A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data.

The evaluation of the quality of data and their use in hazard and risk assessment as a systematic approach is described. Definitions are proposed for reliability, relevance, and adequacy of data. Reliability is differentiated into four categories. Criteria relating to international testing standards for categorizing reliability are developed. A systematic documentation of evaluating reliability especially for use in the IUCLID database is proposed. This approach is intended to harmonize data evaluation processes worldwide. It may help the expert in subsequent assessments and should increase the clarity of evaluation.

Conditional gene expression in secretory tissues and skin of transgenic mice using the MMTV-LTR and the tetracycline responsive system.

Molecular mechanisms of development and disease can be studied in transgenic animals. Controlling the spatial and temporal expression patterns of transgenes, however, is a prerequisite for the elucidation of gene function in the whole organism. Previously we reported that mice carrying a tetR/VP16 hybrid gene (tTA), under the control of the human cytomegalovirus immediate early 1 (HCMV-IE1) gene promoter, can be used to temporally activate the expression of transgenes under the control of a promoter containing tetop sequences. We now show that the MMTV-LTR can be used to target expression of tTA to the epithelial cells of secretory organs and skin in transgenic mice. Notably, nearly uniform expression of a tetop-lacZ transgene was found in seminal vesicle, salivary gland, and Leydig cells of mice carrying also the MMTV-tTA transgene. More heterogeneous patterns of gene expression were observed in mammary epithelial cells and basal cells of the epidermis. Different MMTV-tTA lines had comparable tissue expression patterns. Transcriptional activation mediated by tTA was up to several hundredfold, and it was abrogated after the administration of tetracycline. The MMTV-tTA mice established in this work will be useful for experiments examining the roles of biological factors at defined developmental stages in the epithelial cells of salivary gland, seminal vesicle, mammary gland, and skin and the Leydig cells of testes. In addition, in combination with the CRE/lox recombination system, these mice wil be useful to achieve gene deletions at defined time points in these organs.

Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

MPTP is a murine homolog of the human T-cell protein tyrosine phosphatase (PTPase) and the rat PTP-S enzyme. Enzymatic activity of this ubiquitously expressed protein was demonstrated in immunoprecipitates from NIH 3T3 cells and in recombinant protein overexpressed in bacteria. Expression of beta-galactosidase-MPTP MPTP chimeric proteins in COS1 cells identified a nuclear localization signal at the carboxyl terminus of the MPTP that was sufficient to direct beta-galactosidase as well as a tagged version of the MPTP to the nucleus. Deletion analysis of amino acids within the nuclear targeting signal showed that this sequence does not conform to the bipartite type of nuclear localization signals. Furthermore, it was shown that the steady-state levels of MPTP RNA fluctuate in a cell cycle-specific manner. On the basis of these experiments, we discuss the possible function of MPTP in the cell cycle and other nuclear processes.

Cloning and characterization of a mouse cDNA encoding a cytoplasmic protein-tyrosine-phosphatase.

A mouse cDNA encoding a non-receptor-type phosphotyrosine phosphatase (PTP; EC 3.1.3.48) has been isolated. The 1570-base-pair cDNA contains a single open reading frame that predicts a 382-amino acid protein with Mr 44,640. The nucleic acid and amino acid sequences are homologous to those of a previously described human T-cell PTP [Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A., Fischer, E. H. & Krebs, E. G. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261]; however, the mouse and human 3' sequences diverge and predict markedly different protein carboxyl termini. The mouse PTP gene is expressed as a 1.9-kilobase message in several stages of murine embryonic development and in a variety of adult tissues. An additional 1.3-kilobase message was found to be expressed specifically in testes. Finally, we report the isolation of a human T-cell PTP cDNA containing a 3' end sequence homologous to the mouse PTP.

Cytotoxicity detected by image analysis: A new method for the quantification of survival, mortality, recovery and growth of mammalian cell cultures.

The use of in vitro methods is increasingly recommended for the assessment and evaluation of cytotoxic effects resulting in cell damage. In most cases, cellular response and reaction of cell populations is determined by endpoint measurements. Even if a battery of such test systems is applied, information about the dynamics of cell damage and recovery is obtained only in part. However, phenomena of damage and recovery can be followed by observing the fate of individual cells and their progeny in culture over several days, which means over several cell cycles, by using light microscopy combined with image analysis. We have developed a recording system for such continuous observation and registration of toxic effects, based on image analysis. Growth rate, generation time, delay or shifting of cell cycle, identification of the progeny (pedigrees) and cellular locomotion can be recorded simultaneously.

cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): isolation and characterization by immunological methods.

An auxin-binding protein (ABP) cDNA clone was selected from a lambda gt11 cDNA library from corn coleoptiles with highly purified IgGanti ABP. The sequence of 794 bp contains an open reading frame (ORF) of 603 bp, coding for a 22 kd protein. There are indications of a signal peptide of 38 amino acids (von Heijne, G. 1983, Eur. J. Biochem., 133, 17-21). A N-glycosylation site can be deduced and a C-terminal KDEL amino acid sequence is detected. An EcoRI fragment containing the beginning portion of the cDNA with about three quarters of the ORF was used to select cDNA clones from an independently produced lambda gt11 cDNA library of corn coleoptiles. Northern blot analysis with in vitro transcribed biotinylated RNA showed a single band of not more than 850 bases. The full-length in vitro transcript directed the in vitro synthesis of a protein which is precipitated by IgGanti ABP. Rabbit antibodies raised against a fusion protein detect the ABP as a double band on Western blots. Only the smaller of the two ABP bands is labeled by two different KDEL-specific IgG preparations.

[Surgically relevant complications in percutaneous transluminal angioplasty]. / Chirurgisch relevante Komplikationen bei perkutaner transluminaler Angioplastik (PTA).

Seven significant surgical complications in 118 patients after PTA-treatment of the lower extremities are presented. In two cases the puncture site in the vessel had to be sutured, in three patients we had to perform a femoro-popliteal bypass, once a TEA with profundaplasty and in another case an embolectomy of the trifurcation. Our experience reveals that with the indication for PTA, the rate of significant complications and their surgical consequences has to be considered too. A close cooperation between Radiologist, Angiologist and Surgeon is of importance.

Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae.

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0. Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas.

When programmed with yeast prepro-alpha-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-alpha-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated alpha-factor precursor (pp-alpha-F3, 27.5 kDa). Glycosylation of the membrane specific pp-alpha-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-alpha-F0), whereas the primary translation product pp-alpha-Fcyt is not affected. Likewise, only the glycosylated pp-alpha-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between pp-alpha-F0 and pp-alpha-Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp-alpha-Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp-alpha-F3 polypeptide indicates that its oligosaccharide chains are processed to presumably Man9-GlcNAc2 structures under the in vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence.(ABSTRACT TRUNCATED AT 250 WORDS)

Relation of actin fibrils to energy metabolism of endothelial cells.

The physiological significance of the association of glycolytic enzymes with actin fibrils was investigated in cell culture. Cytochalasin D (CD) was used to induce the known actin-based sequence of events in a culture of an endothelial-cell line (XTH-2) derived from hearts from tadpoles of Xenopus laevis. 1 min following addition of CD, ruptures in the cortical fibrillar meshwork and in stress fibres are seen. At the same time the cellular ATP level decreases by ca. 25%. This and the following reactions resulting in a kind of arborization depend on a continuous supply with metabolic energy. As shown by measurements of oxygen consumption, cells with intact energy metabolism provide the ATP needed from glycolysis; ATP produced by oxidative phosphorylation is not utilized as long as lactate dehydrogenase (LDH) reoxidizes NADH2. After inhibition of LDH, respiration in XTH-2 cells doubles. CD treatment induces a transient increase in oxygen consumption, indicating an increased energy supply by respiration. From these results we conclude: The energy needed by the actomyosin system is - under normal metabolic conditions -supplied from ATP phosphorylated in glycolysis. The processes of energy metabolism seem to be highly compartmentalized; ATP is not a parameter that is kept constant in time intervals of minutes up to one hour.

[Catheter embolization of the thoracic arteries in the treatment of lung hemorrhage]. / Katheterembolisation von Thoraxarterien bei der Behandlung von Lungenblutungen.

In cases of acute, life-threatening pulmonary haemorrhage, immediate angiographic localisation of the source of haemorrhage is mandatory. Instead of a surgical intervention, transcatheter angiographic therapeutic embolisation may be performed. Embolisation is an effective method with only minimal complications, in favourable contrast to the 23 to 30% mortality encountered with surgical procedures. Embolisation, therefore, should be the first procedure in patients presenting with acute pulmonary haemorrhage. It must be emphasised that in cases of chronic pleuro-pulmonary diseases bleeding occurs not only from bronchial arteries. In 64% of these cases, the bleeding artery originates from a thoracic or diaphragmatic artery. The authors were able to treat 26 of 34 pulmonary haemorrhages (76%) by transcatheter embolisation. In 4 of these patients recurrent haemorrhage was treated by transcatheter embolisation. Severe complications were not noted in any case.

[Catheter embolization: parenchyma-preserving therapy of renal bleeding of nonmalignant etiology]. / Katheterembolisation: Die parenchymschonende Therapie der Nierenblutungen nicht-maligner Atiologie.

9 patients with life-threatening renal bleeding of non-malignant origin, including trauma, AV fistulas, pseudoaneurysms and polycystic kidneys, were embolised after angiographic demonstration of the leakage. In all cases, the bleeding was stopped and in one case only nephrectomy was necessary 3 days after the initial embolisation procedure. Transcatheter renal embolisation should be performed as selectively as possible. With this technique most of the renal parenchyma can be saved. Embolisation is a safe and inexpensive procedure which also can be performed in critically ill patients.

[Arterial aneurysm of the liver with arterio-portal fistula: treatment using embolization]. / Arterielles Aneurysma der Leber mit arterio-portaler Fistel: Behandlung mittels Embolisation.

Aneurysms of the hepatic artery are rare and may present multiform clinical pictures. Prompt diagnosis is important because life threatening complications can be treated by relatively simple measures. A case of posttraumatic intrahepatic aneurysm of the hepatic artery with arterioportal fistula and successful transcatheter embolisation is reported. The present state of the art is outlined and indications for diagnosis and therapy are presented.

Interaction of metabolic inhibitors with actin fibrils.

The dependence of the arrangement of fibrillar actin in cultured endothelial cells on metabolic conditions was investigated with cellular elements derived from the heart of Xenopus laevis tadpoles. Either primary culture or an established cell line (XTH-2) were used in these studies. The metabolic stage of the cells was influenced by inhibiting respiration and lactate production. The actin pattern was revealed either by indirect immunofluorescence or by tetramethylrhodaminyl (TRITC)-phalloidin fluorescence. Total block of energy supply causes in all cases a distinct loss of actin fibrils, while inhibition of respiration alone increases the variability of actin organization. In primary XTH cells but not in XTH-2 cells cyanide disintegrates most of the actin fibres during 3 h of treatment. This effect is independent of the inhibition of respiration, since actin gels prepared from skeletal muscle also undergo destruction in the presence of cyanide. It is concluded that the actin fibrils of the primary cells and the established line behave differently to changing metabolic conditions and to application of KCN.

Antibodies against the alpha-factor pheromone of Saccharomyces cerevisiae.

The mating pheromone of baker's yeast, the alpha-factor, is a dodeka-/tridecapeptide, which is not antigenic by itself. It was coupled to succinylated thyroglobulin by the carbodiimide procedure to facilitate selective coupling of the alpha-factor mainly by its N-terminal region. Antibodies against this conjugate were raised in rabbits. After selective precipitation of the rabbit antiserum with succinylated carrier prior to the radial double diffusion test (Ouchterlony) specific antibodies against the coupled alpha-factor could be detected.