Search PubMed⌕ Search

Biomedical subjects

J Roth

Publications and source records attributed to J Roth.

At least 55 records · Page 3Linked to original sources
PubMed1991

Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos.

Insulin and insulin-like growth factor I (IGF-I) initiate their metabolic, growth, and differentiation effects through binding to the insulin receptor and the IGF-I receptor, two members of the tyrosine kinase family of receptors. To study the role of these peptides and receptors in early development, we used the polymerase chain reaction and embryo-derived RNA to generate partial cDNA sequences of the insulin receptor and IGF-I receptor from the amphibian Xenopus laevis. Three unique tyrosine kinase-related sequences were obtained. Two of the nucleotide sequences, XTK 1a and XTK 1b, corresponded to peptide that share 92% amino acid identity, and each is 89% identical to the human insulin receptor. The third sequence, XTK 2, corresponds to a peptide that has 92% amino acid identity with the human IGF-I receptor but only 80% identity with XTK 1a and XTK 1b. On the basis of these similarities, the pattern of conserved amino acids, and the tetraploid nature of the Xenopus genome, we suggest that XTK 1a and XTK 1b most likely represent the product of two different nonallelic insulin receptor genes, while XTK 2 may be one of the probable two Xenopus IGF-I receptor genes. By reverse transcription-polymerase chain reaction and gene-specific hybridization, expression of the three XTK sequences was detected in the oocyte, unfertilized egg, and embryos through gastrulation, neurulation, and tailbud stages. Competition binding assays with Xenopus membrane preparations demonstrated insulin receptors and IGF-I receptors in older tadpoles. IGF-I receptors were also present in oocytes, eggs, and gastrula embryos. By contrast, insulin binding was present but atypical in oocytes and was barely detected in eggs and gastrula embryos. The expression of receptors for insulin and IGF-I in early Xenopus embryos and their apparent distinct developmental regulation suggest that these molecules and their ligands may be important in early Xenopus development.

Amino Acid Sequence↗
PubMed1991

Calcium-dependent complex assembly of the myeloic differentiation proteins MRP-8 and MRP-14.

MRP-8 and MRP-14 are calcium-binding proteins belonging to the S-100 protein family which have been shown to be associated with specific stages of myeloic/monocytic cell differentiation. Members of this protein family are shown to form homo- and heterodimers. Complex formation has also been observed in preliminary experiments for MRP-8 and MRP-14. To evaluate the in vivo relevance of the MRP complex formation and the stoichiometric ratio of individual components complexes were isolated from granulocytes and monocytes by immunoaffinity chromatography using monospecific antibodies. The purified fraction of the MRPs was found to contain monomers and dimers as shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by silver staining and immunoblotting. Similar results were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of crude cell extracts. The existence of the MRP complexes in vivo was demonstrated by chemical cross-linking and subsequent isolation of complexes by immunoaffinity chromatography. Two new, highly abundant complexes were found in addition to the heterodimer, but neither monomers nor homodimers were detected. The two larger protein complexes (35.0 and 48.5 kDa) were identified as [MRP-8)2.(MRP-14] trimer and [MRP-8)2.(MRP-14)2) tetramer, respectively. All complexes could be shown to be noncovalently associated in vivo. Furthermore, the association of MRPs was shown to be Ca2+ dependent.

Blotting, Western↗
PubMed1991

pp75: A novel tyrosine-phosphorylated protein that heralds differentiation of HL-60 cells.

The human promyeloid cell line HL-60 differentiates toward monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or dibutyryl cAMP, respectively. When nondifferentiated cells were incubated for 20 min with 2 mM H2O2 and 0.1 mM sodium orthovanadate to inhibit their protein-tyrosine-phosphatase activity (Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) J. Biol. Chem. 265, 2896-2902), we found marked tyrosine phosphorylation of a single major protein of 53 kDa. Induction of differentiation of HL-60 cells was accompanied by the appearance of an additional major cytosolic tyrosine-phosphorylated protein of 75 kDa (pp75). In dibutyryl cAMP-treated cells, tyrosine phosphorylation of pp75 peaked after 24 h and then declined rapidly. In 1,25(OH)2D3-treated cells, increased tyrosine phosphorylation was detected as early as 2 h and peaked after 3 days, whereas the presence of differentiated phenotypes, assessed by the capacity of the cells to reduce nitro blue tetrazolium, was detected no earlier than 24 h. Doses of 1,25(OH)2D3 as low as 1 nM induced the appearance of pp75 at a stage where almost no differentiation measured by nitro blue tetrazolium reduction was detected. Phosphorylation of pp75 was not stimulated by adriamycin, which induced growth arrest without initiation of differentiation. pp75 could also be detected in U-937, a monocytic cell line that is more advanced in its differentiation state, and also in terminally differentiated circulating human monocytes treated with H2O2/vanadate. pp75 underwent in vitro tyrosine phosphorylation in cytosolic extracts derived from 1,25(OH)2D3-induced HL-60 cells, but not in extracts derived from uninduced cells. Our results raise the possibility that tyrosine phosphorylation of pp75 may be a common early event that heralds the differentiation of HL-60 cells into both the monocytic and granulocytic pathways.

Blotting, Western↗
PubMed1991

Genes for the insulin receptor and the insulin-like growth factor I receptor are expressed in the chicken embryo blastoderm and throughout organogenesis.

The early expression of insulin and insulin-like growth factor I (IGF-I) in the chicken embryo suggests that these peptides play an important role in early development. The receptors for insulin and IGF-I, however, had not been studied at the molecular level in this model. We report two chicken sequences that, by comparison with known tyrosine kinases, appear to correspond to the tyrosine kinase domain of the insulin receptor homologue (CTK-1) and the IGF-I receptor homologue (CTK-2). Using reverse-transcription of RNA, amplification with the polymerase chain reaction (RT-PCR), and gene-specific hybridization, we demonstrate that the two genes, CTK-1 and CTK-2, are expressed in embryos at least as early as the blastoderm (Day 0), during neurulation (Day 1), and in early (Days 2-3) and late (Day 9) organogenesis.

Amino Acid Sequence↗
PubMed1991

Localization of glycosylation sites in the Golgi apparatus using immunolabeling and cytochemistry.

This review summarizes data on the distribution of certain glycosylation steps in the Golgi apparatus as revealed by immunolabeling and lectin techniques. The methodical basis for such investigations was provided by the introduction of the colloidal gold marker system for immunolabeling and the development of new means of tissue processing such as the low-temperature embedding technique using Lowicryl K4M. The application of these techniques together with highly specific antibodies has provided much of the basis for our current understanding of the Golgi apparatus in functional terms. Thus, in many cell types, three Golgi apparatus compartments can be distinguished, whereas in others no such functional subdivision is evident. Investigations on sialyltransferase distribution have also provided direct evidence that GERL is structurally and functionally part of the Golgi apparatus.

Animals↗
PubMed1991

Influence of noradrenergic input into the hypothalamic paraventricular nucleus on fever in the guinea-pig.

The febrile response of guinea-pigs to a bacterial pyrogen was tested under different experimental conditions: (1) during electrical stimulation of the hypothalamic paraventricular nucleus (PVN), (2) after destruction of noradrenergic afferents into the PVN by 6-hydroxydopamine (6-OHDA), (3) during a microinfusion of noradrenaline (NA) into the PVN. Electrical stimulation of the PVN neurons by implanted microelectrodes reduced the febrile response to 45% of the control values. This confirmed the proposed antipyretic function of these neurons. Chronic destruction of noradrenergic afferents to the PVN by microinjected 6-OHDA also resulted in a significant reduction of febrile responses to 38% of the control values. A microinfusion of NA into the PVN enhanced the febrile responses to bacterial endotoxin by 39% in comparison to animals microinfused with the solvent (0.9% NaCl). Immunoreactivity to an antiserum against arginine vasopressin (AVP) was compared in PVN neurons of 6-OHDA-treated and of control animals. The number of AVP-immunoreactive perikarya and the intensity of immunoreactivity were increased in the animals treated with 6-OHDA, especially in the medial part of the PVN. Since fever was increased by microinfused NA and decreased by 6-OHDA treatment, we assume an inhibitory influence of noradrenergic brain stem afferents on the proposed antipyretic vasopressinergic system of the PVN.

Afferent Pathways↗
PubMed1991

NCAM in developing mouse gonads and ducts.

The expression of the Neural Cell Adhesion Molecule, NCAM, in mouse gonads and ducts was studied from fetal life to maturity. The methods used were immunocytochemical staining and Western blotting. The immunocytochemical studies showed that the only structures that remain NCAM-positive throughout life were the mesonephric-derived rete ovarii and rete testis. Also in the fetal gonads some somatic cell lining the groups of differentiating germ cells were stained. In the immature as well as in the mature ovary the granulosa cells and oocytes of growing and large follicles--but not of small follicles--were stained. A particularly strong staining of the cytoplasm of the oocyte, healthy as well as atretic, was seen. All cells of the testis remained negative except for weakly stained residual bodies and late spermatids. At all ages the male ducts showed only weak staining, whereas in the female Müllerian duct the epithelium became strongly positive at puberty. The stroma of the Müllerian duct was positive during a transitory period around day 16 of fetal life in both sexes. One-dimensional gel immunoblotting of total protein from gonads, rete and ducts from immature and mature mice showed that only the two largest isoforms of NCAM (NCAM-A and NCAM-B) were present. The gonads and the rete of both sexes and the adult uterus expressed only NCAM-B, whereas NCAM-A was also detected in the adult epididymis. The present findings suggest that NCAM may be involved in the normal development and formation of both the gonads and ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The severity of irritant contact dermatitis in various strains of mice correlates with endothelial expression of migration inhibitory factor (MIF).

Irritant contact dermatitis to croton oil in BALB/cByJ, C57Bl/6J and six recombinant inbred CxB strains of mice was investigated in relation to variations in endothelial migration inhibitory factor (MIF) reactivity. MIF has been shown to be a mediator of cellular immunity and operates as a differentiation signal inducing an inflammatory type of macrophage. The intensity of the ear swelling response reached a maximum 8 h after induction of contact dermatitis, with highest values in BALB/cByJ and CxB4/ByAH mice and weak reactions in CxB2/ByAE, CxB7/ByAK, C57Bl/6J and CxB1/ByAD mice. After the same time period (8 h) cryostat sections were immunostained for capillary endothelium expressing MIF. The most pronounced MIF expression was observed in BALB/cByJ mice, and CxB4/ByAH mice showed intermediate reactions and the other strains weak reactions. Endothelial MIF expression correlated well with the intensity of ear swelling (Pearson's correlation coefficient 0.82). Patterns of endothelial MIF expression in recombinant inbred strains suggest that endothelial MIF expression is not under the control of a single gene. Our data support the hypothesis that endothelial MIF expression plays a prominent role in inflammatory events and correlates with the severity of inflammation.

Animals↗
PubMed1991

Prognosis of patients with ventricular tachycardia or fibrillation and a normal electrophysiologic study.

The outcome of 26 patients with sustained ventricular tachycardia (n = 16) or ventricular fibrillation (n = 10) and no inducible ventricular tachycardia (less than or equal to 10 beats) by baseline programmed stimulation was studied. Coronary artery disease was present in 14 patients, dilated cardiomyopathy was seen in seven, valvular heart disease was present in two, and no apparent cardiac abnormalities were found in three. The mean left ventricular ejection fraction was 53 +/- 14%. During the follow-up period of 24 +/- 16 months, actuarial survival rates at 1 and 2 years were 95% and 89% for sudden death and 95% and 83% for total cardiac death, respectively. No patients with a known ejection fraction greater than 30% died suddenly during the follow-up. Noninducibility by programmed stimulation in patients with sustained ventricular tachycardia or fibrillation is associated with a relatively preserved ventricular function. It may predict a low risk of sudden death in patients with preserved ventricular function.

Actuarial Analysis↗
PubMed1991

Ligase-free subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a single day.

Often, it is convenient to subclone polymerase chain reaction (PCR) products into a plasmid vector for subsequent replication in bacteria, but conventional subcloning methods often fail. We report a rapid and versatile method to subclone PCR products directionally into a specific site of virtually any plasmid vector. The procedure requires only four primers, does not require DNA ligase, and may be accomplished in a single day. Ligase-free subcloning is performed by incorporating into the PCR primers sequences at the 5' ends that result in PCR products whose 3' ends are complementary to the 3' ends of the recipient linearized plasmid. The PCR product and the linearized plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3' ends (ligation by overlap extension). Denaturation followed by heterologous reannealing and cyclization results in a cyclic recombinant plasmid with two nicks that may be used directly to transform competent Escherichia coli. In our hands, ligase-free subcloning is rapid, and offers many advantages over existing strategies.

Bacteriophage lambda↗
PubMed1991

Expression of polysialylated N-CAM during rat heart development.

Developmental patterns of immunoreactivity for the neural cell adhesion molecule (N-CAM) and alpha 2.8-linked polysialic acid (PSA) were identified in embryonic and postnatal rat heart by immunocytochemistry and immunoblotting. Polyclonal antibodies against N-CAM and a monoclonal antibody which recognises only polymers of PSA with a chain length greater than eight units were used. Gold- and alkaline-phosphatase-labelled antibodies were used for detection. The N-CAM polypeptide isoform pattern seen by immunoblotting after endoneuraminidase treatment changed as development progressed. During embryonic development a 160-kDa polypeptide isoform was predominant. Around birth, 130-, 160- and 170-kDa polypeptide isoforms were found. The expression of the 130- and 170-kDa isoforms diminished until finally, in the adult, weak immunoreactivity for bands of 120-, 130- and 160-kDa was seen. In general the extent and intensity of PSA and N-CAM immunostaining in rat heart increased until birth and declined thereafter. Early in development prominent immunostaining for PSA and N-CAM was seen in the epicardium while later in development this area was only weakly stained. Initially myocardial cells, endocardial cells and some cells in the atrioventricular cushions were immunoreactive for both PSA and N-CAM. Later in development N-CAM immunostaining was more prominent than PSA immunoreactivity, reflecting a decrease in N-CAM polysialylation, which was also seen by immunoblotting. During innervation of the heart, nerve fibres were strongly immunostained for PSA and N-CAM, and this was the only immunostaining seen in adult heart.

Animals↗
PubMed1991

Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

Adenocarcinoma↗
PubMed1991

Normal coding sequence of insulin gene in Pima Indians and Nauruans, two groups with highest prevalence of type II diabetes.

The nucleotide sequence of the insulin gene was determined in American Pima Indians and Micronesian Nauruans, two populations in whom the prevalence of non-insulin-dependent (type II) diabetes mellitus is the highest in the world. The insulin gene was amplified by the polymerase chain reaction to generate single-stranded DNA suitable for direct sequencing. The nucleotide sequences of the coding and adjacent regions of the insulin gene in six Pima Indians and two Nauruans with type II diabetes were identical to previously published insulin gene sequences of nondiabetic subjects.

Adult↗
PubMed1991

Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids.

The neural cell adhesion molecule (NCAM) exists in various types of neuroendocrine cells and their tumors. A typical feature of NCAM is polysialic acid, of which the chain length is developmentally regulated. The authors have performed a comparative immunohistochemical study on small cell lung carcinomas and bronchial as well as gastrointestinal carcinoids with the monoclonal antibody (MAb) 735 reactive with the long-chain form of polysialic acid. The small cell lung carcinomas, irrespective of their histological type, were positive for polysialic acid. Metastatic tumor cell complexes also exhibited immunostaining. The tumor cell-surface-associated immunostaining for polysialic acid was sensitive to endoneuraminidase. The mature and atypical bronchial and gastrointestinal carcinoids were not immunoreactive for polysialic acid. Cytoplasmic staining in groups of cells of carcinoids (2 of 28 cases) was due to nonspecific antibody binding, which could be prevented by increased ion strength. These data indicate that neuroendocrine tumors of the lung can be distinguished by their content of highly sialylated NCAM.

Antibodies, Monoclonal↗
PubMed1991

Polysialic acid as a marker of both immature and mature neural tissue in human teratomas.

The neural cell adhesion molecule (NCAM) is involved in cell-cell interaction during neural development. We employed a monoclonal antibody directed against the long chain polysialic acid moiety of NCAM to evaluate its usefulness as a marker of primitive neural elements in teratomas. This marker was compared with other neural markers, S-100, glial fibrillary acidic protein (GFAP), neurofilament protein (NFL), nerve growth factor receptor (NGFR), as to its effectiveness in labeled neural tissue in human teratomas. The anti-polysialic acid antibody was the only reagent that consistently marked all types of neural tissue, both mature and immature in these lesions. Immature neural elements alone have prognostic significance in teratomas. Our results indicate that anti-polysialic acid antibodies are the most sensitive and useful markers of immature neural elements in these lesions.

Antibodies, Monoclonal↗