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Biomedical subjects

H Riedel

Publications and source records attributed to H Riedel.

At least 19 recordsLinked to original sources
PubMed1997

The adapter protein Grb10 associates preferentially with the insulin receptor as compared with the IGF-I receptor in mouse fibroblasts.

To identify receptor-associated proteins that may contribute to the specificity of insulin and IGF-I signaling responses, a mouse embryo library was screened using the yeast two-hybrid system. Multiple receptor-interactive clones encoding the SH2 domain of the adapter protein Grb10 were isolated. Subsequent cloning of the full-length Grb10 sequence from a mouse fat cDNA library defined a previously unknown Grb10 variant, that appears to be the predominant isoform in mouse tissues. Receptor-deficient R- cells (fibroblasts from mice with homologous disruption of the IGF-I receptor gene) and transfected R- cells expressing either insulin receptors (R-IR cells) or IGF-I receptors (R+ cells) were used to investigate the specificity of Grb10 interaction with the two related receptors. Hormone-activated insulin receptors in R-IR cells coprecipitated with three species, all recognized as Grb10 isoforms by specific Grb10 antibody. Under the same conditions, Grb10 was essentially undetectable in IGF-I receptor immunoprecipitates from stimulated R+ cells. Grb10 association with insulin receptors was maximal at 10 nM insulin stimulation and sustained from 5-10 min after hormone stimulation in R-IR cells. In conclusion, Grb10 interacts preferentially with insulin vs. IGF-I receptors in intact cells and, thus, may have a role in mediating insulin receptor-specific cellular responses.

Animals↗
PubMed1996

Interaction between the Grb10 SH2 domain and the insulin receptor carboxyl terminus.

Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor beta-subunit using the yeast two-hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.

3T3 Cells↗
PubMed1996

Activation of conventional mammalian protein kinase C isoforms expressed in budding yeast modulates the cell doubling time--a potential in vivo screen for protein kinase C activators.

Conventional mammalian protein kinase C (PKC) isoforms alpha, beta 1, and gamma were expressed in Saccharomyces cerevisiae and resulted in a differential increase in the yeast doubling time in response to distinct classes of PKC activators. Mutants were created in the regulatory domain of PKC alpha to map the interaction with the different activators. The macrocyclic lactone bryostatin 5 preferentially regulated PKC alpha activity through the second cysteine-rich sequence (CYS2) of Cl, while regulation by the diterpene ester mezerein displayed strong preference for the first cysteine-rich sequence (CYS1) of Cl. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA) regulated PKC enzymatic activity equally potently via CYS1 or CYS2 albeit at reduced levels compared with native PKC alpha. For the diterpene ester ingenol-3, 20-dibenzoate and the indol alkaloids (-)-7-octyl-indolactam V and (-)-indolactam V, no responses were observed for mutants lacking either CYS1 or CYS2 whereas native PKC alpha activity was regulated. These in vivo results were complemented by in vitro binding and catalytic assays which showed correlation between PKC enzymatic activity and the cell growth characteristics. The observed phenotype can be exploited to screen natural compounds in vivo for their PKC regulatory potential and to map the underlying interactions.

Animals↗
PubMed1996

[Phase prevention in bipolar affective disorder with nimodipine. A case report].

A 56-year-old female patient had a history of more than 10 years' duration of a bipolar affective disorder manifest mainly as depressive episodes. These episodes used to occur once or twice each year, frequently leading to hospital admission. On average, the episodes lasted for about 2 months, and they tended to be followed by brief periods of hypomania. Only once, in 1986, did a manic episode make hospitalization necessary. Attempted prophylaxis with lithium at therapeutic plasma levels did not prove effective. Treatment with carbamazepine was discontinued because of leukopenia. The most recent stay in hospital became necessary because of a depressive episode that lasted for 5 months and did not respond to therapy. On admission the patient's score on the Hamilton depression scale was 23. When the calcium antagonist nimodipine was given at a dosage quickly escalated to 360 mg daily, the patient could be discharged in a state of complete remission after 26 days. For the first time in many years she has been emotionally stable for almost 1 year with single agent nimodipine therapy at 180 mg daily.

Bipolar Disorder↗
PubMed1995

Deletion analysis of protein kinase C inactivation by calphostin C.

Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-alpha (a Ca(2+)-dependent conventional isoform) and PKC-epsilon (a Ca(2+)-independent novel isoform) are similarly inactivated by calphostin C (75-100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-alpha required for inactivation by calphostin C. A series of N-terminal-truncation mutants of bovine PKC-alpha expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-alpha by 80-90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND140) was inactivated by only 20%. These findings imply that the aa sequence 92-140 is a structural determinant of PKC-alpha inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102-144), which is highly conserved among most PKC isoforms including PKC-epsilon. In addition to aa 92-140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca2+) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect to calphostin C action is discussed.

Animals↗
PubMed1995

Differential protein kinase C ligand regulation detected in vivo by a phenotypic yeast assay.

The molecular dissection of protein kinase C (PKC) action has been based in part on time-consuming functional assays such as the mouse skin model for testing the tumor promoter activity of phorbol esters and related PKC activators. To help overcome the limitations imposed by the complexity of such assays, we developed the yeast Saccharomyces cerevisiae as an alternative, rapid, and simple experimental system. This model has a specific phenotype, an increase in the cell doubling time, that is proportional to the level of enzymatic activity of expressed mammalian PKC isoforms. We used this phenotype to assay and compare the regulation of native bovine PKC alpha and mutants in the conserved regulatory region C1 in vivo by various activators: two diterpenes, the phorbol ester phorbol-12-myristate-13-acetate (PMA) and mezerein, and the indole alkaloid indolactam V. We found that PMA activated PKC mutants lacking either Cys-rich, zinc finger-like repeat of the conserved region C1 to comparably reduced levels, whereas indolactam V activated native PKC alpha but none of the mutants at normal doses. In contrast, mezerein activated native PKC alpha and a mutant lacking the second Cys repeat equally well but mutants lacking the first Cys repeat of C1 at a greatly reduced level. These differential responses were supported by the observed in vitro PKC catalytic activities. Therefore, PMA regulates PKC alpha activity comparably well via either Cys repeat, whereas mezerein regulation predominantly occurs via the first Cys repeat of C1. Indolactam V activation was less potent, it was greatly reduced in the absence of either Cys repeat, and displayed no preference. We introduce this phenotypic assay as a rapid and general screen for the PKC-activating or possibly inhibitory potential of drug candidates and to identify the PKC regulatory sites involved in these interactions.

Animals↗
PubMed1994

Ligand regulation of bovine protein kinase C alpha response via either cysteine-rich repeat of conserved region C1.

Based on the finding by others that the conserved region C1 of conventional protein kinase C isoforms carries two independent, cysteine-rich phorbol ester binding sites, we have mapped the structural elements of the C1 region for their role in the phorbol ester- and phospholipid regulation of PKC alpha responses. We have prepared two amino terminal truncation mutants of bovine PKC alpha, ND91 lacking the first Cys-repeat of C1, and ND153 lacking both Cys-repeats of C1, as well as two internal deletion mutants, D162-245 lacking most of C2, and D109-263 lacking most of C2 and the second Cys-repeat of C1. The mutants were expressed in the yeast Saccharomyces cerevisiae which allows the rapid biochemical and physiological characterization of mammalian PKC isoforms. We found that all mutants displayed an elevated basal level of enzymatic activity in vitro but retained the basic catalytic PKC characteristics: regulation by Ca2+ and (except for ND153) by phospholipid or phorbol ester. In vivo we observed proportional physiological responses, the stimulation of Ca2+ uptake, and an increase in the cell doubling time for all mutants upon phorbol ester stimulation (constitutive for ND153) similar to the response of normal PKC alpha. Our findings indicate that after partial PKC activation by deletion mutagenesis, the presence of either Cys-repeat in C1 still allows phospholipid- and phorbol ester regulation of protein kinase C alpha responses.

Animals↗
PubMed1994

Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.

3T3 Cells↗
PubMed1993

Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR).

We have significantly improved a method originally developed by Liang and Pardee [Science 257 (1992) 967-971] to display a broad spectrum of expressed genes and to detect differences in expression between different cell types. We have analysed various aspects of the technique and have modified it for both, the application to fast and efficient identification of genes and the use with automatic analysis systems. Based on the mathematical background we have devised the appropriate number of optimal PCR primers. We have also introduced nondenaturating gels for separating double stranded fragments as single bands. By applying the method to regenerating mouse liver, we have identified, out of a total of 38,000 bands, about 70 fragments where the expression of the corresponding genes seems to be differentially regulated at different time points. Application of the method to an automatic DNA sequencer was successfully done. Thus, we have confirmed the usefulness and increased the power of the RNA display technique, which we named differential display reverse transcription PCR (DDRT-PCR), and have extended the range of its application.

Animals↗
PubMed1993

Stimulation of calcium uptake in Saccharomyces cerevisiae by bovine protein kinase C alpha.

Ca2+ plays essential roles as a second messenger often in synergism with the calcium- and phospholipid-dependent phorbol ester receptor, protein kinase C (PKC), which stimulates Ca2+ influx in various cell types in a potential positive feedback mechanism. To address the compatibility of these mechanisms between lower eukaryotes and mammals, we have stably expressed bovine PKC alpha in the yeast Saccharomyces cerevisiae. We find that phorbol ester binding sites are created which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro. Phorbol ester activation in vivo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology. This may represent activation of a putative PKC-mediated signaling pathway utilized by functional yeast homologs of mammalian PKC isoforms. These are suggested by some protein data; however, their genes have not yet been characterized (Simon, A. J., Milner, Y., Saville, S. P., Dvir, A., Mochly-Rosen, D., and Orr, E. (1991) Proc. R. Soc. Lond. B 243, 165-171). Our findings indicate that bovine PKC alpha is functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells, which suggests compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.

Amino Acid Sequence↗
PubMed1993

Phorbol ester activation of functional rat protein kinase C beta-1 causes phenotype in yeast.

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. The molecular dissection of its mechanisms of action has been limited in part by the genetic inaccessibility and complexity of signaling in mammalian cells. Here we present a novel approach to study rat PKC beta-1 action in yeast, a simple lower eukaryotic genetic model. Expression of its cDNA in Saccharomyces cerevisiae introduces novel phorbol ester binding sites which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro consistent with a fully functional protein which phosphorylates cellular yeast proteins in vivo. Phorbol ester activation of PKC beta-1 in vivo results in biological responses which include stimulation of extracellular calcium uptake, changes in cell morphology, and an increase in the cell doubling time. These PKC functions are not affected by truncation of 12 amino terminal amino acids; however, they are completely abolished by truncation of 15 or more carboxyl terminal amino acids which likely result in inactivation of the kinase. The increase in the yeast doubling time caused by PKC beta-1 activation provides a phenotype which can be exploited as a screen for the activity of random PKC cDNA mutations. Our findings indicate that rat PKC beta-1 is functional in yeast and leads to biological responses which suggest compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.

Amino Acid Sequence↗
PubMed1993

Tedisamil (KC 8857) is a new specific bradycardic drug: does it also influence myocardial contractility? Analysis by the conductance (volume) technique in coronary artery disease.

To determine whether inotropism influences the bradycardic action of tedisamil, hemodynamic assessment was performed in 13 patients with ischemic coronary artery disease including analysis of end-systolic pressure-volume relationships after an infusion of tedisamil, 0.3 mg/kg, at rest, and during paced tachycardia stress. Slope Emax fell by 14% at rest (13 patients) and by 10% during tachycardia (6/13 patients), whereas loops of end-systolic pressure-volume relationships moved rightward; all parameter changes indicated a lack of significant inotropism loss with tedisamil (p > 0.05). Although the mean heart rate decreased from 77.5 to 64.7 beats/min and QTc duration increased by 14% (p < 0.05), filling pressure and dp/dtmin remained unchanged and vascular resistance increased by 30%. Parameters of left ventricular pump function (ejection fraction, stroke volume, left ventricular efficiency) decreased slightly (between 3% and 13%), whereas left ventricular volumes increased (end-diastolic volume by 6%, end-systolic volume by 23%). The respective parameter changes during tachycardia were comparable in tendency, and angina could no longer be induced during postdrug pacing stress. We concluded that the bradycardic effects of tedisamil are selectively generated without impairing either ventricular pump function or contractility in a clinically relevant fashion, whereas the postdrug anginal threshold appears elevated. Thus tedisamil can be used safely in ischemic coronary artery disease.

Aged↗
PubMed1993

Reconstitution of protein kinase C alpha function by the protein kinase C beta-I carboxy terminus.

The Ca(2+)- and phospholipid-dependent Ser/Thr kinase protein kinase C (PKC) plays important roles in the transduction of cellular signals. Various PKC isoforms exist in mammalian cells which share conserved and variable regions as defined by cDNA sequence comparisons. To test whether carboxyl (C) terminal sequences of distinct isoforms can complement each other to yield functional chimeric molecules, we have constructed a PKC chimera in which amino acids 595-672 at the C-terminus of bovine PKC alpha (a) were replaced with the corresponding C-terminal amino acids (598-671) of rat PKC beta-I (b) to yield the chimera alpha/beta-I (ab). The chimera was then characterized biochemically and functionally, and compared with the parental isoforms. Since structure/function analysis of PKC in mammalian experimental systems is complicated by multiple PKC isoforms and by cellular complexity, we stably introduced the PKC constructs into the yeast Saccharomyces cerevisiae, a simple, lower eukaryote with a short doubling time and well established molecular genetics. In yeast, the faithfully expressed PKCab chimera and normal PKC isoforms bound radiolabelled phorbol ester and were recognized on immunoblots by PKC-specific antibodies. The chimera phosphorylated substrate peptides in a PMA- and Ca(2+)-dependent manner, and, upon activation, increased the cell doubling time and the rate of Ca2+ uptake into cells. In addition, PKCab displayed characteristics distinct from normal PKCb, but virtually indistinguishable from normal PKCa. Our findings indicate the reconstitution of PKCa function by the PKCb carboxyl terminus.

Animals↗
PubMed1993

Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable to identity rare, randomly altered but active mammalian PKC mutants. It quantifies their catalytic and biological activities in response to PKC activators or inhibitors for a systematic mapping of PKC structure and function or PKC-drug interaction.

Animals↗
PubMed1993

Functional carboxyl terminal deletion map of protein kinase C alpha.

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. Based on the predicted amino acid (aa) sequence homology of more than ten distinct PKC gene coding sequences, four highly conserved regions C1-C4 and five variable regions V1-V5 have been defined for the different PKC subtypes. Some of these regions, such as C1 and C3/V4/C4, have been correlated with specific PKC functions, such as activator binding and enzymatic activity, respectively, while the biological role of others is unknown. The biological significance of the PKC carboxyl terminus is unclear and the predicted boundary of the catalytic C4 region is controversial due to different interpretations of aa sequence comparisons. We explored the PKC alpha carboxyl terminal requirement for basic PKC function and mapped the boundary of the sequences essential for enzymatic activity based on functional criteria. cDNAs encoding normal and random carboxyl terminal truncations of bovine PKC alpha were introduced into Saccharomyces cerevisiae, allowing its rapid functional expression and characterization for catalytic as well as biological activity. We found that deletion of up to 11 carboxyl terminal aa still results in a phorbol ester-responsive, biologically active enzyme in vivo which is dependent on calcium and phospholipids for catalytic activation in vitro. Deletion of 15 and 23 aa results in marginal and total loss of catalytic activity, respectively, and in complete loss of biological activity for both truncations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗