D1-negative cell bodies, consistent with a presynaptic modulatory role for D1 receptors. Larger neurons, which may be GABAergic or cholinergic interneurons, were also labeled. By sequential labeling first with rhodamine-Sch-23390 and then rhodamine-NAPS, we found that 38 +/- 6% of medium-sized neurons express both D1- and D2-like receptors, indicating that D1-D2 interactions may occur at the level of single postsynaptic neurons.

Animals↗
PubMed1998

Steroidogenic acute regulatory protein (StAR) transcripts constitutively expressed in the adult rat central nervous system: colocalization of StAR, cytochrome P-450SCC (CYP XIA1), and 3beta-hydroxysteroid dehydrogenase in the rat brain.

Steroidogenic acute regulatory protein (StAR) is a 30-kDa protein involved in the transport of cholesterol to the inner mitochondrial membrane and thus plays a key role in steroid biosynthesis. To clarify the implications of this protein in neurosteroid biosynthesis, we examined the possible expression of a StAR transcript in the adult rat CNS and detected it. cDNA cloning and sequencing analysis revealed that two forms of StAR mRNAs are expressed in the brain in the same manner as in the adrenal gland, indicating that they are fully functional and not minor gene transcripts. An RNase protection assay quantitatively revealed that the amount of the rat StAR transcript in brain was two to three orders of magnitude lower than that in the adrenal gland. An in situ hybridization study, involving antisense riboprobes, revealed that StAR transcripts were abundant in the cerebral cortex, hippocampus, dentate gyrus, olfactory bulb, cerebellar granular layer, and Purkinje cells. Furthermore, other steroidogenic enzymes, side-chain cleavage cytochrome P-450SCC (CYP XIA1) and 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (EC 1.1.1.145), were found to be coexpressed in the hippocampus, dentate gyrus, cerebellar granular layer, and Purkinje cells. These findings strongly indicate that neurosteroids are synthesized in a region-specific manner in the brain.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

The cellular prion protein colocalizes with the dystroglycan complex in the brain.

The function of PrP(C), the cellular prion protein (PrP), is still unknown. Like other glycophosphatidylinositol-anchored proteins, PrP resides on Triton-insoluble, cholesterol-rich membranous microdomains, termed rafts. We have recently shown that the activity and subcellular localization of the neuronal isoform of nitric oxide synthase (nNOS) are impaired in adult PrP(0/0) mice as well as in scrapie-infected mice. In this study, we sought to determine whether PrP and nNOS are part of the same functional complex and, if so, to identify additional components of such a complex. To this aim, we looked for proteins that coimmunoprecipitated with PrP in the presence of detergents either that completely dissociate rafts, to identify stronger interactions, or that preserve the raft structure, to identify weaker interactions. Using this detergent-dependent immunoprecipitation protocol we found that PrP interacts strongly with dystroglycan, a transmembrane protein that is the core of the dystrophin-glycoprotein complex (DGC). Additional results suggest that PrP also interacts with additional members of the DGC, including nNOS. PrP coprecipitated only with established presynaptic proteins, consistent with recent findings suggesting that PrP is a presynaptic protein.

Actins↗
PubMed2001

Colocalization of the viral interleukin-6 with latent nuclear antigen-1 of human herpesvirus-8 in endothelial spindle cells of Kaposi's sarcoma and lymphoid cells of multicentric Castleman's disease.

Human herpesvirus-8 (HHV-8) also called Kaposi's sarcoma-associated herpesvirus infects spindle cells in Kaposi's sarcoma (KS) and lymphoid cells in multicentric Castleman's disease (MCD). In KS cells, HHV-8 is mainly latent with the expression of latent nuclear antigen-1 (LNA-1), whereas in MCD both lytic and latent antigens are produced by lymphoid cells. We show by immunohistochemical labeling that in KS viral interleukin-6 (vIL-6) is expressed in rare spindle cells, whereas in MCD, vIL-6 is detectable in lymphoid cells around lymphoid follicles but also within the follicular dendritic reticulum cell network. The staining of apoptotic bodies with anti IL-6 antibody suggests the achievement of a complete lytic cycle in a subset of lymphoid cells. Interestingly, in MCD, some areas contained vascular spindle cells latently infected by HHV-8 on the basis of LNA-1 expression. This finding might imply that in MCD, both vascular and lymphoid cells proliferate in response to the viral infection. Double immunostaining with anti LNA-1 and anti vIL-6 in MCD and KS identifies 2 subsets of HHV-8 infected (vascular and lymphoid) cells, some with exclusive expression of LNA-1 and some with coexpression of vIL-6 and LNA-1. This suggests that in vivo the regulation of the expression vIL-6 and LNA-1 protein varies with the cell type. In addition, the detection of infected endothelial cells in MCD may indicate that these cells belong to the reservoir for HHV-8.

Apoptosis↗