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PubMed2005

Expression of a wild-type CFTR maintains the integrity of the biosynthetic/secretory pathway in human cystic fibrosis pancreatic duct cells.

The structural integrity of the Golgi complex is essential to its functions in the maturation, sorting, and transport of plasma membrane proteins. Previously, we demonstrated that in pancreatic duct CFPAC-1 cells, which express DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator), the intracellular trafficking of carbonic anhydrase IV (CA IV), a membrane protein involved in HCO(3)(-) secretion, was impaired. To determine whether these abnormalities were related to changes in the Golgi complex, we examined the ultrastructure and distribution of Golgi compartments with regard to the microtubule cytoskeleton in CFPAC-1 cells transfected or not with the wild-type CFTR. Ultrastructural and immunocytochemical analysis showed that in polarized CFPAC-1 cells, Golgi stacks were disconnected from one another and scattered throughout the cytoplasm. The colocalization of CA IV with markers of Golgi compartments indicated the ability of stacks to transfer this enzyme. This Golgi dispersal was associated with abnormal microtubule distribution and multiplicity of the microtubule-organizing centers (MTOCs). In reverted cells, the normalization of Golgi structure, microtubule distribution, and MTOC number was observed. These observations suggest that the entire biosynthetic/secretory pathway is disrupted in CFPAC-1 cells, which might explain the abnormal intracellular transport of CA IV. Taken together, these results point to the fact that the expression of DeltaF508 CFTR affects the integrity of the secretory pathway.

Carbonic Anhydrase IV↗

PubMed2001

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways.

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.

Animals↗

PubMed2004

Relevance of CD6-mediated interactions in T cell activation and proliferation.

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed1999

Immunohistochemical evidence for the brevican-tenascin-R interaction: colocalization in perineuronal nets suggests a physiological role for the interaction in the adult rat brain.

Brevican is one of the most abundant chondroitin sulfate proteoglycans in the adult rat brain. We have recently shown that the C-type lectin domain of brevican binds fibronectin type III domains 3-5 of tenascin-R. Here we report strong evidence for a physiological basis for this interaction. Substantial brevican immunoreactivity was detected in a number of nuclei and in the reticular formations throughout the midbrain and hindbrain, including, but not limited to, the deep cerebellar nuclei, the trapezoid body, the red nucleus, the oculomotor nucleus, the vestibular nucleus, the cochlear nucleus, the gigantocellular reticular nucleus, the motor trigeminal nucleus, and the lateral superior olive. Most of the brevican immunoreactivity exhibited pericellular and reticular staining patterns. In almost all of these sites, brevican immunoreactivity colocalized with that of tenascin-R, which was also substantially codistributed with versican, another member of the lectican family. Detailed analysis revealed that the pericellular staining of brevican resembled that in perineuronal nets in which tenascin-R has been localized. Immunoelectron microscopy identified brevican immunoreactivity in the intercellular spaces surrounding presynaptic boutons and on their surfaces, but not in the synaptic clefts or in their immediate vicinity, a distribution pattern consistent with perineuronal nets. Taken together, our results provide strong evidence that the previously reported interactions between brevican and tenascin-R may play a functional role within the perineuronal nets.

Age Factors↗

PubMed2005

Stimulation of transforming activity of DJ-1 by Abstrakt, a DJ-1-binding protein.

DJ-1 was identified by us as a novel oncogene in cooperation with activated ras. Although over-expression of DJ-1 has been reported in several cancer cells, including cells in breast cancer, lung cancer and prostate cancer, the precise mechanism underlying transformation has not been clarified. In this study, we screened proteins by a yeast two-hybrid method and identified Abstrakt as a DJ-1-binding protein. Abstrakt is an RNA helicase, but it has not yet been characterized. Northern blot analysis showed that human Abstrakt was expressed ubiquitously in all tissues. Abstrakt was then found to bind to and to be colocalized in the nucleus with DJ-1 in human cells. Furthermore, Abstrakt was found to stimulate transforming activity of DJ-1 in rat 3Y1 cells transfected with DJ-1 with activated ras. These findings suggest that Abstrakt is a positive regulator for DJ-1.

Animals↗

PubMed1994

A novel epitope of entactin is present at the mammalian neuromuscular junction.

The extracellular matrix (ECM) at the neuromuscular junction (NMJ) is biochemically and functionally specialized, and bears molecules that can regulate both the formation and function of this peripheral synapse. We have previously purified one synaptic component of the muscle ECM--a unique laminin isoform named s-laminin--from a rat schwannoma cell line (Chiu et al., 1992). To develop new probes for the ECM, monoclonal antibodies were generated against other components produced by this cell line. One of these new antibodies, 9H6, binds selectively at the synaptic cleft of NMJs in adult rats, but not at extrasynaptic sites on the muscle surface. On Western blots, 9H6 recognizes a 150 kDa band that colocalizes, and copurifies with the laminin-binding, ECM glycoprotein entactin under both reducing and nonreducing conditions. N-terminal sequence analysis also indicates that the 9H6 antigen is related to entactin. However, polyclonal antibodies to entactin stain both synaptic and extrasynaptic sites. Thus, 9H6 appears to identify an entactin epitope with a very restricted distribution. Treatment with N-glycanase reduces the molecular mass of entactin and eliminates 9H6 binding, suggesting that the 9H6 epitope at synapses is dependent on glycosylation. Recent studies have shown that novel isoforms of laminin, collagen IV, agrin, and AChE are selectively sequestered at the NMJ. Our results indicate that the entactin present at the synaptic cleft also differs from entactin present outside the synapse. The synaptic form of entactin may contribute to the unique functions of the ECM at the neuromuscular synapse.

Amidohydrolases↗

PubMed2003

Human recombinant anti-La (SS-B) autoantibodies demonstrate the accumulation of phosphoserine-366-containing la isoforms in nucleoplasmic speckles.

Using the recombinant La (SS-B) protein or a phosphorylated peptide derived thereof 27 La-specific human recombinant autoantibodies were selected from anti-La-positive systemic lupus erythematosus and systemic sclerosis patient-derived combinatorial phage display antibody libraries. Binding of these anti-La antibodies to various isoforms of the La protein present in normal and apoptotic cell extracts was analysed by Western blotting. Twenty-four of the selected antibodies recognize most, if not all isoforms of La, whereas three are exclusively reactive with the protein phosphorylated at serine-366. Sequence analysis of the selected antibodies showed a restricted spectrum of diversity in their VH germline gene usage. Remarkably, the recombinant antibodies recognizing exclusively the phosphoserine-366-containing isoform of La displayed a spleckled nucleoplasmic staining pattern in immunofluorescence analysis of HeLa and HEp-2 cells. This pattern differed markedly from those obtained with anti-La antibodies recognizing all isoforms of the La protein. Colocalization experiments with marker antibodies for spliceosomal UsnRNPs and RNA polymerase III subunits revealed that the anti-phosphorylated La antibodies stain the same nucleoplasmic speckles as anti-UsnRNP antibodies. In contrast to anti-UsnRNP antibodies the anti-phosphorylated La antibodies did not stain the Cajal bodies. In addition, no colocalization of phosphorylated La with RNA polymerase III was observed. Potential functional implications of the accumulation of phosphorylated La in nucleoplasmic speckles are discussed.

Amino Acid Sequence↗

PubMed1999

Characterization of E-cadherin-containing junctions involving skin-derived dendritic cells.

Adherens junctions are sites of contact between epithelial cells in which adhesion is mediated by homophilic interactions of classical cadherins that are linked to the cytoskeleton via catenins. E-cadherin constitutes the major adhesion molecule in adherens junctions of keratinocytes (KC) and mediates the binding of Langerhans cells to KC in vitro. To characterize structures responsible for E-cadherin-mediated binding of Langerhans cells, we utilized Langerhans cells-like fetal skin-derived dendritic cells (FSDDC) capable of E-cadherin-mediated adhesion. Confocal microscopy of FSDDC aggregates demonstrated colocalization of E-cadherin and catenins to areas of cell-cell contact. Immunoprecipitation confirmed a physical association of E-cadherin with intracellular catenins (alpha-, beta-, gamma-catenin/plakoglobin and p120CAs). Transmission electron microscopy of FSDDC aggregates revealed structures with features of adherens junctions in areas of cell-cell contact, and post-embedding immunoelectron microscopy localized beta-catenin to these regions. To characterize junctions that accounted for the adhesion of Langerhans cells-like dendritic cells and KC, disaggregated FSDDC were cocultured with primary murine KC. Transmission electron microscopy analysis of cocultures demonstrated FSDDC-KC contacts that were analogous to those seen in FSDDC aggregates. Confocal microscopy demonstrated focal accumulations of E-cadherin and colocalization of beta-catenin in areas of contact between KC and immature (Langerhans cell-like) dendritic cells, but not in areas of contact between KC and mature (lymph node dendritic cell-like) dendritic cells. E-cadherin in Langerhans cells appears to be localized in structures that resemble adherens junctions formed by nonpolarized epithelial cells. Loss of ability to form or maintain these structures after the induction of Langerhans cells activation/ maturation likely results in the attenuation of Langerhans cells-KC adhesion that precedes Langerhans cells emigration from the epidermis.

Animals↗

PubMed1994

Major retinal cell components recognized by onchocerciasis sera are associated with the cell surface and nucleoli.

PURPOSE: Cellular localization of the components recognized by onchocerciasis autoantibodies has not been investigated in any detail in cultured retinal cells. This study sought to examine, in cultured retinal cells, the subcellular localization of major components that cross-react with onchocerciasis sera. METHODS: Immunofluorescence confocal laser scanning microscopy and Western blot analysis were carried out on adult pig retinal cells. RESULTS: The onchocerciasis sera contain antibodies cross-reacting strongly with components of the surface and nucleoli in both the cultured retinal pigment epithelial and neural retinal cells. These epitopes are not recognized by the control sera obtained from noninfected individuals residing in an onchocerciasis hyperendemic area, and from those with or without ocular disease who have never been in any of the onchocerciasis hyperendemic countries. Double-labeling immunofluorescence microscopy does not detect any colocalization of a putative onchocerciasis autoantigen, calreticulin, and those cellular components recognized by onchocerciasis sera in either cell type. Furthermore, none of the onchocerciasis sera tested recognized recombinant calreticulin by Western blot analysis. CONCLUSIONS: Major epitopes for onchocerciasis anti-retinal autoantibodies are associated with the surface and nucleolus components of retinal cells. Interaction of the onchocerciasis antibodies with the retinal cell surface molecules may play an important role in the development of ocular diseases initiated by the damage of retinal cells. Furthermore, the finding that the cellular components recognized by onchocerciasis sera do not colocalize with calreticulin, taken together with the observation of lack of recognition of recombinant calreticulin by these sera on Western blots, suggests that calreticulin is not a major onchocerciasis autoantigen.

Animals↗

PubMed2006

DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions.

We isolated and characterized a cluster of platypus DMRT genes and compared their arrangement, location, and sequence across vertebrates. The DMRT gene cluster on human 9p24.3 harbors, in order, DMRT1, DMRT3, and DMRT2, which share a DM domain. DMRT1 is highly conserved and involved in sexual development in vertebrates, and deletions in this region cause sex reversal in humans. Sequence comparisons of DMRT genes between species have been valuable in identifying exons, control regions, and conserved nongenic regions (CNGs). The addition of platypus sequences is expected to be particularly valuable, since monotremes fill a gap in the vertebrate genome coverage. We therefore isolated and fully sequenced platypus BAC clones containing DMRT3 and DMRT2 as well as DMRT1 and then generated multispecies alignments and ran prediction programs followed by experimental verification to annotate this gene cluster. We found that the three genes have 58-66% identity to their human orthologues, lie in the same order as in other vertebrates, and colocate on 1 of the 10 platypus sex chromosomes, X5. We also predict that optimal annotation of the newly sequenced platypus genome will be challenging. The analysis of platypus sequence revealed differences in structure and sequence of the DMRT gene cluster. Multispecies comparison was particularly effective for detecting CNGs, revealing several novel potential regulatory regions within DMRT3 and DMRT2 as well as DMRT1. RT-PCR indicated that platypus DMRT1 and DMRT3 are expressed specifically in the adult testis (and not ovary), but DMRT2 has a wider expression profile, as it does for other mammals. The platypus DMRT1 expression pattern, and its location on an X chromosome, suggests an involvement in monotreme sexual development.

Amino Acid Sequence↗

PubMed1999

A novel simple satellite DNA is colocalized with the Stalker retrotransposon in Drosophila melanogaster heterochromatin.

In the T(1:2)dor(var7) multibreak rearrangement the distal 1A-2B segment of the X chromosome of Drosophila melanogaster is juxtaposed to an inverted portion of the heterochromatin of chromosome 2. Analysis of mitotic chromosomes by a series of banding techniques has permitted us precisely to locate the heterochromatic breakpoint of this translocation in the h42 region of 2R. Cloning and sequencing of the eu-heterochromatic junction revealed that the translocated 1A-2B fragment is joined to (AACAC)n repeats, which represent a previously undescribed satellite DNA in D. melanogaster. These repeated sequences have been estimated to account for about 1 Mb of the D. melanogaster genome. The repeats are located mainly in the Y chromosome and in the heterochromatin of the right arm of chromosome 2 (2Rh), where they are colocalized with the Stalker retrotransposon.

Animals↗

PubMed2005

Colocalization of corticotropin-releasing hormone and oestrogen receptor-alpha in the paraventricular nucleus of the hypothalamus in mood disorders.

Oestrogens may modulate the activity of the hypothalamic-pituitary-adrenal (HPA) axis. The present study was to investigate whether the activity of the HPA axis in mood disorders might be directly modulated by oestrogens via oestrogen receptors (ORs) in the corticotropin-releasing hormone (CRH) neurons of the human hypothalamic paraventricular nucleus (PVN). Brains of 13 subjects ranging in age between 45 and 79 years suffering from major depression/major depressive disorder (eight cases) or bipolar disorder (five cases) and of 13 controls, matched for sex, age, brain weight, post-mortem delay, fixation time and season and clock time at death, were studied with double-label immunocytochemistry. The total number of CRH-immunoreactive (IR) neurons, CRH neurons that colocalized ORalpha in the neuronal nucleus and the number of only nuclear ORalpha-containing neurons in the PVN were measured using an image analysis system. In addition, the volume of the PVN delineated on the basis of CRH neurons was determined. It was found that the total number of CRH-IR neurons in patients with mood disorders was nearly 1.7 times higher than in controls (P = 0.034). A novel finding was that the total number of CRH-IR neurons and the number of CRH-nuclear ORalpha double-staining neurons in the PVN were strongly correlated both in controls and in patients with mood disorders (P < 0.001 and P = 0.022, respectively). The ratio of the CRH-nuclear-ORalpha double-staining neurons to the total CRH-IR neurons in patients with mood disorders was similar to that in the controls (P = 0.448). The volume of the sub-region of the PVN that was delineated on the basis of CRH neurons was significantly larger in patients with mood disorders than in controls (P = 0.022). Another novel finding was the large population of extra-hypothalamic CRH neurons that was found in the thalamus. In summary, oestrogens may directly influence CRH neurons in the human PVN. The increased numbers of neurons expressing CRH in mood disorders is accompanied by increased ORalpha colocalization in the nucleus of these neurons. These changes seem to be trait- rather than state-related.

Aged↗

PubMed2026

Integrative Multi-Omics Mendelian Randomization Analysis Identifies NIT2 as a Potential Metabolic Risk Gene in Hepatocellular Carcinoma.

BACKGROUND: Metabolic pathways are crucial in hepatocellular carcinoma (HCC) pathogenesis, but causal metabolic genes remain unclear. This study used Summary data-based Mendelian Randomization (SMR) and colocalization to identify metabolism-related genetic loci influencing HCC risk. METHODS: Differentially expressed genes in hepatic malignancy phenotype versus normal tissues from TCGA and GTEx were analyzed. Metabolism-related candidates were examined via SMR and colocalization using multi-omics data: methylation (mQTL), expression (eQTL), and protein (pQTL) quantitative trait loci. RESULTS: Multi-omics integration identified NIT2 as a key metabolic regulator for HCC. The cg13016775 locus of NIT2 was associated with elevated HCC risk at gene (OR = 1.618, 95% CI: 1.199-2.182) and protein (OR = 4.432, 95% CI: 1.783-11.018) levels. Colocalization supported a shared causal variant (PPH4 > 0.6), linking NIT2 to hepatocarcinogenesis via metabolic regulation. CONCLUSIONS: This study provides multi-omics evidence for NIT2 as a potential causal gene in HCC, enhancing understanding of metabolic contributions to HCC pathogenesis and highlighting integrative genomics for uncovering causal relationships.

Carcinoma, Hepatocellular↗

PubMed2026

Genetically Proxied Inhibition of Cholesterol-Lowering Drug Targets and Survival in HPV-Positive and Non-HPV-Driven Head and Neck Cancer: A Multicentre MR Study.

BACKGROUND: Cholesterol pathways may influence head and neck squamous cell carcinoma (HNSCC) progression, but evidence on prognosis is inconsistent. We used Mendelian randomization (MR) to test whether genetically proxied inhibition of major low-density lipoprotein cholesterol (LDL-C)-lowering drug targets and circulating lipid traits affects overall survival (OS) in HPV-positive and non-HPV-driven HNSCCs. METHODS: We proxied lifelong LDL-C lowering using 55 cis-acting single-nucleotide polymorphisms in HMGCR, NPC1L1, PCSK9, and LDL-receptor (LDLR) from the updated 2021 Global Lipids Genetics Consortium and instrumented circulating lipid traits. Two-sample MR estimated effects on OS in 4,869 multicentre HNSCC cases (1,291 HPV-positive; 3,578 non-HPV-driven) using minimally adjusted Cox models. Sensitivity analyses additionally adjusted for tumor stage and treatment, assessed collider bias using an external HNSCC incidence genome-wide association study, and examined between-center heterogeneity and colocalization. RESULTS: Using the updated Global Lipids Genetic Consortium 2021 instruments, genetically proxied HMGCR inhibition showed a directionally protective but nonsignificant association with OS in HPV-positive oropharyngeal HNSCC in the primary analysis [inverse variance weighted (IVW) HR = 0.19; 95% confidence interval (CI), 0.03-1.18; P = 0.08], with directionally concordant weighted median results. No corresponding protective association was observed for HMGCR in non-HPV-driven disease (IVW HR = 1.68; 95% CI, 0.78-3.63; P = 0.19). No clear evidence of association was observed for NPC1L1, PCSK9, LDLR, or circulating lipid traits in either HPV stratum. Colocalization did not support a shared causal variant. CONCLUSIONS: These analyses provide suggestive evidence that genetically proxied HMGCR inhibition may influence survival in HPV-positive oropharyngeal HNSCC. IMPACT: HMGCR-related pathways may be relevant to prognosis in HPV-positive oropharyngeal HNSCC, whereas clear survival effects of other cholesterol-lowering targets were not supported.

Humans↗

PubMed1996

Organization of ventrolateral periolivary cells of the cat superior olive as revealed by PEP-19 immunocytochemistry and Nissl stain.

Ventrolateral periolivary cell groups, through their descending projections to the cochlear nucleus (CN) and local projections to principal nuclei of the superior olive, may participate in brainstem mechanisms mediating such tasks as signal detection in noisy environments and sound localization. Understanding the function of these cell groups can be improved by increased knowledge of the organization of their synaptic inputs in relation to their cellular characteristics. Immunocytochemistry for PEP-19 (a putative calcium binding protein) reveals four patterns of immunolabeling within the ventrolateral periolivary region. Three of the patterns, which have distinct fiber and punctate labeling characteristics, help to define three subdivisions of the lateral nucleus of the trapezoid body (LNTB). The fourth pattern defines two other nuclei, the anterolateral periolivary nucleus (rostral) and the posterior periolivary nucleus (caudal), which display many immunoreactive cell bodies but little fiber and punctate labeling. One of the subdivisions of the LNTB contains large PEP-19 immunolabeled puncta arranged in pericellular nests. Analysis of Nissl-stained sections reveals a neuronal population that resembles globular cells of the ventral cochlear nucleus (VCN) and which colocalizes with pericellular nests of large immunolabeled puncta. Cell counts reveal that roughly 10,000 neurons constitute the cat ventrolateral periolivary region, 9,000 of which are found in the LNTB. Three-dimensional reconstructions of auditory brainstem nuclei clarify the complex spatial relationships among these structures.

Animals↗

PubMed2001

Differential expression of catecholamine biosynthetic enzymes in the rat ventrolateral medulla.

Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis.

Animals↗

PubMed1992

Nonradioactive in situ hybridisation of the translocation t(1;7) in myeloid malignancies.

Bone marrow cells of four patients with t(1;7) and myelodysplasia or acute myeloid leukemia were analyzed using nonradioactive in situ hydridisation. As probes, centromeric alphoid DNA sequences of chromosomes 1 and 7, a satellite DNA probe for 1q12, and chromosome-specific libraries of chromosomes 1 and 7 were used. The breakpoints of the t(1;7)(p11;p11) as determined by banding analysis could be studied more accurately, and the recently proposed designation t(1;7)(cen;cen) was confirmed in all four cases. Colocalization of alphoid DNA sequences of chromosomes 1 and 7 by double target in situ hybridisation was demonstrated in metaphase cells and also in interphase nuclei. The in situ hybridisation method described is applicable for the screening of peripheral blood cells or archival material.

Acute Disease↗

PubMed2005

Spreading of embryologically distinct urothelial cells is inhibited by SPARC.

The AON epitope of secreted protein acidic and rich in cysteine (SPARC) is a conserved motif expressed by human SPARC in a variety of human cell types. Through the use of a monoclonal antibody that recognizes this epitope, transitional epithelium was found to restrict expression of SPARC to the suprabasal and intermediate layer. Such intracellular expression was defined by immunoreactive signals that localized to the apical plasma membranes of suprabasal and intermediate cells. Polarization of SPARC to apical plasma membranes of suprabasal cells was retained in vitro by a subpopulation of cells that exhibited characteristics of suprabasal cells--cell-cycle quiescence, large cell volumes, and multiple nuclei. In contrast, the basal layer of transitional epithelium in vivo and cycling cells in vitro did not exhibit this apical staining pattern, but instead sequestered the SPARC polypeptide within urothelial cytoplasm and/or nuclei, as revealed by immunohistochemical analysis. Elution of soluble proteins and DNA from urothelial cells revealed the presence of SPARC within the nuclear matrix--and that SPARC colocalized with the nuclear matrix Ki-67 antigen. rSPARC activity was demonstrated and quantified with a rounding assay whereby the spreading of freshly plated cells was inhibited by recombinant SPARC in a concentration- and time-dependent manner. Inhibition of spreading was observed in urothelial cells derived from endoderm (bladder) and mesoderm (ureter) germ layers. Statistically significant differences were seen between urothelial cells from these two layers. Mesodermal cells recovered more slowly from the inhibitory effects of rSPARC, such that at hour 6 endodermal cells underwent significantly more spreading, as shown by a rounding index (RI). These experiments provide new insights about the matricellular trafficking of SPARC and suggest that intra- and extra-cellular localization patterns influence the development, homeostasis, and differentiation of transitional epithelium.

Cells, Cultured↗