[Developing the integration-oriented participation into working life]. / Berufliche Teilhabe integrationsorientiert gestalten.

The pilot project RehaFuturReal of the Deutsche Rentenversicherung Westfalen (DRV) has been in progress since 1st April, 2011 and constitutes the implementation of the results of the na-tionwide development process RehaFutur in the consultation practice of the DRV Westfalen. In order to achieve and secure employability, this pilot project aims at sustained implementation of improved individualisation, flexibility and orientation towards integration, in the framework of the rehabilitation process. The participants are mostly recruited (75%) from different fields of medical rehabilitation (foremost orthopaedy, cardiology). They all meet the 3 selection criteria, which are present employment, current problems concerning occupational integration as well as a demand for occupational rehabilitation. They all have received support and guidance by individualised and structured rehab-management. The average age of the participants was 47.3 years (age range: 33-57 years), which is a rather old age for occupational rehabilitation. In spite of enduring physical disadvantages (83%), it turned out, that the participants had a high level of motivation concerning occupational rehabilitation services and the continuation of their employment, By offering intensive consultation and support, as well as accompanying vocational training, an occupational perspective either with the old employer or with a new employer could be reached for a large number of participants (74%). In the framework of close cooperation between the funding social security institutions, the vocational training institutions, the employers and the receivers of the services, a network has been created, which allows individualised, modularised and flexible integration solutions. In line with the pilot project RehaFuturReal of DRV Westfalen it became apparent, that in order to make occupational rehabilitation future-proof and sustainable, the already developed concepts should be transferred in actual acts of implementation consequently and interdisciplinary.

A fast emergency department triage score based on mobility, mental status and oxygen saturation compared with the emergency severity index: a prospective cohort study.

BACKGROUND: Waiting for triage in overburdened emergency departments (ED) has become an increasing problem, which endangers patients. A fast triage system to rapidly identify low-acuity patients should divert care and resources to more urgent cases. AIM: The objective of this study was to compare the performance of the Kitovu Hospital fast triage (KFT) score with the Emergency Severity Index (ESI), using mortality and hospital admission as proxies for the patients' acuity. DESIGN: This is a prospective observational study of consecutive patients presenting to a Swiss academic ED. METHODS: Patients were prospectively triaged into one of five ESI strata and retrospectively assessed by the KFT score, which awards one point each for altered mental status, impaired mobility and oxygen saturation <94%. RESULTS: The KFT score had a lower discrimination than the ESI for hospital admission, but a higher discrimination for mortality from 24 h to 1 year after ED presentation. A total of 5544 (67%) patients were assigned to the lowest acuity by the KFT score compared with 2374 (28.7%) by the ESI; there was no significant difference in the 24-h mortality of patients who were deemed low acuity by either score. CONCLUSION: Compared to the ESI, the KFT score identifies more than twice as many patients at low risk of early death. Therefore, this score might help to identify patients who could be managed through alternative pathways. This may be particularly helpful in situations of ED crowding and access block.

[Results and recommendations for vocational rehabilitation from the development project RehaFutur]. / Ergebnisse und Empfehlungen zur beruflichen Rehabilitation aus dem Entwicklungsprojekt RehaFutur.

The overall project RehaFutur, which has been initiated and funded by the Federal Ministry of Labour and Social Affairs and which involves all the actors of the vocational rehabilitation system, is aimed at advancing the development of the vocational rehabilitation system, so as to prepare it for the challenges ahead. The project started with a group of scientific experts, who formulated 8 fields of action for further developing the vocational rehab system. On this basis, 4 working groups were set up in the framework of a development project to elaborate concrete recommendations on how to implement the necessary changes in vocational rehabilitation. The topics dealt with by the working groups were "self-determination and self-responsibility", "regulation of the overall process", "occupation and working environment" as well as "research". The process was supervised by a steering group, the results were moreover discussed in workshops. Recommendations have been made for 10 subject areas: "consultation and information", "joint service points", "use of the internet", "quality assurance", "individualisation and greater flexibility", "orientation towards the working environment", "participation of the beneficiaries", "research", "legislation", "cooperation of the actors". The next step of the RehaFutur overall project is implementation of those recommendations. This needs to be done in an interdisciplinary manner and conjointly by the agencies and providers involved, including the beneficiaries as experts. As different framework conditions have to be taken into account, the process is a complex one and needs to be supported by systematic research.

A chimeric, ligand-binding v-erbB/EGF receptor retains transforming potential.

Comparison of amino acid sequences from human epidermal growth factor (EGF) receptor and avian erythroblastosis virus erbB oncogene product suggests that v-erbB represents a truncated avian EGF receptor gene product. Although both proteins are transmembrane tyrosine kinases, the v-erbB protein lacks most of the extracellular ligand-binding domain and a 32-amino acid cytoplasmic sequence present in the human EGF receptor. To test the validity of the proposed origin of v-erbB and to investigate the functional significance of the deleted extracellular sequences, a chimeric gene encoding the extracellular and the transmembrane domain of the human EGF receptor joined to sequences coding for the cytoplasmic domain of the avian erbB oncogene product was constructed. When expressed in Rat1 fibroblasts, this reconstituted gene product (HER-erbB) was transported to the cell surface and bound EGF. Its autophosphorylation activity was stimulated by interaction with the ligand. Expression of the HER-erbB chimera led to anchorage-independent cell growth in soft agar and EGF-induced focus formation in Rat1 monolayers. Thus, it appears that v-erbB protein sequences in the chimeric receptor retain their transforming activity under the influence of the human extracellular EGF-binding domain.

[Shaping the future of vocational rehabilitation of adults: eight fields of action as starting points for a cross-actor innovation process]. / Die Zukunft der beruflichen Rehabilitation Erwachsener gestalten: Acht Handlungsfelder als Ausgangspunkt für einen akteursübergreifenden Innovationsprozess.

Established by the Federal Ministry of Labour and Social Affairs (BMAS) in October 2007, the Scientific Expert Group RehaFutur had been commissioned to elaborate cornerstones for the medium- and long-term development of vocational rehabilitation of adults with disabilities (re-integration). Initial questions inter alia were as follows: Which function should vocational rehabilitation have in a service- and knowledge-oriented working world that will increasingly be affected by demographic change? How can disabled persons' right to occupational participation by way of vocational rehabilitation, a right stipulated both under the German constitution and in German law, be realized as needed also in the future? Various fields of action have been derived on the basis, for one, of an investigation of the factors, social law, social and education policy as well as European, influencing vocational rehabilitation and, for the other, of an evaluation of current labour market and demographic developments. Dealt with in the fields of action outlined are the aspects: equitable opportunities of access, developmental and needs orientation, closeness to the real occupational and working world, as well as the role of self-determination and self-responsibility. The fields of action are to be understood as framework concept for shaping a cross-actor innovation process. Sustainable vocational rehabilitation is characterized in particular by the fact that it is specifically targeted at promoting disabled persons' self-determination and self-responsibility actively using these in the process and that it strengthens an independent lifestyle, ensures social participation by inclusive structures; also, it facilitates continued participation in working life by ongoing education involving holistic development of professional and personal competencies oriented towards the individual's resources and potentials, safeguarding it by systematic networking with companies. The concept presented for vocational rehabilitation of adults with disabilities encompasses a change of paradigms which service carriers and providers will have to face jointly and including the service users, the rehabilitants.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas.

BACKGROUND: Cholangiocellular carcinomas and gallbladder carcinomas are highly aggressive tumours with a poor prognosis and are generally regarded as chemoresistant tumours. Overexpression of ATP-binding cassette transporters of the multidrug resistance protein (MDR) and multidrug resistance-related protein (MRP) family in cancer cells is a major cause for the multidrug resistance phenotype in vitro and in vivo. To further define the role of MRP family members in biliary tract cancer, we studied the expression and localization of MRP2 and MRP3 in cholangiocellular carcinomas and gallbladder carcinomas. MATERIALS AND METHODS: The expression and cellular localization of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas and gallbladder carcinomas were analysed by immunohistochemistry using isoform-specific antibodies. Expression of MRP isoforms was studied in vitro in Mz-ChA-1 cells derived from gallbladder adenocarcinoma by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and immunofluorescence microscopy. RESULTS: Mz-ChA-1 cells constitutively expressed MDR P-glycoproteins, MRP1, MRP2 and MRP3 by RT-PCR, immunoblotting and immunofluorescence microscopy. MRP2 and MRP3 are expressed in the respective apical and basolateral membrane domains. MRP3 was the predominant MRP isoform in gallbladder carcinomas (93%) and cholangiocellular carcinomas (57%), whereas MRP2 expression was detected in only 29% of gallbladder carcinomas and was undetectable in cholangiocellular carcinomas. CONCLUSIONS: Our findings suggest that the intrinsic multidrug resistance of cholangiocellular and gallbladder carcinomas seems to be independent of MRP2 expression while the expression of MRP3 may contribute to the MDR phenotype.

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.

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.

Grb10, a positive, stimulatory signaling adapter in platelet-derived growth factor BB-, insulin-like growth factor I-, and insulin-mediated mitogenesis.

Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth factor I (IGF-I) receptor (IGF-IR). Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cell context has been implicated. We have tested other mitogenic receptor tyrosine kinases as putative Grb10 partners and have identified the activated forms of platelet-derived growth factor (PDGF) receptor beta (PDGFRbeta), hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor as candidates. We have mapped Y771 as a PDFGRbeta site that is involved in the association with Grb10 via its SH2 domain. We have further investigated the putative role of Grb10 in mitogenesis with four independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adaptor in normal fibroblasts. (i) Complete Grb10 expression from cDNA with an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I, and insulin- but not epidermal growth factor (EGF)-induced DNA synthesis in an ecdysone dose-responsive fashion. (ii) Microinjection of the (dominant-negative) Grb10 SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis. (iii) Alternative experiments were based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. A cell-permeable Grb10 SH2 domain similarly interfered with PDGF-BB-, IGF-I-, and insulin-induced DNA synthesis. In contrast, a cell-permeable Grb10 Pro-rich putative SH3 domain binding region interfered with IGF-I- and insulin- but not with PDGF-BB- or EGF-induced DNA synthesis. (iv) Transient overexpression of complete Grb10 increased whereas cell-permeable Grb10 SH2 domain fusion peptides substantially decreased the cell proliferation rate (as measured by cell numbers) in normal fibroblasts. These experimental strategies independently suggest that Grb10 functions as a positive, stimulatory, mitogenic signaling adapter in PDGF-BB, IGF-I, and insulin action. This function appears to involve the Grb10 SH2 domain, a novel sequence termed BPS, and the Pro-rich putative SH3 domain binding region in IGF-I- and insulin-mediated mitogenesis. In contrast, PDGF-BB-mediated mitogenesis appears to depend on the SH2 but not on the Pro-rich region and may involve other, unidentified Grb10 domains. Distinct protein domains may help to define specific Grb10 functions in different signaling pathways.

PSM, a mediator of PDGF-BB-, IGF-I-, and insulin-stimulated mitogenesis.

PSM/SH2-B has been described as a cellular partner of the FcepsilonRI receptor, insulin receptor (IR), insulin-like growth factor-I (IGF-I) receptor (IGF-IR), and nerve growth factor receptor (TrkA). A function has been proposed in neuronal differentiation and development but its role in other signaling pathways is still unclear. To further elucidate the physiologic role of PSM we have identified additional mitogenic receptor tyrosine kinases as putative PSM partners including platelet-derived growth factor (PDGF) receptor (PDGFR) beta, hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor. We have mapped Y740 as a site of PDGFR beta that is involved in the association with PSM. We have further investigated the putative role of PSM in mitogenesis with three independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adapter in normal NIH3T3 and baby hamster kidney fibroblasts. (1) PSM expression from cDNA using an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I-, and insulin- but not EGF-induced DNA synthesis in an ecdysone dose-responsive fashion; (2) Microinjection of the (dominant negative) PSM SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis; and (3) A peptide mimetic of the PSM Pro-rich putative SH3 domain-binding region interfered with PDGF-BB-, IGF-I-, and insulin- but not with EGF-induced DNA synthesis in NIH3T3 fibroblasts. This experiment was based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. These experimental strategies independently suggest that PSM functions as a positive, stimulatory, mitogenic signaling mediator in PDGF-BB, IGF-I, and insulin but not in EGF action. This function appears to involve the PSM SH2 domain as well as the Pro-rich putative SH3 domain binding region. Our findings support the model that PSM participates as an adapter in various mitogenic signaling mechanisms by linking an activated (receptor) phospho-tyrosine to the SH3 domain of an unknown cellular partner.

ATP and the processivity of Xenopus laevis DNA polymerase alpha.

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase alpha from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase alpha to DNA and conclude that ATP increases the processivity of the enzyme.

Tissue distribution and cDNA cloning of a human fatty acid transport protein (hsFATP4).

Fatty acid uptake is partly controlled by the FATP gene family, of which at least five members are known in mice. Using the mmFATP1 cDNA as hybridization probe, a 1.6 kb partial cDNA clone was isolated from a human heart cDNA library. With 5' and 3' RACE procedures, the complete cDNA was isolated. Sequence comparisons with its mouse homologues identified this clone as hsFATP4.

Organization of the immunity region immI of bacteriophage P1 and synthesis of the P1 antirepressor.

The immI region of bacteriophage P1 includes the ant/reb gene, which encodes the antirepressor protein, and the c4 gene, which encodes a repressor molecule that negatively regulates antirepressor synthesis. The antirepressor interferes with the activity of the P1 repressor of lytic function, the product of the c1 gene. We have determined the DNA sequences of the immI region of P1 wild-type and the mutants virs, ant16, ant17, and reb22. Using suitable P1 immI DNA subfragments cloned into a vector of the T7 bacteriophage RNA polymerase expression system the antirepressor protein(s) was overproduced. On the basis of positions of immI mutations and the sizes of ant gene products, the following organizational feature of the P1 immI region is suggested: (1) the genes c4 and ant are cotranscribed in that order from the same promoter in the clockwise direction of the P1 genetic map; (2) an open reading frame for an unknown gene is located in between c4 and ant; (3) the site at which the c4 repressor acts is located within the c4 structural gene; (4) two antirepressor proteins of molecular weights 42,000 and 32,000 are encoded by a single open reading frame, with the smaller protein initiating at an in-frame start codon; (5) transcription of immI is regulated via a c1-controlled operator, Op51, indicating a communication between the immunity systems immC and immI.

The antirepressor of phage P1. Isolation and interaction with the C1 repressor of P1 and P7.

Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in-frame start codon. Another open reading frame, icd, 5' upstream of and overlapping ant1 is required for ant1 expression. Using appropriate ant gene-carrying plasmids we have overproduced and purified Ant1/2 in the form of a protein complex and Ant2 as a single protein. Sequence analysis confirmed the N-terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N-terminal methionine is missing in the Ant2 protein. Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co-precipitate with the Ant1/2 complex but not with Ant2 protein alone. The results suggest that the antirepressor may exert its C1-inactivating function by a direct protein-protein interaction.

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.

PSM, an insulin-dependent, pro-rich, PH, SH2 domain containing partner of the insulin receptor.

Insulin stimulation results in a considerable spectrum of cellular responses, only part of which have been firmly correlated with the activation of established insulin receptor (IR) targets such as IRS-1, IRS-2, and Shc. Many responses may be transduced by alternative direct IR targets, some of which may still be unknown, may act in parallel to but independently of IRS-1, IRS-2, and Shc, and may be members of the growing family of SH2 domain-containing signaling adaptors. An SH2 domain-coding region of a protein termed PSM was cloned based on its interaction with an activated IR cytoplasmic fragment in a yeast two-hybrid screen. When used as a hybridization probe this region led to the isolation of a protein-coding cDNA which is expressed with a wide tissue distribution and exists in several variant forms. A pleckstrin homology domain and three Pro-rich regions including a putative SH3 domain binding site were identified in addition to the SH2 domain in the deduced 756 amino acid sequence. They imply a role of PSM in tyrosine kinase and phosphatase-mediated signaling pathways. A similar sequence termed SH2-B had been reported in an earlier study, which may represent the rat homolog of PSM. A role of PSM specifically in insulin action is suggested by the interaction of its SH2 domain with an activated but not with an inactive catalytic fragment of the IR in the yeast two-hybrid system in vivo, by the insulin-dependent association of a glutathione S-transferase (GST) PSM SH2 domain fusion protein with purified IR in vitro, and by the insulin-dependent association of GST PSM SH2 with the IR in cell extracts. In contrast, PSM was not found to associate with the established IR substrate IRS-1 under any conditions and appears to act independently of IRS-1. All of our findings are compatible with a putative role of PSM in insulin action.

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.

Deletion analysis of protein kinase Calpha reveals a novel regulatory segment.

Using a combined pharmacological and genetic approach, we have identified aa 260-280 in the C2 region as a critical factor in the catalytic function of protein kinase Calpha (PKCalpha). Progressive truncations from the N-terminus as well as selected internal deletion mutants were expressed in Saccharomyces cerevisiae and tested for altered sensitivity to dequalinium, a PKC inhibitor whose target site was previously mapped to the catalytic domain. PKC mutants representing truncations of up to 158 amino acid residues (aa) from the N-terminus (ND84 and ND158) displayed 60-63% inhibition of kinase activity by 50 microM dequalinium, somewhat more sensitive than the wild-type PKCalpha enzyme (45% inhibition). Mutant ND262, lacking N-terminal aa 1-262, was inhibited by almost 72% with 50 microM dequalinium, but mutant ND278, which lacked an additional 16 aa, was inhibited by only 9% of total activity. This result suggests that a C-terminal segment of the C2 region (aa 263-278) influences inhibition by dequalinium at low micromolar concentrations. An internal deletion mutant (D260-280) which retains the entire primary structure of PKCalpha except for aa 260-280, was similarly inhibited by only 4% with 50 microM dequalinium. In the absence of dequalinium and despite the presence of a nearly complete regulatory domain, this mutant exhibited constitutive activity (both in vitro and in a phenotypic assay with S. cerevisiae) that could not be further stimulated even by the potent activator TPA. Taken together, our findings suggest that, in the native structure of PKCalpha, the segment described by aa 260-280 regulates PKCalpha activity and influences the sensitivity of PKCalpha to dequalinium.

Contribution of stack gases and solid process wastes to the organic pollutant output of thermal waste treatment plants.

Within the scope of fundamental investigations as well as individual research projects (W. Knorr, B. Hentschel, C. Marb. S. Schädel, M. Swerev, O. Vierle, J.-P. Lay, 1999. Rückstände aus der Müllverbrennung-Chancen für eine stoffliche Verwertung von Aschen und Schlacken. Initiativen zum Umweltschutz, 13 ed., Deutsche Bundesstiftung Ulmwelt, Erich Schmidt, Berlin), the Bavarian State Office for Environmental Protection performs emission measurements at thermal waste treatment plants to optimize operation, to accompany and support development of new technologies, and to study the effect of this kind of waste treatment technology on the environment. Based on recent studies (October 1995-July 1999) at six municipal solid waste incinerators (MSWI) in Bavaria all emission streams (solid and gas) are characterized with respect to organic pollutant contents and compared to the emissions of waste pyrolysis. The significant ranges of pollutant concentration as well as the specific congener patterns observed are similar for all MSWI, regardless of differences in technical design and waste input, but differ markedly from those of the pyrolysis products. The overall approach, including the sampling of all output streams and the determination of mass streams and volume flow rates, allows the calculation of the total output of different organic pollutants for waste incineration plants aand to estimate the relative contribution of each of the emission streams to the total pollutant load. Removal efficiencies are also calculated for the air pollution control (APC) systems of the different MSWI plants.

An initial comparison of coagulation techniques of sterilization.

For nearly 50 years reports have been published on laparoscopic sterilization techniques. However, the different procedures vary greatly with regard to the extent of the tube destroyed and the magnitude of damage to the mesosalpinx. In 1981 we proved that a clear relationship exists between the extent of destruction of the circulation and nervous system within the mesosalpinx and the occurrence of menstrual disorders as well as menopausal symptoms. Because two-thirds of the ovarian blood supply passes through the ramus tubarius of the ateria uterina, after destruction of larger areas of the mesosalpinx striking disturbances within the ovarian metabolism must be expected. With numerous animal experiments we are currently studying the expansion of the coagulated area following the use of mono- and bipolar high-frequency current, endocoagulation and CO2 laser coagulation. With the aid of conventional histologic techniques, in addition to enzyme histochemical and electron microscopic examinations, attempts are being made not only to determine the extent of the destroyed zone but also to gain additional information on damage of the vascular and nervous systems of the tubes and ovaries. Although these studies have not yet been completed, it is possible at this time to state that the unipolar high-frequency coagulation technique, because of its tremendous primary and secondary complications, should not be used for laparoscopic female sterilization.