Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 55 records · Page 3Linked to original sources

PubMed1992

Natural protein proteinase inhibitors and their interaction with proteinases.

The substrate-like 'canonical' inhibition by the 'small' serine proteinase inhibitors and the product-like inhibition by the carboxypeptidase inhibitor have provided the only atomic models of protein inhibitor--proteinase interactions for about 15 years. The recently published structures of cystatin/stefin--papain complexes and of hirudin--thrombin complexes reveal novel non-substrate-like interactions. In addition, the structure of pro-carboxypeptidase shows a model of inactivation which bears resemblance to proteinase/protein inhibitor systems. Considerable progress in understanding the transition between native and cleaved states of the serpins has also been made by several recent structural studies.

Animals↗

PubMed1992

Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.

Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.

Amino Acid Sequence↗

PubMed1992

Centromere detection in vinblastine- and radiation-induced micronuclei of cytokinesis-blocked mouse cells by using in situ hybridization with a mouse gamma (major) satellite DNA probe.

Non-isotopic in situ hybridization using a mouse gamma (major) satellite probe DNA was applied to detect centromeres in micronuclei, which were induced in vitro in mouse liver cells by ionizing radiation and by vinblastine sulfate. In a cytokinesis-blocked micronucleus assay a dose-dependent induction of micronuclei was found for both agents. After vinblastine exposure the observed micronuclei showed centromere-positive hybridization signals in an order of magnitude of 70-90%, but after radiation exposure the magnitude was only 10-20%. Since the in situ hybridization technique detects centromeric DNA directly, it can be used in a cytokinesis-blocked micronucleus assay for a rapid and reliable discrimination between aneuploidy-inducing and clastogenic agents.

Animals↗

PubMed1992

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B.

The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11-76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1.A. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.

Amino Acid Sequence↗

PubMed1992

Right-to-die responses from a random sample of 200.

Seeking public attitudes toward the right-to-die, 200 telephone interviews were conducted with a random sample of residents in a midwestern city in the summer of 1990. Overall, 90% favored some kind of personal control over death circumstances. With semantics as an independent variable (euthanasia, mercy killing, physician-assisted suicide, or some form of personal control over death) only the group to whom "physician-assisted suicide" had been presented as a choice said "no" or "probably not" to a legalization question (p = < .0001).

Adult↗

PubMed1992

Intra- and inter-individual variation of background and radiation-induced micronucleus frequencies in human lymphocytes.

Serial blood samples were taken from four healthy individuals (three males, one female, aged between 26 and 51 years) in 3-monthly intervals during 1 year. Leucocyte suspensions were prepared and exposed to 3 Gy of 137Cs gamma-rays or left unirradiated as controls. In a cytokinesis-blocked (CB) micronucleus (MN) assay significant inter- and intra-donor variations of background and radiation-induced MN incidences became apparent. The two sources of variation lead to an extra variance sigma I2, in addition to the sample variance sigma e2 of MN incidences. The contributions of the different components to the total variance were estimated by means of a variance component model. The deviation sigma I for the mean background MN level of 1.53 x 10(-2) MN/CB cell was +/- 0.67 x 10(-2) and for the mean radiation-induced MN level of 0.53 MN/CB cell it was +/- 0.10. The contribution of the intra-individual variance to sigma I2 was about 50% for background MN levels and 75% for radiation-induced MN frequencies. With respect to the application of the CB-MN assay as a biological dosimetry system, the consequences of the present findings for calibration purposes and low-dose estimation are discussed. The calculation of the variance components is explained in an appendix, which serves also as an example for the adaptation of analysis of variance techniques to the evaluation of data derived from scoring of MN, as well as from scoring of metaphase chromosomal aberrations.

Adult↗

PubMed1992

The Long-Term Care Ombudsman Program: what does the complaint reporting system tell us?

Using paid staff and/or volunteers, long-term care ombudsman programs are charged with resolving complaints and solving problems that affect elderly persons in a variety of long-term care settings. This paper reports the results of a content analysis of annual ombudsman program reports sent to the Administration on Aging from 49 states in 1990. We found substantial variation in the documented information at both state and local levels and recommend revising the reporting system.

Aged↗

PubMed1992

Quantitative histological study of the optic nerve in species of minnows (Cyprinidae, Teleostei) inhabiting clear and turbid water.

Quantitative analysis of the optic nerve of minnows using light- and electron microscopy demonstrated that anatomical characteristics of the visual system are closely related to habitat turbidity. Species in the genera Notropis and Cyprinella inhabiting predominantly clear water had larger eyes and almost twice as many optic nerve fibers compared to minnows of turbid habitats. No differences were detected in the thickness of myelination, the axon diameter profile, or the number of optic nerve fibers per retinal area, indicating that the relative number of fibers, as well as their anatomical characteristics, are similar in all species and independent of habitat turbidity. It is therefore hypothesized that quantitative differences in the number of visual elements available for sampling and processing in the retina, optic nerve, and optic tectum are sufficient to account for presumed differences in visual performance.

Animals↗

PubMed1992

Pancreatic procarboxypeptidases: their activation processes related to the structural features of the zymogens and activation segments.

The molecular events leading to the complete activation of pancreatic procarboxypeptidases A and B have been investigated. For both proteins the activation process follows a similar general scheme: trypsin is responsible for the first cleavage that separates the active enzyme from the activation segment, the degradation of the activation segment proceeds only from its C-terminal end, and activity release can be correlated with the disappearance of the long forms of the activation segment. In both systems, trypsin and the released carboxypeptidase participate in the trimming of the severed activation regions. However, the rate of enzymatic activation is much faster in the case of procarboxypeptidase B. This phenomenon may be explained by some structural differences in the connecting region which acts as a linker between the globular domain of the activation segment and the N-terminal end of carboxypeptidases and also by the higher efficiency of carboxypeptidase B for the digestion of its own activation segment. It is not due to unfolding of the activation domain, since the isolated activation domain retains its globular conformation in solution.

Amino Acid Sequence↗

PubMed1992

Direct injection/h.p.l.c. methods for the analysis of drugs in biological samples.

1. Direct injection h.p.l.c. methods for zaprinast, and pantoprazole and its sulphone metabolite were developed. 2. Optimal recovery of pantoprazole and its sulphone metabolite was effected by the absence of transfer losses and the effective adjustment of sample pH on-line. 3. Acetonitrile reduced the recovery of pantoprazole and its sulphone metabolite at acetonitrile concentrations greater than 5% in serum. 4. Direct injection h.p.l.c. methods minimize sample handling losses, reduce human contact with biological samples and are sufficiently accurate and reproducible to be used to support pharmacodynamic and toxicokinetic studies.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1992

[Ultrasound assessment of reconstructed rotator cuffs].

The evaluation of recurrent shoulder symptoms in patients who have had rotator cuff repair is a diagnostic challenge. Pain and limitation of motion may be caused by a recurrence of the rotator cuff tear. Arthrography is not considered to be helpful in postoperative cases, since it is false-positive in most of them. Pre-operative ultrasonography of the shoulder is regarded as a highly accurate diagnostic tool for rotator cuff tears. The diagnostic criteria used are: the continuity of the rotator cuff, its contour, its thickness and its echogenicity. Our study was aimed at determining which ultrasonographic criteria were significant for a retear. In addition, the "normal" postoperative sonographic appearance of the rotator cuff was established. Of 133 patients with a full thickness rotator cuff tear, 110 were evaluated 4-48 months (mean in 21 months) after surgery. A total of 85 cuffs were reconstructed, in 46 cases by direct suture, in 33 cases by suture to the major tubercle and in 6 cases by tendon transfer (Cofield technique). In 25 cases cuffs the could not be reconstructed. Subacromial decompression was performed routinely. The patients were evaluated clinically by range of movement, force and isometric and impingement tests. The subjective outcome was assessed by the algo-functional index of Patte. Ultrasonography was performed using a 7.5-MHz linear scanner. Each sonographic criterion was referred to the clinical and subjective findings. Ultrasonographic evaluation of the rotator cuffs that could not be reconstructed revealed non-visualization of the tendons. In 13 of the 85 patients in whom reconstruction of the cuff was possible a normal sonographic pattern was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Annexin V-crystal structure and its implications on function.

Annexins constitute a family of cytosolic, water soluble proteins, which bind to negatively charged phospholipids in a calcium-dependent manner. They display structural and functional features of both soluble and integral membrane proteins. The annexins face the hydrophilic as well as the hydrophobic phase (Janus-faced proteins) and mediate ion transport in vitro. We present the refined structure and molecular model of annexin V at 2.0 A resolution. The molecule is almost entirely alpha-helical, and each of the four repeats of annexin V is folded into a compact domain of similar structure. The four domains are arranged in an almost planar, cyclic array. In the center of the molecule, one can find a prominent hydrophilic pore, which we associate with the calcium-selective channel found in annexin V. Annexin V has an overall flat, slightly curved shape with two faces, one convex and one concave. The three calcium binding sites Ca1 to Ca3, all located at the convex face of the molecule, are assumed to be phospholipid binding sites, as suggested by their structural similarity to the calcium site of phospholipase A2. Soluble and membrane-bound annexin have closely similar structures, as shown by electron microscopic analysis. Several other observations provide evidence that the membrane-anchoring region of the annexin V molecule is located on the convex face. In the last part of this article, the electrophysiology of the annexins is described. Ion permeation occurs in discrete conductance states and is regulated by voltage across the membrane. A model for the annexin V-membrane interaction, the ion channel formation, and the ion conduction pathway is proposed.

Amino Acid Sequence↗

PubMed1992

[Presence of inhibitors of activated partial thromboplastin time (TTPA). Clinical repercussion in obstetric patients].

Partial thromboplastin time, activated with kaolin (TTPA) is a qualitative test used to find defects of some factor of intrinsic via of coagulation or to rule out the presence of some circulating inhibitor. The lupus anticoagulant (LA) is part of a group of several auto-antibody with pathogenic potential in several branches of medicine, mainly rheumatology, hematology and gyneco-obstetrics. In this last area the LA has been associated with different obstetrical repercussions. The purpose of this study was to determine the main obstetrical events associated with patients with circulating anticoagulants identified by TTPA with kaolin. Ninety six patients were included in cases-control study. Group I (n = 48) cases and Group II (n = 48) controls, were selected from the same population and information source. The cases were included when presenting prolongation of more than 4 seconds of TTPA activated with kaolin regarding a control with lack of correction with normal plasma. A registration sheet for data captation, was designed specially for this study. The comparability of both groups was established, as there were no differences (significant) among the variable considered as basal. The group of cases presented with a greater frequency of habitual abortion, neonatal death and thrombotic phenomena. This relationship of autoimmunological pictures with recurrent fetal loss and thromboembolic incidents has been consistently described in literature. To this respect, several immunological abnormalities. Including positive anticardiolipin antibodies and VDRL falsely positive. The incidence, in this country of these entities, is unknown. These observations show the need of a methodologic superation.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Habitual↗

PubMed1992

Lack of pharmacokinetic interaction as an equivalence problem.

The demonstration that concomitant administration of drug B does not affect the pharmacokinetics of drug A can be adequately handled as an equivalence problem. Administration of drug A alone serves as reference and simultaneous administration of drugs A and B as test situation. The range of clinically acceptable variation in the pharmacokinetic characteristics of drug A defines the equivalence range. This will usually correspond to the bioequivalence range accepted for the comparison of different formulations of drug A. Equivalence, i.e. lack of pharmacokinetic interaction, is concluded if the 90%-confidence interval for the ratio (difference) of the expected medians for test and reference is entirely within the equivalence range. This decision procedure ensures that the consumer risk of incorrectly concluding "lack of interaction" is limited to 5%. Moreover, the producer risk of incorrectly concluding "interaction" can be controlled by appropriate sample sizes.

Drug Interactions↗

PubMed1991

Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants.

Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg, Met358----Arg]alpha 1-PI) results in complete loss of its inhibitory activity against human alpha-thrombin; whereas an exchange of residue Met351 (P8) by Glu [( Met351----Glu, Met358----Arg]alpha 1-PI) does not alter activity. [Thr345----Arg, Met358----Arg]alpha 1-PI is rapidly cleaved by thrombin, while [Met358----Arg]alpha 1-PI and [Met351----Glu, Met358----Arg]alpha 1-PI form stable proteinase-inhibitor complexes. The stability of [Thr345----Arg, Met358----Arg]alpha 1-PI against guanidinium chloride denaturation is significantly enhanced compared to wild-type alpha 1-PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345----Arg amino acid exchange had been derived. [Met351----Glu, Met358----Arg]alpha 1-PI and [Met358----Arg]alpha 1-PI resemble the wild-type protein in this respect. The CD spectra of intact and cleaved alpha 1-PI variants do not compare well with the wild-type protein, probably reflecting local structural differences. Insertion of a synthetic peptide, which corresponds to residues Thr345----Met358 of human alpha 1-PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild-type protein.

Amino Acid Sequence↗

PubMed1991

Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip.

The X-ray crystal structure of recombinant wild-type azurin from Pseudomonas aeruginosa was determined by difference Fourier techniques using phases derived from the structure of the mutant His35Leu. Two data sets were collected from a single crystal of oxidized azurin soaked in mother liquor buffered at pH 5.5 and pH 9.0, respectively. Both data sets extend to 1.93 A resolution. The two pH forms were refined independently to crystallographic R-factors of 17.6% (pH 5.5) and 17.5% (pH 9.0). The conformational transition previously attributed to the protonation/deprotonation of residue His35 (pKa(red) = 7.3, pKa(ox) = 6.2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36-Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole N delta 1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated N delta 1 acts as an acceptor of a weak hydrogen bond from HN Gly37. The structure of the remainder of the azurin molecule, including the copper binding site, is not significantly affected by this transition.

Aurintricarboxylic Acid↗

PubMed1991

Refined structure of the hirudin-thrombin complex.

The structure of a recombinant hirudin (variant 2, Lys47) human alpha-thrombin complex has been refined using restrained least-squares methods to a crystallographic R-factor of 0.173. The hirudin structure consists of an N-terminal domain folded into a globular unit and a long 17-peptide C-terminal in an extended chain conformation. The N-terminal domain binds at the active-site of thrombin where Ile1' to Tyr3' penetrates to the catalytic triad. The alpha-amino group of Ile1' of hirudin makes a hydrogen bond with OG of Ser195 of thrombin, the side-chains of Ile1' and Tyr3' occupy the apolar site, Thr2' is at the entrance to, but does not enter, the S1 specificity site and Ile1' to Tyr3' form a parallel beta-strand with Ser214 to Gly219. The latter interaction is antiparallel in all other serine proteinase-protein inhibitor complexes. The extended C-terminal segment of hirudin, which is abundant in acidic residues, makes many electrostatic interactions with the fibrinogen binding exosite while the last five residues are in a 3(10) helical turn residing in a hydrophobic patch on the thrombin surface. The precision of the complementarity displayed by these two molecules produces numerous interactions, which although independently generally weak, together are responsible for the high degree of affinity and specificity. Although hirudin-thrombin and D-Phe-Pro-Arg-chloromethyl ketone-thrombin differ in conformation in the autolysis loop (Lys145 to Gly150), this is most likely due to different crystal packing interactions and changes in circular dichroism between the two are probably due to the inherent flexibility of the loop. An RGD sequence, which is generally known to be involved in cell surface receptor interactions, occurs in thrombin and is associated with a long solvent channel filled with water molecules leading to the surface from the end of the S1 site. However, the RGD triplet does not appear to be able to interact in concert in a surface binding mode.

Amino Acid Sequence↗

PubMed1991

Correlation of metal decoration and topochemistry on protein surfaces.

On the surface of protein molecules the formation of metal clusters during vacuum condensation is controlled by topochemical features of the substrate and by specific properties of the decorating material. The resulting metal distribution (decoration pattern) can be mapped by electron microscopy in conjunction with image processing. We have applied this technique to freeze-etched crystals of the lumazine synthase-riboflavin synthase complex and its derivative obtained by binding of the heteropolytungstate (NaP5W30O110)(NH4)14.31 H2O. The decoration pattern of the free protein and its heteropolytungstate derivative showed marked differences. The correlation of these data with the X-ray structure of the protein showed an increased affinity of both gold and silver to the location of heteropolytungstate. Decoration sites can, but do not need to, be close to the protein surface. Actually, two of the observed decoration sites are located on a layer of ice as thick as 20 A, which apparently transmits underlying topochemical features. Preferential affinity of a site to a given metal must be seen as a property that depends on specific interaction with the decorating material but also on the differential affinities in adjacent areas.

Bacillus subtilis↗