Biomedical subjects

E Krüger

Publications and source records attributed to E Krüger.

At least 19 recordsLinked to original sources

PubMed2001

Clp-mediated proteolysis in Gram-positive bacteria is autoregulated by the stability of a repressor.

The heat shock proteins ClpC and ClpP are subunits of an ATP-dependent protease of Bacillus subtilis. Under non-stressed conditions, transcription of the clpC and clpP genes is negatively regulated by CtsR, the global repressor of clp gene expression. Here, CtsR was proven to be a specific substrate of the ClpCP protease under stress conditions. Two proteins of former unknown function, McsA and McsB, which are also encoded by the clpC operon, act as modulators of CtsR repression. McsA containing zinc finger motifs stabilizes CtsR under non-stressed conditions. McsB, a putative kinase, can inactivate CtsR by modification to remove the repressor from the DNA and to target CtsR for degradation by the ClpCP protease during stress. Thus, clp gene expression in Gram-positive bacteria is autoregulated by a novel mechanism of controlled proteolysis, a circuit of down-regulation by stabilization and protection of a transcription repressor, and induction by presenting the repressor to the protease. Thereby, the ClpC ATPase, a member of the Hsp100 family, was identified as a positive regulator of the heat shock response.

Adenosine Triphosphatases↗

PubMed2001

Physiological responses to mixing in large scale bioreactors.

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Acetic Acid↗

PubMed2000

Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes.

Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits. A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein). We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome. Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma. To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied. In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent. Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes. As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells.

Amino Acid Sequence↗

PubMed2000

Staged operations for basal cell carcinoma of the face.

211 patients (88 female, 123 male, mean age 63 years) presented with 279 basal cell carcinomas (191 primary tumours, 88 recurrent tumours) in the head and neck area that were excised. The resected margins were examined microscopically and the defect repaired at a second operation. The excision margin was 1-2 mm. The recurrence rate was 3%, which is lower than is achieved by other methods, and unaffected structures are optimally conserved.

Adolescent↗

PubMed2000

The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins.

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1999

Monitoring of genes that respond to process-related stress in large-scale bioprocesses.

In large-scale aerobic fed-batch processes, cells are exposed to local zones of high glucose concentrations that can also cause local oxygen limitations at high cell densities. The mRNA levels of four stress genes (clpB, dnaK, uspA, and proU) and three genes responding to oxygen limitation or glucose excess (pfl, frd, and ackA) were investigated in an industrial 20-m(3) Escherichia coli process and in a scale-down reactor with defined high-glucose and low-oxygen zones. The mRNA levels of ackA and proU were high during the batch growth phase, but declined drastically when glucose became limited, whereas the mRNA levels of the other stress genes were relatively constant throughout the process. In the industrial-scale reactor, the stress gene mRNA levels were, in most cases, highest in the middle part and at the top of the reactor, where the substrate was fed. Cells passing through the high glucose zone of the scale-down reactor had elevated mRNA levels for the oxygen limitation genes and had also elevated heat-shock gene mRNA levels. Both responses to stress occurred within seconds. The approach presented in this study offers a tool for monitoring process-related changes in the transcriptional regulation of genes.

Animals↗

PubMed1999

Disruption of male sex hormones with regard to pesticides: pathophysiological and regulatory aspects.

Several pesticides used as herbicides, insecticides and fungicides are known to be endocrine disrupting chemicals (EDCs). In three pair-matched studies we found changes in sex hormone concentrations and T-lymphocytes in relation to acute and chronic pesticide exposure. After acute exposure, 1 day later the concentrations of testosterone and especially estradiol decreased. T4- and T8-lymphocytes slightly increased. Effects of chronic occupational pesticide exposure were expressed by a higher level of testosterone and a larger ratio of T4-/T8-lymphocytes in comparison to control persons. Concentrations of LH in exposed men were higher after exposure than before. We assume an inhibition of the aromatase system by testosterone metabolites. The studies show two effects with regard to the duration of exposure: a hormonal and immune suppression after acute exposure and an activation of both systems following chronic exposure.

CD4-Positive T-Lymphocytes↗

PubMed1999

Fungicide residues in strawberry processing.

The fate of three fungicides (dichlofluanid, procymidone, and iprodione) applied under field conditions was studied during strawberry processing to juice, wine, and jam. An untreated control was compared to raw material treated with fungicides according to recommended doses and to a sample with 6-fold higher application rates. The highest residue values were found in the pomace after pressing. Residue values in readily produced juices and fruit wines were very low and did not exceed legally required maximum residue levels. Generally, processing steps such as pressing and clarification diminished fungicide residues from 50 to 100%. If the whole fruit is processed, as in fruit preparations or jam, the residue levels remain higher due to missing processing steps.

Aminoimidazole Carboxamide↗

PubMed1999

[Acute paranoid hallucinatory psychosis following mefloquine prophylaxis (Lariam)].

Mefloquine is a drug of choice for malaria prophylaxis in Africa because of the spread of chloroquine resistant plasmodium falciparum. On the other hand there are some reports about severe neuropsychiatric side effects associated with the intake of mefloquine medication. In our paper we present a case-report of a patient suffering for the first time from an acute paranoid psychosis induced by mefloquine prophylaxis.

Acute Disease↗

PubMed1998

A new coculture-system of bronchial epithelial and endothelial cells as a model for studying ozone effects on airway tissue.

In order to study the inflammatory potential of ozone on the airway tissue, we developed an in vitro model system in which human bronchial epithelial cells (BEAS 2B) and human umbilical vein endothelial cells (ECV304) were able to communicate with each other. The BEAS 2B cells were grown on filter supports which were inserted into six-well culture dishes. Endothelial cells were cultivated on the bottom of the basolateral compartment. The upper epithelial cells were exposed to 0.15 ppm ozone for 90 min. Supernatants were collected after 1, 4 and 24 h and were quantified for IL-6 and IL-8 secretion. At the same time points we measured the expression of ICAM-1 on the umbilical vein endothelial cells. Exposure of the coculture-system to air or ozone induced the production of IL-6 as well as IL-8 that exceeded the sum of the amounts produced by the two cell types when exposed separately. At 24 h after ozone exposure the IL-6 and IL-8 levels were significantly elevated compared with the air treated cells. Concerning the ICAM-1 expression on ECV304 cells we found elevated ICAM-1 levels on cells which had been cocultured with BEAS 2B cells compared with cells cultured alone. This might be a hint for the secretion of a soluble factor that acts as a mediator in amplifying the response of epithelial cells.

Bronchi↗

PubMed1998

In vitro effects of nitrogen dioxide on the release of nitric oxide by bovine alveolar macrophages.

Bovine alveolar macrophages (BAM) were assessed for nitric oxide-production after exposure to synthetic air or NO2 [0.2 ppm] for 2 h and subsequent treatment with lipopolysaccharide (LPS) [1 microg/ml]. Nitric oxide (NO) release, measured as nitrite in the supernatant, was quantified at different time points. LPS-stimulated BAM showed elevated nitrite levels after 24 h. Pre-treatment with NO2 resulted in higher nitrite levels in comparison to air exposed BAM. Some of the BAM preparations resulted in pre-activated macrophages that produce high amounts of NO without LPS treatment. Within these cells LPS treatment reduced the nitrite levels in comparison to untreated control incubations from the same preparation. These pre-activated BAM exhibited no uniform effect after NO2-exposure. In some experiments nitrite levels of pre-activated control BAM were decreased, while in others no influence could be observed. NO-production by pre-activated BAM or after LPS-treatment could be inhibited by N(G)-monomethyl-L-arginine (L-NMMA) [1 mM]. In order to investigate, whether NO2-exposure alters inducible NO-synthase mRNA expression, we now perform reverse transcription polymerase chain reaction (RT-PCR) of iNOS mRNA. This method will be a good tool to elucidate whether NO2-exposure can modulate iNOS mRNA expression.

Animals↗

PubMed1998

Stress induction of the Bacillus subtilis clpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance.

The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced. The amount of clpP-specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin. Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by sigmaA and sigmaB transcriptional factors of the B. subtilis RNA polymerase respectively. Transcription initiation occurred predominantly at the putative sigmaA-dependent promoter in exponentially growing cells and was induced under stress conditions. After exposure to stress, initiation of transcription also increased at the sigmaB-dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter-controlled subgroup of class III general stress genes in B. subtilis. In a sigB mutant strain, clpP remained heat and stress inducible at the sigmaA-dependent promoter. BgaB-reporter gene fusions, carrying either the sigmaA- or the sigmaB-dependent promoter, showed a higher bgaB induction at the sigmaA-dependent promoter, whereas a significantly lower level of induction was measured at the sigmaB-dependent promoter. The sigmaA-dependent promoter appeared to be crucial for the heat-inducible transcription of clpP. A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites. Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type. Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation. Comparison of two-dimensional protein gels from wild-type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern. Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1998

The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes.

The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter. When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.

ATP-Dependent Proteases↗

PubMed1997

[Care of the diabetic patient].

Explore the source record for details and available documents.

Adaptation, Psychological↗

PubMed1996

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon.

clpC of Bacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (PA and PB) were mapped upstream of the first gene. PA resembles promoters recognized by the vegetative RNA polymerase E sigma A. The other promoter (PB) was shown to be dependent on sigma B, the general stress sigma factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a sigma B-dependent manner. This is the first evidence that sigma B in B. subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by sigma 32 in Escherichia coli, indicating a new function of sigma B-dependent general stress proteins. PB deviated from the consensus sequence of sigma B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the sigma B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the sigma A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream sigma A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.

Autoradiography↗

PubMed1994

Frequency and distribution of numerical chromosomal aberrations in prostatic cancer.

Prostatic cancer frequently shows striking morphological heterogeneity and multifocal growth. To better understand the relationship between chromosomal changes and pathological characteristics, 31 routinely processed radical prostatectomy specimens were studied for the presence of numerical chromosomal aberrations by in situ hybridization with centromeric nucleic acid probes specific for chromosomes 7, 10, 17, X, and Y. In 24 of the cases preoperative core biopsy specimens were available and were examined with the probe for the X chromosome. In eight of the prostatectomy specimens chromosome numbers consistent with a normal male karyotype were found. Three cases, besides diploid chromosome numbers, showed a focal doubling of hybridization signals, consistent with tetraploidy. The other 20 cases displayed numerical chromosomal aberrations to a various degree. In this group the appearance of numerical chromosomal aberrations often showed considerable local heterogeneity, generally coinciding with morphological dedifferentiation, and was significantly correlated with tumor stage (P = .0004) as well as primary (P = .0068), worst (P = .0002), and combined (P < .0001) Gleason grades, total tumor volume (P = .0448), and the volume of tumor with Gleason grades 4 or 5 (P < .0001). In four of the 24 core biopsy specimens no residual tumor tissue was left for cytogenetic examination. In the remaining 20 biopsy specimens the presence or absence of numerical changes matched the result obtained on the corresponding prostatectomy specimen. We conclude that in prostatic cancer the presence of numerical chromosomal aberrations is associated with advanced disease. Especially in low differentiated tumors local heterogeneity in 2 chromosome numbers can be very marked. It is possible to forecast the presence or absence of numerical chromosomal changes on preoperative core biopsy specimens.

Adenocarcinoma↗

PubMed1994

Immunohistochemical detection of p53 protein in human prostatic cancer.

Tissue sections from 73 radical-prostatectomy specimens were studied immunohistochemically for the presence of p53 protein. In seven specimens numerous tumor cells showed a strong nuclear immunostaining. An additional 27 revealed a more discrete and focal accumulation of p53 protein. Comparison of the pathologic characteristics of the p53-negative and -positive groups showed that the presence of p53 protein closely correlated with more advanced tumor stages (p < 0.00001), with higher primary (p = 0.0004), combined (p < 0.0001) and worst (p < 0.0001) Gleason grades, and with larger total (p = 0.0001) and high-grade (p < 0.0001) tumor volumes. No staining was found in areas of benign hyperplasia or in well-differentiated tumor zones. Our results suggest that the accumulation of p53 protein to immunohistochemically detectable concentrations is not a feature of low-grade cancer. This finding implies that abnormal p53 accumulation might be involved in the process of prostatic cancer progression.

Aged↗

PubMed1994

Stress induction of clpC in Bacillus subtilis and its involvement in stress tolerance.

A member of the clpC subfamily of stress response-related Clp ATPases was cloned from Bacillus subtilis. The B. subtilis clpC gene was induced in response to various stresses, including heat shock. Its product was identified as a general stress protein (Gsp12) described previously. A dramatic increase in the amount of clpC mRNA immediately after exposure to multiple stresses suggested regulation on a transcriptional level. Induction by heat shock was independent of the alternative sigma factor SigB, indicating a new mechanism of heat shock induction in B. subtilis. A clpC insertional mutant had an impaired tolerance for heat shock and salt stress. Furthermore, the mutation triggered the formation of elongated cells, a phenomenon particularly pronounced during stress.

Adaptation, Biological↗