Biomedical subjects

S Zimmermann

Publications and source records attributed to S Zimmermann.

At least 73 records · Page 4Linked to original sources

PubMed1998

RFLP and sequence analysis of the cytochrome b gene of selected animals and man: methodology and forensic application.

To identify common animal species by analysis of the cytochrome b gene a method has been developed to obtain PCR products of a large domain of the cytochrome b gene (981 bp out of 1140 bp) in humans, selected mammals and birds using the same specifically designed primers. Species-specific RFLP patterns are generated by co-restriction with the restriction endonucleases ALU I and NCO I. The RFLP patterns obtained are conclusive even in mixtures of two or more species. The results were confirmed by sequence analysis which in addition explained intraspecies variations in the RFLP patterns. The method has been applied to forensic casework studies where the origin of roasted meat, stomach contents and a bone sample has been successfully identified.

Animals↗

PubMed1998

Validation of computer systems: practical testing of a standard LIMS.

In recent years the introduction of computer systems for data handling in the pharmaceutical industry has increased. A standard LIMS (laboratory information management system) is software commercially available from different suppliers not only to facilitate data handling in laboratories but also to cover GMP-requirements. Computer systems introduced in GMP-areas of pharmaceutical companies have to be validated. For a standard LIMS, the general validation of the program is performed by the supplier. Nevertheless, the user is always required to cover all phases of a validation. The objective of this paper is to discuss suitable test procedures for the most critical functions of a standard LIMS needed during the verification step of the validation process.

Clinical Laboratory Information Systems↗

PubMed1998

Characterization of SKT1, an inwardly rectifying potassium channel from potato, by heterologous expression in insect cells.

A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K+in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than -60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated K+in currents half-maximally with an inhibitor concentration (IC50) of 105 microM. An almost identical high Cs+ sensitivity (IC50 = 90 microM) was found for the potato guard-cell K+in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.

Amino Acid Sequence↗

PubMed1998

Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1.

The Leydig insulin-like gene (Ley I-L), a member of the insulin-related gene family, is specifically expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. To determine the functional region of the mouse Ley I-L promoter and factors controlling the Ley I-L gene expression, we used 2.1 kb of the 5'-flanking region of the mouse Ley I-L gene to generate chimeric constructs with the chloramphenicol acetyltransferase gene (CAT). Transient transfections of MA10 Leydig cells, LTK- fibroblasts, and F9 embryonic cells by a series of 5'-deleted mouse Ley I-L promoter-CAT constructs revealed that the sequence between nucleotides -157 to +4 directs the transcription of the reporter gene in MA10 but not in LTK- and F9 cells, indicating that the determinants of Leydig cell-specific expression reside within this region. Deoxyribonuclease I (DNase I) footprint analysis revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase I-protected regions are homologous to the consensus binding site of the steroidogenic factor-1 (SF-1). Competition and antibody studies showed that the three SF-1-binding sites in the Ley I-L promoter have similar binding affinities for SF-1. Furthermore, transient transfections of MA10 cells with mutant reporter constructs, in which SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1-binding sites are required for SF-1-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing expression vector together with a Ley I-L promoter-CAT construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicated that SF-1 can transactivate the Ley I-L promoter. These data demonstrate an essential role of SF-1 in transcriptional activation of the Ley I-L promoter.

Animals↗

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

Association of plant K+(in) channels is mediated by conserved C-termini and does not affect subunit assembly.

Inward rectifying potassium (K+(in)) channels play an important role in turgor regulation and ion uptake in higher plants. Here, we report a previously unrecognized feature of these proteins: K+(in) channel C-terminal polypeptides mediate channel protein interactions. Using a C-terminal fragment of potato guard cell K+(in) channel KST1 in a yeast two-hybrid screen two novel putative K+(in) channel proteins (SKT2 and SKT3) were identified by interaction of their C-termini which contained a conserved domain (K(HA)). Interactions were confirmed by Western blot-related assays utilizing K+(in) channel C-termini fused to green fluorescence protein. Although deletion of the K(HA)-domain abolished these interactions, K+(in) currents were still detectable by patch-clamp measurements of insect cells expressing these KST1 mutants, indicating that formation of a functional channel does not depend on this C-terminal domain.

Amino Acid Sequence↗

PubMed1997

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of 'two-pore' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.

Amino Acid Sequence↗

PubMed1997

Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense.

Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley.

Journal Article↗

PubMed1997

Mouse Leydig insulin-like (Ley I-L) gene: structure and expression during testis and ovary development.

Leydig insulin-like protein (Ley I-L) is a novel member of the insulin-like hormone superfamily. We report here the isolation and expression of the mouse Ley I-L gene. The gene encodes a polypeptide of 122 amino acids that shows a relatively weak homology (54%) to human and porcine prepro-Ley I-L. However, the predicted B and A chain of the mature mouse Ley I-L exhibit similarities of 73% and 71% with human and porcine Ley I-L, respectively. Alignment of the 5' flanking region of the mouse gene with those of human and porcine did not exhibit any significant sequence homology. However, it contains the conserved sequence of the Ad4 binding site that is present in all promoter regions of steroidogenic P-450 genes and the Müllerian inhibitor substance gene and is recognized by steroidogenic factor 1. The Ley I-L gene is expressed at a high level in the testis and at a much lower level in the ovary. No transcripts could be detected in placenta prepared between days 10 and 19 of pregnancy. Ley I-L transcripts were first detected in fetal testis at 13.5 dpc. After birth, transcript levels remain constant during the following 3 weeks, increasing at the stage in which the first wave of round spermatids undergo spermiogenesis suggesting a functional role of the Ley I-L in early stages of spermatogenesis and germ-cell maturation. In the ovary, the expression of Ley I-L was first detected at day 6 after birth. The pattern of Ley I-L expression at various stages of the estrous cycle and during pregnancy showed a correlation with follicle development.

Amino Acid Sequence↗

PubMed1997

Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines.

Certain bacterial immunostimulatory (i.s.) DNA sequences containing unmethylated CpG motifs stimulate antigen-presenting cells (APC) to express a full complement of costimulatory molecules and to produce cytokines including interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha. While IL-12 is key to their T helper cell (Th)1-promoting adjuvant activity, secretion of toxic levels of TNF-alpha is harmful in that it promotes toxic shock. Given the beneficial as well as harmful consequences of i.s. DNA, we investigated the possibility of identifying DNA sequences, i.e. CpG oligodeoxynucleotides (ODN) which differentially activate IL-12 versus TNF-alpha cytokine production in APC. Here, we describe an i.s. DNA sequence with these characteristics. While its potential to induce IL-12 is preserved, its ability to trigger TNF-alpha release is strongly curtailed both in vitro and in vivo. I.s. DNA could be segregated into lethal and non-lethal in a mouse toxic shock model. The non-toxic i.s. DNA was useful as an adjuvant, thus allowing cytotoxic T cell responses to the soluble protein ovalbumin and conferring a resistant Th 1 phenotype to BALB/c mice lethally infected with Leishmania major. This i.s. CpG motif may thus be prototypic for a useful immunostimulating DNA sequence that lacks harmful side effects.

Adjuvants, Immunologic↗

PubMed1997

Outbreak of Escherichia coli O157:H7 infection in a large family.

An outbreak of bloody and nonbloody diarrhoea caused by Escherichia coli O157:H7 including one case of haemolytic uraemic syndrome (HUS) and two cases of haemolytic anaemia, in five siblings (aged 2.5 to 11.3 years) and their playmate was investigated. Using sorbitol-MacConkey agar, colony blot hybridisation, and immunomagnetic separation, Shiga toxin 2-producing Escherichia coli O157:H7 was isolated from all children but the HUS patient; however, this patient had high immunoglobulin M antibody titres against Escherichia coli O157 lipopolysaccharide. Escherichia coli O157 isolates from all patients were indistinguishable in serotype, virulence properties, and genomic background, indicating that the same strain caused the infections. These data confirm the importance of person-to-person transmission.

Anemia, Hemolytic↗

PubMed1997

A novel immunotoxin recognising the epithelial glycoprotein-2 has potent antitumoural activity on chemotherapy-resistant lung cancer.

Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer. Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer. The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas. An immunotoxin composed of MOC31 and a recombinant from of Pseudomonas exotoxin A lacking the cell-binding domain (ETA252-613) was prepared and its effect on lung cancer cell lines examined. MOC31 ETA252-613 was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM. Moreover, the immunotoxin reduced the number of clonogenic tumour cells from cultures by factors of 10(4) and 10(5) during a 24-h and a 3-week exposure respectively. In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm3) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm3). This finding indicates that MOC31-ETA252-613 may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease.

ADP Ribose Transferases↗

PubMed1997

Epidemiological relatedness and clonal types of natural populations of Escherichia coli strains producing Shiga toxins in separate populations of cattle and sheep.

Two separate animal populations consisting of a herd of cattle (19 animals) and a flock of sheep (25 animals) were investigated for strains of Escherichia coli producing Shiga toxins (STEC) over a time period of 6 months. Thirty-three STEC were isolated from 63.2% of cattle and grouped into 11 serotypes and eight electrophoretic types (ETs) by multilocus enzyme analysis. In sheep, 88% of the animals excreted STEC (n = 67 isolates) belonging to 17 different serotypes and 12 different ETs. STEC from cattle and sheep differed with respect to serotype, and only 4 of the 16 ETs occurred in both animal populations. In cattle, ET14 (O116:H21) strains predominated, whereas other STEC serotypes occurred only sporadically. The predominating STEC types in sheep were ET4 (O125 strains), ET11 (O128:H2 and others), and ET14 (O146:H21). In contrast to their diversity, STEC originating from the same animal population were similar with respect to Shiga toxin (stxy genes. Almost all STEC isolated from cattle were positive for stx2 and stx2c; only one was positive for stx1. In sheep, almost all STEC isolated were positive for stx1 and stx2, whereas stx2c was not found. XbaI-digested DNAs of genetically closely related O146:H21 strains have different restriction profiles which were associated with size alterations in XbaI fragments hybridizing with stx1- and stx2-specific DNA probes. Our results indicate that stx-encoding bacteriophages might be the origin of the genetic heterogeneity in STEC from animals.

Animals↗

PubMed1997

["Patients with genetic diseases can, by surgical intervention, be sterilized..." Mandatory sterilization in Germany during the era of national socialism].

The ethical problem of permissibility of sterilisation for eugenic reasons is being discussed since the end of the last century. For many years restriction and rejection were the predominant opinions. A bill passed July, 1933--"Gesetz zur Verhütung erbkranken Nachwuchses" ("Act of prevention of heredopathic progeny")--imposed juridical regulations to prevent offspring with inherited diseases in National Socialist's Germany. No major protests had been made against such compulsion put upon the medical profession. Papers dealing with questions of surgical techniques had been published in Zentralblatt für Gynäkologie predominantly in 1935 and 1936. The significance of the scientific analysis of mandatory sterilisations for so-called eugenic reasons performed in almost 400,000 women and men in Germany between 1933 and 1945 has only recently began to be recognised by the scientific community in the last decade addressing predominantly the regional impact of this jurisdiction.

Eugenics↗

PubMed1996

Immune response to non-O157 Vero toxin-producing Escherichia coli in patients with hemolytic uremic syndrome.

The incidence and time course of the immune response to Vero toxin-producing Escherichia coli (VTEC) other than O157 (O26, O55, O111, O128) were determined in 107 children with enteropathic hemolytic uremic syndrome (HUS) by ELISA and compared to antibody profiles of 125 healthy pediatric controls and 100 children with community-acquired uncomplicated diarrhea. Six of 8 HUS patients with non-O157 VTEC isolates exhibited a serologic response to the homologous lipopolysaccharide antigen similar to that of patients infected with E. coli O157. In addition, elevated IgM or IgG antibodies were demonstrated in 7 of 19 culture- and O157 serology-negative HUS patients. Serogroup-specific antibodies decreased below cutoff levels within several months after convalescence. Anti-non-O157 antibodies were found in 14% of the diarrheic controls. Unexpectedly, a high proportion (27/82) of anti-O157 lipopolysaccharide antibody-positive HUS samples reacted with LPS from E. coli O55:B5, suggesting shared epitopes with endemic VTEC O157 strains.

Adolescent↗

PubMed1996

Rapid detection and isolation of shiga-like toxin (verocytotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay.

By combining the enterohemolysin test and the VTEC-RPLA test (specific for the detection of Shiga-like toxin I [SLT-I], SLT-II, and SLT-IIc), single colonies of SLT-producing Escherichia coli were found to constitute between 0.03 and 68.1% of the coliform flora in human stool cultures and were isolated and characterized within 72 to 96 h.

Agar↗

PubMed1996

[Not Available].

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Anthropology, Cultural↗

PubMed1996

The structures of the bovine and porcine proacrosin genes and their conservation among mammals.

Sperm acrosin is a serine protease that is involved in the recognition, binding and penetration of the sperm of the zona pellucida of the ovum. The bovine and porcine genes were cloned and characterized. Alignment of the intron/exon structure of both genes with the previously characterized human, rat and mouse genes and with other serine protease genes reveals that the coded sequence of the mammalian proacrosin is distributed in 5 exons and the splice junction types are identical to the exons encoding the catalytic domain of other serine protease genes. A comparison of the bovine, porcine, human, guinea pig, rabbit, rat and mouse preproprotein sequences shows that the catalytic domain is highly conserved, while the sequence of the proline rich domain is very variable among the species, ranging from 28.9% to 68.8%.

Acrosin↗