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Biomedical subjects

R Seger

Publications and source records attributed to R Seger.

At least 91 records · Page 5Linked to original sources
PubMed1995

BMP-4 regulates the dorsal-ventral differences in FGF/MAPKK-mediated mesoderm induction in Xenopus.

Recent studies on Xenopus development have revealed an increasingly complex array of inductive, prepatterning, and competence signals that are necessary for proper mesoderm formation. In this study, we establish that fibroblast growth factor (FGF) signals through mitogen-activated protein kinase kinase (MAPKK) to induce mesodermal gene expression. We demonstrate that a partially activated form of MAPKK restores expression of the mesodermal genes Xcad-3 and Xbra, eliminated by the dominant-negative FGF receptor (delta FGFR). Similar to the results reported earlier with delta FGFR, expression of a dominant-negative form of MAPKK (MAPKKD) preferentially eliminates the dorsal expression of Xcad-3 and Xbra. We tested whether the regional localization of bone morphogenetic protein-4 (BMP-4) could explain why both MAPKKD and delta FGFR eliminate the dorsal and not the ventral expression of Xcad-3 and Xbra. We show that ectopic expression of BMP-4 is sufficient to maintain the dorsal expression of Xcad-3 and Xbra in embryos containing delta FGFR and that expression of a dominant-negative BMP receptor reduces the dorsal-ventral differences in delta FGFR embryos. These results indicate that regional localization of BMP-4 is responsible for the dorsal-ventral asymmetry in FGF/MAPKK-mediated mesoderm induction.

Animals↗
PubMed1995

Signal transduction of the gonadotropin releasing hormone (GnRH) receptor: cross-talk of calcium, protein kinase C (PKC), and arachidonic acid.

1. The decapeptide neurohormone gonadotropin releasing hormone (GnRH) is the first key hormone of the reproductive system. Produced in the hypothalamus, GnRH is released in a pulsatile manner into the hypophysial portal system to reach the anterior pituitary and stimulates the release and synthesis of the gonadotropin hormones LH and FSH. GnRH, a Ca2+ mobilizing ligand, binds to its respective binding protein, which is a member of the seven transmembrane domain receptor family and activates a G-protein (Gq). 2. The alpha subunit of Gq triggers enhanced phosphoinositide turnover and the elevation of multiple second messengers required for gonadotropin release and biosynthesis. 3. The messenger molecules IP3, diacylglycerol, Ca2+, protein kinase C, arachidonic acid and leukotriene C4 cross-talk in a complex networks of signaling, culminating in gonadotropin release and gene expression.

Animals↗
PubMed1995

The MAPK signaling cascade.

The transmission of extracellular signals into their intracellular targets is mediated by a network of interacting proteins that regulate a large number of cellular processes. Cumulative efforts from many laboratories over the past decade have allowed the elucidation of one such signaling mechanism, which involves activations of several membranal signaling molecules followed by a sequential stimulation of several cytoplasmic protein kinases collectively known as mitogen-activated protein kinase (MAPK) signaling cascade. Up to six tiers in this cascade contribute to the amplification and specificity of the transmitted signals that eventually activate several regulatory molecules in the cytoplasm and in the nucleus to initiate cellular processes such as proliferation, differentiation, and development. Moreover, because many oncogenes have been shown to encode proteins that transmit mitogenic signals upstream of this cascade, the MAPK pathway provides a simple unifying explanation for the mechanism of action of most, if not all, nonnuclear oncogenes. The pattern of MAPK cascade is not restricted to growth factor signaling and it is now known that signaling pathways initiated by phorbol esters, ionophors, heat shock, and ligands for seven transmembrane receptors use distinct MAPK cascades with little or no cross-reactivity between them. In this review we emphasize primarily the first MAPK cascade to be discovered that uses the MEK and ERK isoforms and describe their involvement in different cellular processes.

Animals↗
PubMed1994

Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII.

Mitogen-activated protein kinase kinase (MAPKK) is a dual specificity protein kinase that exhibits a high degree of specificity toward its downstream target, mitogen-activated protein kinase (MAPK). In this study, we used stable overexpression of MAPKK and its mutants in NIH 3T3 cells to study effects on downstream components of the MAPK signaling cascade and to correlate them to physiological responses. We have mutated the potential regulatory serine residue 222 to alanine (S222A) or to glutamate (S222E) and serines 212 and 218 together to alanine residues (S212A,S218A). Lysine 97 was mutated to alanine (K97A) to provide an inactive enzyme. Overexpression of the wild type MAPKK had no effect on any of the parameters examined. The K97A and S222A mutants served as dominant negatives by suppressing MAPKK, MAPK, and p90rsk activation in vivo. S222E enhanced all of these activities, and S212A,S218A had a small inhibitory effect. A similar trend was observed when cellular proliferation was examined and the different effects were accompanied by altered cellular shape. Taken together, our results demonstrate a direct linkage between the MAPK signaling pathway and the control of cellular proliferation and morphology and also establish that phosphorylation of serine 222 is essential for MAPKK activation together with the phosphorylation of an additional serine(s) (probably serine 218).

3T3 Cells↗
PubMed1994

Purification, cloning, and expression of a murine phosphoprotein that binds the kappa B motif in vitro identifies it as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein. Description of a novel DNA-dependent phosphorylation process.

The kappa B enhancer element regulates expression of many genes involved in immune responses and other processes. kappa B motif binds a number of proteins, some but not all, are related to the NF-kappa B family of transcription factors. We have previously identified a 65-kDa phosphoprotein that is specifically recognized by the kappa B motif (Ostrowski, J., Sims, J. E., Sibley, C. H., Valentine, M. A., Dower, S. K., Meier, K. E., and Bomsztyk, K. (1991) J. Biol. Chem. 266, 12722-12733). This protein is closely associated with a serine/threonine kinase that is responsive to treatment of cells with interleukin-1 and other agents. We report here purification, cloning, and expression of this kappa B motif-binding phosphoprotein. The primary structure deduced from the isolated murine cDNA, identifies the protein as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein. Antipeptide antibodies and expression of the cloned cDNA in Escherichia coli, demonstrated that the K protein is the authentic phosphoprotein that binds the kappa B motif in vitro. We also demonstrate that the in vitro phosphorylation of the natural and the recombinant K proteins by the associated kinase is stimulated by the kappa B motif.

Amino Acid Sequence↗
PubMed1994

Autosomal recessive chronic granulomatous disease with absence of the 67-kD cytosolic NADPH oxidase component: identification of mutation and detection of carriers.

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to kill certain bacteria and fungi. This is caused by deficiencies in one of the components of NADPH oxidase, the enzyme in phagocytic leukocytes that generates superoxide. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of NADPH oxidase (p67-phox) is missing. Until now, mutations in the gene coding for this protein have not been identified. We now report on a 10-year-old girl with lymph node and liver abscesses who was recognized as an A67(0) CGD patient by lack of NADPH oxidase activity in her granulocytes, a cytosolic defect in a cell-free oxidase system, and lack of immunoreactive material with an antiserum against the p67-phox protein. mRNA for this protein was present in normal amounts in her monocytes. This p67-phox mRNA was reverse-transcribed, and the coding region was amplified by polymerase chain reaction in six overlapping fragments and was sequenced. The patient appeared to be homozygous for a G-233-->A mutation, resulting in a nonconservative amino acid change (78Gly-->Glu). This mutation was also found in the genomic DNA of this patient but not in that of 38 normal donors. Both parents and a sister proved to be carriers of the disease, as deduced from the mutation in only one allele. The carrier state was also manifested by intermediate superoxide production by their intact granulocytes and in the cell-free system.

Amino Acid Sequence↗
PubMed1994

Juvenile chronic arthritis: imaging of the knees and hips before and after intraarticular steroid injection.

Intraarticular steroid therapy in juvenile chronic arthritis (JCA) is performed because of high local efficacy with few side effects. Imaging is used for initial evaluation and for monitoring of treatment response. The aim of this study was to compare imaging findings in diseased hips and knees before and after therapy. A prospective study was performed on 10 patients (15 joints) scheduled for intraarticular therapy. Pretherapeutic assessment included clinical work-up, radiographs, ultrasound (US), and magnetic resonance imaging (MRI) of affected joints. Following therapy, clinical and sonographic examinations were performed at 1 week and 1 month. MRI was repeated at 1 month. MRI and US demonstrated pannus formation and effusion, but differentiation was less distinct on US. Popliteal cysts and lymph nodes were visible in both modalities. MRI additionally revealed articular cartilage loss and subchondral cysts, not shown by US. Epiphyseal overgrowth and osteopenia were best seen radiographically. At present MRI is the best tool to assess the inflammatory changes of the joints in JCA. Initial staging of the joints may be done with plain films and MRI. US is useful to assess effusion and pannus and may be used to monitor treatment response.

Administration, Topical↗
PubMed1994

Sensitive detection and early prognostic significance of p24 antigen in heat-denatured plasma of human immunodeficiency virus type 1-infected infants. Swiss Neonatal HIV Study Group.

Immune complex formation causes underdetection of p24 antigen in human immunodeficiency virus (HIV) infection. Briefly boiling diluted plasma releases all complexed antigen, which can then be measured by some commercial assays. In a retrospective pediatric cohort study, the specificity of this procedure in 390 uninfected samples was 96.9% after initial testing and 100% after neutralization. Sensitivity among 125 postnatal infected samples was, at a detection of 2 pg/mL, 96.0% (97% neutralizable) compared with 47.7% for regular antigen (76% neutralizable), 96% for polymerase chain reaction, and 77% for viral culture. The high sensitivity and specificity of heat-denatured antigen was confirmed by prospectively testing 113 additional samples. Quantitative analysis of samples from infected infants showed low levels of p24 antigen in 29% of cord blood sera, a postnatal increase to levels that were during the first 6 months of life inversely associated with survival, and persistence of antigenemia thereafter independent of clinical status. Prevalence and antigen levels were significantly lower in mothers. The persistent antigenemia in children indicates that their immune systems cannot restrict HIV expression as efficiently as those of adults.

Age Factors↗
PubMed1994

Processing and presentation of tetanus toxin by antigen-presenting cells from patients with chronic granulomatous disease (CGD) to human specific T cell clones are not impaired.

The capacity of peripheral blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed B cell lines from CGD patients to process and present tetanus toxin (tt)-specific epitopes was assessed using various tt preparations and human tt-specific T cell clones. PBL from all of the donors were able to process and present either native tt and/or denatured tt to human T cell clones specific for various tt epitopes. Furthermore, no difference was found in the antigen requirement when normal or CGD EBV-B cell lines were used as antigen-presenting cells (APC). These results suggest that the deficiency in oxygen metabolism in CGD cells does not affect tt processing and presentation.

Amino Acid Sequence↗
PubMed1994

IgG, IgM, and IgA response to HIV in infants born to HIV-1 infected mothers. Swiss Neonatal HIV Study Group.

Children born to HIV-1-positive mothers were prospectively tested for HIV-reactive IgG, IgM, and IgA by Western blot, in order to study the children's humoral immune response in the background of passively transferred maternal IgG. In infected infants, a response was first seen at 1-3 months for env-reactive IgM and IgA, as well as gag-reactive IgM and IgG. This was followed by production of IgG to env, IgA to pol and to gag p17 and p55 at 7-9 months, and IgG to pol at 10-12 months. IgG Western blot positivity by all interpretation guidelines in all infected infants was found by 10-12 months. Subsequently, only IgG to env and p24, and IgA to env were maintained in all, whereas IgG to pol and p17 disappeared again in a significant fraction. A considerable proportion of uninfected infants also produced gag-reactive antibodies: IgM at 1-3 months, followed by IgG, which persisted in 10-20% and were also found in children born to uninfected mothers. These antibodies were, however, present at lower titers than in infected infants and were apparently produced in response to agent(s) different from HIV. Maternal antibodies to env disappeared significantly faster in infected than uninfected infants. Traces of HIV-reactive IgG were present for up to 21 months in children who subsequently seroreverted completely.

Age Factors↗
PubMed1993

Identification of an inducible 85-kDa nuclear protein kinase.

To identify inducible protein kinases localized exclusively in the nucleus, nuclear and cytosolic extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to an Immobilon-P membranes were incubated in phosphorylation buffer containing [gamma-32P]ATP. Autoradiographs of the membranes revealed an 85-kDa 32P-labeled band; the intensity of this band was transiently increased in nuclear but not in cytosolic extracts from interleukin-1 alpha-treated cells. Incorporation of 32P label into a blotted protein band suggested the presence of an interleukin-1 alpha-responsive 85-kDa nuclear protein kinase. Fractionation of nuclear extracts by Mono Q failed to separate the kinase activity from the substrate, indicating that the 85-kDa band identified on the Immobilon-P membrane represents a protein kinase that undergoes autophosphorylation. Phosphoamino acid analysis of the 85-kDa band showed that this enzyme is a serine/threonine kinase. Purified pp90RSK could not be identified by the denaturation-renaturation method, indicating that the 85-kDa kinase identified here is not pp90RSK. This observation, nuclear but not cytoplasmic localization, and the fact that antibodies to known protein kinases kinase failed to recognize it suggest that the enzyme identified here is a novel protein kinase.

Amino Acid Sequence↗
PubMed1993

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Amino Acid Sequence↗
PubMed1993

Metabolic labeling of mitogen-activated protein kinase kinase in A431 cells demonstrates phosphorylation on serine and threonine residues.

Mitogen-activated protein (MAP) kinase kinase is an enzyme that activates the growth factor-regulated MAP kinase in vitro by a mechanism that involves direct phosphorylation of MAP kinase on tyrosine and threonine residues. MAP kinase kinase is stimulated by growth factor treatment of cells and has been shown to be inactivated with protein phosphatases, suggesting that it is regulated by protein phosphorylation. Analysis of two epidermal growth factor-stimulated forms of MAP kinase kinase, purified from 32P-labeled A431 cells, shows that the kinase is phosphorylated on serine and threonine residues and that treatment with protein phosphatases leads to serine dephosphorylation. Under conditions that lead to complete inactivation, only partial dephosphorylation of MAP kinase kinase is observed. Consistent with this finding, inactive forms of MAP kinase kinase, which separate from active forms during the course of purification, are also observed to be phosphorylated in intact cells.

Cell Line↗
PubMed1993

The management of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease which results from absence of the NADPH oxidase in the professional phagocytic cells neutrophils, monocytes, macrophages and eosinophils. Deficiency of this oxidase renders the patient liable to infection by bacteria and fungi, and, as the name of the disease suggests, to chronic granulomatous inflammation. These patients present with a great variety of infections and other complications of their disease, which often tax the clinical and therapeutic skill of the doctors responsible for their care. Collectively we look after, or advise on the management of, over 100 of these subjects, and have developed experience in the diagnosis and management of the infections and other clinical problems they present. We thought that it might be timely to provide guidelines for their management based upon this experience. The numbers of patients are still relatively small, and the clinical presentations very varied, so it is impossible to provide clear statistical proof of the veracity of this advice. It does, however, reflect the working practise of the physicians caring for many of these patients in Europe.

Anti-Bacterial Agents↗
PubMed1993

The frequency of pretransplant donor cytotoxic T cell precursors with anti-host specificity predicts survival of patients transplanted with bone marrow from donors other than HLA-identical siblings.

Transplantation with bone marrow from other than genotypically HLA-identical donors is associated with an increased incidence and severity of graft-versus-host disease (GvHD). The precise influence of HLA incompatibilities is not easy to analyze as even perfectly matched, HLA-identical unrelated donors might still express HLA differences that remain undetected by conventional typing. To measure T cell activity against serologically detectable and nondetectable HLA antigens, we analyzed the frequencies of CTL precursors (CTLp) between 11 unrelated HLA-matched and five related haploidentical donor/recipient pairs in graft-versus-host direction. Our results show that whenever HLA class I disparities could be identified by serology, high precursor frequencies (1/28,000-1/94,000) were measured. In contrast, in donor/recipient pairs that differed for class II only, no precursors were detected. CTLp were elevated in two out of eight fully matched donor/recipient combinations. These combinations displayed activities as high (1/21,000; 1/52,000) as the combinations that were serologically HLA class I disparate. The incompatibilities detected by the cellular assay were highly significant for the clinical results after transplantation. High CTLp frequencies before transplantation correlated with unfavorable clinical results independent of the incidence of detected HLA differences. Five out of the six patients with high (> 1/100,000) CTLp frequencies died within 120 days after transplantation. GvHD IV was the cause of death for all (3/5) patients who had received an unmanipulated bone marrow. In the group with intermediate or undetectable CTLp frequencies, eight out of 10 patients are alive, seven (CTLp frequency undetectable) without GvHD more severe than grade II, while one patient (CTLp frequency = 1/180,000) suffered from GvHD grade III. One patient rejected the graft and was rescued by an autologous BMT.

Adolescent↗