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Thomas Steiner

Publications and source records attributed to Thomas Steiner.

15 recordsLinked to original sources
PubMed2006

X-ray crystallographic structures of enamine and amine Schiff bases of pyridoxal and its 1:1 hydrogen-bonded complexes with benzoic acid derivatives: evidence for coupled inter- and intramolecular proton transfer.

Crystal structures of Schiff bases containing pyridoxal (PL), N-(pyridoxylidene)-tolylamine, C(15)H(16)N(2)O(2) (I), N-(pyridoxylidene)-methylamine, C(9)H(12)N(2)O(2) (III), and their 1:1 adduct with 2-nitrobenzoic acid, (I)(+) C(7)H(4)NO_4;- (II), and 4-nitrobenzoic acid, (III)(+) C(7)H(4)NO_4;- (IV), serve as models for the coenzyme pyridoxal-5'-phosphate (PLP) in its PLP-dependent enzymes. These models allow the study of the intramolecular OHN hydrogen bond of PL/PLP Schiff bases and the H-acceptor properties of their pyridine rings. The free base (I) forms hydrogen-bonded chains involving the hydroxyl side groups and the rings of adjacent molecules, whereas (III) forms related hydrogen-bonded cyclic dimers. The adducts (II)/(IV) consist of 1:1 hydrogen-bonded complexes, exhibiting strong intermolecular bonds between the carboxylic groups of the acids and the pyridine rings of (I)/(III). In conclusion, the proton in the intramolecular O-H...N hydrogen bond of (I)/(III) is located close to oxygen (enolamine form). The added acids protonate the pyridine ring in (II)/(IV), but only in the latter case does this protonation lead to a shift of the intramolecular proton towards the nitrogen (ketoimine form). All crystallographic structures were observed in the open form. In contrast, the formation of the pyridinium salt by dissolving (IV) leads to the cyclic aminal form.

Benzoates↗
PubMed2005

A four-subunit cytochrome bc(1) complex complements the respiratory chain of Thermus thermophilus.

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc(1) complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc(1) subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc(1) complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c(1) carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c(552), mediating electron transfer to the ba(3) oxidase. Identification of this cytochrome bc(1) complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.

Amino Acid Sequence↗
PubMed2005

Long-term prognosis of patients after kidney transplantation: a comparison of those with or without diabetes mellitus.

BACKGROUND: Compared with non-diabetic subjects, patients with type 2 diabetes and end-stage renal disease (ESRD) have seldom been selected for renal transplantation. It was the aim of this study to compare the long-term prognoses of the two groups of patients after transplantation and to identify factors associated with allograft rejection. METHODS: In a retrospective analysis, we studied all 333 consecutive patients who received a kidney transplant at our centre since 1992. Mean follow-up in 302 out of 333 patients (91%) was 3.3+/-1.5 (0.1-11.7) years. At the time of transplantation, diabetes mellitus (type 1, n=3; type 2, n=46) was known in 49 patients. RESULTS: Patients with diabetes mellitus were older [patients without diabetes (n=253) vs patients with diabetes (n=49), 52.2+/-12.6 vs 58.8+/-13.1 years, respectively; P=0.002], but they had very good diabetes control [haemoglobin A1c (HbA1c) of patients with diabetes 6.3+/-0.9% vs those without diabetes 5.2+/-1.0%, P=0.03]. Even during their follow-up, patients with diabetes showed a tendency to further improvement (HbA1c for patients with diabetes 5.7+/-0.9% vs those without diabetes 5.5+/-0.9%, P=0.30). At the end of follow-up also, there were no differences between the groups with respect to blood pressure control (patients with diabetes 135.3+/-28.2/79.6+/-17.2 mmHg vs patients without diabetes 130.9+/-28.7/78.8+/-17.1 mmHg, P=0.33/0.78) and renal function (creatinine, 142.9+/-61.6 vs 151.8+/-68.2 micromol/l, P=0.38; glomerular filtration rate, 63.1+/-23.3 vs 59.1+/-24.0 ml/min/1.73 m(2), respectively, P=0.30). In total, 26 patients had acute transplant rejections [eight patients with diabetes (prevalence 16.3%) vs 18 patients without diabeteses (prevalence 7.1%), P=0.11]. In multivariate analysis, the most important parameter associated with the incidence of transplant rejections was the preceding fasting blood glucose (R2=0.044, beta=0.21, P=0.009). All other parameters included in the model (body mass index, time since transplantation, diabetes duration, immunosuppressive therapy, HbA1c and HLA mismatch) revealed no associations. CONCLUSIONS: Following kidney transplantation, the prevalence of rejections in patients with diabetes mellitus is slightly but not significantly higher than in non-diabetic subjects. One of the most important risk factors seems to be fasting blood glucose. Hence, following renal transplantation, treatment strategies should focus not only on optimal immunosuppressive therapy and HLA matching, good HbA1c and blood pressure control, but also on maintaining near-normal fasting blood glucose levels.

Aged↗
PubMed2005

Post-transplant diabetes mellitus: risk factors, frequency of transplant rejections, and long-term prognosis.

BACKGROUND: Estimates of the incidence of new-onset diabetes after renal transplantation vary between 2% and 54%. It was the aim of the present trial to study the prevalence of post-transplant diabetes mellitus (DM), the risk factors, the frequency of transplant rejections, and the long-term prognosis. METHODS: We studied all consecutive patients with endstage renal disease, but without DM who received kidney transplantation at our center since 1992 (n = 253; age, 52.2 +/- 12.6 years; body mass index, 22.0 +/- 7.9 kg/m2). Follow up was 3.3 +/- 1.6 years (range, 0.1-17.7) years. RESULTS: In total, 43/253 patients (17%) developed new-onset DM after transplantation. Patients with new-onset diabetes were significantly older (58.3 +/- 11.4 vs 50.9 +/- 12.5 years; P < 0.01) and had a tendency to a higher body mass index (24.0 +/- 8.5 vs 21.6 +/- 7.8 kg/m2; P = 0.077). There were no differences between the groups in respect of blood pressure control (137.7 +/- 19.0/81.8 +/- 14.2 vs 137.1 +/- 21.9/83.9 +/- 13.1 mmHg; P = 0.89/0.39), glomerular filtration rate (58.0 +/- 28.1 vs 64.1 +/- 22.1 ml/min per 1.73 m2; P = 0.13), steroid dosage (4.5 +/- 1.2 [n = 21] vs 4.6 +/- 2.2 [n = 135] mg/day; P = 0.13), or the frequency and dosage of immunosuppressive drugs such as cyclosporine, tacrolimus, and sirolimus during the follow up. However, more patients with post-transplant diabetes received steroids (83.7% vs 64.3%; P = 0.021) and azathioprine (41.9% vs 24.3%; P = 0.030). Patients with new-onset diabetes had higher serum creatinine values (163.4 +/- 67.9 vs 138.7 +/- 59.5 micromol/l; P = 0.017). The mean hemoglobin (Hb)A1c in patients with DM was 6.28 +/- 1.29% (Tosho HPLC; mean normal, 5.15%). In 18 patients (7.1%) transplant rejections occurred (16 patients without DM [7.6%] vs 2 patients with new-onset DM [4.7%]; P = 0.39). On performing multivariate analysis, the only parameter found to be associated with new-onset DM was the body mass index (R2 = 0.05; beta = 0.23; P = 0.02), and the only factor associated with transplant rejection was fasting blood glucose (R2 = 0.07; beta = 0.28; P = 0.02). None of the other parameters included in the models (age, duration after transplantation, diabetes duration, immunosuppressive therapy, HbA1c, HLA mismatches) showed any associations. CONCLUSIONS: The prevalence of new-onset DM after renal transplantation was 17%. The most important parameter associated with new-onset diabetes was a higher body mass index, and the most important parameter associated with transplant rejection was an elevated fasting blood glucose level. To prevent transplant rejections and to improve patients' outcome, in addition to providing optimal immunosuppressive therapy and HLA matching, good blood pressure control and HbA1c, but also near normal fasting blood glucose levels, should be achieved.

Aged↗
PubMed2004

Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis.

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.

Adenine↗
PubMed2004

Prognostic significance of tumour size in patients after tumour nephrectomy for localised renal cell carcinoma.

OBJECTIVES: Staging of the primary tumour is accepted as the most important prognostic factor in organ confined renal cell carcinoma. METHODS: The outcome of 286 patients with non-metastatic RCC treated by radical nephrectomy at our institution between 1968 and 1992 was evaluated retrospectively. The median follow-up was 114 +/- 62.6 months. In T1/T2 tumours, the validity of tumour size cut-off points for predicting survival outcome was tested. RESULTS: According to the 1997 TNM classification, 168 patients (59%) showed pathological stage T1 (72 stage T1a, 96 stage T1b), 30 (10%) showed stage T2, 84 patients (29%) demonstrated T3 tumours (53 stage T3a, 31 stage T3b), and 4 patients (2%) presented with T4 tumours. The median survival estimated by Kaplan-Meier analysis for T1a, T1b, T2, T3a, T3b and T4 tumours was over 300 months, 187.0 +/- 32.76; 177.0 +/- 1.21; 121.0 +/- 2.57; 124.0 +/- 11.82 and 52.0 +/- 18.38 months, respectively. Regarding survival in T1/T2 tumours Cox regression analysis yielded the highest significance level for a tumour size cut-off point at 4 cm (p = 0.003; 95%CI 1.511-6.991), but no prognostic value for a cut-off point at 7 cm (T1 vs. T2) (p = 0.375; 95%CI 0.655-3.071). CONCLUSIONS: Tumour size is an important prognostic factor in patients with organ confined RCC. The recently specified new cut-off point of 4 cm for T1a/T1b tumours is feasible for separating groups with different survival after tumour nephrectomy and should be considered as the new boundary between T1 and T2 stages. Hence, a more accurate prediction of prognostic differences between these groups should be possible.

Adult↗
PubMed2004

Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code.

The expanded genetic code in combination with site-directed mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange ('atomic mutation') and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphur-aromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.

Aequorin↗
PubMed2002

Crystal structures of transcription factor NusG in light of its nucleic acid- and protein-binding activities.

Microbial transcription modulator NusG interacts with RNA polymerase and termination factor rho, displaying striking functional homology to eukaryotic Spt5. The protein is also a translational regulator. We have determined crystal structures of Aquifex aeolicus NusG showing a modular design: an N-terminal RNP-like domain, a C-terminal element with a KOW sequence motif and a species-specific immunoglobulin-like fold. The structures reveal bona fide nucleic acid binding sites, and nucleic acid binding activities can be detected for NusG from three organisms and for the KOW element alone. A conserved KOW domain is defined as a new class of nucleic acid binding folds. This module is a close structural homolog of tudor protein-protein interaction motifs. Putative protein binding sites for the RNP and KOW domains can be deduced, which differ from the areas implicated in nucleic acid interactions. The results strongly argue that both protein and nucleic acid contacts are important for NusG's functions and that the factor can act as an adaptor mediating indirect protein-nucleic acid associations.

Amino Acid Sequence↗
PubMed2002

A neutral molecule in a cation-binding site: specific binding of a PEG-SH to acetylcholinesterase from Torpedo californica.

The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 A resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH(2) groups of the inhibitor, which are engaged in C-H...pi interactions that structurally mimic the cation-pi interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H...O and C-H...pi types.

Acetylcholinesterase↗
PubMed2002

Hydrogen bonds from water molecules to aromatic acceptors in very high-resolution protein crystal structures.

Short contacts of water molecules with the pi-faces of aromatic residues were studied in a set of 75 very high resolution (<1.1 A) protein X-ray crystal structures. For 18 water molecules found at distances to aromatic midpoints <3.5 A, it was attempted to assign the hydrogen bond configuration (without experimental knowledge of the H-atom positions) by inspection of the surrounding. For approximately one-quarter of the cases, evidence for an O-H...pi hydrogen bond was found, another one-quarter does not form such a hydrogen bond, and for the remaining half, no conclusive assignment could be made. The results confirm the relatively frequent occurrence of aromatic hydrogen bonding in biomolecular hydration, but also underline difficulties in hydrogen bond assignment without reliable knowledge of the H-atom positions.

Amino Acids, Aromatic↗
PubMed2002

Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism.

The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.

Binding Sites↗
PubMed2002

The hydrogen bond in the solid state.

The hydrogen bond is the most important of all directional intermolecular interactions. It is operative in determining molecular conformation, molecular aggregation, and the function of a vast number of chemical systems ranging from inorganic to biological. Research into hydrogen bonds experienced a stagnant period in the 1980s, but re-opened around 1990, and has been in rapid development since then. In terms of modern concepts, the hydrogen bond is understood as a very broad phenomenon, and it is accepted that there are open borders to other effects. There are dozens of different types of X-H.A hydrogen bonds that occur commonly in the condensed phases, and in addition there are innumerable less common ones. Dissociation energies span more than two orders of magnitude (about 0.2-40 kcal mol(-1)). Within this range, the nature of the interaction is not constant, but its electrostatic, covalent, and dispersion contributions vary in their relative weights. The hydrogen bond has broad transition regions that merge continuously with the covalent bond, the van der Waals interaction, the ionic interaction, and also the cation-pi interaction. All hydrogen bonds can be considered as incipient proton transfer reactions, and for strong hydrogen bonds, this reaction can be in a very advanced state. In this review, a coherent survey is given on all these matters.

Journal Article↗
PubMed2002

Gain in chromosome 8q correlates with early progression in hormonal treated prostate cancer.

OBJECTIVE: Development and especially hormone refractant progression of prostate cancer are incompletely understood. Clinical studies evaluating genetic aberrations of prior therapy biopsies in correlation with progression data in patients receiving hormone therapy for prostate cancer have not been performed until now. METHODS: After DNA isolation from histological sections of primary prostate cancer biopsies, comparative genomic hybridization (CGH) was performed according to standard protocols. Primary staging, clinical course and PSA levels of the patients were assessed. RESULTS: CGH was performed on 28 primary prostate cancer samples. After a mean follow-up of 36 months 11 (39%) of the patients showed progression of disease under hormonal treatment. In patients without and with progression we found the following results, respectively: losses of 6q (41/36%), 8p (41/45%), 16q (23/18%), 18q (30/9%), and gains of 8q (12/64%; P < 0.0001) and 17 (47/26%). CONCLUSIONS: Gain of 8q is found predominantly in primary core biopsies of local advanced or metastasized prostate cancers. It shows in univariate analysis significant correlation with progression in hormone treated prostate cancer. This fact suggests that gain in 8q represents a marker of aggressiveness in prostate cancer.

Aged↗