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Biomedical subjects

W Keck

Publications and source records attributed to W Keck.

At least 73 records · Page 4Linked to original sources
PubMed1990

Cloning and characterization of mepA, the structural gene of the penicillin-insensitive murein endopeptidase from Escherichia coli.

The putative structural gene mepA of the penicillin-insensitive murein endopeptidase from Escherichia coli was cloned and sequenced. N-terminal sequence determination with the isolated endopeptidase protein showed that this enzyme is coded by the mepA gene and that it is synthesized initially with an N-terminal signal peptide. No significant sequence homology with the other (penicillin-sensitive) murein endopeptidase (dacB) or any other protein was found. The precise chromosomal mapping position of mepA relative to two other genes, aroC and fabB, was shown to be 50.4 min. E. coli strains carrying multicopy plasmids with the mepA gene produced 5-6-fold more endopeptidase and secreted it into the periplasm, where it appeared to function normally in vivo since the release of cell wall peptides into the medium increased in parallel. The transformed cells were, however, not unusually sensitive to penicillin and their murein had a normal degree of cross-bridges.

Amino Acid Sequence↗
PubMed1990

Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies.

We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.

Acyltransferases↗
PubMed1990

Rotatory evoked cortical potentials in normal subjects and patients with unilateral and bilateral vestibular loss.

Rotatory evoked late potentials following clockwise and counterclockwise rotational acceleration were measured in subjects with normal labyrinthine function and in patients with unilateral and bilateral vestibular loss. In the normal subjects the threshold of the evoked cortical responses ranged between 4 degrees/s2 and 7 degrees/s2, independent of the direction of rotation. For patients with unilateral vestibular loss thresholds were found to be dependent on the direction of rotation. The difference in threshold between clockwise and counter-clockwise rotation was at least 5 degrees/s2. The threshold for rotational accelerations in the direction of the healthy labyrinth was higher for patients with compensated unilateral losses. Those patients who still exhibited lesion-associated nystagmus manifested a higher threshold in the direction of the defective labyrinth. In those patients with bilateral vestibular loss, thresholds were found to be independent of the direction of rotation and were of the order of 20 degrees/s2. The results indicate that the late potentials reflect the interaction of somatosensory and vestibular stimulation. The difference in threshold (approx. 15 degrees/s2) between the normal subjects and patients with bilateral vestibular loss suggests that the vestibular component predominates at low accelerations, with the somatosensory component assuming significance at higher acceleration levels.

Acceleration↗
PubMed1988

Properties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12.

Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.

Bacterial Proteins↗
PubMed1988

Conduction of thermal stimuli in the human temporal bone.

Temperature changes at different locations in the labyrinth were measured in human temporal bone preparations after syringing with water. In order to simulate physiological conditions, the preparations were placed in a water bath at 37 degrees C. The maximum temperature changes in the horizontal semicircular canal after syringing with temperatures symmetrical to body temperature (44 degrees or 30 degrees C) were found to be clearly asymmetrical (with mean values of 0.6 and -0.3K). From measurements in the external auditory meatus, findings showed that the reference temperature was 34 degrees C in front of the tympanic membrane, which explains the asymmetry recorded. Measurements at different locations showed that the temperature first changes in the regions of the ampullae of the horizontal and the superior semicircular canals. In the vestibule the onset and decay of the temperature change is delayed. The time courses of the temperature difference between locations demonstrate that the temperature difference across the horizontal semicircular canal, which would be responsible for any convective effect in the endolymph, is of shorter duration than the absolute temperature change, which would be responsible for any temperature-mediated volume changes.

Adult↗
PubMed1988

[Effect of the stimulus medium--water or air--in thermal evaluation of the vestibular apparatus].

The influence of the stimulus medium in thermal vestibular tests has been investigated by many authors, comparing nystagmus reactions after air and water irrigations. We first measured temperature changes in the ear canal and in the horizontal semicircular canal after both kinds of stimuli, using human temporal bone preparations, which were placed in a water bath of 37 degrees C. Irrigations were done with air of 45 and 27 degrees C with a flow of 5 l/min during 45 s and with 100 ml water of 44 and 30 degrees C during 30 s. The maximum temperature changes in the ear canal are almost equal after both kinds of irrigation (see Figs. 2 and 3). The change of temperature, however, lasts longer after water irrigation. The time constants are 20 and 57 s for the decay after warm and cold water irrigation and 9 and 20 s after air irrigation, respectively. The maximum temperature change in the lateral part of the horizontal semicircular canal is 0.4 and -0.3 degree after warm and cold irrigation with water. The corresponding values after irrigation with air are 0.1 and -0.15 degree. In addition, we investigated the effect of irrigating temporal bone preparations with perforated tympanic membranes (Fig. 4). After irrigation with air of 45 degrees C, the temperature in the horizontal semicircular canal decreased due to evaporation of humidity in the tympanic cavity. The equipment of choice for the irrigation of such ears is a stimulator reported by Scherer, where water flows through a silicon bubble which coats the ear canal.(ABSTRACT TRUNCATED AT 250 WORDS)

Air↗
PubMed1987

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.

Acyltransferases↗
PubMed1985

Comparison of two hydrolytic murein transglycosylases of Escherichia coli.

Escherichia coli has two murein transglycosylases, which are found in the soluble and the particulate fraction, respectively. The enzymes have been purified and have been shown to differ in some of their molecular properties [Mett, H., Keck, W., Funk, A. & Schwarz, U. (1980) J. Bacteriol. 144, 45-52]. We improved and simplified the purification procedure for the membrane-derived transglycosylase and characterized the two enzymes in more detail by peptide mapping and by immunological procedures. The peptide pattern obtained after tryptic digestion of the purified enzymes differed for the two enzymes. Antisera to the transglycosylases reacted only with their own antigen as shown by specific inhibition of the enzymatic activity, double immunodiffusion and by immunochemical staining of protein blots on nitrocellulose filters. Thus we conclude that the transglycosylases are two distinct proteins and that the one is not a precursor of the other.

Antibodies↗
PubMed1985

Sequences of the active-site peptides of three of the high-Mr penicillin-binding proteins of Escherichia coli K-12.

The amino acid compositions of the radioactive peptides obtained from trypsin digestion of [14C]benzylpenicillin-labeled penicillin-binding proteins (PBPs) 1A, 1B, and 3 of Escherichia coli have been obtained. Complete digestion of these peptides with a combination of aminopeptidase M and carboxypeptidase Y showed that benzylpenicillin was bound to a serine residue in each of these proteins. Comparison of the compositions of the penicillin-labeled peptides with the complete amino acid sequences of PBPs 1A, 1B, and 3 showed that the acylated serine occurs near the middle of each of the proteins, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The sequence around the acylated serine of these high Mr PBPs shows little similarity to that around the acylated serine of the low-Mr PBPs (D-alanine carboxypeptidases) or of the class A or class C beta-lactamases, except that in all of these enzymes which interact with penicillin the acylated serine residue occurs within the sequence Ser-Xaa-Xaa-Lys.

Amino Acid Sequence↗
PubMed1983

[Agreement of automatically and manually determined parameters of the electronystagmogram].

Prerequisite for clinical use of programs for automatic analysis of nystagmus is a similar accuracy in determining parameters of electronystagmograms in comparison with manual evaluation. For examination of accuracy and reliability of our program NYSLYS, we compared maximal values of the slow phase velocity (SPV) and the number of beats in 10 s of responses from thermal tests, with 100 patients with equilibrium disturbances. The average difference between the automatically and manually evaluated values for SPV and the number of beats in 10 s is in the range of +/- 15%. Correlation coefficients are 0.97 for SPV and 0.95 for the number of beats in 10 s. Similar correlation coefficients are obtained for the ratio of responses after left and right ear stimulation. These results show that our automatic analysis can replace tedious manual evaluation in most cases. In addition the temporal course of SPV is obtained as a good aid in the judgement of nystagmus. Automatic analysis, however, cannot fully replace the ability to examine electronystagmograms. Especially if curves are disturbed by many artifacts or in cases with very irregular reactions the original electronystagmogram must be considered, too.

Caloric Tests↗
PubMed1981

Exoenzymatic activity of transglycosylase isolated from Escherichia coli.

The possibility that murein transglycosylase of Escherichia coli may function as an exoenzyme to cleave the murein sacculus in a systematic fashion was investigated. Two molecular species of this hydrolytic enzyme have been isolated and characterized: one is associated with the soluble fraction and the other with the envelope fraction of ruptured E. coli cells. The soluble enzyme was employed to digest murein sacculi that had been uniformly labeled with [3H]diaminopimelic acid. The analysis of the reaction product indicated that the enzyme did not cleave the glycan chains randomly. To determine whether transglycosylase released muropeptide first from the N-acetylglucosaminyl or the 1,6-anhydromuramyl ends of the glycan chains, the [3H]diaminopimelate-labeled sacculi were further radiolabeled at their N-acetylglucosaminyl ends with [14C]galactose by a galactosyl transferase reaction. The transglycosylase released galactose-labeled X + X' muropeptides early during the course of digestion, suggesting exoenzymatic cleavage of the glycan chains preferentially from the N-acetylglucosaminyl ends. (X = N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelic acid; X' = X-D-alanine.) The kinetics of the activity of the membrane-bound enzyme were found to be identical to that of the soluble enzyme, indicating that both molecular species of transglycosylase function as exoenzymes in vitro.

Escherichia coli↗
PubMed1980

Oriented fragmentation of Escherichia coli sacculi by sonication.

Isolated murein sacculi of Escherichia coli PA3092 were physically ruptured by sonication. Electron microscopical analysis showed that the rupturing occurred preferentially prependicular to the long axis of the cell. The fragmentation of the sacculi increased with longer sonication times. It was found that the extent of cross-linkage was more affected by the sonication than was the average glycan chain length. These data confirm earlier observations and indicate that the glycan chains run predominantly perpendicular to the long axis of the cell. By contrast, the peptide cross-links are more dispersely oriented.

Bacterial Proteins↗
PubMed1980

Two different species of murein transglycosylase in Escherichia coli.

We demonstrated that Escherichia coli murein transglycosylase exists in two forms. After mechanical disruption of the cells, one form was found in the soluble fraction and the other, in the cell envelope. The two enzymes differed with respect to molecular weight, isoelectric point, solubility in aqueous buffers, and to some extent in their requirements for maximal catalytic activity. The molecular weight of the membrane-bound transglycosylase (35,000) was half that of the soluble enzyme. Whether the high-molecular-weight soluble protein is a precursor of the membrane-bound enzyme species remains to be elucidated.

Cell Membrane↗
PubMed1979

Escherichia coli murein-DD-endopeptidase insensitive to beta-lactam antibiotics.

A novel endopeptidase degrading the peptide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. The enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of beta-lactam antibiotics. In addition, it is exceptional in being inhibited by single-stranded deoxyribonucleic acid and by some polynucleotides. The possible role of the enzyme in cell division is discussed.

Endopeptidases↗