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Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 289 records · Page 16Linked to original sources
PubMed1997

Inhibition of carboxypeptidase A by excess zinc: analysis of the structural determinants by X-ray crystallography.

Pancreatic metallocarboxypeptidases are inhibited by a millimolar excess of zinc together with other exo- and endometalloproteases. We have analyzed the structure of bovine carboxypeptidase A inhibited by an excess of zinc ions using X-ray crystallography at 1.7 A overall resolution. Under these conditions, a second zinc is observed to bind to the enzyme active site, establishing a distorted tetrahedrally coordinated complex which involves Glu-270 (the general base for catalysis), a water molecule, a chloride ion, and a hydroxide ion. This hydroxide ion forms a 114 degrees angular bridge between the inhibitory and the catalytic zinc ions, which are at a distance of 3.3 A from one another. The inhibitory zinc holds the hydroxide at nearly the same location as a previously observed active site water molecule (W571) and probably perturbs the substrate positioning and stereochemical rearrangements required for substrate cleavage during catalysis.

Animals↗
PubMed1997

Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster, Homarus americanus.

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A approximately 90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas.

5,7-Dihydroxytryptamine↗
PubMed1997

Three-dimensional structure of annexins.

Annexins constitute a family of structurally related calcium- and phospholipid-binding proteins whose molecular structure has been investigated in detail in the crystalline and membrane-bound form. Their polypeptide chain is folded into four or eight alpha-helical domains of similar structure with a central hydrophilic pore. Bound to phospholipid membranes, the four-domain arrangement of the annexin molecule is conserved. A peripheral binding mode has been well documented by electron microscopy and a variety of other techniques.

Annexins↗
PubMed1997

Annexin V and vesicle membrane electroporation.

The method of membrane electroporation (ME) has been used as an analytical tool to quantify the effect of membrane curvature on transient electric pore formation, and on the adsorption of the protein annexin V (M(r)= 35,800) to the outer surface of unilamellar lipid vesicles (of radii 25 < or = a/nm < or = 200). Relaxation kinetic studies using optical membrane probes of the diphenylhexatriene type suggest that electric pore formation is induced by ionic interfacial polarization causing entrance of the (more polarizable) water into the lipid bilayer membrane yielding (hydrophobic and hydrophilic) pore states with a mean stationary pore radius rp = 0.35 (+/-0.05) nm. Extent and rate of ME, compared at the same induced transmembrane voltage, were found to decrease both with increasing vesicle radius and with increasing protein concentration. This 'inhibitory' effect of annexin V is apparently allosteric and saturates at about [ANT]sat = 4 microM annexin V for vesicles of a = 100 nm at 1 mM total lipid concentration, 0.13 mM total Ca2+ concentration and at T = 293 K. Data analysis in terms of Gibbs area-difference-elasticity energy suggests that the bound annexin V reduces the gradient of the lateral pressure across the membrane. At [ANT]sat, about 20% of the vesicle surface is covered by the bound protein, but it is only 0.01% of the surface of the outer lipid leaflet in which a part of the protein, perhaps the aromatic residue of the tryptophan (W 187), is inserted. Insertion leads to a denser packing of the lipid molecules in the outer membrane leaflet. As a consequence, the radius of the electropores in the remaining membrane part, not covered by annexin V decreases (rp/nm = 0.37, 0.36 and 0.27) with increasing adsorption of the protein ([ANT] = 0, 2 and 4 microM, respectively).

Adsorption↗
PubMed1997

Biological monitoring of hospital pharmacy personnel occupationally exposed to cytostatic drugs: urinary excretion and cytogenetics studies.

For evaluation of the risk borne by hospital pharmacy personnel exposed to antineoplastic agents, the incorporation of cyclophosphamide, ifosfamide, and platinum-containing drugs was quantified by the determination of urinary concentrations. In addition, the induction of micronuclei (MN) and sister-chromatid-exchange (SCE) rates in peripheral blood lymphocytes were studied for correlation with the urinary excretion of cytostatic drugs. Cyclophosphamide and ifosfamide were determined in 24-h urine samples using gas chromatography with electron capture (detection limit 2.5 micrograms/l). Voltammetric analysis enabled the determination of platinum concentrations of 4 ng/l. Heparinized blood (20 ml) was drawn and lymphocytes were cultured for MN and SCE studies. In all, 13 hospital pharmacists and pharmacy technicians regularly involved in the preparation of cytostatic drugs participated in this investigation (7 persons represent a follow-up group). All subjects applied standard safety precautions, including the use of a vertical laminar air-flow hood, protective gowns, and latex gloves. On the day of urine sampling an average of 4,870 mg cyclophosphamide, 5,580 mg ifosfamide, and 504 mg platinum-containing drugs were handled. The excretion of 5 and 9 micrograms cyclophosphamide/l urine was measured in two samples, respectively. An elevated level of urinary platinum was found in one pharmacist (22.3 ng/g creatinine) in comparison with a nonexposed control group. Mean frequencies of MN and SCE did not differ significantly between the drug exposed group and control group. The employees who had incorporated chemotherapeutic agents were part of the follow-up group and, thus, particularly cautious and sensitive to a possible hazard. The results emphasize the necessity of improving personal protection of hospital pharmacy personnel occupationally exposed to cytostatic drugs and support the importance of biological monitoring. In an ongoing project in our department the sources of contamination are being investigated parallel to biological monitoring so as to determine critical situations and improve personal protection.

Adult↗
PubMed1997

Structure and function of molybdopterin containing enzymes.

Molybdopterin containing enzymes are present in a wide range of living systems and have been known for several decades. However, only in the past two years have the first crystal structures been reported for this type of enzyme. This has represented a major breakthrough in this field. The enzymes share common structural features, but reveal different polypeptide folding topologies. In this review we give an account of the related spectroscopic information and the crystallographic results, with emphasis on structure-function studies.

Coenzymes↗
PubMed1997

Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: thermodynamic studies and structural interpretation.

This paper provides further understanding of the thermodynamic and structural features determining the stability of Bacillus licheniformis alpha-amylase (BLA) at two crucial positions, His133 and Ala209. Results of protein modelling and saturated site-directed mutagenesis at position 133 and 209 have been reported in a previous paper (Declerck et al., 1995, Prot. Engng, 8, 1029-1037). In the first part of the present work, evidence is presented supporting the hypothesis that the stabilizing mutations reduce the rate of initial unfolding of the enzyme during the reversible step of the inactivation reaction and do not modify the irreversible processes undergone subsequently by the unfolded molecules. In the second part, we have examined the three-dimensional structure of BLA which has been determined recently by X-ray analysis (Machius et al., 1995, J. Mol. Biol., 246, 545-559). This analysis showed that our previous predictions made from molecular modelling were partly correct. At position 209, the effect of the stabilizing substitutions can be explained by a groove-filling effect reinforcing the hydrophobic packing between two helices of the central domain, while preserving a well-ordered water structure at the surface. At position 133, the stabilizing substitutions must compensate the loss of the hydrogen bond network in which the original histidine side-chain is involved; this compensation could be achieved through enhanced hydrophobic side-chain interactions within the beta-sheet where residue 133 is located, which correlates with the propensity of the residue to form and maintain a beta-strand conformation of the main chain at this position.

Alanine↗
PubMed1997

Organic solutes in hyperthermophilic archaea.

We examined the accumulation of organic solutes under optimum growth conditions in 12 species of thermophilic and hyperthermophilic Archaea belonging to the Crenarchaeota and Euryarchaeota. Pyrobaculum aerophilum, Thermoproteus tenax, Thermoplasma acidophilum, and members of the order Sulfolobales accumulated trehalose. Pyrococcus furiosus accumulated di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate and (beta)-mannosylglycerate, Methanothermus fervidus accumulated cyclic-2,3-bisphosphoglycerate and (beta)-mannosylglycerate, while the only solute detected in Pyrodictium occultum was di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate. Methanopyrus kandleri accumulated large concentrations of cyclic-2,3-bisphosphoglycerate. On the other hand, Archaeoglobus fulgidus accumulated three phosphorylated solutes; prominent among them was a compound identified as di-glycerol-phosphate. This solute increased in concentration as the salinity of the medium and the growth temperature were raised, suggesting that this compound serves as a general stress solute. Di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate accumulated at supraoptimal temperature only. The relationship between the accumulation of unusual solutes and high temperatures is also discussed.

Journal Article↗
PubMed1997

Microhabitat use, trophic patterns, and the evolution of brain structure in African cichlids.

The species assemblages of cichlids in the three largest African Great Lakes are among the richest concentrations of vertebrate species on earth. The faunas are broadly similar in terms of trophic diversity, species richness, rates of endemism, and taxonomic composition, yet they are historically independent of each other. Hence, they offer a true and unique evolutionary experiment to test hypotheses concerning the mutual dependencies of ecology and brain morphology. We examined the brains of 189 species of cichlids from the three large lakes: Victoria, Tanganyika, and Malawi. A first paper demonstrated that patterns of evolutionary change in cichlid brain morphology are similar across taxonomic boundaries as well as across the three lakes [van Staaden et al., 1995 ZACS 98: 165-178]. Here we report a close relationship between the relative sizes of various brain structures and variables related to the utilization of habitat and prey. Causality is difficult to assign in this context, nonetheless, prey size and agility, turbidity levels, depth, and substrate complexity are all highly predictive of variation in brain structure. Areas associated with primary sensory functions such as vision and taste relate significantly to differences in feeding habits. Turbidity and depth are closely associated with differences in eye size, and large eyes are associated with species that pick plankton from the water column. Piscivorous taxa and others that utilize motile prey are characterized by a well developed optic tectum and a large cerebellum compared to species that prey on molluscs or plants. Structures relating to taste are well developed in species feeding on benthos over muddy or sandy substrates. The data militated against the existence of compensatory changes in brain structure. Thus enhanced development of a particular function is generally not accompanied by a parallel reduction of structures related to other modalities. Although genetic and environmental influences during ontogeny of the brain cannot be isolated, this study provides a rich source of hypotheses concerning the way the nervous system functions under various environmental conditions and how it has responded to natural selection.

Africa↗
PubMed1997

Biogenic amines and aggression: experimental approaches in crustaceans.

This review summarizes our experimental approaches attempting to link amines and their metabolites to aggression in crustaceans. The results demonstrate (i) that agonistic behavior in crustaceans can be quantified, (ii) that the amines themselves have telling and subtle effects on the fighting behavior of animals, (iii) that pharmacological interventions are possible that might allow a biochemical dissection of the underlying mechanisms involved in processes like decision making in these animals, and (iv) that selective metabolites of amines are excreted in the urine of lobsters where they may serve behavioral roles. Many of the studies presented here are preliminary. Nonetheless, we believe the results are provocative and nicely complement previous detailed physiological, morphological and biochemical studies exploring the roles of amines in aggression in crustaceans. We expect that the continued use of this invertebrate model system will allow us to gain considerable insight into, and understanding of, the role served by biogenic amines in a complex behavioral process like aggression.

Aggression↗
PubMed1997

Synthesis, kinetic characterization and X-ray analysis of peptide aldehydes as inhibitors of the 20S proteasomes from Thermoplasma acidophilum and Saccharomyces cerevisiae.

A comparative kinetic characterization of the peptide aldehydes Ac-Leu-Leu-X-H [X = Trp, Tyr and Tyr(tBu)] and Z-Gly-Pro-Gly-Gly-Leu-Leu-Nle-H as inhibitors of the chymotryptic activity of 20S proteasomes from the archaebacterium T. acidophilum and yeast S. cerevisiae revealed significantly differentiated inhibitory potencies that can be rationalized on the basis of X-ray crystallographic data.

Aldehydes↗
PubMed1997

[Women in surgery--the status at universities in North-Rhine-Westphalia].

As a part of the survey of "women in surgery" of the German Association of Surgeons data were collected of Nordrhein-Westfalen to characterize the participation of women in academic surgery: 1996 only 6 women held a faculty appointment, 4 were head physicians, 49 were residents and 14 interns.

Career Choice↗
PubMed1996

Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor.

The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C-terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.

Amino Acid Sequence↗
PubMed1996

The 2.8 A crystal structure of Gla-domainless activated protein C.

The structure of the Gla-domainless form of the human anticoagulant enzyme activated protein C has been solved at 2.8 A resolution. The light chain is composed of two domains: an epidermal growth factor (EGF)-like domain modified by a large insert containing an additional disulfide, followed by a typical EGF-like domain. The arrangement of the long axis of these domains describes an angle of approximately 80 degrees. Disulfide linked to the light chain is the catalytic domain, which is generally trypsin-like but contains a large insertion loop at the edge of the active site, a third helical segment, a prominent cationic patch analogous to the anion binding exosite I of thrombin and a trypsin-like Ca[II] binding site. The arrangement of loops around the active site partially restricts access to the cleft. The S2 and S4 subsites are much more polar than in factor Xa and thrombin, and the S2 site is unrestricted. While quite open and exposed, the active site contains a prominent groove, the surface of which is very polar with evidence for binding sites on the primed side, in addition to those typical of the trypsin class found on the non-primed side.

Binding Sites↗
PubMed1996

Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis.

Maturation of eukaryotic 20S proteasomes involves the processing of beta-subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta-subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly-1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild-type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome-specific inhibitor. While the processing of inhibitor-treated wild-type proprotein was completely prevented, the site-directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8-10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self-activation of proteasomal beta-subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta-subunits proceeds via a novel ordered two-step mechanism involving autocatalysis.

Amino Acid Sequence↗
PubMed1996

X-ray structure of active site-inhibited clotting factor Xa. Implications for drug design and substrate recognition.

The 3.0-A resolution x-ray structure of human des-Gla-coagulation factor Xa (fXa) has been determined in complex with the synthetic inhibitor DX-9065a. The binding geometry is characterized primarily by two interaction sites: the naphthamidine group is fixed in the S1 pocket by a typical salt bridge to Asp-189, while the pyrrolidine ring binds in the unique aryl-binding site (S4) of fXa. Unlike the large majority of inhibitor complexes with serine proteinases, Gly-216 (S3) does not contribute to hydrogen bond formation. In contrast to typical thrombin binding modes, the S2 site of fXa cannot be used by DX-9065a since it is blocked by Tyr-99, and the aryl-binding site (S4) of fXa is lined by carbonyl oxygen atoms that can accommodate positive charges. This has implications for natural substrate recognition as well as for drug design.

Anticoagulants↗