Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 361 records · Page 20Linked to original sources

PubMed1996

Pantoprazole has no influence on steady state pharmacokinetics and pharmacodynamics of metoprolol in healthy volunteers.

The new H+/K+ ATPase inhibitor pantoprazole, a substituted benzimidazole, is metabolized by the hepatic cytochrome P450 enzymes 2C19 or 3A4 and subsequently undergoes phase II metabolism. The widely used beta 1-adrenoreceptor-blocking agent metoprolol is metabolized via CYP2D6. The influence of pantoprazole on the pharmacokinetics of an orally administered zero order kinetics formulation (ZOK) of metoprolol was the objective of this study. Eighteen volunteers (9 female, 9 male, age 20-44 years) completed the randomized, double-blind crossover study. Each subject received either 95 mg metoprolol and placebo (reference (R)) or 95 mg metoprolol and 40 mg pantoprazole (test (T)) as oral doses (sid) for 5 consecutive days. On day 5 of each period serum concentrations of R- and S-metoprolol were determined and a treadmill ergometry was performed. The primary pharmacokinetic characteristics for extent and rate of absorption were AUC(0-24h) and % PTF, respectively. To assess the pharmacodynamic effect of metoprolol the excess area under the effect vs. time curve was calculated for heart rate during ergometry (AUCexHR). Point estimate and 90% confidence limits (CI) for the ratios of population medians of T and R were given. The respective point estimates (90% CI) of the ratios of both primary and secondary characteristics were entirely within the equivalence range of 0.80 and 1.25 for both enantiomers of metoprolol. Moreover, equivalence could be shown for the pharmacodynamic characteristic AUCexHR. Hence, it was concluded that pantoprazole does not interact with metoprolol pharmacokinetics or pharmacodynamics. Therefore, no dose adjustment of metoprolol during therapy with pantoprazole is necessary.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1996

Pantoprazole does not interact with nifedipine in man under steady-state conditions.

The new H+/K+ -ATPase inhibitor pantoprazole is extensively metabolized by the liver. As substituted benzimidazoles can interact with the cytochrome P450 system, the influence of pantoprazole on the steady-state pharmacokinetics of the calcium antagonist nifedipine was investigated. Nifedipine is widely used in the treatment of cardiovascular diseases and is mainly metabolized in the liver by CYP3A4. Additionally possible influence of gastric pH on the absorption of nifedipine is discussed. Twenty-four healthy volunteers (13 m/11 f) completed a randomized crossover study. As test they received orally 40 mg pantoprazole s.i.d. for 10 days and concomitantly 20 mg nifedipine sustained release (SR) b.i.d. from day 6 to 10. During the reference period 20 mg nifedipine SR were dosed b.i.d. for 5 days. Nifedipine and pantoprazole serum concentrations were measured over one dosing interval on the last day of each period. Lack of pharmacokinetic interaction was handled as an equivalence problem. The 90%-confidence intervals (CI) of the ratios of the primary characteristics AUC and Cmax of nifedipine were entirely within the equivalence range of 0.8 - 1.25. Hence no influence of pantoprazole on the pharmacokinetics of nifedipine was concluded, either by competition with the CYP3A4 or by the reduction of gastric acid secretion. As secondary criterion nifedipine had no relevant influence on the pantoprazole pharmacokinetic characteristics. All treatments were safe and well tolerated. No dose adjustment is required during concomitant treatment with nifedipine and pantoprazole.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1996

[Value of regional lymph node excision for prognosis of advanced malignant melanoma treated by perfusion of the extremity].

Staging by means of preoperative clinical screening will always stage melanoma study patients too favourably. Only a postoperative histopathological staging by means of lymphadenectomy will allow a correct stage-correlated patient prognosis. Lymphadenectomy does not prolong overall survival (95% confident intervals +/-6.6% to +/-25%) however prolongs recurrence free survival.

Antineoplastic Agents↗

PubMed1995

Cloning and sequencing of the gene coding for topoisomerase I from the extremely thermophilic eubacterium, Thermotoga maritima.

A 2767 bp fragment containing a gene coding for a topoisomerase I from the extremely thermophilic eubacterium Thermotoga maritima (Tm TopA) has been cloned and sequenced. The protein is composed of 633 amino acids with a calculated molecular mass of 72,695 Da. It shares significant similarity with the topoisomerases I of mesophilic eubacteria. The highest score is obtained with Bacillus subtilis (44% identity); in particular, T. maritima and B. subtilis possess an insertion of 7-8 amino acids in the vicinity of the active site, that is absent in topoisomerases of other organisms. A specific feature of T. maritima topoisomerase I is its low cysteine content compared to its mesophilic homologs. It contains 5 cysteine residues, of which 4 could constitute a zinc finger motif. Finally, analysis of the regions flanking the gene reveals that Tm TopA is surrounded by two other ORFs, suggesting the occurrence of a polycistronic transcriptional unit.

Amino Acid Sequence↗

PubMed1995

Active site topology and reaction mechanism of GTP cyclohydrolase I.

GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.

Amino Acid Sequence↗

PubMed1995

Structural and functional consequences of mutations in 6-pyruvoyltetrahydropterin synthase causing hyperphenylalaninemia in humans. Phosphorylation is a requirement for in vivo activity.

Four naturally occurring mutants with single amino acid alterations in human 6-pyruvoyltetrahydropterin synthase (PTPS) were overexpressed and characterized in vitro. The corresponding DNA mutations were found in patients with hyperphenylalaninemia and monoamine neurotransmitter insufficiency due to lack of the tetrahydrobiopterin biosynthetic enzyme PTPS. To predict the structure of the mutant enzymes, computer modeling was performed based on the solved three-dimensional structure of the homohexameric rat enzyme. One mutant (delta V57) is incorrectly folded and thus unstable in vitro and in vivo, while a second mutant (P87L) has substantial activity but enhanced sensitivity to local unfolding. Two other mutants, R16C and R25Q, form stable homomultimers and exhibit significant activity in vitro but no activity in COS-1 cells. In vivo 32P labeling showed that wild-type PTPS, P87L, and R25Q are phosphorylated, while R16C is not modified. This strongly suggests that the serine 19 within the consensus sequence for various kinases, RXXS, is the site of modification. Our results demonstrate that PTPS undergoes protein phosphorylation and requires additional, not yet identified post-translational modification(s) for its in vivo function.

Alcohol Oxidoreductases↗

PubMed1995

Crystal structure of the xanthine oxidase-related aldehyde oxido-reductase from D. gigas.

The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

Aldehyde Oxidoreductases↗

PubMed1995

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin.

Crystal structures of DX9065a and a related bisamidino-aryl inhibitor specific for the blood-clotting factor Xa have been solved in complex with bovine beta-trypsin to a resolution of 1.9 A. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibitors. Few direct contacts (predominantly in the S1 pocket) are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three-site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobic surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific '60-insertion loop', thus conferring selectivity against thrombin.

Amino Acid Sequence↗

PubMed1995

Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non-covalent complex between recombinant rhodniin and bovine alpha-thrombin reveals that the two Kazal-type domains of rhodniin bind to different sites of thrombin. The amino-terminal domain binds in a substrate-like manner to the narrow active-site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy-terminal domain, whose distorted reactive-site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 x 10(-13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino-terminal domain towards the active-site cleft, and of the electrostatic interactions between the carboxy-terminal domain and the exosite.

Amino Acid Sequence↗

PubMed1995

6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding.

6-Pyruvoyl tetrahydropterin synthase (PTPS) is an enzyme involved in tetrahydrobiopterin biosynthesis, the cofactor for several aromatic amino acid monooxygenases and the nitric oxide synthases. The crystal structure of PTPS was recently solved and showed a homohexameric enzyme composed of a dimer of trimers. A transition metal binding site formed by the three histidine residues 23, 48 and 50 was found in each subunit. We showed by metal analysis and reconstitution of apo-PTPS that Zn(II) was the bound transition metal and responsible for the enzymatic activity. Site-directed mutagenesis of each of these three histidine residues resulted in a complete loss of metal binding and enzymatic activity. The three residues, Cys42, His89 and Glu133, located close to the metal binding site, were previously postulated to be involved in the catalytic reaction. We altered these residues and found a complete loss of enzymatic activity for the mutant C42A. The two mutants, H89N and E133Q, showed 4.3% and 1.3% enzymatic activity, respectively, but had similar KM values for the substrate as compared to wild-type PTPS. Based on these results we propose a model of the substrate fitted into the active site and we described a novel intersubunit catalytic triad motif composed of the amino acid residues Cys42, His89 and Asp88. Different from most other catalytic triads that catalyse the hydrolysis of an amide or ester bond, the catalytic triad in the active site of PTPS seems to be involved in the deprotonation of the substrate's side-chain carbons. Our model also proposes Zn(II) as the coordination site for the two substrate side-chain hydroxy groups as well as the involvement of Glu133 as putative stereospecific proton server.

Alcohol Oxidoreductases↗

PubMed1995

Studies on the lumazine synthase/riboflavin synthase complex of Bacillus subtilis: crystal structure analysis of reconstituted, icosahedral beta-subunit capsids with bound substrate analogue inhibitor at 2.4 A resolution.

The lumazine synthase/riboflavin synthase of Bacillus subtilis is a bifunctional enzyme complex catalysing the formation of riboflavin from 5-amino-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and L-3,4-dihydroxy-2-butanone-4-phosphate via 6,7-dimethyl-8-ribityllumazine. The complex is composed of 3 alpha (riboflavin synthase) subunits and 60 beta (lumazine synthase) subunits and has a relative mass of 1 MDa. The 60 beta subunits are arranged in an icosahedral capsid enclosing the alpha trimer in the central core. The protein was crystallised, and an X-ray structure of the icosahedral capsid was obtained at 3.3 A resolution with a crystallographic R-factor of 0.33. Hollow, icosahedral capsids consisting of 60 beta subunits can be obtained by inhibitor-driven renaturation of isolated beta subunits. They catalyse the formation of 6,7-dimethyl-8-ribityllumazine at the same rate as the native alpha 3 beta 60 complex and can be crystallised in two different hexagonal and one monoclinic form. Crystallographic intensity data of the monoclinic crystals to a resolution of 2.4 A were obtained using synchrotron radiation and an image plate detector system. The orientation of the icosahedral molecules in the monoclinic cell was deduced by real space vector search procedures from a 3.5 A Patterson map. Phases were calculated from the model of the alpha 3 beta 60 protein and were extended by cyclic averaging exploring the 30-fold redundancy of the electron density. The 2.4 A map allowed us to refine the existing atomic model of lumazine synthase. The refined model includes 154 amino acid residues, one inhibitor molecule, 58 water molecules and one phosphate ion. Applying non-crystallographic-symmetry restraints the crystallographic R-factor is 16.7% for 100,092 reflections between 10 and 2.4 A. The chain folding of the beta subunits is closely similar to the native alpha 3 beta 60 enzyme. The lumazine synthase bears resemblance to the sugar binding proteins. The significantly higher resolution compared to the alpha 3 beta 60 structure determination allows a detailed description of the substrate analogue binding site. The environment of the 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione inhibitor is particularly rigid, and the chain segments involved in forming the active site are highly conserved for lumazine synthases of different species. A residual density feature in the final map is interpreted as a bound phosphate which mimics the binding of the second substrate. We discuss the reaction mechanism on this structural basis.

Amino Acid Sequence↗

PubMed1995

Elucidation of crystal packing by X-ray diffraction and freeze-etching electron microscopy. Studies on GTP cyclohydrolase I of Escherichia coli.

A monoclinic crystal modification of GTP cyclohydrolase I (space group P2(1), a = 204.2 A, b = 210.4 A, c = 71.8 A, alpha = gamma = 90 degrees, beta = 95.8 degrees) was studied by freeze-etching electron microscopy and by Patterson correlation techniques. The freeze-etched samples were either shadowed with Pt/C or decorated with monolayers of gold, silver or platinum. Correlation averaged electron micrographs of decoration replicas indicated 5-fold molecular symmetry. In conjunction with the molecular mass of the active GTP cyclohydrolase I enzyme complex of about 210,000 Da, which had been reported in the literature, and a molecular mass of the protomers of 24,700 Da, the electron microscopic observation suggests that the enzyme is a decamer with 5-fold symmetry. The processed images of decorated crystal surfaces also showed that the four protein multimers in the crystal unit cell are related by 4-fold pseudosymmetry. A Patterson analysis of the X-ray data showed two non-crystallographic 5-fold axes, inclined at 12 degrees to each other, thus confirming and extending the electron microscopic findings. Additionally, local 2-fold axes were found in planes perpendicular to the 5-fold particle axes. Thus, the combined X-ray and electron microscope data indicate that GTP cyclohydrolase I is a decamer with D5 symmetry. A procedure for hkl assignments of the crystal planes observed in electron micrographs was developed. On this basis, it was possible to determine the approximate molecular positions in the ab plane. Independent information on the crystal packing was obtained by single isomorphous replacement and electron density averaging. The 5-fold averaged 6 A electron density shows that the GTP cyclohydrolase I decamer is torus-shaped with an approximate diameter of 100 A and a thickness of 65 A. The study demonstrates that the combination of freeze-etching electron microscopy with Patterson analysis of X-ray data is a powerful approach for the solution of complex crystallographic problems. The procedure for this analysis as well as possible pitfalls are discussed in detail.

Crystallization↗

PubMed1995

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

Amino Acid Chloromethyl Ketones↗

PubMed1995

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.

Animals↗

PubMed1995

Crystal structure of desulforedoxin from Desulfovibrio gigas determined at 1.8 A resolution: a novel non-heme iron protein structure.

The crystal structure of desulforedoxin from Desulfovibrio gigas, a new homo-dimeric (2 x 36 amino acids) non-heme iron protein, has been solved by the SIRAS method using the indium-substituted protein as the single derivative. The structure was refined to a crystallographic R-factor of 16.9% at 1.8 A resolution. Native desulforedoxin crystals were grown from either PEG 4K or lithium sulfate, with cell constants a = b = 42.18 A, c = 72.22 A (for crystals grown from PEG 4K), and they belong to space group P3(2)21. The indium-substituted protein crystallized isomorphously under the same conditions. The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete beta-barrel with the two iron centers placed on opposite poles of the molecule. Each iron atom is coordinated to four cysteinyl residues in a distorted tetrahedral arrangement. Both iron atoms are 16 A apart but connected across the 2-fold axis by 14 covalent bonds along the polypeptide chain plus two hydrogen bonds. Desulforedoxin and rubredoxin share some structural features but show significant differences in terms of metal environment and water structure, which account for the known spectroscopic differences between rubredoxin and desulforedoxin.

Amino Acid Sequence↗

PubMed1995

Determination of hemihedral twinning and initial structural analysis of crystals of the procarboxypeptidase A ternary complex.

The initial structural analysis of the ternary complex of procarboxypeptidase A from hemihedrally twinned crystals diffracting up to 2.8 A is described. Detection of twinning by different techniques is presented, including biochemical and intensity statistics approaches. The structure was initially solved using Patterson-search techniques, and the three positioned search models were used to effectively deconvolute the twinned data.

Journal Article↗

PubMed1995

Three-dimensional crystals of cytochrome-c oxidase from Thermus thermophilus diffracting to 3.8 A resolution.

The ba3-type cytochrome-c oxidase from Thermus thermophilus has been crystallized in its native form. Crystallization was achieved by the batch and the vapour diffusion sitting drop methods using polyethylene glycol monomethyl ether 2000 as precipitating agent in the presence of octyl-beta-D-thioglucoside as detergent. The crystals diffract to 3.8 A, belong to the space group P2 or P2(1) and have unit cell dimensions of a = 80.7 A; b = 116.0 A; c = 156.9 A and beta = 104.4 degrees. The asymmetric unit contains two ba3-type oxidase molecules.

Crystallography, X-Ray↗

PubMed1995

Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis.

A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities.

Archaea↗