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Biomedical subjects

C Rommel

Publications and source records attributed to C Rommel.

17 recordsLinked to original sources
PubMed2000

Retroviral gene transfer of dominant negative raf-1 mutants suppresses ha-ras-induced transformation and delays tumor formation.

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.

3T3 Cells↗
PubMed1999

Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt.

Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways were shown to exert opposing effects on muscle cell hypertrophy. Furthermore, the PI3K-Akt pathway was shown to inhibit the Raf-MEK-ERK pathway; this cross-regulation depended on the differentiation state of the cell: Akt activation inhibited the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlated with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex.

Animals↗
PubMed1999

Psychophysical measures of sensitization to tonic heat discriminate chronic pain patients.

Sensitization to continued nociceptive stimulation is supposed to be involved in the development of chronic pain at several levels of the CNS, but experimental studies investigating the perceptual dynamics of sensitization in humans are rare, and the diagnostic validity of experimental pain models is not known. The present study used a tonic heat paradigm to assess early sensitization (15-100 s) to experimental pain in 30 chronic pain patients (15 musculoskeletal/back pain, 15 headache) and 23 healthy controls. Change in pain sensation during prolonged stimulation was measured by a dual sensitization method which combines subjective ratings and behavioural responses in an indirect psychophysical protocol protected against response bias. Phasic and tonic pain thresholds were measured for control purposes. The degree of sensitization was linearly related to stimulus temperature, and groups differed significantly in this 'sensitization gradient': chronic pain patients sensitized earlier and stronger than healthy subjects, musculoskeletal pain patients showed the strongest effect. Pain thresholds were lowered in headache patients only. Discriminant analysis demonstrated good sensitivity and specificity of individual sensitization measures for distinguishing pain syndromes, particularly in combination with pain thresholds. The results are in accordance with current models of spinal plasticity contributing to pathological pain states. They argue for the diagnostic value of psychophysical measures of sensitization.

Adolescent↗
PubMed1998

Ras--a versatile cellular switch.

With the number of known roles played by Ras proteins increasing rapidly, finding answers to how the diverse cellular responses are triggered is becoming increasingly pertinent. Although our understanding of the control of specificity of signal transduction is still small, the combination of biochemical, structural and genetic analyses is starting to reveal how the cell-specific responses to Ras activation are controlled.

Animals↗
PubMed1997

14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction.

14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 zeta on Thr-233. This protein kinase has been identified as casein kinase Ialpha (CKIalpha) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 tau possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 zeta is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 zeta associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 zeta at the CKIalpha site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.

14-3-3 Proteins↗
PubMed1997

MEK1 mediates a positive feedback on Raf-1 activity independently of Ras and Src.

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1 indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. MEK1 can however synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism which allows fast signal amplification and could prolong activation of Raf-1.

Cell Line, Transformed↗
PubMed1997

The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila.

The heat shock protein Hsp90 has been shown to associate with various cellular signalling proteins such as steroid hormone receptors, src-like kinases and the serine/threonine kinase Raf. While the interaction between steroid hormone receptors and Hsp90 appears to be essential for ligand binding and activation of the receptors, the role of Hsp90 in Raf activation is less clear. We have identified mutations in the hsp83 gene, the Drosophila homologue of hsp90, in a search for dominant mutations that attenuate signalling from Raf in the developing eye. The mutations result in single amino acid substitutions in the Hsp83 protein and cause a dominant-negative effect on the function of the wild-type protein. We show that both wild-type and mutant forms of Hsp83 bind to the activated Drosophila Raf but the mutant Hsp83 protein causes a reduction in the kinase activity of Raf. Our results indicate that Hsp83 is essential for Raf function in vivo.

Amino Acid Sequence↗
PubMed1997

Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo.

In the developing eye of Drosophila the protein kinase D-Raf controls the specification of the R7 photoreceptor cells. We show that overexpression of wild-type D-Raf inhibits the formation of R7 cells in a dose-dependent manner. Conversely, overexpression of mutant D-Raf proteins in which the conserved S388 is replaced by A or by D promotes the formation of supernumerary R7 cells, indicating increased D-Raf activity in vivo. S388 in D-Raf corresponds to S259 in c-Raf; shown to be involved in binding of 14-3-3. We show that analogous substitutions of S259 in c-Raf prevent binding of 14-3-3 zeta to the amino terminus of c-Raf and cause a Ras-independent constitutively increased c-Raf kinase activity. Binding of 14-3-3 zeta to the second binding site at the carboxy terminal catalytic domain was unaffected by these mutations. These results suggest that the increased kinase activity of mutant D-Raf is caused by the selective loss of 14-3-3 binding to its amino terminus. Therefore, binding of 14-3-3 to the amino terminus of Raf appears to negatively regulate Raf kinase activity in vivo.

14-3-3 Proteins↗
PubMed1996

Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1.

The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf-1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1.

14-3-3 Proteins↗
PubMed1995

Direct interaction and N-terminal phosphorylation of c-Jun by c-Mil/Raf.

c-Mil is the avian homologue of the mammalian serine/threonine kinase c-Raf-1. c-Mil/Raf is a mediator of signal transduction leading to gene expression via the c-Jun DNA-binding site, AP-1. Here we show that c-Mil immunopurified from MC29-virus-transformed quail fibroblasts phosphorylates c-Jun in vitro near its N terminus (Ser-63 and -73). Furthermore, the viral oncogene product Gag-Mil of the avian wild-type retrovirus MH2 phosphorylates c-Jun in vitro. A contribution by other known kinases phosphorylating c-Jun, such as the mitogen-activated protein kinases (MAPKs) and the c-Jun N-terminal kinases, was excluded by control reactions. c-Raf-1 and c-Jun directly interact in vitro as shown by various immobilized glutathione S-transferase-Raf fusion proteins which specify the cysteine-rich region of c-Mil/Raf as the major N-terminal binding site. An additional minor binding site is located in the C-terminal region. The biological relevance of these results is demonstrated by coimmunoprecipitation of c-Jun and c-Mil from 32P-labeled MC29- and MH2-transformed fibroblasts as well as normal quail embryo fibroblasts, whereby c-Jun was identified by tryptic phosphopeptide analysis. The complexed c-Jun exhibits a decreased electrophoretic mobility corresponding to a more highly phosphorylated state. Cell fractionation analyses indicate that the c-Mil/c-Jun complex is located in the cytoplasm. The data demonstrate that c-Jun can be a direct target of the protein kinase c-Mil/Raf, suggesting an alternative pathway, which leads to c-Jun phosphorylation independent of the MAPKs and MAPK-related proteins.

Animals↗
PubMed1994

Phosphorylation of c-Raf-1 by protein kinase A interferes with activation.

c-Raf-1 is a serine/threonine-specific protein kinase which is regulated by phosphorylation. A putative c-AMP dependent protein kinase PKA phosphorylation site with the consensus sequence RRXS, Ser43, and a predominant phosphorylation site of c-Raf-1, Ser259, can be phosphorylated by PKA in vitro as shown by comparison of phosphopeptide maps of recombinant wild-type c-Raf-1 and the corresponding mutants. In vivo stimulation of the PKA pathway by treatment of A431 cells with Forskolin results in increase of phosphorylation in Ser43. Forskolin reduces the upshift of c-Raf-1 induced by EGF-treatment. It inhibits the EGF-activation of the c-Raf-1 protein kinase activity tested in vitro with a peptide substrate.

Adenosine Triphosphate↗
PubMed1994

CT-boxes are involved in control of the rat insulin II gene expression.

Expression of the rat insulin II gene is controlled mainly at the level of transcription initiation by multiple factors binding to specific cis-acting DNA-elements in the regulatory region. We have shown that two elements (CT-motifs) located between nucleotides -83 and -76 (CT-1) and -204 and -197 (CT-2) are involved in transcriptional regulation in the insulin-producing cell line HIT M2.2.2. Transient expression analysis of 5'-deletion as well as block replacement mutants revealed that CT-1 and CT-2 are mutational sensitive. Gel mobility shift assays showed that both motifs bind similar nuclear factors. Our results suggest the involvement of a third CT-motif located directly upstream of CT-2 on the complementary strand.

Animals↗
PubMed1987

[Effect of tricuspid annuloplasty on the clinical course following mitral or aortic valve operation].

To assess the effect of additional tricuspid annuloplasty during mitral/aortic valve surgery on the clinical postoperative course in patients with severe preoperative tricuspid insufficiency, 64 patients were investigated pre- and 11 +/- 4 months postoperatively. Extent of left-side heart failure was graded as well as severity of right-side heart failure using a defined clinical score. Using preoperative biplane angiography of the right ventricle the patients were assigned to three different groups: group I (n = 30) with no preoperative tricuspid insufficiency (TI), group II (n = 19) with preoperative TI and without tricuspid annuloplasty, group III (n = 15) with preoperative TI and with annuloplasty of the tricuspid valve. The patients of all three groups postoperatively improved from an average of NYHA class III to class II. The clinical score of right-side heart failure in gr. III and gr. II was 1.4 +/- 1.0 and 1.5 +/- 1.0, respectively, and was significantly (p less than 0.05) higher than in gr. I (0.8 +/- 0.8). In all three groups there was a postoperatively significant decrease: gr. I: 0.3 +/- 0.5 (p less than 0.01); gr. II: 0.6 +/- 0.9 (p less than 0.02); gr. III: 0.7 +/- 0.8 (p less than 0.05). Mortality was 3% in gr. I; 5% in gr. II and 6% in gr. III. 3% of patients in gr. I, 30% in gr. II and 6% in gr. III had early postoperative hemodynamic complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1981

[Sublingual structures in primates. Part 1: Prosimiae, Platyrrhini and Cercopithecinae].

1. The sublingual structures of primates have been studied light-microscopically. There are 3 different sublingual structures in the species studied. The plica sublingualis occurs in all primates. The sublingual organ is a topographically modified plica sublingualis which occurs exclusively in Callicebus. A sublingua is present only in the prosimians. 2. The plica sublingualis contains the excretory ducts of the submandibular and sublingual salivary glands. The sublingua is ventrally adherent to the body of the tongue and is, with a few exceptions in Tupaia, characterized by a skeleton of cartilage tissue. A sublingua never exhibits excretory ducts or salivary glands. 3. In some Platyrrhini (Ateles, Aotus, Lagothrix, Alouatta, Callicebus), there are taste buds in the epithelium of the plica sublingualis. They are especially concentrated near the orifices of the salivary glands. 4. The fresh saliva of the submandibular and sublingual gland can be tested by the taste buds on the plica sublingualis, because there is a topographical coincidence. 5. There is a complete absence of taste buds at the plica sublingualis of the prosimians and the Cercopithecinae. 6. There are no taste buds in the epithelium of the sublingua. In the Lorisiformes and in the Lemuriformes the sublingua is a cleaning device of the anterior dentition, most probably in connection with a tactile sensibility. In the Tupaiformes and in the Tarsiiformes the sublingua is less developed. 7. There is no anatomical connection between the skeleton of cartilage tissue in the sublingua and the lytta, or the skeleton of the hyoideum. 8. In some Cercopithecinae (Macaca, Papio) a glandula apicis linguae is present.

Animals↗
PubMed1981

[Sublingual structures of primates. II. Hominoidea, review, summary and literature].

1. In Homo and the great apes (Pongidae) there occurs, besides the plica sublingualis a plica fimbriata at the ventral surface of the tongue. This duplicature of the mucosa does not occur in the Hylobytidae and in the other primates. 2. Some taste buds could be found in the epithelium of the plica sublingualis of the Pongidae. 3. There are many taste buds in the epithelium of the plica fimbriata of the Pongidae. On this sublingual structure there were counted 1776 taste buds in Pongo, 592 in Gorilla and 280 in Pan. A few taste buds could also be found on the plica fimbriata of a human newborn. 4. A glandula apicis linguae occurs in Homo, Pan, Gorilla and Pongo. 5. The fresh saliva of the glandula apicis linguae and the saliva on the floor of the mouth can be tested by the taste buds in the epithelium of the plica fimbriata, of papillae lenticulares and of areae gustatoriae at the ventral surface of the tongue. 6. It might be the function of the sublingual taste buds to taste the fresh saliva as a gradient for the central nervous comparison with the taste of the saliva on the dorsal surface of the tongue. 7. Because of the complete absence of a sublingua in the Platyrrhini and in the Cercopithecinae it is unlikely that the plica fimbriata of Homo and the great apes can be interpreted as a homalogon of the sublingua in the prosimians. 8. Because of the absence of a sublingua in other ordines of the Mammalia (Insectivora, Carnivora, Rodentia, Chiroptera, Ungulata) it is unlikely as well that the sublingua in the prosimians can be interpreted as a homologon of the tongues of the lower vertebrates. The sublingual structures occuring in the Marsupialia have to be investigated. 9. Because of these reasons the new development of the sublingua in the prosimians and the plica fimbriata in the Hominoidea, in complete independence from one another, seems to be a better explanation of the 2 structures and less contradictionary to anatomical and phylogenetic arguments. The different function of both structures in the recent primates gives a hint for the possible reason for their development during the process of evolution.

Animals↗
PubMed1979

[Taste buds in the epithelium of the plica sublingualis of New World monkeys (author's transl)].

The Plica sublingualis of new world monkeys was studied microscopically. In Ateles there are approximately 1700 taste buds in the epithelium of the Plica sublingualis, they are fewer in number in Alouatta, Lagothrix, Aotus and are absent in Saimiri and Cebus. The taste buds are situated consistently at the tip of the Plica sublingualis and near the orifices of the submandibular and sublingual salivary glands. From this topographical coincidence it is inferred that the function of the taste buds at the Plica sublingualis might permit a comparison between the fresh saliva with the general saliva in the cavum oris along a gradient.

Animals↗