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K Engel

Publications and source records attributed to K Engel.

At least 37 records · Page 2Linked to original sources
PubMed1995

MAPKAP kinase 2 is activated by heat shock and TNF-alpha: in vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade.

The activation of MAPKAP kinase 2 was investigated under heat-shock conditions in mouse Ehrlich ascites tumor cells and after treatment of human MO7 cells with tumor necrosis factor-alpha (TNF-alpha). MAPKAP kinase 2 activity was determined using the small heat-shock proteins (sHsps) Hsp25 and Hsp27 as substrates. In both cell types, about a threefold increase in MAPKAP kinase 2 activity could be detected in a time interval of about 10-15 min after stimulation either by heat shock or TNF-alpha. Phosphorylation of MAPKAP kinase 2, but not the level of MAPKAP kinase 2 mRNA, was increased after heat shock in EAT cells. It is further shown that activation of MAPKAP kinase 2 in MO7 cells is accompanied by increased MAP kinase activity. These data strongly suggest that increased phosphorylation of the sHsps after heat shock or TNF-alpha treatment results from phosphorylation by MAPKAP kinase 2, which itself is activated by phosphorylation through MAP kinases. Hence, we demonstrate that MAPKAP kinase 2 is responsible not only for phosphorylation of sHsps in vitro but also in vivo. The findings link sHsp phosphorylation to the MAP kinase cascade, explaining the early phosphorylation of sHsp that is stimulated by a variety of inducers such as mitogens, phorbol esters, thrombin, calcium ionophores, and heat shock.

Animals↗
PubMed1994

Characterization of the proline-rich region of mouse MAPKAP kinase 2: influence on catalytic properties and binding to the c-abl SH3 domain in vitro.

The primary structure of mouse MAP kinase-activated protein (MAPKAP) kinase 2 contains a proline-rich N-terminal region which might function as a src-homology 3 (SH3) domain-binding motif in vivo. To demonstrate the ability of this region to bind SH3 domains, we analyzed the interaction of the SH3 domain of the protein tyrosine kinase c-abl with MAPKAP kinase 2. It is demonstrated, that the proline-rich region specifically binds c-abl-SH3 domain in vitro. Furthermore, it is shown, that deletion of this proline-rich region does not significantly influence the substrate binding properties of the enzyme when analyzed with the substrate small heat shock protein Hsp25. The data suggest that the proline-rich region of MAPKAP kinase 2 could interact with proteins containing SH3-domains also in vivo regulating its cellular localization and/or modulating its enzymatic properties.

Amino Acid Sequence↗
PubMed1994

Interleukin-3 and granulocyte-macrophage colony-stimulating factor induce activation of the MAPKAP kinase 2 resulting in in vitro serine phosphorylation of the small heat shock protein (Hsp 27).

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have previously been reported to induce rapid phosphorylation of the mitogen-activated protein (MAP) kinase. However, little is known about signaling events initiated by both hematopoietins that occur downstream of the MAP kinase. MAP kinase has been shown to phosphorylate the AP-1 transcription factor and also to activate two kinases designated insulin-stimulated protein kinase-1 and MAP kinase-activated protein (MAP-KAP) kinase 2. We show here that IL-3 and GM-CSF induce MAPKAP kinase 2 activity in the human megakaryoblastic leukemia cell line MO7 and phosphorylate the human small heat shock protein Hsp 27 on serine residues in vitro. GM-CSF also induced Hsp 27 phosphorylation in neutrophils in a range similar to that observed in MO7 cells, suggesting that MAPKAP kinase 2-mediated Hsp 27 activation occurs independently of proliferation. Hsp 27 phosphorylation was dose-dependent, occurred as early as 5 minutes after factor exposure, and was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A. Furthermore, the protein phosphatase A2 abolished IL-3- and GM-CSF-induced serine phosphorylation of Hsp 27. Taken together, our findings indicate that tyrosine phosphorylation of MAP kinase is a prerequisite for serine phosphorylation of Hsp 27, which is mediated by MAPKAP kinase 2. Hsp 27 has shown activation-dependent translocation from the cytosolic to the nuclear region and has been linked to the cellular stress response. However, its precise function is largely unknown. Our data identify Hsp 27 as a target of the IL-3/GM-CSF stimulation pathway that involves MAP kinase and MAPKAP kinase 2. In addition, our results indicate that Hsp 27 may be target of phosphorylation events not only in the stress response but also in unstressed cells responding to cytokine stimulation.

Benzoquinones↗
PubMed1994

Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance.

Small heat shock proteins (sHsps) show a very rapid stress- and mitogen-dependent phosphorylation by MAPKAP kinase 2. Based on this observation, phosphorylation of sHsps was thought to play a key role in mediating thermoresistance immediately after heat shock, before the increased synthesis of heat shock proteins becomes relevant. We have analysed the phosphorylation dependence of the chaperone and thermoresistance-mediating properties of the small heat shock protein Hsp25. Surprisingly, overexpression of Hsp25 mutants, which are not phosphorylated in the transfected cells, confers the same thermoresistant phenotype as overexpression of wild type Hsp25, which is either mono- or bis-phosphorylated at serine residues 15 and 86 within the cells. Furthermore, in vitro phosphorylated Hsp25 shows the same oligomerization properties and the same chaperone activity as the nonphosphorylated protein. No differences between phosphorylated and nonphosphorylated Hsp25 are detected in preventing thermal aggregation of unfolding proteins and assisting refolding of denatured proteins. The results suggest that chaperone properties of the small heat shock proteins contribute to the increased cellular thermoresistance in a phosphorylation-independent manner.

3T3 Cells↗
PubMed1993

The MAP kinase-activated protein kinase 2 contains a proline-rich SH3-binding domain.

The protein sequence of MAP kinase-activated protein kinase 2 (MAPKAP kinase 2) deduced from mouse cDNA sequence reveals structural features of the enzyme, which could be of importance for its function: a proline-rich SH3-binding domain N-terminal to the catalytic region, a MAP kinase phosphorylation site and a bipartite nuclear targeting sequence located C-terminal to the catalytic region. The catalytic domain itself has the strongest homology to calcium/calmodulin-dependent protein kinase II. Northern blot analysis demonstrates a 3.5 kb MAPKAP kinase 2 transcript which is ubiquitously expressed and, hence, co-expressed with the mRNA of the recently identified substrate Hsp25 in all tissues analysed. However, the functional consequences of the nuclear targeting sequence present in MAPKAP kinase 2 suggest the existence of further substrates for the enzyme in the nucleus.

Amino Acid Sequence↗
PubMed1993

Small heat shock proteins are molecular chaperones.

Small heat shock proteins (sHsp) with a molecular mass of 15-30 kDa are ubiquitous and conserved. Up to now their function has remained enigmatic. Increased expression under heat shock conditions and their protective effect on cell viability at elevated temperatures suggest that they may have a function in the formation or maintenance of the native conformation of cytosolic proteins. To test this hypothesis we studied the influence of murine Hsp25, human Hsp27, and bovine alpha-B-crystallin (an eye lens protein homologous to sHsps) on the unfolding and refolding of citrate synthase and alpha-glucosidase in vitro. Here we show that all sHsps investigated act as molecular chaperones in these folding reactions. At stoichiometric amounts they maximally prevent the aggregation of citrate synthase and alpha-glucosidase under heat shock conditions and stabilize the proteins. Furthermore, they promote the functional refolding of these proteins after urea denaturation similar to GroE and Hsp90. The interaction both with unfolding and refolding proteins seems to be ATP-independent.

3T3 Cells↗
PubMed1993

Pathophysiology of incomplete renal tubular acidosis in recurrent renal stone formers: evidence of disturbed calcium, bone and citrate metabolism.

Urinary acidification, bone metabolism and urinary excretion of calcium and citrate were evaluated in 10 recurrent stone formers with incomplete renal tubular acidosis (iRTA), 10 recurrent stone formers with normal urinary acidification (NUA) and 10 normal controls (NC). Patients with iRTA had lower plasma standard bicarbonate after fasting (P < 0.01) and lower urinary excretion of titratable acid (P < 0.05) and citrate (P < 0.01) compared with NUA patients and NC, and higher urinary excretion of ammonia (P < 0.05) compared with NC (P < 0.05). Hypercalciuria was found in 6 of 10 patients with iRTA compared with 3 of 10 with NUA, and 0 of 10 NC. The citrate/calcium ratio in urine was significantly reduced in iRTA compared with the value in NUA (P < 0.01), and in NUA compared with NC (P < 0.05). Biochemical markers of bone formation (serum osteocalcin) and bone resorption (urinary hydroxyproline) were significantly increased in iRTA compared with NUA and NC (P < 0.01), indicating increased bone turnover in stone formers with iRTA. Stone formers with iRTA thus presented with disturbed calcium, bone and citrate metabolism--the same metabolic abnormalities which characterize classic type 1 RTA. Mild non-carbonic acidosis during fasting may be a pathophysilogical factor of both nephrolithiasis and disturbed bone metabolism in stone formers with iRTA.

Acid-Base Equilibrium↗
PubMed1993

[Chlamydia trachomatis: screening within the scope of prenatal care and rapid diagnosis in threatened premature labor].

Cervical infection with Chlamydia trachomatis during pregnancy is related to the incidence of preterm labour and preterm rupture of membranes, causing elevated neonatal morbidity and mortality. The present study reports our two-years-experiences with a general screening on Chlamydia infection in prenatal care as well as diagnostic possibilities in case of imminent preterm delivery. In the examined population of totally n = 263 pregnant women attended in our prenatal care unit, the prevalence of cervical Chlamydia infection was 3.4%. Among the screened asymptomatic women (n1 = 179) 2.8% had positive findings. Among n2 = 84 patients with preterm labour and/or preterm rupture of membranes 4.8% revealed cervical infection. Specially in the acute situation of imminent preterm delivery, an immunological bedside assay (Clearview Chlamydia, R) tested in 203 cases versus the tissue culture as reference method proved helpful. Its sensitivity was 0.8, specificity 0.99. The test allowed to prove or exclude chlamydial infection within only 30 minutes.

Adult↗
PubMed1992

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins.

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.

Amino Acid Sequence↗
PubMed1992

Dephosphorylation of the small heat shock protein hsp25 by calcium/calmodulin-dependent (type 2B) protein phosphatase.

The dephosphorylation of the mouse small heat shock protein hsp25 within an extract obtained from Ehrlich ascites tumor cells is inhibited by the calcium chelator EGTA and at concentrations of microcystin-LR which are characteristic for inhibition of calcium/calmodulin-dependent (2B type) protein phosphatases. Furthermore, the dephosphorylation of hsp25 in the cell-free system derived from Ehrlich ascites tumor could be increased specifically by addition of the calcium/calmodulin-dependent (2B type) protein phosphatase calcineurin. Dephosphorylation of the heat shock protein hsp25 is also obtained in an in vitro system containing phosphorylated recombinant hsp25, 1 mM Ca2+, calmodulin, and calcineurin specifying hsp25 as the direct substrate for this enzyme. The expression of two isoforms of the catalytic subunit of the mouse calcium/calmodulin-dependent (2B type) protein phosphatases in Ehrlich ascites tumor cells is demonstrated by polymerase chain reaction using specific oligonucleotide primers to the catalytic and calmodulin-binding domain, respectively. Northern blot analysis using the amplified fragments as probes shows that the mRNA of one isoform of the mouse calcium/calmodulin-dependent protein phosphatase is of medium abundance in EAT cells. These data suggest a calcium/calmodulin-dependent dephosphorylation of the small stress protein in EAT cells also in vivo. Since it is known that heat shock increases the intracellular calcium level and that thermotolerance is influenced by calcium chelators, ionophores, and anti-calmodulin drugs, the changes in the degree of hsp25 phosphorylation induced by thermal stress resulting in an altered thermoresistance could be explained at least partially by the calcium/calmodulin-dependent dephosphorylation through protein phosphatases 2B.

Amino Acid Sequence↗
PubMed1992

[Recurrence of vulvar cancer--treatment, experiences and results].

At the University Hospitals of Heidelberg (1970-1988) and Homburg/Saar (1988-1990), 182 patients with vulvar carcinoma were treated. 51 patients had a recurrence of vulvar carcinoma and 21 patients showed a persistence of this tumour. 19 women had a second recurrence of vulvar disease. In these cases, therapy ranged from local surgery to exenteration, depending on the site and tumour extension. 18 patients underwent successful reconstructive surgery. Prognosis was better in cases with local recurrence in comparison to distant sites of metastatic progression (i.e. inguinal nodes or disseminated disease) (p less than 0.001). The five-year survival (after diagnosis of recurrent disease) for patients with early onset of recurrence (less than 20 months after initial therapy) was 28% compared to 68% for patients with late onset of recurrent disease (p less than 0.01).

Female↗
PubMed1992

[Fetomaternal macrotransfusion after successful internal version from breech presentation by moxibustion].

In a primigravida with a fundal/anterior wall placenta, a successful cephalic version was noted at 39 weeks after repeated moxibustion of the point Zhiyin (bl 67). Since routine foetal heart rate monitoring showed a sinusoidal pattern with severe decelerations, immediate Caesarean section was performed. Foetomaternal macrotransfusion of about 300 ml of blood was found. In view of this complication, possible risks of the method are discussed. Moxibustion does not seem to be suitable as self-therapy without close medical follow-up.

Adult↗
PubMed1991

[Effect of surgical procedure and adjuvant therapy on cosmetic results after breast conserving therapy in breast cancer].

A total of 207 patients undergoing breast conserving therapy (BCT, excision of tumour with clear margins, radiotherapy, sequential chemotherapy for node-positive patients) were clinically evaluated after a median follow-up of 11 months (min 2/max 22). The aim of the study was to analyse the impact of various individual factors, surgical techniques and adjuvant therapies on the cosmetic result. In 85.7% of all cases the objective outcome was satisfactory (very good: 48.8%, good: 36.9%) and in 14.3% unsatisfactory (fair: 13%, poor: 1.3%). Objective cosmetic results were rated significantly better by the patients (p = 0.01), in cases with biopsy weights of greater than 100 grms. (large/hyperplastic breast, p = 0.01), non-reconstructed mammary glands (simply closure of subcutis and skin, p = 0.01), non suction drainage of the breast (p - 0.04) and normal healing of the wound (p = 0.01). There was a trend towards better cosmetic results in the premenopause in patients with a tumour localised in the upper outer quadrant, in cases of a curvilinear as compared to a radial incision (upper/outer quadrant) and in small or average-sized breasts with biopsy weights of less than 50 grms. Cosmetic results were unaffected by a "two-step" procedure, standardised postoperative radiotherapy (45-55 Gy) and by sequential chemotherapy (3x CHT/radiotherapy/3x CHT). Unsatisfactory cosmetic short-term results should primarily be regarded as a surgical complication of BCT. Results can be improved by taking into consideration the importance of the relation of breast/biopsy-weight as well as standardising the operative procedure (curvilinear incision, closure of subcutis and skin, drainage without suction, perioperative antibiotic prophylaxis).

Adult↗
PubMed1991

[Breast saving treatment of breast cancer: results, risks, new developments].

In the Departments of Gynaecology and of Gynaecological Radiology of the University of Heidelberg, breast conserving therapy was carried out in 1330 patients with breast cancer between 1975 and 1990. The tumour size was up to 3 cm and 28% showed positive nodes. The median age was 47.6 years, segmental resection was the standard operation and whole breast irradiation with 50 Gy and an additional boost of 10 Gy was the standard irradiation schedule. After 5 years (n = 307), the following results were observed: local failure: 6.8%, regional lymph node recurrence: 2.1%, overall survival: 88.3%, disease-free survival: 81.2%. 5 out of 36 of the cases of mortality died without having had a recurrence. Significant factors for local failure were following: 1. lymphangiosis of more than 1 cm in size around the tumour (p = 0.03) 2. intra-ductal non-invasive cancer of more than 1 cm in size around the primary tumour (p = 0.01) 3. intra-ductal non-invasive cancer reaches the margin of resection (p less than 0.00001) With segmental resection (2 cm margin macroscopically free of tumour), tumour beyond the margins--so-called residuals, showed in 19% histologically. In the other 3 quadrants, additional secondary primaries in 25% (multicentric cancers) of macroscopical size could be confirmed in an additional study. In case of high risk for local failure, more radical surgery as well as more intense irradiation is recommended.

Adult↗
PubMed1991

Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein.

The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.

Actin Cytoskeleton↗
PubMed1991

Generation of antibodies against human hsp27 and murine hsp25 by immunization with a chimeric small heat shock protein.

A hybrid protein containing the N-terminal part of the murine stress protein hsp25 (amino acids 1 to 110) and the C-terminal part of the human stress protein hsp27 (amino acids 111 to 208) was expressed in E. coli using a T7 polymerase/promoter system. The recombinant hybrid protein was purified and used for immunization of rabbits. In contrast to immunization experiments using hsp25 and hsp27 alone, immunization with the hybrid protein hsp25/27 leads to antibodies which can be used for detection of both hsp25 and hsp27.

Amino Acid Sequence↗