Biomedical subjects

T Stein

Publications and source records attributed to T Stein.

At least 37 records · Page 2Linked to original sources

PubMed1998

[Circumscribed apical left ventricular hypertrophy. Dynamic development and long term progression].

HISTORY AND CLINICAL FINDINGS: A 64-year-old obese man had for 15 years suffered from exercise-independent retrosternal pressure sensation, radiating to the neck and back. Shortly after the onset of these symptoms he had undergone coronary angiography with negative results. But at that time the resting ECG showed discrete T wave negativity in the left precordial leads. INVESTIGATIONS: At the present admission the ECG showed deeply inverted T waves in the left precordial and limb leads and a positive Sokolow-Lyon index of 4.8 mV. Left ventricular angiography demonstrated in enddiastole a circumscribed myocardial hypertrophy limited to the apex and of typical "ace of spade" shape. DIAGNOSIS, TREATMENT AND COURSE: Left-heart catheterization and angiocardiography provided the diagnosis of circumscribed apical left ventricular hypertrophy (ALVH). As the patient had only minor symptoms no treatment was given. CONCLUSION: Circumscribed ALVH can show marked dynamic development in long-term observations. If there is marked T wave negativity, even with previously normal LV angiography, circumscribed ALVH should be included in the differential diagnosis. Patients with atypical angina pectoris and increasingly suggestive ECG changes should, even if previous coronary angiography had been negative, undergo transthoracic echocardiography with a high-frequency transducer, special attention being paid to muscular changes at the LV apex.

Angina Pectoris↗

PubMed1998

Separation and Characterization of Surfactin Isoforms Produced by Bacillus subtilis OKB 105

Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a beta-hydroxy fatty acid with chain lengths of 13-15 carbon atoms. The lipopeptide biosurfactant was produced by Bacillus subtilis OKB 105 and part of the material subjected to esterification of its Glu and Asp residues. High-resolution preparative reversed phase HPLC on EnCaPharm 100 of surfactin and its monomethyl and dimethyl esters yielded 44 fractions which were characterized by NMR and MS methods. Among the separated isoforms are the known surfactin variants with l-Leu, l-Val, or l-Ile in position 7 of the peptide ring and three hitherto unknown variants showing replacements of the leucine residues in position 2 and/or 7 by l-Val and l-Ile. Our work makes available lipoheptapeptide compounds with modified structures and different hydrophobicities which promise to have potential for biotechnological and pharmaceutical applications. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Characterization of the binding site of the tripeptide intermediate D-Phenylalanyl L-prolyl-L-valine in gramicidin S biosynthesis.

The tripeptide intermediate D-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of either L-[14C]phenylalanine or L-[14C]valine in an in vitro biosynthetic assay. The gramicidin S synthetase 2-tripeptide complex was first digested with CNBr and subsequently by Staphylococcus aureus V8 protease. The active site peptide carrying the radioactively labeled tripeptide was isolated in pure form by reversed phase high performance liquid chromatography technology and analyzed by liquid phase sequencing, mass spectrometry, and amino acid analysis. It was demonstrated that D-Phe-Pro-Val is attached to the 4'-phosphopantetheine cofactor at the thiolation center for valine of gramicidin S synthetase 2. In this way the attachment site of a peptide intermediate in nonribosomal peptide biosynthesis was identified for the first time. Our results are in full agreement with the multiple carrier model of nonribosomal peptide biosynthesis (Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R., and Morris, H. R. (1996) J. Biol. Chem. 271, 15426-15435), which predicts that the growing peptide chain in the elongation process should always be bound to the thiotemplate site specific for its C-terminal amino acid component.

Amino Acid Isomerases↗

PubMed1998

[Noninvasive determination of coronary flow reserve with signal enhanced high resolution transthoracic Doppler color echocardiography].

UNLABELLED: The feasibility of non-invasive assessment of coronary flow reserve (CFR) in the left anterior descending artery (LAD) using echo-enhanced high-resolution transthoracic color Doppler echocardiography (TTCD) was investigated. The results were compared with the degree of coronary diameter-stenosis obtained during cardiac catheterization. CFR has proven to be useful in the selection of patients undergoing invasive treatment of coronary artery disease and in estimating their prognosis. However, CFR could only be determined in everyday practice invasively during catheterization procedures. Recent development of high-resolution TTCD allows transthoracic visualization of distal LAD and supra-apical intramyocardial perforator branches, and non-invasive measurement of CFR. CFR was determined by measuring the ratio of pulsed-wave Doppler time velocity integral during adenosine-induced hyperemia (140 micrograms/kg/min i.v.) to baseline value. If Doppler signal of LAD flow was insufficiently at basal condition, an echo enhancer (Levovist) was used. 45 patients were examined by TTCD (7 MHz B-mode, 5 MHz color Doppler, 3.5 MHz PW Doppler) after coronary angiography had been performed. Group I consisted of 15 patients without heart disease, Group II of 15 patients with 40 to 70% isolated LAD diameter stenosis, and Group III of 15 patients with > 70% LAD diameter stenosis. Peripheral LAD coronary flow at baseline condition was assessed in 40 patients (88%) using TTCD. CFR could be quantified in 36/45 patients (80%), in 18 patients without echo enhancer, and in 18 patients with echo-enhancing agent. In 9/45 patients CFR could not be assessed. CFR in Group I was 3.13 +/- 0.57, in Group II 2.23 +/- 0.20 (vs Group I p < 0.01) and in Group III 1.64 +/- 0.30 (vs Group II p < 0.02). CONCLUSION: CFR of LAD can be determined in 80% of patients by the synergistic use of high-resolution TTCD combined with intravenous given ultrasound echo-enhancing agent.

Adult↗

PubMed1998

Azf1p is a nuclear-localized zinc-finger protein that is preferentially expressed under non-fermentative growth conditions in Saccharomyces cerevisiae.

In previous studies the AZF1 gene has been identified as a second high-copy number suppressor for a special mutant of the gene for the mitochondrial core enzyme of RNA polymerase. The first high-copy number suppressor of this mutant turned out to be the specificity factor MTF1 for mitochondrial transcription. Up to now, the influence of AZF1 on mitochondrial transcription, its precise localization in the cell and the regulation of its expression has not been determined. The putative protein contains a long stretch of poly-asparagine amino acids and a typical zinc-finger domain for DNA binding. These characteristic structural features were used to create the abbreviation AZF1 (Asparagine-rich Zinc Finger protein). An initial computer analysis of the sequence gave no conclusive results for the presence of a mitochondrial import sequence or a typical nuclear-targeting sequence. A recent more-detailed analysis identified a possible nuclear localization signal in the middle of the protein. Disruption of the gene shows no effect on plates with glucose-rich medium or glycerol. In this report a specific polyclonal antibody against Azf1p was prepared and used in cell-fractionation experiments and in electron-microscopic studies. Both of these clearly demonstrate that the AZF1 protein is localized exclusively in the nucleus of the yeast cell. Northern analysis for the expression of the AZF1 messenger RNA under different growth conditions was therefore performed to obtain new insights into the regulation of this gene. Together with the respective protein-expression analysis these data demonstrate that Azf1p is preferentially synthezised in higher amounts under non-fermentable growth conditions. Over-expression of Azf1p in the yeast cell does not influence the expression level of the mitochondrial transcription factor Mtf1p, indicating that the influence of Azf1p on the suppression of the special mitochondrial RNA polymerase mutant is an indirect one. Subcellular investigation of the deletion mutant by electron microscopy identifies specific ultrastructural cell-division defects in comparison to the wild-type.

Carrier Proteins↗

PubMed1998

A randomized biomechanical study of zone II human flexor tendon repairs analyzed in a linear model.

Komanduri et al showed that dorsal tendon repairs using Kessler and Bunnell techniques were stronger than the standard volar repair (J Hand Surg 1996;21 A:605-611). They concluded that when testing in the anatomic curvilinear mode, the differences in strength were due to tension banding. Soejima et al challenged that concept by stating that the difference in strength was in the biomechanics of the dorsal tendon itself (J Hand Surg 1995;20A:801-807). We set out to confirm Soejima et al's theory by using more core suture techniques. We compared the tensile strength at 2-mm gap and the ultimate tensile strength of Kessler, Strickland, Robertson, and modified Becker sutures. Ten repairs of each suture type were placed either dorsally or volarly in matched human cadaver flexor tendons. There was no statistical difference between volar and dorsal placement for either maximum tensile force or force at 2-mm gap. Our study does not confirm Soejima et al's in any of the four suturing techniques.

Biomechanical Phenomena↗

PubMed1998

A randomized biomechanical study of zone II human flexor tendon repairs analyzed in an in vitro model.

To study the effect of dorsally placed flexor tendon repairs on human cadaver tendons, we used the Kessler, Strickland, Robertson, and modified Becker techniques. The strengths of the repairs were tested in a previously described human cadaveric curvilinear model. There were statistically significant increases in dorsal versus volar grasping strength with the Kessler repair (38 N vs 33 N) and the Robertson repair (51 N vs 43 N). There were no differences with the locking Strickland and modified Becker repairs. One may infer that the locking techniques are intrinsically tighter and may not be as susceptible to the different forces between the tensile and compressive surfaces of a dynamic flexor tendon.

Cadaver↗

PubMed1997

[Signal improvement of pulmonary venous Doppler flow prifile after intravenous injection of levovist].

The analysis of the pulmonary venous flow Doppler pattern can assist in the determination of the severity of mitral regurgitation and, in conjunction with transmitral flow pattern, the assessment of left ventricular diastolic dysfunction. In about one third of the cases, however, transthoracic ultrasonography is not able to record an adequately analyzable pulmonary venous flow pattern. The aim of the study was to examine and compare the effect of the echo-enhancing agent Levovist on the pulsed-wave Doppler flow quality of the transthoracically (TTE) and transesophageally (TEE) recorded pulmonary venous flow. In 26 consecutive patients, a qualitative (score system) and quantitative analysis of the pulmonary venous flow pattern was obtained before and after peripheral venous injection of Levovist at concentrations of 200 mg/ml (low dose) and 400 mg/ml (high dose). The number of measurable studies for the antegrade pulmonary venous flow increased after Levovist from 85% to 96% for TTE and from 96% to 100% for TEE. The retrograde flow as seen by TTE was adequately analyzable in only 45% before and in 73% after injection of Levovist (p < 0.02). Before any contrast enhancement, the retrograde pulmonary venous flow recorded by TEE could be analyzed in 77% of the patients with the percentage increasing to 88% and 92% after administration of a low and high dose of Levovist, respectively (p < 0.05). In particular, the quality score of the retrograde flow was significantly altered by the administration of Levovist (increase from 1.8 +/- 1.0 to 2.6 +/- 1.1 (low dose Levovist), p < 0.05 and to 2.7 +/- 1.3 (high dose Levovist). p < 0.05). The pulsed-wave Doppler evaluation by TTE without Levovist underestimated the velocities of the antegrade and retrograde pulmonary venous flow After administration of Levovist, the recorded values are comparable to those obtained by TEE. An analogous pattern is encountered when quantifying the duration of the retrograde flow component. Thus, the peripheral venous injection of Levovist leads to an improved quality of the pulmonary venous flow Doppler signal recorded by TTE. Qualitatively and quantitatively the values recorded by TTE after administration of Levovist are comparable to those of the TEE technique without an echo-enhancing agent.

Adult↗

PubMed1997

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Amino Acid Isomerases↗

PubMed1996

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↗

PubMed1996

Oriented binding of a lipid-anchored cell adhesion protein onto a biosensor surface using hydrophobic immobilization and photoactive crosslinking.

The carboxymethyl-dextran surface of a biosensor instrument was modified to couple, in an active state, the lipid-anchored contact site A (csA) glycoprotein, a homophilic adhesion molecule of aggregating cells of Dictyostelium discoideum. The carboxy groups were modified by heptyl residues for hydrophobic binding of the molecule with its lipid anchor to the dextran matrix. Alternatively, the protein was fixed in a similar orientation by covalent linkage through a perfluorophenylazide-derived hydrophobic crosslinker. Titration of the bound csA molecules with antibodies that recognize either the native or the denatured glycoprotein verified that the csA molecules were coupled in a native state to the sensor surface. Interaction of the immobilized csA protein with csA in solution established that the bound molecules are capable of taking part in homophilic interactions.

Animals↗

PubMed1996

A new point mutation in the nuclear gene of yeast mitochondrial RNA polymerase, RPO41, identifies a functionally important amino-acid residue in a protein region conserved among mitochondrial core enzymes.

The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

Amino Acid Sequence↗

PubMed1996

[An absent correlation between antioxidant blood concentrations and the remission response of preoperatively treated breast carcinomas].

PURPOSE: Deleterious effects of ionizing radiation and some chemotherapeutic agents are predominantly caused by reactive oxygen agents which are detoxified by antioxidants. This study was designed to evaluate the modifying effects of vitamin A-, vitamin E- and selenium serum concentrations and glutathione peroxidase activity on preoperative radio- and chemotherapy of breast cancer. PATIENTS AND METHODS: Tumor volume, vitamin A-, vitamin E-, selenium serum concentrations and glutathione peroxidase activity in circulating erythrocytes were determined in 40 patients with breast cancer before treatment. Interstitial radiohyperthermia was given initially using a single dose of 10 Gy (HDR) combined with hyperthermia between 43.5 to 44.5 degrees C over 60 minutes followed by external beam radiotherapy with 50 Gy in 5 weeks. All patients received anthracyline or anthrachinone containing chemotherapy. Tumor response was determined by histopathological examination. Patients with complete and incomplete remissions were compared using the Wilcoxon test. Pre- and posttreatment tumor-volume differences were correlated with antioxidant concentrations (Spearman correlation coefficient). RESULTS: Twenty patients (50%) achieved a complete histopathologic tumor regression. This high complete remission rate was not related to the antioxidants under investigation (vitamin A: p = 0.32, vitamin E: p = 0.44, selenium: p = 0.68, glutathione peroxidase: p = 0.3). There was no correlation to pre- and posttreatment tumor-volume-differences either (vitamin A: p = 0.89, vitamin E: p = 0.67, selenium: p = 0.41, glutathione peroxidase: p = 0.87). CONCLUSIONS: In this study serum concentrations of antioxidants had no modifying influence on tumor response in breast cancer patients following induction radio-chemotherapy and subsequent surgery. Further studies measuring tissue levels could clarify if there is any modifying influence of antioxidants on tumor remission.

Adult↗

PubMed1995

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↗

PubMed1995