Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 235 records · Page 13Linked to original sources

PubMed1998

Cutting at the right place--the importance of selective limited proteolysis in the activation of proproteinase E.

Proteinase E is a proteolytic enzyme which belongs to a distinct subfamily of chymotrypsin-like serine endopeptidases. Its proform from the bovine pancreatic system has been structurally analyzed by X-ray crystallography for the intact native form, with a 11-residue N-terminal activation peptide, in a ternary complex with chymotrypsinogen C and procarboxypeptidase A [Gomis-Rüth, F. X., Gómez, M., Bode, W., Huber, R. & Avilés, F. X. (1995) The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C, EMBO J. 14, 4387-4394]. Also for a N-terminally truncated form, lacking the first 13 residues and called subunit III, a crystal structure is available [Pignol, D., Gaboriaud, C., Michon, T., Kerfelec, B., Chapus, C. & Fontecilla-Camps, J. C. (1994) Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E, EMBO J. 8, 1763-1771]. Both structures are well defined by electron density, except for the first 7 residues of subunit III. However, both structures present large deviations of up to 2 nm in several regions, indicating that they correspond to two quite distinct states of low free energy, influenced by very few contacts made via the N-terminal segment. As no structure of an active proteinase E is known so far, pancreatic porcine elastase has been chosen as a model for this enzyme and an activation mechanism for this distinct serine endopeptidase subfamily is proposed.

Amino Acid Sequence↗

PubMed1998

Interactions of benzodiazepine derivatives with annexins.

Human annexins III and V, members of the annexin family of calcium- and membrane-binding proteins, were complexed within the crystals with BDA452, a new 1,4-benzodiazepine derivative by soaking and co-crystallization methods. The crystal structures of the complexes were analyzed by x-ray crystallography and refined to 2.3- and 3.0-A resolution. BDA452 binds to a cleft which is located close to the N-terminus opposite to the membrane binding side of the proteins. Biophysical studies of the interactions of various benzodiazepine derivatives with annexins were performed to analyze the binding of benzodiazepines to annexins and their effects on the annexin-induced calcium influx into phosphatidylserine/phosphatidylethanolamine liposomes. Different effects were observed with a variety of benzodiazepines and different annexins depending on both the ligand and the protein. Almost opposite effects on annexin function are elicited by BDA250 and diazepam, its 7-chloro-derivative. We conclude that benzodiazepines modulate the calcium influx activity of annexins allosterically by stabilizing or destabilizing the conducting state of peripherally bound annexins in agreement with suggestions by Kaneko (Kaneko, N., Ago, H., Matsuda, R., Inagaki, E., and Miyano, M. (1997) J. Mol. Biol., in press).

Amino Acid Sequence↗

PubMed1998

Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V.

Residue-specific bioincorporation of 1,3-thiazolidine-4-carboxylic acid [thiaproline, Pro(S)], a non-natural amino acid analog of proline, into human recombinant annexin V was achieved with a proline-auxotrophic Escherichia coli strain by fermentation procedures in minimal medium. Quantitative replacement of proline with thiaproline was confirmed by mass-spectrometric, amino acid, and x-ray crystallographic analyses. The wild-type protein and its per-Pro(S) mutant were found to crystallize isomorphously and to show identical three-dimensional structures in crystals. In solution the dichroic properties of the wild-type and per-Pro(S) protein confirmed nearly identical overall folds. From thermal denaturation experiments, however, a reduced Tm (-4.5 K) value was determined whereas the van't Hoff enthalpy and entropy were not significantly affected. Therefore, protein mutants containing bioactive amino acid analogs like thiaproline at multiple sites would be expected to fully retain their functional properties, including immunogenicity, and thus could serve as promising vehicles for targeted drug delivery.

Annexin A5↗

PubMed1998

Crystallization of phycoerythrin 545 of Rhodomonas lens using detergents and unusual additives.

Phycoerythrin 545 from the cryptomonad alga, Rhodomonas lens, has been crystallized under a wide variety of conditions. Although this type of photosynthetic light-harvesting protein is water soluble, detergents were always required for crystallization. The crystals were typically poorly ordered, or ordered in only two dimensions. However, crystals that were well-ordered in three dimensions could be obtained under two different conditions. Both used polyethylene glycol as precipitant and the detergent lauryldimethylaminoxide, but the additives that were critical for obtaining well-ordered crystals were propionamide in one case and Cs+/Br- in the other. Crystals obtained in the presence of propionamide have the space group P2(1)2(1)2(1), with cell constants of a = 85.6 angstroms, b = 108.2 angstroms, and c = 131.0 angstroms, and contain two dimers [i.e., 2 x (alpha2beta2)] in the asymmetric unit. They show diffraction to at least 3.0 angstroms resolution. The crystals grown with Cs+/Br- are nearly isomorphous. Both types of crystals show intense, strongly polarized fluorescence, suggesting that energy transfer in the crystals is highly efficient. This should provide a basis for quantitative investigation of the role of exciton interactions in energy transfer in cryptomonad phycobiliproteins.

Buffers↗

PubMed1998

Structure of malonic acid-based inhibitors bound to human neutrophil collagenase. A new binding mode explains apparently anomalous data.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases, which have been implicated in various disease processes. Various classes of MMP inhibitors, including hydroxamic acids, phosphinic acids, and thiols, have been previously described. Most of these mimic peptides, and most likely bind analogous to the corresponding peptide substrates. Among the hydroxamic acids, malonic acid derivatives have been used as MMP inhibitors, although optimization of their inhibition potency was not successful. Here we report the design of malonic acid-based inhibitors using the X-ray structure of a collagenase/inhibitor complex, which revealed a nonsubstrate-like binding mode. The proposed beta-type turn-like conformation for the improved inhibitors was confirmed by X-ray crystallography. The observation of nonsubstrate-like binding confirms the original strategy for structure-based modeling of improved malonic acid inhibitors, and explains kinetic data that are inconsistent with substrate-like binding. Detailed interactions for the improved inhibitors seen in the crystal structure also suggest possibilities for further modifications in cycles of structure based drug design. Indeed, we have designed nonpeptidic inhibitors with approximately 500-fold improved inhibition based on these structures.

Collagenases↗

PubMed1998

Effect of melatonin on sleep and brain temperature in the Djungarian hamster and the rat.

The effect of a single dose of melatonin (3-5 mg/kg intraperitoneally) on sleep, electroencephalographic power density, and cortical temperature (TCRT) was investigated. Melatonin was administered to Djungarian hamsters 4 h or 12 h after lights on in a 16-h light:8-h dark cycle (LD 16:8) and to rats at dark onset in a LD 12:12. The effects in both species were short lasting and depended on the time of day. Sleep latency was prolonged in the late light period, sleep fragmentation was enhanced in the early light period, and TCRT was elevated in all three conditions. Rapid eye-movement sleep was reduced in the first postdrug hour after the late light period treatment in the hamsters and in postdrug hours 2 and 3 after dark onset treatment in the rat. Therefore, we have no evidence for a sleep inducing effect of 3-5 mg/kg of melatonin in the hamster or rat. In view of some data that indicate that melatonin may exert a sleep inducing effect in humans, it is suggested that melatonin induces changes that are typical for the dark period of each species, i.e., waking in the nocturnal Djungarian hamster and rat, and sleepiness in the diurnal human.

Animals↗

PubMed1998

The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action.

The serine proteinase plasmin is the key fibrinolytic enzyme that dissolves blood clots and also promotes cell migration and tissue remodeling. Here, we report the 2.65 A crystal structure of a ternary complex of microplasmin-staphylokinase bound to a second microplasmin. The staphylokinase 'cofactor' does not affect the active-site geometry of the plasmin 'enzyme', but instead modifies its subsite specificity by providing additional docking sites for enhanced presentation of the plasminogen 'substrate' to the 'enzymes's' active site. The activation loop of the plasmin 'substrate', cleaved in these crystals, can be reconstructed to show how it runs across the active site of the plasmin 'enzyme' prior to activation cleavage. This is the first experimental structure of a productive proteinase-cofactor-macromolecular substrate complex. Furthermore, it provides a template for the design of improved plasminogen activators and plasmin inhibitors with considerable therapeutical potential.

Amino Acid Sequence↗

PubMed1998

After-effects of human-computer interaction indicated by P300 of the event-related brain potential.

After-effects of human-computer interaction (HCI) were investigated by using the P300 component of the event-related brain potential (ERP). Forty-nine subjects (naive non-users, beginners, experienced users, programmers) completed three paper/pencil tasks (text editing, solving intelligence test items, filling out a questionnaire on sensation seeking) and three HCI tasks (text editing, executing a tutor program or programming, playing Tetris). The sequence of 7-min tasks was randomized between subjects and balanced between groups. After each experimental condition ERPs were recorded during an acoustic discrimination task at F3, F4, Cz, P3 and P4. Data indicate that: (1) mental after-effects of HCI can be detected by P300 of the ERP; (2) HCI showed in general a reduced amplitude; (3) P300 amplitude varied also with type of task, mainly at F4 where it was smaller after cognitive tasks (intelligence test/programming) and larger after emotion-based tasks (sensation seeking/Tetris); (4) cognitive tasks showed shorter latencies; (5) latencies were widely location-independent (within the range of 356-358 ms at F3, F4, P3 and P4) after executing the tutor program or programming; and (6) all observed after-effects were independent of the user's experience in operating computers and may therefore reflect short-term after-effects only and no structural changes of information processing caused by HCI.

Adolescent↗

PubMed1998

Pharmacokinetics of pantoprazole in patients with end-stage renal failure.

BACKGROUND: Pantoprazole is a selective inhibitor of the gastric H+/K+-ATPase with a low potential to interact with the cytochrome P450 enzyme system. Since pantoprazole is metabolized in the liver to metabolites which are mainly cleared by the renal route, it was the aim of this study to investigate its pharmacokinetics in patients with end-stage renal failure undergoing regular haemodialysis. METHODS: Eight patients with end-stage renal failure (creatinine clearance < 5 ml/min, age 45-65 years) on regular haemodialysis (duration of haemodialysis 4-5 h, cuprophan-dialyser Hemoflow E3, surface 1.3 m2) were given single i.v. doses of 40 mg pantoprazole one day before haemodialysis (A) and on a haemodialysis day immediately before the start of the haemodialysis (B). Concentrations of pantoprazole and metabolite M2 were determined in plasma and urine over 24 h and in timed samples of the dialysis fluid by HPLC. The protein binding was determined using equilibrium dialysis. RESULTS: The pharmacokinetic characteristics of pantoprazole AUC, t(1/2), CL and V(d area) (geometric means) were 2.4 mgxh/l, 0.63 h, 0.227 l/h/kg and 0.206 l/kg on day A (without dialysis) and 2.3 mgxh/l, 0.8 h, 0.237 l/h/kg and 0.273 l/kg on day B (with dialysis), respectively. The protein binding was 96%. Pantoprazole was found in small amounts in the dialysis fluid (max. 2.1% of the dose) but not in the urine. Pantoprazole was well tolerated. In particular, there were no clinically relevant changes in blood count, electrolytes or liver enzymes. CONCLUSIONS: Haemodialysis has no influence on the pharmacokinetic characteristics of pantoprazole. Thus, pantoprazole is not dialysed to any relevant degree, and therefore no dose-adjustment is required for patients with end-stage renal failure undergoing regular haemodialysis treatment.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1998

Sulfur-inhibited Thermosphaera aggregans sp. nov., a new genus of hyperthermophilic archaea isolated after its prediction from environmentally derived 16S rRNA sequences.

Recently, a new procedure was developed which allowed for the first time the isolation of a hyperthermophilic archaeum tracked by 165 rRNA analysis from a terrestrial hot solfataric spring ('Obsidian Pool', Yellowstone National Park, WY, USA). This novel isolate is characterized here. Cells are round cocci with a diameter of 0.2-0.8 micron, occurring singly, in pairs, short chains and in grape-like aggregates. The aggregates exhibit a weak bluish-green fluorescence under UV radiation at 420 nm. The new isolate is an anaerobic obligate heterotroph, using preferentially yeast extract for growth. The metabolic products include CO2, H2, acetate and isovalerate. Growth is observed between 65 and 90 degrees C (optimum: 85 degrees C), from pH 5.0 to 7.0 (optimum: 6.5) and up to 0.7% NaCl. The apparent activation energy for growth is about 149 kJ mol-1. Elemental sulfur or hydrogen inhibits growth. The core lipids consist mainly of acyclic and cyclic glycerol diphytanyl tetraethers. The cell envelope contains a cytoplasmic membrane covered by an amorphous layer of unknown composition; there is no evidence for a regularly arrayed surface-layer protein. The G + C content is 46 mol%. On the basis of 165 rRNA sequence comparisons in combination with morphological, physiological and biochemical properties, the isolate represents a new genus within the Desulfurococcaceae, which has been named Thermosphaera. The type species is Thermosphaera aggregans, the type strain is isolate M11TLT (= DSM 11486T).

Bacteriological Techniques↗

PubMed1998

Thermocrinis ruber gen. nov., sp. nov., A pink-filament-forming hyperthermophilic bacterium isolated from yellowstone national park

A novel hyperthermophilic bacterium was isolated from pink filamentous streamers (pink filaments) occurring in the upper outflow channel (temperature, 82 to 88 degreesC) of Octopus Spring in Yellowstone National Park, Wyo. The gram-negative cells grew at low salinity at temperatures up to 89 degreesC in the neutral to alkaline pH range. Depending on the culture conditions, the organisms occurred as single motile rods, as aggregates, or as long filaments that formed streamer-like cell masses. The novel isolate grew chemolithoautotrophically with hydrogen, thiosulfate, and elemental sulfur as electron donors and oxygen as the electron acceptor. Alternatively, under aerobic conditions, formate and formamide served as sole energy and carbon sources. The novel isolate had a 16S rRNA sequence closely related to the 16S rRNA sequence obtained from uncultivated pink filaments. It represents a new genus in the order Aquificales, the type species of which we name Thermocrinis ruber (type strain, OC 1/4 [= DSM 12173]).

Journal Article↗

PubMed1998

Differences in pH-dependent activation rates of substituted benzimidazoles and biological in vitro correlates.

Gastric proton pump inhibitors (PPIs) are substituted benzimidazole prodrugs that require an acid-induced activation. Its rate depends on the reactivity of the molecule relative to the environmental pH and determines the drug's tissue selectivity. Factors affecting the exposure of moderately acidic tissues to the activated PPI are the area under the serum concentration-time curve (AUC), serum protein binding, the partition coefficient logP and the serum elimination half-life relative to the chemical activation half-life at a critical tissue pH of about 5. These parameters have therefore been determined in a comparative fashion in the present study. The data shows that pantoprazole is less likely to undergo unwanted activation at moderately acidic targets as opposed to the parietal cell, compared to omeprazole. Actually, although 40 mg pantoprazole (steady state) gave a slightly higher serum AUC of the total parent compound than 40 mg omeprazole (10.5 vs. 7.1 micromol x h x l[-1]), a higher serum protein binding of pantoprazole versus omeprazole (98 vs. 96%) reversed the AUC values for the free drug in favor of a lower value for pantoprazole (0.19 vs. 0.28 micromol x h x l[-1]). It is the free parent compound that equilibrates across cell membranes to be activated in acidic tissue compartments. At pH 5.1, the activation half-life of pantoprazole was 4.7 versus 1.4 h for omeprazole, the latter being in the order of the common serum elimination half-life determined in an intraindividual comparison (1.24 vs. 1.25 h). Thus, pantoprazole is eliminated faster from blood than it is activated at a pH of about 5, while omeprazole is as quickly activated at this pH as it is eliminated from blood. Biological in vitro experiments confirmed that pantoprazole displays a lower liability to interfere with unwanted biological targets. This has been demonstrated in vitro for inhibition of both renal Na+/K+-ATPase, lysosomal acidification and the production of reactive oxygen species by neutrophils.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1998

Mechanisms of thyroid hormone action: insights from X-ray crystallographic and functional studies.

This review summarizes the studies conducted in our laboratory on the mechanisms of thyroid hormone action over the past two decades. We have attempted to place our studies on thyroid hormone receptors (TRs) in perspective with the work conducted by other investigators that established their nuclear localization, DNA-binding properties, DNA response elements, and the role of other proteins involved in TR-mediated regulation of gene transcription. Recently, our crystallographic studies of the TR ligand binding domain (LBD) revealed that the ligand has a structural role in the folding of the receptor's hydrophobic core. The analysis of the structure led to biochemical and genetic studies that have defined the surfaces on the TR LBD required for dimerization and binding of coactivator proteins. Placement of the mutations found in patients with the syndrome of generalized resistance to thyroid hormone on the TR LBD revealed that they were restricted to amino acids in the vicinity of the binding pocket for thyroid hormone. The insights gained from the elucidation of the TR LBD structure will provide the basis for the design of compounds with selective agonistic or antagonistic activities.

Animals↗

PubMed1998

Pantoprazole does not interact with the pharmacokinetics of carbamazepine.

OBJECTIVE: Pantoprazole is a H+/K+-ATPase inhibitor with a minimized potential of interaction with the cytochrome P450 system. Imidazole derivatives such as cimetidine and omeprazole have been shown to markedly interact with carbamazepine, a major anticonvulsant with a narrow therapeutic range. Therefore, the influence of steady-state pantoprazole on the pharmacokinetics of carbamazepine was investigated. SUBJECTS AND METHODS: N = 20 healthy volunteers (12 male/8 female) completed a double-blind, placebo-controlled, randomized crossover study. During the test period they received 40 mg pantoprazole p.o. once daily for 11 days and concomitantly a single oral dose of 400 mg carbamazepine on day 5. In the reference period placebo was administered instead of pantoprazole. RESULTS: Serum concentrations of carbamazepine and its active metabolite carbamazepine-10,11-epoxide were measured until day 11. Geometric means of AUC (extent characteristic) and Cmax/AUC (rate characteristic) of carbamazepine were 292 and 287 mgxh/l, and 0.0150 and 0.0144 l/h (reference and test), respectively. Point estimates and 90% confidence intervals of the ratios were 0.98 (0.95, 1.01) for AUC, and 0.96 (0.92, 1.00) for Cmax/AUC, respectively. Since the 90% confidence intervals of the primary characteristics, AUC and Cmax/AUC were entirely within the predefined equivalence range of 0.80 - 1.25, lack of interaction of pantoprazole with the pharmacokinetics of carbamazepine was demonstrated. Equivalence was also demonstrated for carbamazepine-10,11-epoxide using the characteristics AUC and Cmax. CONCLUSION: No dose adjustment of carbamazepine is therefore required during concomitant treatment with pantoprazole.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1997

Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.

BACKGROUND: Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS: The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS: The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Amino Acid Sequence↗

PubMed1997

The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 A in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain alpha/beta-fold with a central four-helix bundle connecting two seven-stranded parallel beta-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1'-carbonyl group near Tyr171 OH and NADP C4'N. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1' and C2' carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.

Alcohol Oxidoreductases↗

PubMed1997

Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure.

DHDPS is the first enzyme unique to the lysine biosynthetic pathway in plants and bacteria and catalyses the formation of (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. It is feedback-regulated in plants by L-lysine. The crystal structure of Nicotiana sylvestris DHDPS with and without inhibitory lysine bound to the enzyme has been solved to a resolution of 2.8 A. The molecule is a homotetramer composed of a dimer of dimers. Comparison with the structure of Escherichia coli DHDPS showed a novel quaternary structure by a profound rearrangement of the dimers forming the tetramer. The crystal structure of the enzyme in the presence of L-lysine revealed substantial changes. These changes together with the novel quaternary structure provide a structural basis for the strong inhibition of plant DHDPS enzymes by L-lysine.

Amino Acid Sequence↗

PubMed1997

Cloning, sequencing, crystallization and X-ray structure of glutathione S-transferase-III from Zea mays var. mutin: a leading enzyme in detoxification of maize herbicides.

Glutathione S-transferases (GSTs) are enzymes that inactivate toxic compounds by conjugation with glutathione and are involved in resistance towards drugs, antibiotics, insecticides and herbicides. Their ability to confer herbicide tolerance in plants provides a tool to control weeds in a wide variety of agronomic crops. GST-III was prepared from Zea mays var. mutin and its amino acid sequence was determined from two sets of peptides obtained by cleavage with endoprotease Asp-N and with trypsin, respectively. Recombinant GST-III was prepared by extraction of mRNA from plant tissue, transcription into cDNA, amplification by PCR and expression. It was crystallized and the crystal structure of the unligated form was determined at 2.2 A resolution. The enzyme forms a GST-typical dimer with one subunit consisting of 220 residues. Each subunit is formed of two distinct domains, an N-terminal domain consisting of a beta-sheet flanked by two helices, and a C-terminal domain, entirely helical. The dimeric molecule is globular with a large cleft between the two subunits. The amino acid sequence of GST-III and its cDNA sequence determined here show differences from sequences published earlier.

Amino Acid Sequence↗