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J Roth

Publications and source records attributed to J Roth.

At least 91 records · Page 5Linked to original sources
PubMed1994

Incretin hormones regulate glucose-dependent insulin secretion in RIN 1046-38 cells: mechanisms of action.

Glucagon-like peptide-1-(7-36) amide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are known incretin hormones, released from enteroendocrine cells in response to food, that enhance insulin secretion, but only in the presence of elevated blood glucose. We used a rat insulinoma cell line, RIN 1046-38, to study the mechanisms underlying the interaction of incretins and glucose. We measured insulin secretion using RIA and the reverse hemolytic plaque assay. GLP-1 stimulates insulin secretion, with a half-maximal concentration of 34 pM. GLP-1 is approximately 2 orders of magnitude more potent than GIP. GLP-1 and GIP have additive effects at submaximal concentrations, but probably not at maximal concentrations, suggesting a common signal transduction pathway. The glucose requirement for GLP-1 action can be replaced by cell membrane depolarization (20 mM KCl in the extracellular medium), suggesting that a rise of intracellular Ca2+ may be an early step required for GLP-1 action. GLP-1 stimulates insulin secretion by significantly increasing the maximum rate of insulin secretion from 10.3 +/- 2.25 to 25.2 +/- 2.94 ng insulin/mg protein.h. GLP-1 acts by recruiting 1.5-fold more cells to secrete insulin as well as enhancing insulin secretion by individual cells. Combinations of stimuli, such as glucose, cell membrane depolarization, and GLP-1, can recruit 90% of RIN 1046-38 cells to secrete insulin.

Animals↗
PubMed1994

Glucagon-like peptide-1(7-36) amide (GLP-1) enhances insulin-stimulated glucose metabolism in 3T3-L1 adipocytes: one of several potential extrapancreatic sites of GLP-1 action.

We investigated the effects of glucagon-like peptide-1(7-36) amide, GLP-1, on glucose metabolism in 3T3-L1 adipocytes and we used polymerase chain reaction to search for the presence of GLP-1 receptors in various rat tissues. GLP-1 at 1 nM significantly increased insulin-mediated 2-deoxyglucose uptake by 40% while having no effect on basal uptake. In conjunction with the elevated uptake, the insulin-dependent incorporation of 14C-glucose into fatty acids was also increased. Moreover, neither glycogen synthesis nor insulin binding to its receptor were affected by GLP-1. In addition to the presence of GLP-1 receptor in pancreas we found messenger RNA for this receptor in brain, kidney, heart, fat, skeletal muscle, liver, and intestine. This study indicates that GLP-1, in addition to its well known effect of stimulating insulin secretion, may improve insulin responsiveness by promoting fatty acid synthesis in adipose cells and possibly modulating insulin signaling in other insulin sensitive tissues.

3T3 Cells↗
PubMed1994

Xenopus laevis oocytes, eggs and tadpoles contain immunoactive insulin.

Insulin is a multifunctional polypeptide hormone that regulates metabolic processes and promotes mitogenesis and differentiation in vitro in the cells and tissues of several species. Its role in vivo during embryogenesis is still poorly understood. We have previously found insulin mRNA in mature Xenopus laevis oocytes and in embryos during neurulation (before organogenesis of the pancreas takes place). We have now measured insulin immunoactivity in mature oocytes, unfertilized eggs and day-2 tadpoles. Using reversed phase high performance liquid chromatography, we found low levels of insulin in extracts of oocytes (stage VI). Both Xenopus insulin I and II were detected in unfertilized eggs. The day-2 tadpoles (stages 31-33) also contained immunoactive insulin, and in swimming tadpoles (stage 46) a few clusters of cells containing insulin immunoactivity could be identified by indirect immunofluorescence. Immunoblot analysis was relatively insensitive, detecting insulin only in the adult Xenopus pancreas. In summary, insulin (from maternal origin and embryonic expression) appears to be present early enough in Xenopus laevis to influence developmental processes such as neurulation.

Animals↗
PubMed1994

Detection of Epstein-Barr virus DNA in Hodgkin- and Reed-Sternberg-cells by single cell PCR.

The Epstein-Barr virus (EBV) can be detected in the majority of lymph nodes involved by Hodgkin's lymphoma using the highly sensitive polymerase chain reaction (PCR). However, the rate of EBV-DNA detection by in-situ hybridisation, which allows allocation of EBV to a defined cell population, i.e. the neoplastic H&RS-cells, is lower. In an attempt to combine the advantages of the high sensitivity of the PCR and the possibility of cellular allocation by in-situ hybridisation, we established a single-cell PCR of Hodgkin- and Reed-Sternberg (H&RS)-cells isolated by micromanipulation from biopsy tissues. We amplified EBV sequences from the BamW-region by single-cell PCR. Using this method we were able to detect EBV-DNA in the H&RS-cells from 4 of 6 patients. In EBV positive cases all H&RS-cells of a given patient were positive, proving the high sensitivity and reproducibility of the method. Other cells in the biopsy tissue involved by EBV-positive H&RS-cells were shown to be negative. This indicates that EBV may have a role in the pathogenesis of many but not all cases of Hodgkin's disease.

Adolescent↗
PubMed1994

Cytochemical staining for beta 1,6 branching of asparagine-linked oligosaccharides in variants of metastatic human colon carcinoma cells.

A positive correlation between tumor progression in human colon and increased beta 1,6 branching in oligosaccharides has recently been demonstrated. The present study was undertaken to elucidate whether such a correlation can be extended to variants of metastasizing human colon carcinoma HCT116 cells. The Phaseolus vulgaris leukoagglutinating lectin, which binds to beta 1,6 branched oligosaccharides, was employed. In blots, a band of approximately 140 kd was detectable in both the HCT116a and HCT116b sublines. However, in the more aggressive subline HCT116a, the intensity of this band was increased by 100%, and additional reactive bands of approximately 100 kd and approximately 170 kd were observed. Analysis by electron microscopy revealed lectin labeling in the Golgi apparatus, lysosomal elements, mucus droplets, cytoplasmic vesicles, and at the plasma membrane. Quantification of the lectin plasma membrane labeling revealed a significantly higher labeling intensity in HCT116a cells than in HCT116b cells. The difference in lectin plasma membrane labeling intensity could also be observed in paraffin sections. Thus, variants of metastatic HCT116 colon carcinoma cells differ quantitatively and qualitatively in glycoproteins carrying beta 1,6 branches.

Asparagine↗
PubMed1994

[Pronounced flush symptoms in carcinoid syndrome without liver metastases].

The case of a 78 years old patient with impressive flush symptoms is reported. 5-Hydroxyindoleacetic acid and serotonin were elevated. The responsible pathomorphological structure is a 8 x 4.5 x 6 cm ileocaecal neoplasm. Pre-, intra- and postoperatively liver metastases could not be detected during a 18 month period. After surgery of the carcinoid flush disappeared, so that the tumour seize resulting in enormous secretion of metabolites with endocrine activity should be claimed responsible for causing this symptom.

Aged↗
PubMed1994

[Tumors--disorders of cell adhesion].

Cell adhesion molecules are of pivotal importance during organogenesis and fulfil important functions in maintaining normal organ architecture in the adult. Furthermore, they are involved in diverse and specialized functions such as migration and activation of leukocytes in immunosurveillance and inflammation, and homophilic as well as heterophilic interactions of cells with others and the extracellular matrix. In tumors, various cell adhesion molecules play a role in the complex process of tumor dissemination including metastatic spread. In addition, immunohistochemical detection of certain cell adhesion molecules in tumors has been shown to be of value in predicting the clinical course of the tumor disease. This short review focuses on the polysialic acid of the neural cell adhesion molecule NCAM, integrins and CD44.

Animals↗
PubMed1994

[Comparison of histology, immunohistochemistry, RT-PCR, in situ hybridization, and in situ RT-PCR for demonstration of hepatitis C virus in paraffin-embedded liver biopsies].

To compare immunohistochemical and molecular methods for the detection of hepatitis C virus (HCV) infection in archival liver biopsies we analyzed formalin-fixed and paraffin-embedded liver specimens of 10 patients with serologically confirmed HCV infection. Methods employed included indirect FITC-immunofluorescence, reverse-transcriptase polymerase chain reaction (RT-PCR) using extracted RNA and Southern blotting with chemiluminescence-based detection, non-radioactive in situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes, direct in situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and indirect in situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. (Gastroenterology 1993; 104-595) together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies are representative; (2) the commercially available mAB TORDJI-22 appears to cross-react with non-HCV epitopes; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in situ RT-PCR or ISH; and (5) direct in situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. Further studies are required to define both optimal methods for sample processing and improvements of protocols, in order to increase detection sensitivity and specificity of HCV infection by immunohistochemical and molecular methods.

Biopsy↗
PubMed1994

Expression of murine VCAM-1 in vitro and in different models of inflammation in vivo: correlation with immigration of monocytes.

VCAM-1 (vascular cell adhesion molecule-1) is a cytokine-inducible adhesion molecule which is known to mediate adhesion of mononuclear cells to endothelial cells in vitro via binding to the integrin VLA-4 (very late antigen-4). To further elucidate the role and regulation of VCAM-1 in vivo, we compared in vitro and in vivo expression of VCAM-1 in response to cytokines and investigated immunohistochemically the expression of VCAM-1 in three murine models of experimental inflammation. These models differed with regard to the pathogenetic mechanism and the subsequent infiltrate: allergic contact dermatitis (ACD) to DNFB as a T cell-controlled, DTH type of inflammation, cutaneous infection with Leishmania major as a chronic granulomatous inflammation and the cauterized cornea as a model for acute inflammation. VCAM-1 was found to be markedly enhanced on vascular endothelia in all types of inflammation and after subcutaneous administration of LPS and TNF-alpha. Administration of IL-4, however, failed to induce VCAM-1 both in vivo and in vitro. The increased VCAM-1 expression in the inflammatory models correlated with the appearance of infiltrating monocytes/macrophages. A concomitant influx of CD4-positive/CD8-positive lymphocytes was only observed in ACD and Leishmaniasis.

Animals↗
PubMed1994

Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing.

The suitability of formalin-fixed and paraffin-embedded tumor material was evaluated for molecular analysis of the RET proto-oncogene. We analyzed exons 10, 11, and 16 for point mutations in seven sporadic and six multiple endocrine neoplasia (MEN) 2A-associated pheochromocytomas by a nonradioactive single-strand conformation polymorphism assay followed by nonradioactive direct sequencing of PCR-amplified DNA using an automated DNA sequencer. All MEN 2A-associated pheochromocytomas contained a heterozygous missense germline mutation within cystine codons of the cysteine-rich extracellular domain encoded by exons 10 and 11. Mutations were located in codon 619 (TGC-->TCC; Cys-->Ser) in one, in codon 635 (TGC-->CGC; Cys--Arg) in three, and in codon 635 (TGC-->TAC; Cys-->Tyr) in two pheochromocytomas. No tumor-specific (somatic) mutations were detected in exons 10, 11, and 16 of the sporadic pheochromocytomas. These data support recent findings that germline point mutations that are clustered in distinct cysteine codons of the RET proto-oncogene are involved in the neoplastic phenotype of the MEN 2A syndrome. Our results demonstrate that both nonradioactive single-strand conformation polymorphism and direct sequencing are suitable methods to detect single base substitutions in DNA extracted from archival material.

Adolescent↗
PubMed1994

[A rare etiology for HBs-Ag negative acute hepatitis B--coinfection by hepatitis B and delta].

An unusual case of a 25-year-old male Italian is reported. The patient endured an acute hepatitis without detectable HBs-antigen by coinfection with hepatitis-B and Delta. Coincidently, a cured hepatitis-C was present. Firstly hepatitis-B-virus DNA could be demonstrated in a small quantity by serodiagnosis (6 pg/ml, hybridization technique). Subsequently, the identification of B-virus DNA was only possible in liver tissue (PCR-technique), but no longer by serodiagnosis. The probable enduring inhibition of hepatitis-B-virus replication by Delta virus resulted in a self limitation of the disease within 2 months (HDV-RNA negative, HBs-Ag and HBe-Ag negative; Anti-HBs negative, Anti-HBe and Anti-HBc positive). In spite of negativation of replication markers for hepatitis-B a subsequent reactivation of the infection was possible by viral material which persisted in liver tissue.

Acute Disease↗
PubMed1994

In vitro and in vivo growth of clonal sublines of human small cell lung carcinoma is modulated by polysialic acid of the neural cell adhesion molecule.

BACKGROUND: Polysialic acid (poly Sia) of the neural cell adhesion molecule (N-CAM) is an oncodevelopmental antigen and is found in small cell lung carcinomas (SCLC) as well as cell lines derived from these tumors. EXPERIMENTAL DESIGN: Cell heterogeneity in poly Sia expression was observed in primary SCLC and cell cultures of SCLC by immunostaining using a directly gold-labeled monoclonal antibody against poly Sia (MAb 735) and antibodies against N-CAM. Clonal sublines of the N-CAM-positive SCLC cell line, NCI-H69 were established to study the basis of this heterogeneity. The resulting sublines were examined for the proportion of cells expressing poly Sia, the stability of poly Sia expression, and the possible involvement of DNA methylation. Two of the sublines that expressed poly Sia on 0 and 95% of the cells were used in three independent in vitro assays to investigate the importance of poly Sia in cell-cell aggregation, disaggregation and cell to substrate adherence. Finally, clonogenic growth of these sublines was studied in soft agar and in the nude mouse. RESULTS: The proportion of cells immunoreactive for poly Sia was stable in serial subculture in these clones and was not affected by reducing DNA methylation. In aggregation and disaggregation assays, poly Sia was shown to modulate both calcium-dependent and independent cell-cell adhesion. No measurable differences in the attachment rates to various substrates (collagen type IV, laminin, heparan sulfate, and poly-L-lysine) were detected between the sublines. Cells from the poly Sia-positive clonal subline formed significantly more colonies in semisolid media and more intracutaneous metastasis in the nude mouse. CONCLUSIONS: Poly Sia does not occur on all N-CAM immunoreactive cells of SCLC. Poly Sia on SCLC cells is a clonable trait and high poly Sia expression correlates with reduced cell-cell adherence, a greater clonogenic ability in semisolid media, and a significantly higher incidence of intracutaneous metastases in nude mice.

Animals↗
PubMed1993

Cataract formation after bone marrow transplantation.

OBJECTIVE: To evaluate the incidence, time course, and factors associated with cataract formation in bone marrow transplant recipients. DESIGN: Prospective cohort study. SETTING: University Hospitals, Basel, Switzerland. PATIENTS: 197 patients treated with allogeneic or autologous bone marrow grafts at least 180 days before the start of the study. INTERVENTION: Three regimens for bone marrow transplant were used: 74 patients received single-dose, total-body irradiation (TBI), 90 patients received fractionated TBI, and 33 received chemotherapy alone. RESULTS: Three and one half years after single-dose TBI, 51 of the 74 patients (69%) were alive and cataracts had developed in all of these 51 patients. Cataracts developed in 18 of the 90 (20%) patients treated with fractionated TBI, with an 83% (95% CI, 63% to 100%) risk for lens opacification at 6 years. Cataracts developed in only 1 of the 33 (3%) patients treated with chemotherapy alone. Incidence of cataracts is higher and lens opacification occurs earlier after single-dose TBI than after fractionated TBI (P < 0.01). With Cox regression analysis, the use of irradiation (relative risk, 21.0), the mode of irradiation (relative risk, 7.4), and the use of steroid treatment (relative risk, 2.9) for more than 3 months after bone marrow transplantation increased the risk for cataract formation. In contrast, age, sex, and chronic graft-versus-host disease did not influence the rate of cataract development. The probability of requiring cataract surgery after 6 years was 85% (CI, 75% to 95%) for the patients treated with single-dose TBI and 20% (CI, 0% to 49%) for those prepared with fractionated irradiation. CONCLUSIONS: Patients treated with TBI, regardless of fractionation, are likely to have cataracts within 10 years, and some will need surgical repair. Long-term steroid treatment accelerates cataract formation. Preventive measures, such as lens shielding during TBI, should be considered.

Adolescent↗
PubMed1993

MRP8 and MRP14, S-100-like proteins associated with myeloid differentiation, are translocated to plasma membrane and intermediate filaments in a calcium-dependent manner.

MRP8 and MRP14 are two Ca(2+)-binding proteins of the S-100 family expressed by myelomonocytic cells. Both proteins assemble to noncovalently associated complexes in a Ca(2+)-dependent manner. Members of the S-100 family are known to play a role in cytoskeletal-membrane interactions; therefore, we investigated the subcellular distribution of MRP8/MRP14 and their complexes in human monocytes. Using differential centrifugation and subsequent Western blot or enzyme-linked immunosorbent assay analysis, we found that MRP8/MRP14 were almost completely translocated from the cytoplasma to membrane and cytoskeletal structures in a Ca(2+)-dependent manner. Using a cross-linking technique, complexed forms of MRP8/MRP14 were found to be associated with the plasma membrane. Analysis of MRP-transfected L132 cells showed that the MRP8 as well as the MRP14 component of the MRP8/MRP14 complex may independently bind to membrane and cytoskeletal structures. Furthermore, immunogold electron microscopy showed a colocalization of MRP8/MRP14 and the intermediate filament type III protein vimentin in A23187-treated monocytes. Our data indicate that, in analogy to other S-100-like proteins, MRP8 and MRP14 play a role in Ca(2+)-dependent cytoskeletal-membrane interactions. Restriction of MRP8/MRP14 expression to distinct stages of myelomonocytic differentiation suggests that these proteins are involved in highly specific pathways of intracellular signaling in phagocytes.

Antibodies, Monoclonal↗
PubMed1993

Expression of calcium-binding proteins MRP8 and MRP14 is associated with distinct monocytic differentiation pathways in HL-60 cells.

MRP8 and MRP14 are two calcium-binding proteins of the S-100 family which are expressed during distinct stages of monocytic maturation. To further investigate their regulation the human leukemic cell line HL-60 which can be induced to differentiate to monocytes/macrophages by 12-O-tetradecanoylphorbol 13-acetate (TPA), 1,25-(OH)2 Vitamin D3 (VD3), tumor necrosis factor-alpha (TNF-alpha) and interferon-g (IFN-g) were analyzed for expression of MRP8/MRP14. Employing Northern blotting, a sandwich enzyme-linked immunosorbent assay and immunocytochemical analysis we determined MRP8/MRP14 mRNA and protein levels, which were found to be elevated after VD3 and reduced after TPA treatment. TNF-alpha and IFN-g did not affect MRP8/MRP14 levels. Western blot analysis revealed that formation of MRP8/MRP14 to biologically active complexes which has previously been shown to be a calcium-mediated process is not dependent on the differentiation stages of HL-60 cells. Restriction of MRP8/MRP14 expression to only distinct pathways of monocytic differentiation in HL-60 cells may thus reflect different functional phenotypes of monocytes/macrophages in vivo.

Calcium-Binding Proteins↗
PubMed1993

A novel chicken homeobox-containing gene expressed in neurulating embryos.

We have isolated a cDNA clone from chicken embryo that contains a homeobox sequence (CHox-cad2). Analysis at the nucleotide and amino acid levels revealed closest similarity to the Xenopus Xcad1 and to other homeoboxes related to Drosophila caudal. RNA blot analysis showed hybridization of CHox-cad2 to two transcripts of 2.6 and 1.5 kb, present at day 1 of embryogenesis (E1). Using the highly sensitive polymerase chain reaction (PCR) to amplify cDNAs from embryonic RNAs from E0 (unincubated blastoderm) to E4, we confirmed the restricted expression of this homeobox sequence to the period of neurulation (E1).

Amino Acid Sequence↗