Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 73 records · Page 4Linked to original sources

PubMed1994

Hemodialysis via the Scribner shunt in childhood: a surgical evaluation.

From 1973 to 1988, 105 children with renal failure (average age, 6.5 years; range, 5 days to 16.2 years) were treated with hemodialysis via surgically implanted Scribner shunts (n = 120). The average duration of dialysis was 16 days (range, 1 to 194 days). Twenty-three patients died during treatment or during the hospital stay because of the underlying disease; no patient died because of shunt complications. The treatment was initially successful for 82 children, but 14 of them died within several months of discharge. Among the initially surviving 82 patients, the shunt was the only means of access for dialysis in 52. In the other 30, the primary Scribner shunt was unsuccessful; it required replacement, or the type of dialysis had to be changed. The shunts were implanted in the forearm in 23%, the groin in 40%, and the ankle in 37%. Early complications were local bleeding (17%) after an average of 37.3 days, shunt occlusion (34%) after an average of 47.3 days, infections (9.3%) after an average of 43.9 days, and decreased blood flow (8.5%) after an average of 47.7 days. Vessels were reconstructed after discontinuation of dialysis in 28 cases, in which the Scribner shunt had been implanted in the groin. No patient experienced immediate ischemic problems. Long-term follow up results were obtained for 60% (n = 40) of the surviving 68 children after an average of 7 years (range, 2.1 to 15.2 years). We found no evidence of arterial or venous complications at the former shunt site.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗

PubMed1994

Acute peritoneal dialysis in preterm newborns and small infants: surgical management.

Experience with acute peritoneal dialysis gathered over the last 10 years shows that, in 63 premature newborns and infants (up to 1 year of age), a standardized surgical procedure using a silicone catheter with a Dacron cuff (individually glued to fit) has a lower complication rate and is more effective than placement of a trocar catheter.

Acute Kidney Injury↗

PubMed1994

Annexin V: the key to understanding ion selectivity and voltage regulation?

Annexin V is a Ca(2+)-dependent membrane-binding protein that forms voltage-dependent Ca2+ channels in phospholipid bilayers and is the first ion channel to be structurally and functionally characterized. Data outlined here indicate that key amino acid residues act as selectivity filters and voltage sensors, thereby regulating the permeability of the channel pore to ions.

Annexin A5↗

PubMed1994

A cryptic miniplasmid from the hyperthermophilic bacterium Thermotoga sp. strain RQ7.

An 846-bp cryptic plasmid has been discovered in the hyperthermophilic bacterium Thermotoga sp. strain RQ7. This is the first plasmid described for an organism from this ancient bacterial lineage and the smallest plasmid described to date for any organism. Nucleotide sequencing revealed a single open reading frame possibly encoding a 25,460-Da basic protein (212 amino acids). Upstream of the putative promoter lie five 11-bp direct repeats, each separated by 1 to 4 bp, while between the promoter and the open reading frame lies an 11-bp palindromic sequence. Its mode of replication is unknown, but its sequence bears similarities to those of plasmids which replicate by a rolling-circle mechanism.

Amino Acid Sequence↗

PubMed1994

Membrane versus bubble oxygenator in hyperthermic regional perfusion: a prospective randomized clinical study.

In a prospective randomized clinical study a routinely used bubble oxygenator (Bentley-5) was compared with a hollow fiber membrane oxygenator (D 701 Masterflo 34) during hyperthermic isolated extremity perfusion. This was done to find out whether there were differences between the two oxygenators in hemolysis, cellular damage, oxygenation and temperature achieved during extremity perfusion. In 30 perfusions blood samples were obtained at defined times: plasma hemoglobin (Hb), serum lactate dehydrogenase (s-LDH), number of erythrocytes, mean corpuscular volume (MCV), hemoglobin and bilirubin were determined for hemolysis, leukocyte count (neutrophils, lymphocytes, monocytes) and platelets as a check for cellular damage, and PO2, PCO2, O2 saturation and pH to define blood oxygenation and CO2 elimination. Maximal increase in temperature after 30 min and perfusion time until maximum tissue temperature were also recorded. The membrane oxygenator yielded better results from the aspect of hemolysis: s-LDH and plasma Hb were significantly different (p < 0.001). Cellular damage was less with the membrane oxygenator: platelet differences were significant (p < 0.01). Oxygenation and hyperthermia were obtained more quickly and were better controllable in membrane oxygenator. Further advantages for the patient were the smaller volume of blood needed for priming in a membrane oxygenator (750 vs. 1,200 ml) and improved safety resulting from a 'closed' perfusion system. On the basis of the clinical prospective randomized trial conducted, we conclude that membrane oxygenators must be adopted as the new standard in isolated hyperthermic extremity perfusion.

Chemotherapy, Cancer, Regional Perfusion↗

PubMed1994

Lipoprotein lipase deficiency due to a 3' splice site mutation in intron 6 of the lipoprotein lipase gene.

In a patient with primary hyperchylomicronemia as a result of lipoprotein lipase (LPL) deficiency, we sequenced all translated exons and intron-exon boundaries of the LPL gene. We found a C-->A mutation in position -3 at the acceptor splice site of intron 6 which caused aberrant splicing. The major transcript showed a deletion of exons 6 through 9 and amounted to about 3% of the normal transcript of a healthy control individual. In addition to this major transcript, we found trace amounts of both a normally spliced LPL mRNA and a second aberrant transcript devoid of exon 7. On the same allele, we detected in the LPL gene of our patient four polymorphic variations, three of which have not as yet been described. A second patient from an unrelated family, but from the same geographic area, was also found to be homozygous for the same mutation. Of the relatives of the two probands studied, 11 were heterozygous and 5 were unaffected by the mutation. LPL activity in postheparin plasma was near zero in the probands and reduced in 4 of the 10 heterozygotes. A third hyperchylomicronemic patient from the same area was found to be a compound heterozygote who carried on one allele the 3' splice site mutation of intron 6 and on the other one an already described missense mutation resulting in Gly188-->Glu substitution.

Alleles↗

PubMed1994

Lack of pantoprazole drug interactions in man.

This review summarizes the results of pharmacokinetic and pharmacodynamic drug interaction studies in man with pantoprazole, a new, selective proton pump inhibitor. Different mechanisms have to be considered as causes for potential drug-drug interactions. Proton pump inhibitors (PPIs) in general may alter the absorption of drugs by increasing the intragastric pH. Due to the presence of an imidazole ring, the PPIs of the class of substituted benzimidazole sulfoxides may interfere with the metabolism of other drugs by altering the activity of drug metabolizing enzymes of the cytochrome P450 system, via either induction or inhibition. With the increasing use of PPIs, their interaction potential gains therapeutic importance as was the case with the first and second generation of H2-blockers (cimetidine and ranitidine, respectively). The enhanced selectivity of pantoprazole to the gastric H+/K(+)-ATPase characterizes the new PPI generation. In comparison to omeprazole and lansoprazole, pantoprazole showed a much lower affinity to cytochrome P450 in vitro and a markedly lower potency in the in vivo rat model for interaction with diazepam. In contrast to omeprazole, pantoprazole does not interact with the cytochrome P450 system in man. In the drug interaction studies conducted so far, pantoprazole did not affect the pharmacokinetics or pharmacodynamics of antipyrine, diazepam, digoxin, a hormonal contraceptive, nifedipine, phenytoin, theophylline and warfarin in man. Also pantoprazole neither induced the drug metabolism of antipyrine nor increased urinary excretion of the induction markers D-glucaric acid and 6 beta-hydroxycortisol. Vice versa, the investigated drugs had no relevant influence on the pharmacokinetics of pantoprazole.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1994

Pantoprazole lacks interaction with antipyrine in man, either by inhibition or induction.

Substituted benzimidazole inhibitors of the gastric H+/K(+)-ATPase may interact with the cytochrome P450 enzyme system and alter the pharmacokinetics of coadministered drugs, as known for omeprazole. The primary aim of the present studies was to determine whether pantoprazole, a new, selective proton pump inhibitor, modifies the plasma concentrations of orally-administered antipyrine, a commonly used marker for mixed hepatic oxidase enzyme activity. In the acute study, 12 healthy male volunteers were given a) a single 30 mg i.v. doses of pantoprazole, b) a single 5 mg/kg oral dose of antipyrine, or c) coadministered pantoprazole and antipyrine according to a randomized three-period change-over design. In the chronic study, another 12 volunteers received 40 mg once-daily oral doses of pantoprazole on day 3 and on days 5-12, and a single oral 5 mg/kg dose of antipyrine on days 1, 12 and 14. Antipyrine plasma concentrations were measured without pantoprazole (day 1), on the last day of chronic dosing with pantoprazole (day 12) and 48 hours after the last dose of pantoprazole (day 14) to differentiate between inhibition and induction, respectively. Both drugs were well tolerated and no adverse events or clinically relevant alterations in vital signs or laboratory parameters were observed during treatment. The point estimates of the respective AUC- and Cmax-ratios for antipyrine with and without pantoprazole were 0.99 and 0.98 in the acute study, and 1.01 and 0.93 on day 12, and 1.04 and 0.99 on day 14 of the chronic study.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1994

Dose linearity of the pharmacokinetics of the new H+/K(+)-ATPase inhibitor pantoprazole after single intravenous administration.

Pantoprazole is a specific inhibitor of the H+/K(+)-ATPase of the gastric parietal cell. The dose-dependency of a range of pantoprazole pharmacokinetic characteristics was studied. Twelve healthy male subjects were given 10, 20, 40 and 80 mg pantoprazole intravenously according to a randomized, single blind, 4-period change-over scheme. The area under the concentration vs time curve (AUC) and the maximum serum concentration (Cmax) showed a linear increase in line with the dose. Apparent volume of distribution (Vd area), clearance (Cl) and terminal half-life (t1/2) were independent of the dose. The dose-independent elimination of pantoprazole was attributed to the lack of interaction of the drug with cytochrome P450. In clinical practice, a good predictable response, as well as a low potential for interaction with other drugs might be expected.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1993

Conformational variability of chicken cystatin. Comparison of structures determined by X-ray diffraction and NMR spectroscopy.

The structural model derived from X-ray crystallography for unphosphorylated wild-type chicken cystatin is compared with two chicken cystatin structures derived from NMR spectroscopy: the phosphorylated wild-type and the genetically engineered variant AEF-SIM-M29I-M89L. The comparison shows the same overall fold, but also significant differences in structurally variable segments of the polypeptide chain. The largest such segment, comprising residues 71 to 89, is a region characteristic of the family 2 cystatin inhibitors which contains a disulphide bridge (71-81) and the phosphorylation site (Ser80) discussed in the accompanying article. In the crystal structure, the segment 71 to 76 is found as a flexible loop, 77 to 85 as an alpha-helical segment, and 86 to 89 is completely undefined. The solution NMR structures on the other hand are disordered in the initial segment 72 to 80, have an extended conformation at 81 to 83 in contact with the beta-sheet, and clearly show a beta-turn at residues 87 to 90. The segment comprising residues 53 to 57, with smaller variability, is of particular interest as the hairpin loop conserved throughout the cystatin superfamily which binds to the cysteine proteinase. In most of the solution NMR structures, this segment adopts a conformation more like that of stefin B, a family 1 cystatin inhibitor, as was observed in the crystal structure of its inhibitory complex with papain. The differences between the structures are rationalized by an examination of the crystal contacts generated by hypothetical crystal packing of the NMR structures. Additionally, the X-ray refinement shows evidence of conformational disorder in the crystal. Joint refinement with NOE restraints and reflection data does not produce a structure to satisfy the restraints of both methods.

Animals↗

PubMed1993

Preliminary X-ray crystallographic study of the proteasome from Thermoplasma acidophilum.

Single cystals of proteasomes from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapor diffusion method. The crystals diffract to better than 3.0 A and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 308.9 A, b = 208.8 A and c = 116.9 A. There is one molecular complex in the asymmetric unit. Two potentially useful heavy-atom derivatives have been obtained. The self-rotation function of the native Patterson map shows local sevenfold symmetry, consistent with the low-resolution structure obtained by electron microscopic techniques. The unit cell dimensions and crystal symmetry together with the shape and size of the proteasome suggest a packing arrangement of proteasome molecules in the unit cell, with their cylinder axis nearly parallel to the crystallographic a-axis.

Crystallization↗

PubMed1993

Activation of snake venom metalloproteinases by a cysteine switch-like mechanism.

The cDNAs of several snake venom zinc endopeptidases code for a putative propeptide, which includes the conserved cysteine-containing sequence PKMCGVT. It has been suggested that binding of the cysteine thiol function to the active-site zinc, resulting in inactivation of the catalytic domain, occurs in a mode similar to the 'cysteine switch' mechanism proposed for matrix metalloproteinases. In order to confirm this hypothesis, inhibition kinetics have been performed on the metalloproteinase adamalysin II of the venom of the snake Crotalus adamanteus using several cysteine peptides. Among these the synthetic hexapeptide PKMCGV-NH2, corresponding to the conserved sequence portion of the known propeptides, was found to be by far the strongest inhibitor of this proteinase with a Ki of 3.4 microM. The inhibitory potencies of an equivalent peptide with the L-Cys replaced by a D-Cys or by an L-Ser as well as of reduced glutathione, cysteine and two unrelated cysteine peptides were by one to two orders of magnitudes lower. These findings strongly support a cysteine switch-like mechanism even for activation of the snake venom metalloproteinases.

Amino Acid Sequence↗

PubMed1993

ATP-independent DNA topoisomerase from Fervidobacterium islandicum.

Thermotogales are thermophilic eubacteria belonging to a very slowly evolving branch in the eubacterial tree. In this report, we describe the purification and characterization of an ATP-independent DNA topoisomerase from the Thermotogale, Fervidobacterium islandicum. The enzyme, a monomer of about 75 kDa, is a type I DNA topoisomerase sharing many properties with the other bacterial topoisomerases I: it absolutely requires Mg2+ for activity, relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA and spermidine. A feature of the F. islandicum ATP-independent DNA topoisomerase I is its thermophily. The optimal temperature for the enzymatic activity is 75 degrees C. Studies about thermostability show that the enzyme is more stable when incubated undiluted in the storage buffer. In these conditions, 60% activity was retained after a 30 min preincubation at 75 degrees C.

DNA Topoisomerases, Type I↗

PubMed1993

Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23-nm resolution and localization of the gamma subunit.

The three-dimensional structure of the light-harvesting pigment-protein b-phycoerythrin from the red alga Porphyridium cruentum has been determined at 0.23-nm resolution. The b-phycoerythrin structure is very similar to the structure of B-phycoerythrin from Porphyridium sordidum. Besides three non-identical residues there are only small differences between b-phycoerythrin and B-phycoerythrin alpha and beta subunits, respectively. In the crystals b-phycoerythrin forms an (alpha beta)6 hexamer (molecular mass: 236 kDa), whereas B-phycoerythrin additionally contains a 30-kDa gamma subunit. The comparison of the b-phycoerythrin and B-phycoerythrin electron-density maps clearly reveals, that the gamma subunit is located inside the (alpha beta)6 aggregate.

Crystallography, X-Ray↗

PubMed1993

Structure-function analysis of the ion channel selectivity filter in human annexin V.

Electrophysiology and structural studies were performed on an annexin V variant containing a mutation of glutamic acid-95 to serine in the center of the pore region. The mutation resulted in a lower single channel conductance for calcium and a strongly increased conductance for sodium and potassium, indicating that glutamic acid-95 is a crucial constituent of the ion selectivity filter. There were only minor differences in the crystal structures of mutant and wild-type annexin V around the mutation site; however, the mutant showed structural differences elsewhere, including the presence of a calcium binding site in domain III unrelated to the mutation. Analysis of the membrane-bound form of annexin V by electron microscopy revealed no differences between the wild type and mutant.

Annexin A5↗

PubMed1993

A rapid and efficient purification method for recombinant annexin V for biophysical studies.

Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.

Animals↗

PubMed1993

Structure of human des(1-45) factor Xa at 2.2 A resolution.

The structure of a large molecular fragment of factor Xa that lacks only a Gla (gamma-carboxyglutamic acid) domain (N-terminal 45 residues) has been solved by X-ray crystallography and refined at 2.2 A resolution to a crystallographic R-value of 0.168. The fragment identity was clearly established by automated Edman degradation. X-ray structure analysis confirmed the biochemical characterization and also revealed that the N-terminal epidermal growth factor (EGF)-like domain is flexibly disordered in crystals. The second EGF module, however, is positionally ordered making contacts with the catalytic domain. The overall folding of the catalytic domain is similar to that of alpha-thrombin, excluding the insertion loops of the latter with respect to simpler serine proteinases. The C-terminal arginine of the A-chain interacts in a substrate-like manner with the S1 specificity site of the active site of a crystallographically neighboring molecule. Based on this interaction and the structure of D-PheProArg methylene-thrombin, a model of the commonly used dansylGluGlyArg methylene inhibitor-factor Xa interaction is proposed. The region of factor Xa corresponding to the fibrinogen recognition site of thrombin has a reversed electrical polarity to the anion binding fibrinogen recognition site of thrombin but possesses a site similar to the Ca2+ binding site of trypsin and other serine proteinases. The structure of the C-terminal EGF domain of factor Xa is the first to be determined crystallographically. Its folding has been comprehensively compared with similar domains determined by NMR. Although the A-chain makes 44 contacts at less than 3.5 A with the catalytic domain, only 16 involve the EGF module. In addition, the A-chain makes 30 intermolecular contacts with a neighboring catalytic domain.

Amino Acid Sequence↗

PubMed1993

Electrostatic evidence for the activation of the glutathione thiol by Tyr7 in pi-class glutathione transferases.

A number of spectrophotometric studies [Graminski, G.F., Kubo, Y. & Armstrong, R.N. (1989) Biochemistry 28, 3562-3568; Liu, S., Zhang, P., Ji, X., Johnson, W.W., Gilliland, G.L. & Armstrong, R.N. (1992) J. Biol. Chem. 267, 4296-4299] have recently shown that the glutathione (GSH) thiol is deprotonated when it is in complex with glutathione S-transferase. Different models have been proposed for the activation of the glutathione S gamma, all pointing out the key role of active-site residue Tyr7. It remains unclear, however, how Tyr7 is actually involved in this process. In this paper we present an analysis of the electrostatic potential in the region of the active site of a pi-class GSH transferase. This analysis provides evidence that the titration behaviour of the absorption band of the E.GSH complex with a pK between 6 and 7 [Liu, S., Zhang, P., Ji, X., Johnson, W.W., Gilliland, G.L. & Armstrong, R.N. (1992) J. Biol. Chem. 267, 4296-4299] should rather be explained by the protonation/deprotonation equilibrium of Tyr7 than by the protonation/deprotonation equilibrium of the GSH thiol group itself. On the basis of this conclusion, a mechanism for activation of GSH is proposed: the Tyr7 OH group is deprotonated by the influence of the protein charge constellation and the peptide dipoles. Thus it acts as a general base, promotes proton abstraction from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity.

Animals↗