Biomedical subjects

K Gruber

Publications and source records attributed to K Gruber.

At least 19 recordsLinked to original sources

PubMed2001

Determination of beta-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system.

A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.

Adult↗

PubMed2001

Elucidation of the mode of substrate binding to hydroxynitrile lyase from Hevea brasiliensis.

The hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) is used as a catalyst in enantiospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones. The catalyzed reaction represents one of the few industrially relevant examples of enzyme mediated C-C coupling reactions. In this work, we modeled Hb-HNL substrate complexes that have as yet proven inaccessible to experimental structure determination and were able to identify two binding modes for the natural substrate acetone cyanohydrin in docking simulations. Discrimination of the two alternatives was achieved by modeling complexes with two different chiral cyanohydrins followed by an analysis of the respective relative binding energies from molecular mechanics and thermodynamic integration. Only for one of the alternative binding modes the experimentally established S-selectivity of the enzyme was correctly predicted. Our results yielded further support for an enzymatic mechanism involving the catalytic triad Ser80, His235, and Asp207 as a general acid/base. A pivotal role was ascribed to Lys236, which seems to be crucial for enzymatic activity at low pH values. In addition, the modeling calculations provided possible explanations for the observed substrate and enantioselectivity of the enzyme that rationalize available mutational data and will be the basis for future protein engineering efforts.

Aldehyde-Lyases↗

PubMed2001

Metal complexes of a biconcave porphyrin with D4-structure--versatile chiral shift agents.

Representative metal complexes of a biconcave D4-symmetric porphyrin were synthesised by metalion insertion into the porphyrin ligand 1. The NMR spectra suggested D4-symmetry for the ZnII and dioxo-RuVI complexes of 1 and C4-symmetry for the unsymmetrically ligated RuII and RhIII complexes. Metal complexes of 1 proved to be versatile chiral 1H NMR shift agents for a broad spectrum of organic amines, alcohols, carboxylic acids, esters, nitriles and nonpolar fullerene derivatives. A practical analysis of chiral substrates with 1 covers enantiomeric excesses beyond 99%. An X-ray structure of (1:1)-cocrystals of an achiral, biconcave CoII porphyrinate and C60 provided the first detailed insights into the structure of such a biconcave metallo-porphyrinate. It also showed remarkable packing of the carbon sphere against the main concave units of the porphyrin and gave clues about the relevant interactions between biconcave porphyrins and fullerenes.

Journal Article↗

PubMed2001

Reinforcement-based outpatient treatment for opiate and cocaine abusers.

A reinforcement-based intensive outpatient treatment was delivered to 37 recently detoxified, inner city, heroin and/or cocaine abusers who did not want methadone treatment. Attendance was scheduled and urine collected daily for the first 2 weeks, four times weekly for the next 2 weeks, and then thrice weekly for the final 8 weeks. As attendance incentives, patients received transportation assistance (bus tokens), and $28-$30 per week in vouchers to be spent on activities/items chosen and agreed upon with their counselor. As abstinence incentives, patients received weekend supported recreational activities, lunches, $42-$45 per week in vouchers, and rent or utilities payment ($150 over 4 weeks). Total potential earnings was $1,435 per patient; actual mean earnings was $583. Forty-three percent (n=16) completed 10 or more weeks of treatment. These 16 long-stay patients submitted 92% (SD=19) opiate- and cocaine-negative urines during their enrollment compared with 56% (SD=42) drug-negative urines submitted by 21 drop-outs, F(1,35)=9.99, p=0.003. Overall, 32% of clients became employed during their treatment episode; 94% of long-stay patients were employed at the end of their treatment episode. Patients who were drug-positive at intake were highly likely to drop out. Treatment outcomes compare favorably with those reported in the literature for outpatient nonmethadone treatment of opiate and cocaine abusers. Continued evaluation of this new treatment appears warranted.

Adult↗

PubMed2001

The hydroxynitrile lyase from almond: a lyase that looks like an oxidoreductase.

BACKGROUND: Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase, a key enzyme of this process, cleaves a cyanohydrin into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, alpha/beta hydrolases, and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. RESULTS: We have determined the crystal structure of a 61 kDa hydroxynitrile lyase isoenzyme from Prunus amygdalus (PaHNL1) to 1.5 A resolution. Clear electron density originating from four glycosylation sites could be observed. As concerns the overall protein fold including the FAD cofactor, PaHNL1 belongs to the family of GMC oxidoreductases. The active site for the HNL reaction is probably at a very similar position as the active sites in homologous oxidases. CONCLUSIONS: There is strong evidence from the structure and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Since key residues implicated in oxidoreductase activity are also present in PaHNL1, it is not obvious why this enzyme shows no oxidase activity. Similarly, features proposed to be relevant for hydroxy-nitrile lyase activity in other hydroxynitrile lyases, i.e., a general base and a positive charge to stabilize the cyanide, are not obviously present in the putative active site of PaHNL1. Therefore, the reason for its HNL activity is far from being well understood at this point.

Aldehyde-Lyases↗

PubMed2001

Blushing and physiological arousability in social phobia.

Blushing is the most prominent symptom of social phobia, and fear perception of visible anxiety symptoms is an important component of cognitive behavioral models of social phobia. However, it is not clear how physiological and psychological aspects of blushing and other somatic symptoms are linked in this disorder. The authors tested whether social situations trigger different facial blood volume changes (blushing) between social phobic persons with and without primary complaint of blushing and control participants. Thirty social phobic persons. 15 of whom were especially concerned about blushing, and 14 control participants were assessed while watching an embarrassing videotape, holding a conversation, and giving a talk. Only when watching the video did the social phobic persons blush more than controls blushed. Social phobic persons who complained of blushing did not blush more intensely than did social phobic persons without blushing complaints but had higher heart rates, possibly reflecting higher arousability of this subgroup.

Adult↗

PubMed2000

XAS spectroscopy reveals X-ray-induced photoreduction of free and protein-bound B12 cofactors.

Crystal structures of several proteins with a B(12) cofactor show abnormally long axial bonds between the cofactor's Co atom and its ;lower' ligand, which is typically a protein-derived imidazole from a histidine residue. X-ray absorption spectroscopy (XAS) experiments were carried out with the following cofactor derivatives to examine the question of whether the bond elongation might be due to an X-ray-induced reduction of the cofactor's cobalt centre: aquocobalamin, cyanocobalamin, methylcobalamin, 5'-desoxyadenosylcobalamin and cob(II)alamin. Each cofactor was investigated at 100 K in a water/glycerol or water/trehalose glass, both as unbound free species and bound to the protein components of the enzyme glutamate mutase. XAS data were collected for each sample around the cobalt absorption edge before and after exhaustive (10 min) irradiation with X-rays of energy 7.76 keV. While XAS spectra for cob(II)alamin, methylcobalamin and 5'-desoxyadenosylcobalamin were the same (within experimental error) before and after irradiation, both in the free and protein-bound state, the spectra of samples with aquocobalamin and cyanocobalamin changed substantially upon irradiation. The spectra of the irradiated samples resembled each other and were similar - but not identical - to the spectrum of the reduced cob(II)alamin. The implications of these observations for the interpretation of the ;long' axial Co-N bonds observed crystallographically in B(12) proteins are discussed.

Journal Article↗

PubMed2000

Reinforcement-based intensive outpatient treatment for inner city opiate abusers: a short-term evaluation.

We evaluated 3-month outcomes for reinforcement-based intensive outpatient treatment (RBT), a new relapse prevention behavior therapy for inner city opiate abusers. The therapy provides abstinence-contingent partial support of housing, food and recreational activities, abstinence-contingent access to social skills and job finding group therapy and non-contingent individual counseling, all in the context of a day treatment program. Heroin abusers (n = 52), contacted at a 3-day detoxification unit, were randomly assigned to RBT (n = 28) or referred to community treatment resources (n = 24) after a staff escort from the detoxification unit. For RBT patients, treatment began on the day of discharge; 61% received partial rent support in a recovery house based on the need for drug-free housing; the remainder were eligible for partial support of utility payments where they lived. Abstinence-based contingencies were in effect for 1 month with three times per week counseling available for an additional 2 months. One month after detoxification, 61% of RBT versus 17% of referral patients were enrolled in outpatient treatment (P < 0.01); RBT patients were significantly less likely than controls to have returned to any drug use; and 50% of RBT versus 21% of controls reported 30 days of abstinence from heroin and cocaine with confirmatory negative urine (P < 0.05). RBT patients had significantly lower scores on the Beck Depression Inventory at 1 month (M = 9.0 versus 17.6 for controls; P < 0.05) and showed evidence of less alcohol use and higher rates of employment. These results establish the short-term efficacy for RBT and support continued development and evaluation of this new outpatient behavioral treatment.

Adolescent↗

PubMed2000

Trigeminal neuralgia: opportunities for research and treatment.

Trigeminal neuralgia was the focus of a recent workshop convened by the National Institute of Neurological Disorders and Stroke (NINDS) and the National Institute of Dental and Craniofacial Research (NIDCR). The workshop brought together basic scientists, clinicians, epidemiologists, and patient advocates. New research directions for epidemiology, diagnosis and assessment, pain mechanisms, and treatment were identified. (The workshop was held in Rockville MD on September 14, 1999, with financial support from NINDS, NIDCR, the NIH Office of Rare Diseases, and the NIH Pain Research Consortium.)

Animals↗

PubMed2000

[Aggressive behavior of mentally incompetent psychiatrically ill criminals during inpatient treatment].

UNLABELLED: OBJECTIVE, METHODS: To assess the amount of violent behaviour among mentally ill offenders NGRI during forensic long-term inpatient-treatment we retrospectively investigated the official incident-reports concerning verbal and physical aggression and damage to foreign property during an 8-year period in Austria's central high-security institution. RESULTS: 29.2% of our patients exhibited violent behaviour with only 7.8% being responsible for 41.1% of all incidents. Mentally impaired patients were significantly overrepresented in the violent group. Physical violence was reported in 25.8% (= 16.48 assaults/100 patients/year). 68% of the total amount of physical violence was directed against fellow patients. Violent behaviour was less driven by psychotic symptoms but rather by current everyday conflicts and stress situations. CONCLUSIONS: The majority of incidents had only minor consequences. Yet, an inquiry concerning the offenders' intentions and the danger experienced by staff members indicated a reasonable violent potential also in minor assaults which appears to be important with respect to ward climate and distress of staff.

Adult↗

PubMed1999

Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights.

BACKGROUND: Glutamate mutase (Glm) equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. Catalysis proceeds with the homolytic cleavage of the organometallic bond of the cofactor to yield a 5'-desoxyadenosyl radical. This radical then abstracts a hydrogen atom from the protein-bound substrate to initiate the rearrangement reaction. Glm from Clostridium cochlearium is a heterotetrameric molecule consisting of two sigma and two epsilon polypeptide chains. RESULTS: We have determined the crystal structures of inactive recombinant Glm reconstituted with either cyanocobalamin or methylcobalamin. The molecule shows close similarity to the structure of methylmalonyl CoA mutase (MCM), despite poor sequence similarity between its catalytic epsilon subunit and the corresponding TIM-barrel domain of MCM. Each of the two independent B12 cofactor molecules is associated with a substrate-binding site, which was found to be occupied by a (2S,3S)-tartrate ion. A 1:1 mixture of cofactors with cobalt in oxidation states II and III was observed in both crystal structures of inactive Glm. CONCLUSIONS: The long axial cobalt-nitrogen bond first observed in the structure of MCM appears to result from a contribution of the species without upper ligand. The tight binding of the tartrate ion conforms to the requirements of tight control of the reactive intermediates and suggests how the enzyme might use the substrate-binding energy to initiate cleavage of the cobalt-carbon bond. The cofactor does not appear to have a participating role during the radical rearrangement reaction.

Clostridium↗

PubMed1999

Structural basis for antibody catalysis of a disfavored ring closure reaction.

The catalysis of disfavored chemical reactions, especially those with no known natural enzyme counterparts, is one of the most promising achievements of catalytic antibody research. Antibodies 5C8, 14B9, 17F6, and 26D9, elicited by two different transition-state analogues, catalyze disfavored endo-tet cyclization reactions of trans-epoxy alcohols, in formal violation of Baldwin's rules for ring closure. Thus far, neither chemical nor enzyme catalysis has been capable of emulating the extraordinary activity and specificity of these antibodies. X-ray structures of two complexes of Fab 5C8 with the original hapten and with an inhibitor have been determined to 2.0 A resolution. The Fab structure has an active site that contains a putative catalytic diad, consisting of AspH95 and HisL89, capable of general acid/base catalysis. The stabilization of a positive charge that develops along the reaction coordinate appears to be an important factor for rate enhancement and for directing the reaction along the otherwise disfavored pathway. Sequence analysis of the four catalytic antibodies, as well as four inactive antibodies that strongly bind the transition-state analogues, suggests a conserved catalytic mechanism. The occurrence of the putative base HisL89 in all active antibodies, its absence in three out of the four analyzed inactive antibodies, and the rarity of a histidine at this position in immunoglobulins support an important catalytic role for this residue.

Amino Acid Sequence↗

PubMed1999

Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.

Aldehyde-Lyases↗

PubMed1998

Heterologous phi X174 gene E-expression in Ralstonia eutropha: E-mediated lysis is not restricted to gamma-subclass of proteobacteria.

E-lysis of Ralstonia eutropha H16, which belongs to the beta-subclass, was undertaken to verify whether transmembrane tunnel formation is possible in bacteria which do not belong to the enterobacteriaceae. For this purpose, a new gene E expression plasmid, pKG12, with two origins of replication, oriV and oriT, from plasmid pRP4, chloramphenicol and kanamycin resistance genes and a casette composed of lambda cI857 and lambda pR gene E was constructed. Temperature upshift of R. eutropha H16 (pKG12) from 28 to 45 degrees C during exponential growth resulted in lysis of the strain with features characteristic of E-mediated lysis of Escherichia coli. The cytoplasmic contents released can easily be separated from the still intact envelope fraction by centrifugation or filtration. As R. eutropha H16 represents an important industrial organism, E-mediated lysis could facilitate procedures for the recovery of intracellular mediators or products like polyhydroxyalkanoates.

Bacteriophages↗

PubMed1998

Aqueous release and purification of poly(beta-hydroxybutyrate) from Escherichia coli.

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon (phaCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% PHB/celldry mass when the cells are grown in Luria-Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.

Bacteriolysis↗

PubMed1998

Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies.

The crystal structure of the thermostable xylanase from Thermomyces lanuginosus was determined by single-crystal X-ray diffraction. The protein crystallizes in space group P21, a = 40.96(4) A, b = 52. 57(5) A, c = 50.47 (5) A, beta = 100.43(5) degrees, Z = 2. Diffraction data were collected at room temperature for a resolution range of 25-1.55 A, and the structure was solved by molecular replacement with the coordinates of xylanase II from Trichoderma reesei as a search model and refined to a crystallographic R-factor of 0.155 for all observed reflections. The enzyme belongs to the family 11 of glycosyl hydrolases [Henrissat, B., and Bairoch, A. (1993) Biochem. J. 293, 781-788]. pKa calculations were performed to assess the protonation state of residues relevant for catalysis and enzyme stability, and a heptaxylan was fitted into the active-site groove by homology modeling, using the published crystal structure of a complex between the Bacillus circulans xylanase and a xylotetraose. Molecular dynamics indicated the central three sugar rings to be tightly bound, whereas the peripheral ones can assume different orientations and conformations, suggesting that the enzyme might also accept xylan chains which are branched at these positions. The reasons for the thermostability of the T. lanuginosus xylanase were analyzed by comparing its crystal structure with known structures of mesophilic family 11 xylanases. It appears that the thermostability is due to the presence of an extra disulfide bridge, as well as to an increase in the density of charged residues throughout the protein.

Amino Acid Sequence↗

PubMed1998

A comparative study of impact dynamics: wobbling mass model versus rigid body models.

We demonstrate that the results of inverse dynamics depend sensitively on the model used to simulate the human body. For this reason we produce "ideal" input data of the ground reaction force and the heel acceleration for a computer simulated drop jump with a wobbling mass model. Then we determine the internal torques and forces in the knee and hip joint via inverse dynamics by employing a rigid body model. It turns out that during the impact phase the analysis with the rigid body model yields completely incorrect internal torques and forces.

Acceleration↗

PubMed1998

[Electrodiagnosis of focal neuropathies].

Electrodiagnostic techniques used in an EMG-Laboratory and the diagnostic value in focal mononeuropathies are presented. The electrodiagnostic techniques include nerve conduction studies and needle electrode examination. Measurement of distal latency, amplitude, conduction velocity and the finding of focal conduction block may localize the focal character of the mononeuropathy. The needle electrode examination gives further information about distribution, extent, activity, or chronicity of the disease, especially in cases with axonal injuries. Some normal electrodiagnostic patterns are presented, accompanied by some illustrative pathological cases.

Electrodes↗