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PubMed1995

Nitric oxide pathway in cat esophagus: localization of nitric oxide synthase and functional effects.

In the cat lower esophageal sphincter (LES) and esophageal body, nitric oxide synthase (NOS) immunoreactive nerves were abundant in the circular smooth muscle layer, especially in the LES region. NADPH diaphorase staining showed an identical pattern. The ability to form L-citrulline from L-arginine corresponded roughly to the distribution of NOS. Confocal microscopic analysis indicated colocalization within neurons of vasoactive intestinal peptide (VIP) in 65% of NOS-positive nerves. In LES circular smooth muscle preparations, electrically induced relaxations (single train stimuli) were generally abolished by NG-nitro-L-arginine (L-NNA). Continuous electrical stimulation for 2 min evoked a relaxation in the presence of L-NNA. This relaxation was inhibited by VIP antiserum and followed by a decrease in guanosine 3',5'-cyclic monophosphate, but not by any consistent change in adenosine 3',5'-cyclic monophosphate levels. K+ (124 mM) induced a biphasic relaxation, with L-NNA inhibiting the first phase but not the second. We conclude that nitric oxide (NO) has a major role as the mediator responsible for relaxation in the cat esophagus. NO seems also to initiate the release and enhance the effect of another transmitter.

Amino Acid Oxidoreductases↗

PubMed2000

Molecular distribution of volume-regulated chloride channels (ClC-2 and ClC-3) in cardiac tissues.

The molecular identification of cardiac chloride channels has provided probes to investigate their distribution and abundance in heart. In this study, the molecular expression and distribution of volume-regulated chloride channels ClC-2 and ClC-3 in cardiac tissues were analyzed and quantified. Total RNA was isolated from atria and ventricles of several species (dog, guinea pig, and rat) and subjected to a quantitative RT-PCR strategy. ClC-2 and ClC-3 mRNA expression were calculated relative to beta-actin expression within these same tissues. The transcriptional levels of ClC-3 mRNA were between 1.8 and 10.2% of beta-actin expression in atria and between 3.4 and 8.6% of beta-actin in ventricles (n = 3 for each tissue). The levels of ClC-2 in both atria and ventricles were significantly less than those measured for ClC-3 (n = 3; P < 0.05). ClC-2 mRNA levels were between 0.04-0.08% and 0.03-0.18% of beta-actin expression in atria and ventricles, respectively (n = 3 for each tissue). Immunoblots of atrial and ventricular wall protein extracts demonstrated ClC-2- and ClC-3-specific immunoreactivity at 97 and 85 kDa, respectively. Immunohistochemical localization in guinea pig cardiac muscle demonstrates a ubiquitous distribution of ClC-2 and ClC-3 channels in the atrial and ventricular wall. Confocal analysis detected colocalization of ClC-2 and ClC-3 in sarcolemmal membranes and distinct ClC-3 immunoreactivity in cytoplasmic regions. The molecular expression of ClC-2 and ClC-3 in cardiac tissue is consistent with the proposed role of these chloride channels in the regulation of cardiac cell volume and the modulation of cardiac electrical activity.

Actins↗

PubMed2004

Inhibition of tumorigenicity and metastasis of human melanoma cells by anti-cathepsin L single chain variable fragment.

We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anti-cathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.

Animals↗

PubMed1997

Thyrotropin-releasing hormone gene expression by anterior pituitary cells in long-term cultures is influenced by the culture conditions and cell-to-cell interactions.

It has been suggested that TRH, synthesized by anterior pituitary (AP) cells in long-term monolayer cultures, may act as a paracrine or autocrine regulator. Because local control through messenger molecules depends on the cellular microenvironment, we were interested in studying the synthesis of TRH by AP cells in different culture systems and under various conditions. When AP cells were cultured as monolayers in medium containing 10% FCS for long periods of time (up to 3 weeks), a considerable increase in TRH content and prepro-TRHmessengerRNA (preproTRHmRNA) levels could be demonstrated by RIA and Northern blot analysis, whereas the cellular content of the TRH-like peptide pyroGlu-Glu-Pro-NH2 decreased with time in culture to undetectable levels. The release of TRH could be stimulated by depolarizing concentrations of K+ (55 mM), by the Ca++ ionophore A23187, and by GnRH, but not by CRH or GRF, indicating that TRH is stored in gonadotropes. Moreover, a combined in situ hybridization and immunocytochemical analysis demonstrated colocalization of LH in preproTRHmRNA-positive AP cells. When AP cells were cultured as reaggregates in the same (FCS-containing) medium, only a marginal increase in TRH content and preproTRHmRNA levels was observed. Irrespective of the culture systems and the culture conditions used, TRH gene expression was not observed when FCS was omitted. These results indicate that TRH gene expression more likely reflects derepression, rather than induction, of the TRH gene.

Animals↗

PubMed2004

Caveolin-1 and MAL are located on prostasomes secreted by the prostate cancer PC-3 cell line.

MAL, BENE and MAL2 are raft-associated integral membrane proteins of the MAL family of proteins involved in membrane trafficking processes. We show here that the human prostate carcinoma PC-3 cell line expresses the transcripts for the three proteins simultaneously. MAL, BENE and MAL2 co-fractionated with caveolin-1 in the raft fraction of PC-3 cells, and immunofluorescence analysis showed colocalization of these proteins with caveolin-1 in a multivesicular intracellular compartment. Markers of the Golgi apparatus, early and recycling endosomes and lipid droplets were excluded from this compartment. Prostate epithelial cells contain vesicular organelles enriched in raft components named prostasomes that are secreted in the prostate fluid. Interestingly, the prostasome fraction isolated from the culture supernatant of PC-3 cells consisted mainly of 30-130 nm cup-shaped vesicles that were positive for MAL, caveolin-1 and CD59, a glycosylphosphatidylinositol-anchored protein previously found in prostasomes. CD63, an integral membrane protein found in multivesicular bodies/lysosomes and secretory granules was also found in PC-3 cell-derived prostasomes. Prostasome secretion was not inhibited by brefeldin A, a compound that blocks the conventional secretory pathway. However, wortmannin, an inhibitor of phosphatidylinositol-3 kinase, reduced the secretion of prostasomes in PC-3 cells. Our results suggest that MAL family proteins are associated with caveolin-1 in a multivesicular compartment that may be involved in prostasomal secretion in PC-3 cells.

Androstadienes↗

PubMed2004

Beta-catenin associates with human parainfluenza virus type 3 ribonucleoprotein complex and activates transcription of viral genome RNA in vitro.

Several studies have indicated that human parainfluenza virus type 3 (HPIV-3) requires polymeric actin for transcription of its genome RNA in vitro and in vivo. In the current study, we have identified beta-catenin, an actin-bound protein, as one of the transcriptional activators for HPIV-3 genome RNA. Beta-catenin was packaged within the purified HPIV-3 virions and was associated with the HPIV-3 ribonucleoproteins (RNP) from infected cells. Moreover, purified beta-catenin interacted with bacterially expressed HPIV-3 nucleocapsid protein (N) and phosphoprotein (P) fused to glutathione S-transferase (GST). Double-labeled immunofluorescent confocal microscopic analysis revealed colocalization of beta-catenin with HPIV-3 RNP at cell periphery in infected cells. The HPIV-3 RNP-associated beta-catenin functioned as a transactivator of HPIV-3 genome, because purified beta-catenin stimulated transcription of viral RNP in an in vitro transcription assay. These results demonstrate that beta-catenin, a multifunctional protein that is involved in cell-cell adhesion and embryogenesis, acts as one of the transcriptional activators of HPIV-3 genome RNA.

Cytoskeletal Proteins↗

PubMed2001

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells.

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the beta(2)-microglobulin (beta(2)m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical Fc gamma Rs: Fc gamma RI, Fc gamma RII, and Fc gamma RIII.

Adult↗

PubMed2003

Inhibition of platelet-derived growth factor receptor phosphorylation by STI571 (Gleevec) reduces growth and metastasis of human pancreatic carcinoma in an orthotopic nude mouse model.

PURPOSE: We evaluated the expression of platelet-derived growth factor (PDGF) ligands and receptors in clinical specimens of human pancreatic adenocarcinomas and determined the therapeutic effect of STI571 (Gleevec), a protein tyrosine kinase inhibitor of PDGF receptor (PDGFR), on human pancreatic carcinoma cells growing in the pancreas and liver of nude mice. EXPERIMENTAL DESIGN: Immunohistochemical staining for PDGF-AA and -BB ligands, PDGFR-alpha and -beta, and phosphorylated PDGFR-alpha and -beta was performed on 31 specimens of human pancreatic cancer and L3.6pl human pancreatic adenocarcinoma cell line. To determine the in vivo effects of STI571, nude mice with L3.6pl cells injected into the pancreas were randomized 7 days later to receive one of the following treatments: sterile water p.o. (control), STI571, gemcitabine, or a combination of STI571 and gemcitabine. RESULTS: In 29 of 31 clinical specimens of human pancreatic adenocarcinoma, both tumor cells and tumor-associated endothelial cells expressed phosphorylated PDGFR-alpha and -beta. L3.6pl cells growing in culture expressed moderate amounts of PDGF-AA and little to no PDGFR-alpha or -beta, whereas L3.6pl cells growing in the pancreas of nude mice expressed a high level of PDGF and receptors. Colocalization immunohistochemical analysis demonstrated expression of activated PDGFR-beta by tumor-associated endothelial cells in both the pancreas and in liver metastases. Tumors of mice treated for 4 weeks with STI571 (50 mg/kg or 100 mg/kg p.o. daily) were slightly smaller than controls. Tumors treated with gemcitabine and STI571 (50 mg/kg) were >70% smaller than tumors in control mice and 36% smaller than those in mice treated with gemcitabine only (P < 0.0002 and P < 0.04, respectively). Combination therapy also inhibited spontaneous metastasis to the liver. Tumors from mice treated with both STI571 and gemcitabine had decreased expression of activated (phosphorylated) PDGFR-alpha and -beta, decreased mean vessel density, decreased cell proliferation, and increased apoptosis of tumor cells. CONCLUSIONS: Collectively, these data show that activated PDGFR on tumor cells and tumor-endothelial cells can be a novel target for therapy of pancreatic carcinoma.

Adenocarcinoma↗

PubMed2026

Dissecting the shared genetic architecture between migraine subtypes and cardiovascular diseases: a multi-layered genomic analysis.

BACKGROUND: Epidemiological studies have linked migraine to an increased risk of cardiovascular disease (CVD); however, the shared genetic basis and putative causal relationships between migraine subtypes and cardiovascular traits remain poorly understood. METHODS: Leveraging large-scale GWAS summary statistics for migraine phenotypes (overall migraine, migraine with aura [MA], and migraine without aura [MO]) from FinnGen R12, along with seven cardiovascular diseases from publicly available consortia, we conducted a multi-layered genetic analysis. This integrative framework encompassed genetic correlation [linkage disequilibrium score regression (LDSC) and high-definition likelihood (HDL)], cross-trait meta-analysis (CPASSOC and PLACO), Bayesian colocalization, summary-data-based Mendelian randomization (SMR) using GTEx v8 eQTL data, and bidirectional two-sample Mendelian randomization (MR). RESULTS: Significant genetic correlations were identified between migraine and multiple cardiovascular traits, with hypertension and coronary artery disease (CAD) showing the most robust associations. MA exhibited broader genetic overlap with cardiovascular diseases than MO, including a notably stronger correlation with ischemic stroke, whereas MO demonstrated a stronger correlation with hypertension. Cross-trait meta-analysis identified 160 pleiotropic loci across 17 of 21 trait pairs. Colocalization analysis confirmed 32 loci harboring shared causal variants, mapped to 13 candidate genes, of which 7 (PHACTR1, LRP1, SOX7, ABO, FHOD3, MEI1, XKR6) were further validated by SMR as exhibiting tissue-specific regulatory effects. Among these, PHACTR1 displayed the broadest pleiotropic profile across migraine phenotypes and vascular diseases. After MR-PRESSO outlier removal, bidirectional MR identified 10 MR-supported associations, two of which (genetic liability to hypertension on overall migraine, and CAD on MA) survived Bonferroni correction, all free of detectable horizontal pleiotropy. Genetic liability to hypertension was associated with increased migraine risk (OR = 1.90, 95% CI 1.25-2.90, P = 2.64 × 10⁻³), atherosclerotic diseases showed subtype-specific effects (inverse for MO, positive for MA), and, in the reverse direction, migraine was associated with increased ischemic stroke risk. CONCLUSIONS: This study provides a comprehensive and systematic characterization of the shared genetic architecture between migraine subtypes and cardiovascular diseases. By identifying pleiotropic genes and bidirectional putative causal relationships with subtype-specific patterns, our findings carry implications for the development of targeted therapeutics and subtype-specific cardiovascular risk stratification.

Humans↗

PubMed2000

Colocalization of parvalbumin and somatostatin-like immunoreactivity in the mouse hippocampus: quantitative analysis with optical dissector.

The colocalization of parvalbumin (PV) and somatostatin (SS)-like immunoreactivity was studied quantitatively in the mouse hippocampus, with particular reference to their areal and dorsoventral differences. The optical disector method was applied by using a confocal laser scanning microscope with immunofluorescent double-labeling. In the present study, we found a particular subpopulation of hippocampal nonprincipal neurons that contained both PV and SS-like immunoreactivity, i.e., PV-immunoreactive (IR)/SS-like immunoreactive (LIR) neurons. In the CA1 region, PV-IR/SS-LIR neurons were restricted to the stratum oriens (SO). In the CA3 region, they were scattered in the SO, stratum pyramidale (SP), and stratum radiatum (SR). However, they were rarely seen in the dentate gyrus (DG). The proportion of PV-IR/SS-LIR neurons in the PV-IR neurons or SS-LIR neurons was about 10% in the CA1 region, 15-30% in the CA3 region, 0-5% in the DG, and 10-20% in total. Laminar analysis revealed that the proportions of PV-IR/SS-LIR neurons in the PV-IR neurons were high in the SO (about 25%) of the CA1 region, and in the SO (about 50%) and SR (30-45%) of the CA3 region. The proportion of PV-IR/SS-LIR neurons in the SS-LIR neurons was low in the SO of the CA1 region (about 10%), but high in the SO (35-65%) and SR (35-45%) of the CA3 region. Morphologically, medium-sized horizontal fusiform and multipolar PV-IR/SS-LIR neurons were frequently observed, and they showed weak immunoreactivity for PV. Large-sized vertical bitufted and triangular PV-IR neurons lacked SS-like immunoreactivity, and most of them showed moderate to intense immunoreactivity for PV. In addition, we provide direct evidence that some PV-IR/SS-LIR neurons projected to the medial septum by using retrograde labeling with Fluoro-Gold injection. These observations indicate that PV-IR/SS-LIR neurons constitute a particular subpopulation of hippocampal nonprincipal neurons.

Animals↗

PubMed2026

Single-cell expression quantitative trait locus Mendelian randomization reveals immune cell-specific causal regulatory networks and actionable targets in polycystic ovary syndrome.

ObjectiveTo systematically investigate whether the pathogenesis of polycystic ovary syndrome (PCOS) is causally related to dysregulated gene expression in specific immune cell subsets, and to evaluate the potential of these causal genes as actionable drug targets.MethodsThis study employed a two-sample Mendelian randomization (MR) framework using publicly available genome-wide association study (GWAS) summary statistics. The participant data included 797 PCOS cases and 140,558 controls (no direct patient recruitment was involved). Instrumental variables were derived from high-resolution immune cell-specific single-cell expression quantitative trait locus (sc-eQTL) data (OneK1K project) across 14 immune cell types. Primary analyses utilized the inverse-variance weighted (IVW) method. Shared causal variants were validated using Bayesian colocalization. Phenome-wide association analysis (PheWAS), external transcriptomic dataset validation (GSE8157), and DrugBank database screening were conducted for pleiotropy assessment and drug repositioning.ResultsMR analysis revealed genome-wide significant causal associations for GLIPR1 in non-classical monocytes (Mono NC) and XBP1 in CD4+ effector memory T cells (CD4 ET) with PCOS risk. Higher GLIPR1 expression was associated with a decreased PCOS risk (OR = 0.669, P = 4.34×10-6), whereas higher XBP1 expression was associated with an increased risk (OR = 1.406, P = 9.53×10-8). Colocalization analysis confirmed that GLIPR1 shares a causal variant with PCOS (PP.H4 = 96.73%). PheWAS and external validation confirmed the safety profile and significant upregulation (P = 0.03) of GLIPR1. Drug repositioning identified SOT-107, a Phase III protein therapy drug, as a potential interacting agent for GLIPR1.ConclusionsThis sc-eQTL MR study reveals immune cell-specific causal regulatory networks in PCOS. GLIPR1 in non-classical monocytes represents a high-confidence protective target, while XBP1 provides suggestive evidence for immune-mediated pathogenesis. The candidate drug SOT-107 highlights theoretical repositioning opportunities, though rigorous preclinical validation remains required.

Female↗

PubMed2001

Quantitative analysis of ER alpha and GAD colocalization in the hippocampus of the adult female rat.

Despite the many effects of estrogen in the hippocampus, there has been little evidence that hippocampal principal cells express nuclear estrogen receptors. In the hippocampus, the alpha form of the nuclear estrogen receptor (ER alpha) has been localized to sparsely distributed cells with the morphological characteristics of inhibitory interneurons. Because inhibitory neurons may be involved in the effects of estrogen on hippocampal principal cells, quantitative description of ER alpha expression in gamma-aminobutyric acid (GABA)ergic (inhibitory) and non-GABAergic cells of the hippocampus is a key step in understanding the mechanism(s) of estrogen action on hippocampal circuitry. We used single and double-label immunohistochemistry for ER alpha and glutamic acid decarboxylase (GAD; a marker of GABAergic neurons) to determine the numbers and distributions of hippocampal GABAergic and non-GABAergic neurons that express ER alpha in the adult female rat. We found many more ER alpha-expressing cells in the hippocampus than any previous study and observed distinct dorsal vs. ventral differences in hippocampal ER alpha expression. In the dorsal hippocampus, most ER alpha-positive cells were also GAD positive; however, ER alpha was expressed in only a subset of GAD-positive cells. Double-labeled cells were concentrated at the border between str. radiatum and str. lacunosum-moleculare. In the ventral hippocampus, we found a very high number of ER alpha-positive cells, the majority of which were not immunoreactive for GAD and are likely to be pyramidal cells. These findings suggest that ER alpha can mediate the effects of estrogen primarily in GABAergic neurons in the dorsal hippocampus and in both GABAergic and non-GABAergic neurons in the ventral hippocampus.

Animals↗

PubMed1988

Immunohistochemical and biochemical analysis of serotonin and substance P colocalization in the nucleus tractus solitarii and associated afferent ganglia of the rat.

In a previous study of afferent projections to the nucleus tractus solitarii (NTS), it was shown that over half of the retrogradely-labelled neurons in the nucleus raphe pallidus contained serotonin-immunoreactivity and over half of these neurons contained substance P-immunoreactivity, suggesting that these two putative neurotransmitters are colocalized in NTS-afferent neurons. The objectives of the present study were to 1) directly determine if varicosities in the NTS, the area postrema (AP), and the dorsal motor nucleus of the vagus nerve (DMN) do contain both transmitters, 2) determine if primary afferent neurons in the nodose and pretrosal ganglia might also colocalize serotonin and substance P, and 3) quantify the amount of substance P that is contained in serotonergic varicosities in the NTS. Distributions and colocalization of substance P and serotonin in the NTS were studied using dual-color immunohistochemistry, while the quantity of substance P in serotonergic varicosities was assessed by radioimmunoassay (RIA) using micropunches from the NTS of 5,7-dihydroxytryptamine-(5,7 DHT-) and vehicle-treated rats. Varicosities that contained both serotonin- and substance P-immunoreactivity were found in the NTS, the DMN, and the AP. Double-labelled varicosities were common in the NTS and DMN (i.e., qualitatively similar to the density seen in the hypoglossal nucleus and in the ventral horn of the cervical spinal cord); however, the vast majority of the varicosities in these autonomic areas only displayed immunoreactivity for one or the other of these transmitters. This paucity of doubly-labelled varicosities, in comparison to the number of singly-labelled varicosities, was reflected in the lack of a significant decrease in substance P levels as determined by RIA of micropunches taken from caudal and intermediate levels of the NTS in 5,7 DHT- and vehicle-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗

PubMed1997

Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells.

Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

Acetylation↗

PubMed2025

From genetic causality to druggable targets: A multiomics framework identifies ZSCAN16 in gout pathogenesis.

ObjectiveGout is a prevalent form of inflammatory arthritis in which many patients respond suboptimally to current therapies. Drug development is hampered by a lack of genetically validated targets, leading to high clinical trial attrition. This study aimed to systematically identify and prioritize novel, druggable targets for gout via a multilayered genetic and functional genomics approach.MethodsWe performed two-sample Mendelian randomization (MR) using cis-expression quantitative trait locus (cis-eQTL) data and dual independent gout genome-wide association study (GWAS) cohorts (openGWAS and FinnGen). The candidate genes were subjected to a rigorous validation pipeline including Bayesian colocalization, phenome-wide association studies (PheWASs) to assess pleiotropy and on-target safety, and single-cell RNA sequencing (scRNA-seq) to delineate the cellular context. Molecular docking was used to evaluate the structural druggability of prioritized targets.ResultsMR analysis revealed 15 genes causally associated with gout. Colocalization analysis (PPH4 > 0.8) prioritized two targets: ZSCAN16 (risk-increasing, OR = 1.04, 95% CI [1.02-1.06]) and TRIM10 (protective, OR = 0.96, 95% CI [0.94-0.98]). Crucially, PheWAS revealed that ZSCAN16 is highly specific to gout, whereas TRIM10 exhibited extensive pleiotropy with hematological and cardiometabolic traits, indicating significant safety risks. Single-cell analysis provided orthogonal validation, demonstrating flare-specific upregulation of ZSCAN16 in cytotoxic T/NK cells. Molecular docking confirmed ZSCAN16 as a structurally druggable target, showing high-affinity binding with known compounds (e.g. digoxin, binding energy = -9.6 kcal/mol).ConclusionsOur study identifies ZSCAN16 as a high-potential, druggable therapeutic target for gout, highlighting its genetic influence on specific immune cell activities during acute flares. Conversely, TRIM10 was deprioritized owing to substantial pleiotropic liabilities and poor chemical tractability. These findings suggest that ZSCAN16 could play a crucial role in the pathogenesis of gout and may provide a valuable lead for future drug discovery efforts.

Humans↗

PubMed2002

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine.

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103+/-25 cells/cm(2), n=3) and that in the antrum (157+/-9 cells/cm(2), n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967+/-188 cells/cm(2), n=3), jejunum (2640+/-140 cells/cm(2), n=3), ileum (2070+/-42 cells/cm(2), n=3), proximal colon (2243+/-138 cells/cm(2), n=3) and distal colon (2633+/-376 cells/cm(2), n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45-55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4+/-5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.

Animals↗

PubMed2000

Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells.

The subcellular colocalization of prostacyclin synthase (PGIS) with prostaglandin H synthase (PGHS) has not been delineated. To test the hypothesis that its colocalization with PGHS is crucial for prostacyclin synthesis, we determined subcellular locations of PGIS, PGHS-1, and PGHS-2 in bovine aortic endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS-1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS and PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cyclooxygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS-2 in cells induced with phorbol 12-myristate 13-acetate where PGHS-2 was visualized primarily in vesicle-like structures. The lack of colocalization was accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Confocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a filamentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synthesized a large quantity of prostacyclin. These findings underscore the importance of colocalization of PGHS and PGIS to ER and NE in prostacyclin synthesis.

Animals↗

PubMed2006

Proteomics of polyglutamine aggregates.

In nine members of polyglutamine (polyQ) diseases, CAG repeat expansions of their responsible genes are observed. The disease is considered to be caused by the formation of polyQ aggregates that sequester proteins essential for cell viability. To understand the pathological process of polyQ diseases, a proteomic approach was used to identify aggregate interacting proteins (AIPs). Constructs were designed to express EGFP-fused, CAG-expanded (150 Q) huntingtin exon1 under the control of an ecdysone-inducible promoter and either lacking or containing a nuclear localization signal (NLS). After induction of a stably transfected Neuro 2A cell line with ecdysone, aggregates form in either the cytoplasm or the nucleus. The aggregates in these two different compartments were isolated with different methods. Cytoplasmic aggregate particles were purified using a fluorescence-activated cell sorter (FACS) by monitoring EGFP fluorescence, whereas nuclear aggregates were purified by using the detergent insoluble nature of aggregates. The resulting highly pure aggregates were subjected to SDS-PAGE followed by Coomassie blue staining. Bands containing AIP candidates were excised, and, after in-gel digestion with trypsin, were analyzed by mass spectrometry to identify the proteins. Novel candidates were confirmed as AIPs by immunocytological analysis to observe colocalization with polyQ aggregates. This chapter describes methods for the establishment of stable mutant cells, the purification of polyQ aggregates, and sample preparation for mass spectrometry analysis in detail.

Animals↗