Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 415 records · Page 23Linked to original sources

PubMed1994

Dose-response relationships of micronuclei in human lymphocytes induced by fission neutrons and by low LET radiations.

The induction of micronuclei in cytokinesis-blocked human lymphocytes of different donors has been measured after G0 irradiation in vitro with a mixed fission neutron-gamma-ray beam, 220 kV X-rays and 60Co gamma-rays. The dose-response relationships for the micronucleus yields were linear-quadratic for both types of low LET radiations. The linear model applied for neutron-induced micronuclei over the dose range 0-0.66 Gy. At higher doses a saturation effect became apparent. For neutron-induced micronucleus yields a limiting RBE value of 12.2 was derived from the ratio of the alpha coefficients of neutrons and 60Co gamma-rays.

Cells, Cultured↗

PubMed1994

Structural and electrophysiological analysis of annexin V mutants. Mutagenesis of human annexin V, an in vitro voltage-gated calcium channel, provides information about the structural features of the ion pathway, the voltage sensor and the ion selectivity filter.

Annexin V binds to phospholipids in a calcium-dependent manner and exhibits calcium channel activity in vitro. We prepared a variety of mutants yielding information about the structure-function relationship of the ion channel activity. All mutants were characterized by X-ray crystallography, electron microscopy and electrophysiological measurements. Their structures are insignificantly changed whereas their electrophysiological properties are drastically different. Glu95, located in the central hydrophilic pore of the molecule, is crucial for the ion selectivity filter as its exchange leads to reduced calcium and increased sodium conductance. The removal of Glu17, located on the protein surface and far from the ion conduction pathway, leads to the appearance of a second conductance level of 9 pS in addition to the conductance level of about 30 pS in the wild-type molecule. This was also the case for Glu78, which is part of a weak calcium binding site. The exchange of Glu17 and Glu78 produced a mutant retaining only the smaller conductance level. We conclude that these two residues influence the angle between the two halves of the molecule, which determines the diameter of the ion conduction pathway, thereby leading to the occurrence of a second conductance level.

Amino Acid Sequence↗

PubMed1994

Sequence and structure comparison suggest that methionine aminopeptidase, prolidase, aminopeptidase P, and creatinase share a common fold.

Amino acid sequence comparison suggests that the structure of Escherichia coli methionine aminopeptidase (EC 3.4.11.18) and the C-terminal domain of Pseudomonas putida creatinase (EC 3.5.3.3) are related. A detailed comparison of the three-dimensional folds of the two enzymes confirms this homology: with an approximately 260-residue chain segment, 218 C alpha atoms of the structures superimpose within 2.5 A; only 41 of these overlapping positions (i.e., 19%) feature identical amino acids in the two protein chains. Notwithstanding this striking correspondence in structure, methionine aminopeptidase binds and is stimulated by Co2+, while creatinase is not a metal-dependent enzyme. Searches of protein data banks using sequence and structure-based profiles reveal other enzymes, including aminopeptidase P (EC 3.4.11.9), prolidase (EC 3.4.13.9), and agropine synthase, that likely share the same "pita-bread" fold common to creatinase and methionine aminopeptidase.

Amino Acid Sequence↗

PubMed1994

Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis.

The crystal structure of rat liver 6-pyruvoyl tetrahydropterin synthase has been solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.4% at 2.3 A resolution. 6-Pyruvoyl tetrahydrobiopterin synthase catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. The functional enzyme is a hexamer of identical subunits. The 6-pyruvoyl tetrahydropterin synthase monomer folds into a sequential, four-stranded, antiparallel beta-sheet with a 25 residue, helix-containing insertion between strands 1 and 2 at the bottom of the molecule, and a segment between strands 2 and 3 forming a pair of antiparallel helices, layered on one side of the beta-sheet. Three 6-pyruvoyl tetrahydropterin synthase monomers form an unusual 12-stranded antiparallel beta-barrel by tight association between the N- and C-terminal beta-strands of two adjacent subunits. The barrel encloses a highly basic pore of 6-12 A diameter. Two trimers associate in a head-to-head fashion to form the active enzyme complex. The substrate-binding site is located close to the trimer-trimer interface and comprises residues from three monomers: A, A' and B. A metal-binding site in the substrate-binding pocket is formed by the three histidine residues 23, 48 and 50 from one 6-pyruvoyl tetrahydropterin synthase subunit. Close to the metal, but apparently not liganding it, are residues Cys42, Glu133 (both from A) and His89 (from B), which might serve as proton donors and acceptors during catalysis.

Alcohol Oxidoreductases↗

PubMed1994

The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity.

Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine. The 2.0 A crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a five-stranded beta-sheet, two alpha-helices, and bridging loops. The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite. Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function.

Crystal structures of cytosolic glutathione S-transferases (EC 2.5.1.18), complexed with glutathione or its analogues, are reviewed. The atomic models define protein architectural relationships between the different gene classes in the superfamily, and reveal the molecular basis for substrate binding at the two adjacent subsites of the active site. Considerable progress has been made in understanding the mechanism whereby the thiol group of glutathione is destabilized (lowering its pKa) at the active site, a rate-enhancement strategy shared by the soluble glutathione S-transferases.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and sequence analysis of the gene of the molybdenum-containing aldehyde oxido-reductase of Desulfovibrio gigas. The deduced amino acid sequence shows similarity to xanthine dehydrogenase.

In this report, we describe the isolation of a 4020-bp genomic PstI fragment of Desulfovibrio gigas harboring the aldehyde oxido-reductase gene. The aldehyde oxido-reductase gene spans 2718 bp of genomic DNA and codes for a protein with 906 residues. The protein sequence shows an average 52% (+/- 1.5%) similarity to xanthine dehydrogenase from different organisms. The codon usage of the aldehyde oxidoreductase is almost identical to a calculated codon usage of the Desulfovibrio bacteria.

Aldehyde Oxidoreductases↗

PubMed1994

Crystal structure analysis and refinement at 2.15 A resolution of amicyanin, a type I blue copper protein, from Thiobacillus versutus.

The crystal structure of the type I blue copper protein amicyanin from Thiobacillus versutus has been determined by Patterson search techniques on the basis of the molecular model of amicyanin from Paracoccus denitrificans, and refined by energy-restrained least-squares methods. Amicyanin crystallizes in the trigonal space group P3(2) with unit cell dimensions of a = b = 87.40 A, c = 38.20 A. The asymmetric unit is composed of three independent molecules centred on the crystallographic 3(2) axes. The final R-value is 17.4% for 15,984 reflections to a resolution of 2.15 A. The polypeptide fold in amicyanin is based on the beta-sandwich structure commonly found in blue copper proteins. Nine beta strands are folded into two twisted beta-sheets that pack together with a filling of non-polar residues between them. The geometry of the copper site is similar to that of plastocyanin. There are four ligands, arranged approximately as a distorted tetrahedron, to the copper atom: His54, Cys93, His96 and Met99. One of the copper ligands, His96, is exposed to the surface and lies in the centre of a cluster of seven hydrophobic residues.

Amino Acid Sequence↗

PubMed1994

Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation.

Overproduction of eukaryotic proteins in microorganisms often leads to the formation of insoluble protein aggregates which accumulate as intracellular inclusion bodies. alpha 1-Proteinase inhibitor (alpha 1-PI) when produced as a cytoplasmic protein in Escherichia coli (E. coli) forms inclusion bodies containing the majority of the inhibitor in an inactive form. Several variants of alpha 1-PI with single amino acid substitutions within their active site loop (amino acids 345-358) were produced in a bioreactor showing that substitution of Met351 with Glu resulted in significantly reduced aggregate formation compared to the other variants and to wild-type protein. In addition, this variant proved to be fully functional as a proteinase inhibitor. Based on these findings and on results of previous structural studies a mechanism for aggregate formation during expression of alpha 1-PI is suggested.

Binding Sites↗

PubMed1994

The amino acid sequences of human and pig L-arginine:glycine amidinotransferase.

We have isolated and sequenced the L-arginine:glycine amidinotransferase of pig kidney mitochondria. Due to endogenous proteolysis, the purified molecules showed some heterogeneity at the N terminus. The longest form recovered had 386 amino acids. Part of the pig amidinotransferase sequence information was used to isolate cDNA clones coding for the human enzyme. The deduced amino acid of the human amidinotransferase was 37 amino acids longer due to the presence of a single sequence. The mature proteins were 94% identical to each other and 36% identical to the sequences of bacterial L-arginine:inosamine phosphate amidinotransferases.

Amidinotransferases↗

PubMed1994

Structural implications for the role of the N terminus in the 'superactivation' of collagenases. A crystallographic study.

For the collagenases PMNL-CL and FIB-CL, the presence of the N-terminal Phe79 correlates with an increase in proteolytic activity. We have determined the X-ray crystal structure of the recombinant Phe79-Gly242 catalytic domain of human neutrophil collagenase (PMNL-CL, MMP-8) using the recently solved model of the Met80-Gly242 form for phasing and subsequently refined it to a final crystallographic R-factor of 18.0% at 2.5 A resolution. The PMNL-CL catalytic domain is a spherical molecule with a flat active site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, that harbours two zinc ions, namely a 'structural' and a 'catalytic' zinc, and two calcium ions. The N-terminal segment prior to Pro86, which is disordered in the Met80-Gly242 form, packs against a concave hydrophobic surface made by the C-terminal helix. The N-terminal Phe79 ammonium group makes a salt link with the side chain carboxylate group of the strictly conserved Asp232. Stabilization of the catalytic site might be conferred via strong hydrogen bonds made by the adjacent, likewise strictly conserved Asp233 with the characteristic 'Met-turn', which forms the base of the active site residues.

Amino Acid Sequence↗

PubMed1994

Environmental behavior of bentazon herbicide.

Bentazon is a postemergence herbicide used in early spring to early summer in many crops, usually at application rates of 1.0 kg a.i./ha. Its selectivity is based on the ability of the crop plants to metabolize bentazon quickly to 6-OH- and 8-OH-bentazon and conjugate these with sugars, while weeds do not, so that photosynthesis is disrupted and the weeds die. Also, there is a further degradation to small fragments, which subsequently are incorporated into natural plant products. Residues in the raw agricultural commodities range from 0.1 to approximately 1.0 mg/kg (straw and other "leftover" plant parts). In the upper soil layer, bentazon is quickly degraded, microbially and aerobically, via the intermediary and instable products 6-OH-, 8-OH-bentazon, and AIBA. These are immediately bound biotically and abiotically to the mostly nonbioavailable soil organic matter fraction. Additionally, a considerable part (24-50%) is mineralized to CO2. Half-lives in field soils ranged from 3 to 21 d with an average of 12 d. Abiotic degradation processes predominantly involve photolyses on plant and soil surfaces and in surface water. The physical-chemical properties and soil column laboratory studies with bentazon would seem, at first glance, to predict a leaching potential. However, several field lysimeter studies unambiguously proved that it does not leach under field situations. Annual averages in the leachates were always < 0.1 microgram/L, also after the second year. Reasons for the favorable field behavior, in contrast to the laboratory studies, are discussed. Reports on bentazon findings in groundwater and drinking water were classified as resulting predominantly from former filtrate along Rhine banks. Since 1988, the bentazon levels in the Rhine River ranged below or near 0.1 microgram/L. Very few isolated point-source contaminations, arising from accidents and other reasons, are marked by sporadic findings of concentrations > 1 microgram/L. Nonvalidated findings at various locations are reported that lie below or near the determination limit. In such situations, it is highly recommended to identify bentazon with a "full" MS-spectrum. The ecotoxicological effects of bentazon lead to "no classification necessary." The small octanol/water partition coefficient precludes bioaccumulation. Bentazon is rapidly excreted by warm-blooded animals without any uptake of residues in edible tissues. Based on its toxicological properties, bentazon was classified as noncarcinogenic ("Group E") by the EPA. The ADI is set at 0.1 mg/kg body weight/d. The WHO drinking water guideline value, based on the toxicological profile of bentazon, was recently raised to 30 micrograms/L.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

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↗