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Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 541 records · Page 30Linked to original sources
PubMed1990

The blue oxidases, ascorbate oxidase, laccase and ceruloplasmin. Modelling and structural relationships.

On the basis of the spatial structure of ascorbate oxidase [Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi-Agro, A. (1989) J. Mol. Biol. 206, 513-529], an alignment of the amino acid sequence of the related blue oxidases, laccase and ceruloplasmin is proposed. This strongly suggests a three-domain structure for laccase closely related to ascorbate oxidase and a six-domain structure of ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationships suggest that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and 3 and ceruloplasmin has mononuclear copper ions in domains 2, 4 and 6 and a trinuclear copper between domains 1 and 6.

Amino Acid Sequence↗
PubMed1990

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.

Amino Acid Sequence↗
PubMed1990

The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.

A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta-sheet wrapped around a five turn alpha-helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal 'trunk' (occupying the 'unprimed' subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1990

New evidence for anticipatory grief.

This exploratory study was designed to learn more about the needs of the survivors of hospice patients. A transitions framework is proposed as a way to conceptualize both hospice care and anticipatory grief, and a new clinical tool--the 10-Mile Mourning Bridge--is presented for survivors' assessments of their progress in bereavement. Survivors perceived that the routine pre-death work of the hospice team had a positive impact on their subsequent well-being in bereavement.

Bereavement↗
PubMed1990

Modeling the intact form of the alpha 1-proteinase inhibitor.

The structure of the intact form of the serpin alpha 1-proteinase inhibitor has been modeled based on the assumption that the central strand s4A of the six-stranded beta-sheet A of the cleaved inhibitor is not incorporated into the sheet of intact alpha 1-proteinase inhibitor. This strand was removed from its position in the center of the sheet by suitable rotations about the backbone dihedrals of Lys343 using molecular graphics. The resulting structure was then annealed using molecular dynamics (MD) while applying progressive distance restraints to the reactive peptide bond (Met358-Ser359) for 50 ps. During this time, the disrupted beta-sheet reformed to create a five-stranded beta-sheet with strands 3 and 5 in a parallel arrangement. This change and accompanying structural rearrangements are largely confirmed by the X-ray structure of plakalbumin, whose structure reflects the overall structure of intact serpins. The successful modeling experiment demonstrates the utility of MD for making gross structural predictions based on related structures. The binding loop of the intact form is modeled to allow docking with serine proteinases, in particular thrombin, which most highly constrains the possible conformations of the binding loop.

Models, Chemical↗
PubMed1990

Single intravenous administration of the H+, K(+)-ATPase inhibitor BY 1023/SK&F 96022--inhibition of pentagastrin-stimulated gastric acid secretion and pharmacokinetics in man.

The effects of H+, K(+)-ATPase inhibitor BY 1023/SK&F 96022 on pentagastrin-stimulated acid secretion have been studied in healthy male volunteers (n = 12). The gastric acid response to submaximal pentagastrin-stimulation (0.6 micrograms/h/kg b.w.) was dose-dependently inhibited. A single dose of 5 mg decreased acid output by 22% while after 60 mg and 80 mg secretion was almost completely abolished. A good dose linearity was observed for AUC (0, infinity) and Cmax over the dose range from 5 to 80 mg. Elimination half-life, total clearance and volume of distribution of the parent compound were independent of the dose. The drug was well tolerated up to the highest dose of 80 mg. No clinically relevant influence was found on either laboratory screen or cardiovascular parameters.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1990

Effect of repeated oral administration of BY 1023/SK&F 96022--a new substituted benzimidazole derivative--on pentagastrin-stimulated gastric acid secretion and pharmacokinetics in man.

Pentagastrin stimulated gastric secretion was measured in 12 healthy male subjects after repeated once daily oral administration of 20 and 40 mg BY 1023/SK&F 96022--a new substituted benzimidazole derivative. Twenty milligrams inhibited acid output compared with placebo by 24% (2.5-3.5 h) and 26% (24.5-25.5 h) after the first oral intake. Inhibition increased to 56% and 50%, respectively, after the seventh oral dose. Forty milligrams inhibited acid output by a mean of 51% (2.5 to 3.5 h) and 52% (24.5-25.5) after the first oral intake. After the seventh dose mean inhibition rose to 85% and 66%, respectively. The drug was well tolerated, no drug-related changes in clinical laboratory, ECG, heart rate and blood pressure were observed. Fasting gastrin serum concentrations tended to increase with both doses, the mean values being within the normal range. AUC, Cmax and t1/2 of the drug after repeated oral intake were not significantly different when compared with a single dose at either 20 mg or 40 mg.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1990

Presence of growth hormone-releasing hormone-like immunoreactivity in human tumors: characterization of immunological and biological properties.

The distribution and physical and biological properties of GH-releasing hormone-like immunoreactivity (GHRH-IR) in human tissues and tumors was investigated using a specific GHRH RIA, gel chromatography, immunoaffinity chromatography, and bioassay with cultured rat anterior pituitary cells. Variable concentrations of GHRH-IR, ranging from 1.4-39.0 ng/g wet wt, were found in normal liver, lung, placenta, and pancreas. In the latter tissue, however, a different chromatographic profile and a marked decrease in GHRH-IR after immunoaffinity occurred, suggesting that GHRH-IR in pancreatic extracts is not native GHRH. In all tumors examined (n = 35) GHRH-IR could be detected, and four tumors (three carcinoids and one jejunal carcinoma) contained a very high amount of GHRH-IR (greater than 1000 ng/g wet wt). Affinity chromatography of tumor extracts led to a significant loss (greater than 50%) of GHRH-IR in nine tumors. The four tumors containing large amounts of GHRH-IR were obtained from two patients with active acromegaly and two patients who had no clinical evidence of acromegaly. Using antibodies with different specificities for GHRH-(1-44) and GHRH shortened at the C-terminus, varying concentrations of GHRH-(1-44) in these tumors were found, ranging from 10-87% of the total GHRH-IR. The biological activity of GHRH in the four tumor extracts was similar to that of synthetic GHRH alone or GHRH added to control tissue subjected to extraction. These results demonstrate the presence of GHRH-IR in the majority of normal tissues and tumors, which, though they may produce large amounts of biologically active GHRH, do not always lead to acromegaly.

Adolescent↗
PubMed1990

Pentagastrin-stimulated gastric acid secretion and pharmacokinetics following single and repeated intravenous administration of the gastric H+, K(+)-ATPase-inhibitor pantoprazole (BY1023/SK&F96022) in healthy volunteers.

The objective of the study was to investigate the pharmacodynamics and pharmacokinetics of the gastric H+, K(+)-ATPase inhibitor pantoprazole in man following repeated i.v. dosing. 8 healthy male volunteers aged from 25 to 31 years (median: 29 years), body weight between 72 and 95 kg (median: 82 kg) entered this single-blind two-period cross-over study. Each subject underwent two treatment periods of 5 days each with daily infusion of 15 mg or 30 mg pantoprazole, respectively. A placebo day preceeded the trial. On the placebo day as well as on days 1, 4 and 5 of each treatment period, gastric secretion was stimulated submaximally by a pentagastrin infusion of 0.6 micrograms/h/kg over a period of 4 h, starting one hour before administration of drug or placebo. Repeated once-daily infusion (15 min) of pantoprazole resulted in a rapidly increasing pharmacodynamic effect: as compared to placebo the mean percent inhibition of acid output measured from 1 to 3 h after start of infusion was 22%, 63% and 78% for the 15 mg dose, and 56%, 97% and 99% for the 30 mg dose on days 1, 4 and 5, respectively. The pH also increased in relation to the dose and the duration of treatment. Mean fasting gastrin serum concentrations increased by about 50%, yet remained within the normal range. Only in one subject, one of the individual values was above the upper limit of the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1990

[Pharmacokinetics of the antirheumatic lonazolac-Ca in humans].

The pharmacokinetics of the non-steroidal, antiinflammatory drug lonazolac-Ca in man was investigated. Lonazolac-Ca (Irritren, arthro akut, Argun-L, Argun-300) (single doses 200 mg or 300 mg) was administered both to healthy, young volunteers and to patients with renal impairment, to elderly and arthritic patients and to nursing women. Serum concentrations were measured for lonazolac and its main serum metabolite M1, and pharmacokinetic characteristics such as Cmax, AUC and t1/2 were calculated after both single and multiple oral doses of film-coated tablets of lonazolac-Ca. In addition also suppositories of lonazolac-Ca (single dose 400 mg) were tested after single and multiple administration. In a study in healthy, normal volunteers, total radioactivity in blood was followed after oral and i.v. administration of 14-C-lonazolac-Ca (-Na). Lonazolac and M1 generally showed a biphasic pattern of elimination, their terminal half-lives being both ca. 6 h in young volunteers. In elderly patients the terminal half-life was about twice as long. Nevertheless, no accumulation of lonazolac on multiple dosing was observed in either volunteers or patients due to the rapid alpha-phase. However, some accumulation occurred for the (pharmacologically inactive) M1 in elderly patients and in patients with renal impairment. Serum profiles were lower and flatter after administration of suppositories as compared to oral administration. Lonazolac concentrations in synovial fluid were about half of those in serum, no lonazolac could be found in breast milk although there was some transfer of M1.

Administration, Oral↗
PubMed1989

Nuclear magnetic resonance solution and X-ray structures of squash trypsin inhibitor exhibit the same conformation of the proteinase binding loop.

A comparison of the solution nuclear magnetic resonance (n.m.r.) structures of squash trypsin inhibitor from seeds of the squash Cucurbita maxima with the X-ray structure of a trypsin complex of the inhibitor shows that the n.m.r. and X-ray structures are similar in terms of the global folding and secondary structure. The average atomic root-mean-square difference between the 36 n.m.r. structures on the one hand and the X-ray structure is 0.96 A for the backbone atoms and 1.95 A for all heavy atoms. The n.m.r. and X-ray structures exhibit extremely similar conformations of the primary proteinase binding loop. Despite the overall similarity, there are small differences between the mean computed structure and the X-ray structure. The n.m.r. structures have slightly different positions of the segments from residues 16 to 18, and 24 and 25. The n.m.r. results show that the inclusion of stereospecific assignments and precise distance constraints results in a significant improvement in the definition of the n.m.r. structure, making possible a detailed analysis of the local conformations in the protein.

Binding Sites↗
PubMed1989

The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein.

Peptide maps obtained by reversed-phase HPLC of tryptic digests of isoelectric form 1 (pI = 6.5) and 2 (pI = 5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser-74-Lys-91). Ser-80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms 1 and 2 was shown by ion-exchange chromatography to cause the disappearance of isoelectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non-phosphorylated form 1.

Amino Acid Sequence↗
PubMed1989

Solution of the phase problem in the X-ray diffraction method for proteins with the nuclear magnetic resonance solution structure as initial model. Patterson search and refinement for the alpha-amylase inhibitor tendamistat.

Patterson search calculations using the three-dimensional structure of the alpha-amylase inhibitor from Streptomyces tendae obtained from experimental nuclear magnetic resonance (n.m.r.) data were performed to study the possibility of solving the phase problem in the X-ray diffraction method with protein structures determined by n.m.r. Using all heavy atoms (C, N, O, S) of the residues 5 to 73 in the best n.m.r. structure of the alpha-amylase inhibitor (520 out of the 558 heavy atoms in the complete polypeptide chain), the maximum of the rotation function corresponded to the correct solution obtained by the previous independent determination of the crystal structure. However, additional local maxima, which are not significantly lower than the global maximum, also showed up. Performing the Patterson search with a model containing the backbone atoms and the heavy atoms of only the interior side-chains (399 atoms), which are much better defined by the n.m.r. data, the correct maximum was significantly higher than all other maxima. A translation search for the best orientation of the latter model yielded the correct solution. The energy-restrained crystallographic refinement was performed with this model to an R-factor of 26%. This corresponds approximately to the R-factor calculated for the X-ray crystal structure previously determined using the isomorphous replacement technique, if the residues 1 to 4 and 74 and all localized solvent molecules were removed from this structure. During the refinement the root-mean-square deviation between the two structures decreased from 1.03 A to 0.26 A for the polypeptide backbone and from 1.64 A to 0.73 A for all heavy atoms. There are no major local conformational differences between the two structures, with the single exception of the side-chain of Gln52.

Bacterial Proteins↗
PubMed1989

Comparison of the high-resolution structures of the alpha-amylase inhibitor tendamistat determined by nuclear magnetic resonance in solution and by X-ray diffraction in single crystals.

The three-dimensional structure of the alpha-amylase inhibitor Tendamistat determined by nuclear magnetic resonance in aqueous solution is compared with the Tendamistat crystal structure refined at 2.0 A resolution. Between the two independently obtained structures the root-mean-square distances are 1.05 A for the backbone atoms N, C alpha and C', 1.25 A for the backbone and the interior side-chains, and 1.84 A for all heavy atoms. These numbers show that the interior of the molecule is nearly identical in the two states. Near the protein surface a small number of local differences between the two structures were identified. In most surface areas the solution structure appears more disordered than the crystal structure, with the exception of Tyr15, which was not observed in the X-ray diffraction.

Magnetic Resonance Spectroscopy↗
PubMed1989

X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands.

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.

Ascorbate Oxidase↗
PubMed1989

The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes.

The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.

Animals↗