Extending the role of breast-conserving surgery by immediate volume replacement.
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Breast Diseases↗
Biomedical subjects
A Otto
Publications and source records attributed to A Otto.
[Significance of double crush in carpal tunnel syndrome].
From June 1992 to October 1995, 224 patients were operated on carpal tunnel syndrome. We treated 71 male and 153 female patients. During our investigations, we found that one third of the patients suffered from a double crush syndrome. 84% of the patients suffered from one or more predisposing accompanying diseases. In most of them (42%) we found rheumatic diseases, 19% diabetes mellitus, 8% hyperuricaemia, and 6% hypothyreosis. In 33%, we observed a very short history and a bilateral carpal tunnel syndrome. According to the preoperative tests, four different groups of patients regarding electrophysiological parameters and the graduation of thenar atrophy were differentiated. The fourth group was composed of patients with double crush syndrome, who continued to complain of considerable pain despite surgery, the operated hand remaining both weak and awkward in its movements. Inspired by the prospective study by Dellon (1992) and our results, we will perform a retrospective analysis of the longterm effect of carpal tunnel release in diabetic neuropathy.
Adult↗
Gene transfer mediated by alpha2-macroglobulin.
alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein.
Gene Transfer Techniques↗
Cadmium-detoxification in the earthworm Enchytraeus: specific expression of a putative aldehyde dehydrogenase.
The ubiquitous earthworm Enchytraeus buchholzi is provided with a very effective, but still unknown, mechanism of Cadmium (Cd)-detoxification, a central role in which has been ascribed to the Cd-inducible crp-gene encoding a novel cysteine-rich, non-metallothionein 25 kDa protein. This study identifies another Cd-responsive gene by differential screening of a cDNA-library constructed from Cd-exposed E. buchholzi. The isolated cDNA-clone designated Ebaldh encodes a putative aldehyde dehydrogenase. Northern blot analysis shows that the Ebaldh-expression is strongly enhanced by Cd, but remains unaffected by other stressors such as Zn, Hg, and H2O2. This Cd-specificity of Ebaldh suggests that Cd-detoxification in E. buchholzi requires low intracellular concentrations of aldehydes which are known to target sulfhydryl groups thus inactivating the Cd-binding capacity of CRP.
Aldehyde Dehydrogenase↗
Characterization of the human small-ribosomal-subunit proteins by N-terminal and internal sequencing, and mass spectrometry.
Reverse-phase HPLC was used to fractionate 40S ribosomal proteins from human placenta. Application of a C4 reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic fraction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40S human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40S proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications.
Amino Acid Sequence↗
The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates.
Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.
Amino Acid Isomerases↗
Biogenesis of the covalently flavinylated mitochondrial enzyme dimethylglycine dehydrogenase.
Rat dimethylglycine dehydrogenase (Me2GlyDH) was used as model protein to study the biogenesis of a covalently flavinylated mitochondrial enzyme. Here we show that: 1) enzymatically active holoenzyme correlated with trypsin resistance of the protein; 2) folding of the reticulocyte lysate-translated protein into the trypsin-resistant, holoenzyme form was a slow process that was stimulated by the presence of the flavin cofactor and was more efficient at 15 degrees C than at 30 degrees C; 3) the mitochondrial presequence reduced the extent but did not prevent holoenzyme formation; 4) covalent attachment of FAD to the Me2GlyDH apoenzyme proceeded spontaneously and did not require a mitochondrial protein factor; 5) in vitro only the precursor, but not the mature form, of the protein was imported into isolated rat liver mitochondria; in vivo, in stably transfected HepG2 cells, both the precursor and the mature form were imported into the organelle; 6) holoenzyme formation in the cytoplasm did not prevent the translocation of the proteins into the mitochondria in vivo; and 7) lack of vitamin B2 in the tissue culture medium resulted in a reduced recovery of the precursor and the mature form of Me2GlyDH from cell mitochondria, suggesting a decreased efficiency of mitochondrial protein import.
Amino Acid Sequence↗
Earthworm gene as indicator of bioefficacious cadmium.
The expression of a novel Cd-inducible gene encoding a 25 kDa non-metallothionein cysteine-rich protein (CRP) in the earthworm Enchytraeus buchholzi is investigated with respect to Cd-specificity and Cd-sensitivity. The amount of CRP-mRNA correlates with the environmental as well as with the intra-worm Cd-concentration. Even the subtoxic concentration of 100 microgram Cd/1 induces CRP-gene expression. CRP-mRNA is expressed already 2 h after the worms were exposed to Cd. Other heavy metals or stress conditions can induce only little or even no CRP-mRNA. The CRP-gene is switched on long before Cd becomes toxic to worms and can be therefore taken as a warning signal of prospective irreversible Cd-damages at an early, still reversible stage.
Amino Acid Sequence↗
Identification of human myocardial proteins separated by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry.
Disease-associated proteins separated by two-dimensional electrophoresis (2-DE) are often in the femtomole range. Identification of 2-DE separated proteins by sequencing and amino acid analysis is limited to the lower picomole range. Identification down to the femtomole range can be achieved by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). We optimized the measurement by MALDI-MS for the analysis of proteolytic digests of 2-DE-separated proteins. The direct analysis of peptide mixtures can be used for rapid and sensitive protein identification. In some cases, more information about the protein can be obtained by separating the peptides by micro high-performance liquid chromatography (HPLC) before employing MALDI-MS analysis. More peptides are found than in the mixtures, and comparison of HPLC patterns can reveal some differences to be post-translational modifications of proteins, even in the case of identical peptide mass fingerprints. Furthermore, carboxy-terminal sequencing by on-target carboxypeptidase P digestion can be used to confirm the obtained result without the need for more material. The search program FRAGFIT was modified and renamed FRAGMOD to include the modifications of methionine and tryptophan oxidation and alkylation of cysteine by acrylamide into the mass search. By applying this procedure, 15 proteins were identified, among them two different putative phosphorylated forms of two proteins, a putative N-terminal blocking group and four dilated cardiomyopathy-associated proteins. The resulting approach for the identification may be used for large-scale investigations of 2-DE-separated proteins.
Amino Acid Sequence↗
Resolution power of two-dimensional electrophoresis and identification of proteins from gels.
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Electrophoresis, Gel, Two-Dimensional↗
Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry.
Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.
Acetyl-CoA C-Acyltransferase↗
High-performance human myocardial two-dimensional electrophoresis database: edition 1996.
The master gel of the human myocardial two-dimensional electrophoresis (2-DE) gel database contains about 3300 protein spots characterized in terms of isoelectric point (pI) and molecular mass. A high-performance technique was applied, using large gels (23 x 30 cm). Isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) was combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 15% acrylamide gels in the second dimension. The range of pI extends from pH 4.5 to 9.6. Seventy proteins were identified by combinations of amino acid analysis, N-terminal and internal sequencing, immunostaining, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting, post-source decay MALDI-MS and ladder sequencing by carboxypeptidase P. The identification of additional proteins, not found in the master gel, was achieved by immunoblotting. Unequivocal identification with high sensitivity and good yield was obtained by combining internal sequencing and MALDI-MS. In-gel digestion, the concentration and purification of peptides in a peptide collecting device, and the improved FRAGMOD program for peptide mass fingerprinting have added to the security and sensitivity of identification. The high-performance human myocardial 2-DE database was built up with proteins detected by the TOPSPOT program. Spots within six sections of the whole pattern are clickable. Protein description includes detailed information about identification, characterization, and links to the related SWISS-PROT, other 2-DE databases and Medline entries. The database is constructed in accordance with four of the rules for a federated database.
Computer Communication Networks↗
Intravenous ondansetron in established postoperative emesis in children. S3A-381 Study Group.
BACKGROUND: In pediatric postsurgical patients, postoperative vomiting is a common occurrence that can delay recovery and result in unplanned hospital admissions after outpatient surgery. This randomized, double-blind, placebo-controlled, multicenter study evaluated the efficacy and safety of ondansetron in the control of established postoperative emesis in outpatients aged 2-12 yr. METHODS: Screened for the study were 2,720 ASA physical status 1-3 children undergoing outpatient surgery during general anesthesia, which included nitrous oxide. Children experiencing two emetic episodes within 2 h of discontinuation of nitrous oxide were given intravenous ondansetron (n = 192; 0.1 mg/kg for children weighing < or = 40 kg; 4 mg for children weighing > 40 kg) or placebo (n = 183). RESULTS: The proportion of children with no emetic episodes and no use of rescue medication was significantly greater (P < 0.001) in the ondansetron group compared with placebo for both 2- and 24-h periods after study drug administration (78% of the ondansetron group and 34% of the placebo group for 2 h; 53% of the ondansetron group and 17% of the placebo group for 24 h). Among patients with at least one emetic episode or with rescue medication use, the median time to onset of emesis or rescue was 127 min in the ondansetron group compared with 58 min in the placebo group (P < 0.001). The median time from study drug administration until discharge was significantly shorter (P < 0.01) in the ondansetron group (153 min, range 44-593 min) compared with the placebo group (173 min, range 82-622 min). The incidence of potentially drug-related adverse events was similar in the ondansetron (3% of patients) and the placebo (4% of patients) groups. CONCLUSION: A single dose of ondansetron (0.1 mg/kg up to 4 mg) is effective and well tolerated in the prevention of further episodes of postoperative emesis in children after outpatient surgery. Administration of ondansetron also may result in a shorter time to discharge.
Antiemetics↗
Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4'-phosphopantetheine.
The biosynthesis of the decapeptide antibiotic gramicidin S in Bacillus brevis ATCC 9999 is catalyzed by a multienzyme system consisting of two multifunctional proteins, gramicidin S synthetase 1 and 2, encoded by the grsA and grsB genes, respectively. Gramicidin S synthetase 1 (phenylalanine racemase, EC 5.1.1.11, GS1) racemizes phenylalanine in the thioester-bound stage. The amount of 4'-phosphopantetheine liberated from highly purified GS1 was determined microbiologically using Lacto-bacillus plantarum as the test organism. It matches exactly with the amount of L-[14C]phenylalanine covalently incorporated by GS1 as thioester. The reaction center of GS1 for L-phenylalanine thiolation and racemization was labeled with [3H]iodoacetic acid. After tryptic fragmentation of the 3H-carboxymethylated enzyme, the active site peptide for thioester binding and racemization of phenylalanine was isolated in pure form by multistep methodology and investigated by sequence, amino acid, and mass spectrometric analysis. A 4'-phosphopantetheine carrier was found to be attached to the active site serine of the consensus motif LGGDSI forming the thiolation site of phenylalanine. These specific properties establish GS1 as a prototype of amino acid racemases using 4'-phosphopantetheine as a cofactor and yield further evidence that multiple Pan carriers are involved in gramicidin S formation. Our results are strong evidence for the "multiple carrier model" as a new concept of nonribosomal peptide biosynthesis at protein templates as recently proposed [Stein, T., et al. (1994) FEBS Lett. 340, 39-44].
Amino Acid Isomerases↗
Efficient catalysis of disulfide formation during protein folding with a single active-site cysteine.
Protein disulfide isomerases (PDIs) catalyze disulfide bond formation during protein folding in vivo and are essential for viability in eukaryotic cells. They share the active-site sequence C-X-X-C that forms a catalytic disulfide. The recent finding that the EUG1 protein, a PDI-related yeast protein, with C-X-X-S sequence at its active sites can complement PDI-deficiency raised the general question of whether disulfide-isomerase activity is essential for cell viability or whether PDI variants with single active-site thiol groups can be catalytically active as disulfide isomerases. We investigated the function of the catalytic cysteine residues in DsbA, a PDI-related protein required for disulfide formation in the periplasmic space of Escherichia coli, by replacing C30 and C33 with alanine. While the mutant C30A and the double mutant CC30/33AA are inactive, C33A catalyzes disulfide-interchange reactions and oxidative renaturation of the reduced, unfolded thrombin inhibitor hirudin with close to wild-type efficiency. Thus, the single active-site thiol group of C30 is sufficient for disulfide-isomerase activity of the DsbA protein.
Base Sequence↗
A novel strategy to identify maternal and paternal inheritance in the mouse.
A novel strategy for identifying proteins which reveal maternal or paternal inheritance in the mouse is presented. Using two-dimensional electrophoresis we investigated protein expression patterns of adult liver and different embryonic and extraembryonic tissue in C57BL/6Crl and in DBA/2Crl mice, as well as in their reciprocal hybrids. We found three groups of protein spots which showed maternal or paternal inheritance of quantitative variations. These proteins were characterized by N-terminal or internal amino acid sequencing, by determination of the amino acid composition, by glycoprotein staining and RNA expression analysis. The three proteins identified were: alpha-enolase, cyclophilin and beta-group hemoglobins. The parental effects observed for alpha-enolase and cyclophilin were found to be due to parent-specific post-translational modifications of these proteins. For the beta-group hemoglobins our results suggested parental effects on the transcriptional level.
Amino Acid Isomerases↗
Partitioning of streptokinase in aqueous two-phase systems.
The partitioning of streptokinase in aqueous two-phase systems, containing poly(ethylene glycol) and dextran or poly(ethylene glycol) and salt was investigated. The protein partitions in favour of the upper, PEG-rich phase, if PEG of low molecular weight and potassium phosphate or ammonium sulphate were used. This property was found to be independent of the degree of purity and was exploited for the partial purification of streptokinase from crude material. The protein was shown to exhibit negligible affinity to diverse triazine dyes applied in affinity partitioning experiments.
Affinity Labels↗
Identification of tumor-associated protein variants during rat hepatocarcinogenesis. Aldose reductase.
Tumor-associated protein variants were detected by two-dimensional gel electrophoresis (2-DE) in soluble proteins from chemically induced rat hepatomas and transformed rat liver cell lines. Among them, a series of 8 protein variants was found localized at similar sites in 2-DE gels (33-35 kDa, with different pI values). We characterized four of them. In situ peptide mapping with limited proteolysis disclosed a significant relationship between these four individual protein spots. Their different position in 2-DE gels might be due to posttranslational modifications: one of the variants was phosphorylated, three others were modified by glycosylation. The most prominent tumor-associated protein variant of this series (spot 17) was further studied by amino acid analysis and internal amino acid microsequencing. It became evident that this variant is identical to aldose reductase (EC 1.1.1.21). This enzyme of the sorbitol pathway is expressed in the liver during embryogenesis, but is absent in adult rat liver. Our results suggest that it is reexpressed and functionally active during liver carcinogenesis.
Aldehyde Reductase↗