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Biomedical subjects

K Büttner

Publications and source records attributed to K Büttner.

At least 19 recordsLinked to original sources
PubMed2002

Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli.

The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response. Here we demonstrate that the general stress response, which is connected to the stress response regulator sigmas (sigma38, rpoS gene product), is suppressed during strong overproduction of a heterologous alpha-glucosidase. The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system. It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system. Furthermore, it is demonstrated that an E. coli c/pP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene. Data from this study suggest that the overexpression of the alpha-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased sigmas level in this mutant background.

Bacterial Proteins↗
PubMed2001

A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis.

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology. This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago. Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified. In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7. The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control. Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot. In a few selected cases evidence for phosphorylation of these proteins is presented. The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B. subtilis proteome such as alkaline proteins as well as extracellular proteins. A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.

Bacillus subtilis↗
PubMed2001

Stress-induced proteins of Agrobacterium tumefaciens.

The pattern of proteins produced by bacteria represents the physiological state of the organism as well as the environmental conditions encountered. Environmental stress induces the expression of several regulons encoding stress proteins. Extensive information about the proteins which constitute these regulons (or stimulons) and their control is available for very few bacteria, such as the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli (gamma-proteobacteria) and is minimal for all other bacteria. Agrobacterium tumefaciens is a Gram-negative plant pathogen of the alpha-proteobacteria, which constitutes the main tool for plant recombinant genetics. Our previous studies on the control of chaperone-coding operons indicated that A. tumefaciens has unique features and combines regulatory elements from both B. subtilis and E. coli. Therefore, we examined the patterns of proteins induced in A. tumefaciens by environmental changes using two-dimensional gel electrophoresis and dual-channel image analysis. Shifts to high temperature, oxidative and mild acid stresses stimulated the expression of 97 proteins. The results indicate that most of these stress-induced proteins (80/97) were specific to one stress stimulon. Only 10 proteins appear to belong to a general stress regulon.

Journal Article↗
PubMed2000

Vascular extracellular superoxide dismutase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation.

BACKGROUND: Increased inactivation of nitric oxide by oxygen free radicals contributes to endothelial dysfunction in patients with coronary artery disease (CAD). We therefore determined the activity of extracellular superoxide dismutase (EC-SOD), the major antioxidant enzyme system of the vessel wall, and its relation to flow-dependent, endothelium-mediated dilation (FDD) in patients with CAD. METHODS AND RESULTS: SOD isoenzyme activity was determined in coronary arteries from 10 patients with CAD and 10 control subjects. In addition, endothelium-bound EC-SOD activity (eEC-SOD), released by heparin bolus injection, and FDD of the radial artery were measured in 35 patients with CAD and 15 control subjects. FDD, determined by high-resolution ultrasound, was assessed at baseline, after intra-arterial infusion of vitamin C, N-monomethyl-L-arginine, and combination of both. EC-SOD activity in coronary arteries (control subjects: 126+/-14; CAD: 63+/-11 U/mg protein; P<0.01) and eEC-SOD activity in vivo (control subjects: 14.5+/-1.1; CAD: 3.8+/-1.1 U. mL(-1). min(-1); P<0.01) were reduced in patients with CAD. Activity of eEC-SOD was positively correlated with FDD (r=0.47; P<0. 01) and negatively with the effect of the antioxidant vitamin C on FDD (r=-0.59; P<0.01). In young individuals with hypercholesterolemia, however, eEC-SOD activity was increased (21. 0+/-1.2 U. mL(-1). min(-1); n=10; P<0.05). CONCLUSIONS: In patients with CAD, vascular EC-SOD activity is substantially reduced. The close relation between endothelium-bound EC-SOD activity and FDD suggests that reduced EC-SOD activity contributes to endothelial dysfunction in patients with CAD. In young hypercholesterolemic individuals, however, endothelium-bound EC-SOD activity is increased and may, in part, counteract impairment of endothelial function as the result of increased formation of oxygen free radicals.

Adult↗
PubMed1999

The amphipathic helix concept: length effects on ideally amphipathic LiKj(i=2j) peptides to acquire optimal hemolytic activity.

In a minimalist approach to modeling lytic toxins, amphipathic peptides of LiKj with i=2j composition and whose length varies from 5 to 22 residues were studied for their ability to induce hemolysis and lipid vesicle leakage. Their sequences were designed to generate ideally amphipathic alpha helices with a single K residue per putative turn. All the peptides were lytic, their activities varying by more than a factor of 103 from the shortest 5-residue-long peptide (5-mer) to the longest 22-mer. However, there was no monotonous increase versus length. The 15-mer was as active as the 22-mer and even more than melittin which is used as standard. Partition coefficients from the buffer to the membrane increased in relation to length up to 12 residues, then weakly decreased to reach a plateau, while they were expected to increase monotonously with peptide length and hydrophobicity as revealed from HPLC retention times. Fluorescence labeling by a dansyl group at the N-terminus, or by a W near the CO-terminus, show that up to 12 residues, the peptides were essentially monomeric while longer peptides strongly aggregated in the solution. Lipid affinity was then controlled by peptide length and was found to be limited by folding and self-association in buffer. The lytic activity resulted both from lipid affinity, which varied by a factor of 20-fold, and from efficiency in disturbing the membrane when bound, the latter steeply and monotonously increasing with length. The 15-residue-long peptide, KLLKLLLKLLLKLLK, had the optimal size for highest lytic activity. The shallow location of the fluorescent labels in the lipids is further evidence for a model of peptides remaining flat at the interface.

Amino Acid Sequence↗
PubMed1999

Dual channel imaging of two-dimensional electropherograms in Bacillus subtilis.

The allocation of proteins to stimulons and regulons is an essential step towards the understanding of the global regulation of the expression of entire genomes. The computer-aided evaluation and matching of two-dimensional protein gels loaded with radioactively labeled proteins from exponentially growing or stressed cells is a useful but time-consuming procedure for the description of stimulons and regulons. This paper describes the dual-channel image analysis that offers the opportunity to visualize the content and synthesis rate of a whole set of bacterial proteins on a single electropherogram. By pulse-labeling with L-[35S]methionine, the protein synthesis pattern (red color) can be directly compared with the protein level pattern (green color). Because matching of other gels can be avoided, this new technique is useful for the rapid search for proteins that belong to different stimulons or regulons. This approach was tested for the identification of proteins of heat stress or oxidative stress stimulons. Proteins that were induced by heat or oxidative stress colored red while proteins whose synthesis was switched off by the stress factor colored green. Proteins that were continuously synthesized before and after the imposition of stress retained their yellow color. The advantages and possible pitfalls of the technique are discussed.

Bacillus subtilis↗
PubMed1998

[Preoperative diagnosis in laparoscopic cholecystectomy: is intravenous cholangiography currently still justified?].

Laparocopic cholecystectomy requires essentially the safe exclusion of choledocholithias. The aim of this study was to compare the intravenous cholangiography and ERCP in addition to a basic program (case history, laboratory results ultrasound) with references to the diagnostic ability and therapeutic consequences in patients with choledocholithiasis. The results show, that the intravenous cholangiography not provides extra important informations after the case history, ultrasound and laboratory findings and therefore its general use is not justified. Instead of the intravenous cholangiography the preoperative ERCP should be performed generously if choledocholithiasis is suspected, especially because the ERCP offers the possibility to extract the stone.

Cholangiography↗
PubMed1997

Copurification of ribosomal protein S2 and DNA-dependent RNA polymerase from heat-shocked cells of Bacillus subtilis.

The DNA-dependent RNA polymerases from heat-shocked and vegetatively grown cells of Bacillus subtilis were isolated and compared. The RNA polymerase from non-stressed cells had the well known alpha, beta, beta' and sigma composition of eubacterial RNA polymerases. The RNA polymerase from heat-shocked cells exhibited one additional band shown by SDS-PAGE. N-terminal sequencing of the first 16 amino acids of the associated protein demonstrated its identity with the ribosomal protein S2.

Amino Acid Sequence↗
PubMed1996

Urinary levels of 1-hydroxypyrene, 1-, 2-, 3-, and 4-hydroxyphenanthrene in females living in an industrial area of Germany.

The concentrations of 1-hydroxypyrene (1-HOPYR), and 1-, 2-, 3-, and 4-hydroxyphenanthrene (HOPHE) as metabolites of pyrene and phenanthrene, were measured in urine samples collected from 124 housewives (27 smokers and 97 non-smokers) living in Bottrop, an industrial city located in the Ruhr area in Germany. The urine samples were analyzed by a very sensitive and practical high-performance liquid chromatographic (HPLC) method using a two-column switching technique and a special precolumn packing material followed by fluorescence detection. The polycyclic aromatic hydrocarbon (PAH) metabolites are selectively enrichéd on the precolumn and separated from the matrix. Therefore, laborious clean-up steps were omitted. The above-mentioned PAH metabolites could be detected in all urine samples investigated. Smokers had significantly higher urine concentrations of 1-HOPYR (median 0.48 microgram/g creatinine), 3-HOPHE (median 0.61 microgram/g creatinine), 2-HOPHE (0.41 microgram/g creatinine) and 4-HOPHE (median 0.10 microgram/g creatinine) than non-smokers (median 0.15 microgram/g creatinine, 0.31 microgram/g creatinine, 0.31 microgram/g creatinine and 0.04 microgram/g creatinine, respectively). The study shows that the influence of smoking is of such an order of magnitude that potential environmental exposure to PAH in this highly industrialized area is obscured by smoking habits. Furthermore, it can be concluded that the determination of 1-HOPYR, 1-, 2-, 3-, and 4-HOPHE in urine is a diagnostically useful method for the biological monitoring of persons environmentally exposed to PAH.

Air Pollutants↗
PubMed1994

The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin.

An original series of 12- to 22-residue-long peptides was developed, they are only constituted by apolar Leu and charged Lys residues periodically located in the sequence in order to general ideal highly amphipathic alpha-helices. By circular dichroism, the peptides are proven to be mainly alpha-helical in organic and aqueous solvents and in the presence of lipids. The peptides are highly hemolytic, their activity varies according to the peptide length. The 15-, 20-, and 22-residue-long-peptides have LD50 approximately 5 x 10(-8) M for 10(7) erythrocytes, i.e. they are 5-10 times more active than melittin, and are indeed several orders of magnitude more active than magainin or mastoparan.

Amino Acid Sequence↗
PubMed1992

Anomalous reversed-phase high-performance liquid chromatographic behavior of synthetic peptides related to antigenic helper T cell sites.

Sets of overlapping synthetic peptides for three well characterized proteins (sperm whale myoglobin, hen egg lysozyme, and the circumsporozoite protein from Plasmodium falciparum) were prepared and examined by reversed-phase high-performance liquid chromatography (RP-HPLC). Using retention coefficients to predict the retention time of each peptide, several peptides in each protein set were found that exhibited anomalous behavior (i.e. eluted significantly later than predicted). Previous work with model peptides has shown that this anomalous behavior can be attributed to specific amphipathic arrangements induced by the lipid stationary phase during the RP-HPLC process. In the current study it was found that although not all of the peptides containing an antigenic T cell site displayed anomalously late behavior, all of the peptides which eluted anomalously late during RP-HPLC included the regions of these proteins known from earlier studies to be antigenic T cell sites.

Amino Acid Sequence↗
PubMed1992

Evaluation of peptide-peptide interactions using reversed-phase high-performance liquid chromatography.

The separation of peptides during RP-HPLC depends mainly upon differential hydrophobic interactions of the individual peptides being separated with the C18 group of the stationary phase. We have examined the behavior of dimeric disulfide-linked model peptides during RP-HPLC in order to study self-induced conformational effects. A set of 18 analogues of the amphipathic alpha-helical sequence Ac-LKLLKKLLKKLKKLLKKL-NH2 was used for this study. These analogues differed only by the successive replacement of each position with a cysteine. Strong peptide-peptide interactions, occurring through interchain hydrophobic forces, resulted in a presenting face to the C18 group, consisting primarily of lysine residues and, in turn, in early retention times. Three homo-dimers were also found to be strongly alpha-helical in water as determined by circular dichroism spectroscopy.

Amino Acid Sequence↗
PubMed1992

Perturbation of peptide conformations induced in anisotropic environments.

Reversed-phase high performance liquid chromatography (RP HPLC) has been found to be a convenient and powerful tool for the study of the secondary structure of peptides. Here, the ability of proline to perturb the secondary structures of peptides induced at aqueous-lipid interfaces and the induced conformation of polyproline peptides were investigated by means of RP HPLC. For these studies, four different complete sets of substitution analogues of model peptides expected to have specific induced conformations were used. In the first two studies, a single lysine was "walked" through two 18-residue polyproline sequences (one N-acetylated, the other not). In the remaining two studies, a proline was "walked" through two different sequences that had been found earlier to be induced into an alpha-helical conformation during RP HPLC (an 18-residue polyalanine sequence and the amphipathic 14-residue sequence Ac-LLKLLKKLLKKLKK-NH2). Sixty-eight individual analogues were synthesized for this study and the effect of the respective substitutions on retention times was determined. The results are consistent with the concept that, upon interaction with the C-18 of the stationary phase during RP HPLC, polyproline is induced into a type II helical conformation, polyalanine into an alpha-helical conformation, and Ac-LLKLLKKLLKKLKK-NH2 into an amphipathic alpha-helical array. In an extension of this study, the antimicrobial activities of Ac-LLKLLKKLLKKLKK-NH2 and its 18 proline substitution analogues were found to be inversely correlated with their RP HPLC retention times.

Amino Acid Sequence↗
PubMed1990

Detection of a 2 mu derivative yeast plasmid with altered properties.

The Saccharomyces cerevisiae (Sacch. cerevisiae) strain RXII, like many others, harbours plasmid DNAs. and one category of them is homologous to the 2 mu plasmid of yeast. DNA-DNA hybridization experiments indicated altered structures of this species as regards the number and distribution of the restriction sites. The efforts made to clone either the whole plasmid in pBR328 or its fragments in pBR322 vectors remained unsuccessful, since deleted plasmids were isolated without insert DNA, and even the loss of vector sequences was observed. The data suggest, that the 2 mu derivative plasmid in strain RXII represent an unique category of this extrachromosomal genetic element.

Autoradiography↗
PubMed1990

Studies on the total synthesis of an A7,B7-dicarbainsulin. III. Assembly of segments and generation of biological activity.

As a further contribution to the synthesis of an insulin analogue with a stable A7-B7 interchain bond, the synthesis of A(8-21) by solution methods, and of B(9-25) as well as [7-(2,7-diaminosuberic acid)]B(1-8) by solid phase methods is described. In the latter compound, the amino group of the diaminosuberic acid residue was acylated with A(1-6), and the resulting "U-peptide" sequentially elongated with the C-terminal A- and finally B-chain sequences. The conversion of the product into the disulfide moiety gave a mixture which could not be resolved by currently available methods. However, the low biological activity of the crude product indicates that the A7-B7 disulfide bond is not crucially important for the activity of insulin.

Adipose Tissue↗
PubMed1988

[Inpatient rehabilitation measures in the early phase following surgery of intervertebral disk displacement as an effective alternative to ambulatory after-care].

Between 1983 and 1986, a total of 1012 patients were treated in our rehabilitation clinic after intervertebral disc surgery, either admitted shortly post op, or at a later time. Males outweighed females in proportion by 56 vs. 44%; the average age at surgery was 44 yrs. for male, and 42 yrs for female patients. 966 patients (95%) had undergone lumbar surgery, 46 (5%) surgery in the cervical region; 12% of the patients were operated twice and 2% for a third time. Except for those admitted shortly post-surgery, an average period of 6 to 7 months elapsed between the family doctor's, or specialist's, application for a rehabilitation measure and admission to the rehabilitation clinic. Average duration of incapacity for work totalled 6.2 months (males 5.6 months, females 6.8 months). Of the 228 patients with "incapable for work" status on admission to the clinic, 160 were discharged home capable for work, another 34 were recommended for work trial. The remaining 34 patients were referred back to the acute clinics for further diagnosis, or renewed surgery. The advantages of in-patient rehabilitation measures, among them post-surgery postural etc. training ("back training"), are set out, along with ways how the rehabilitative procedures should be dovetailed with the course of management.

Adolescent↗
PubMed1986

BCG immunostimulation in primary pulmonary cancer (a clinical, immunological and radiological study).

The authors reported the results of a clinical, radiological and immunological study of 147 primary pulmonary cancer cases who were immunostimulated with BCG vaccine over a period of three years between 01.01.76 and 31.12.78. Compared with the same number and stage of cancerous patients treated with the same cytostatic drugs and/or surgically without BCG immunostimulation, the survival rate of the immunostimulated group was higher than that of the control group. The radiological picture did not change during the study time in 45% of the patients, regression was observed in 34%, and progression in 21% in the Stage I vaccinated group, while in the control Stage I group regression was only noticed in 21%, and progression was observed in 50%. In the Stage II vaccinated group regression was noted in 48%, in the control group in 23%. No favourable changes were noted in Stage III groups, neither in the vaccinated nor in the nonvaccinated cases. The more favourable prognosis of the immunostimulated group was reflected in the results of the skin tests and in the cellular and humoral immunological responses too. The modification of the BCG immunostimulated pulmonary cancer patients gives a statistically significant prolongation of survival time and a beneficial improvement of life quality and expectation.

BCG Vaccine↗