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Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 145 records · Page 8Linked to original sources
PubMed2000

Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation.

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.

Amino Acid Motifs↗
PubMed2000

Protein binding of NADH on chemical preconditioning.

Chemical preconditioning, an emerging neuroprotective strategy described in recent years, results in preserved energy metabolism during hypoxia via yet unknown mechanisms. The hypoxic increase of NADH content is attenuated by preconditioning. The goal of the present study was to investigate whether attenuation of the hypoxic NADH increase is due to a shift between free and protein-bound NADH. NADH in solution has a fluorescence maximum at 469.2 nm. In untreated mouse hippocampal slices, lambda(control onset) is 456.2 +/- 5.3 nm in CA1 (mean +/- SD; p < 0.01 vs. solution) and 454.6 +/- 6.1 nm in CA3 [p < 0.01 vs. solution, not significant (NS) to lambda(control onset) in CA1]. In slices prepared from animals pretreated in vivo with 20 mg/kg 3-nitropropionate, lambda(preconditioning onset) is 439.2 +/- 5.0 nm (p < 0.001 vs. control) in CA1 and 434.2 +/- 6.4 nm in CA3 (p < 0.001 vs. control; NS to lambda(preconditioning onset) in CA1). In controls, the fluorescence maximum shifts to lambda(control hypoxia) 458.2 +/- 1.3 nm in CA1 (NS vs. onset) and 456.0 +/- 3.6 nm in CA3 (NS vs. onset). On preconditioning with 3-nitropropionate, lambda(preconditioning hypoxia) shifts to 446.4 +/- 4.3 nm in CA1 (p < 0.03 vs. onset) and 438.6 +/- 6.9 nm in CA3 (p < 0.03 vs. onset). Posthypoxic decay of free and protein-bound NADH is diminished after preconditioning. We conclude that the free NADH level is reduced on an increase of hypoxic tolerance by chemical preconditioning. Reduction of free NADH content is maintained during hypoxia after preconditioning.

Animals↗
PubMed2000

Hypereosinophilia induced by high-dose intratumoral and peritumoral mistletoe application to a patient with pancreatic carcinoma.

UNLABELLED: A patient with inoperable adenocarcinoma of the pancreas was treated with intraperitumoral and peritumoral injections of a Viscum album L. extract containing 5,700 ng/mL mistletoe lectin, mainly mistletoe lectin 1 (Abnobaviscum Quercus 2) for 5 weeks (1 injection per week). After the third injection (day 22), a marked eosinophilia was observed (1,800 per microliter) that rose to 3,268 per microliter after the fifth injection (day 42). Furthermore, histology performed on day 28 revealed accumulation of eosinophils in ductal lesions and adjacent stroma in addition to the features of ductal adenocarcinoma. In order to investigate whether eosinophilia correlated with immunological features, we analyzed cytokine production of peripheral blood mononuclear cells (PBMC) from this patient after stimulation with antigens known to "unmask" an individual's predisposition for defined immunoreactions, namely purified protein derivative (PPD) as a stimulator of T-helper (TH1)1-cells and tetanus toxoid (TT) as an activator of TH2-cells. PBMC of the patient showed a strong proliferation and production of interleukin (IL)-5 and IL-10 after incubation with TT indicating a type-2 response. Simultaneously, PBMC were induced to proliferate and produce interferon gamma (IFN-gamma) by incubation with PPD suggesting also a type-1 response. These data would readily explain the eosinophilia because eosinophils are effector cells of type-2 reaction but also require type-1 cytokines. Although the overall clinical course of the patient was rapidly progressive, temporary stabilization of the patient's general condition during mistletoe treatment was observed. It is, however, still an open question whether this transient benefit was due to the induction of eosinophilia by a type-2 response. CONCLUSION: Before high-dose intratumoral and peritumoral treatment with a Viscum album L. extract containing mistletoe, lectin 1 can be associated with hypereosinophilia and strong production of TH1 as TH2 cytokines as well.

Adenocarcinoma↗
PubMed2000

Selection bias in clinical trials with antipsychotics.

Although the selection of patients is known to be a powerful factor affecting the results of clinical trials, little is known about recruitment issues. Many patients with schizophrenia who are screened for a clinical trial of an investigational antipsychotic are ultimately not included in the study. Therefore, the question arises of whether the results obtained by studying a selected group of patients are really representative of the general population of patients with schizophrenia. The authors studied possible reasons for selective sampling in 200 patients who were consecutively admitted to inpatient units of Innsbruck's Department of Psychiatry with a diagnosis of schizophreniform or schizophrenic disorder over a time period of 33 months. Apart from demographic data and a psychopathologic rating (using the Brief Psychiatric Rating Scale), the authors recorded whether or not a patient was included in a phase III study and whether or not those were not included would have theoretically been eligible for such a study. Twenty-seven patients were finally recruited for a clinical trial. These patients were younger, on average, had a more recent onset of illness, and had experienced fewer psychotic episodes in the past. A history of noncompliance with previous treatment and the refusal of consent were the most common reasons for not including theoretically eligible patients in a clinical trial.

Adolescent↗
PubMed2000

L-Isoaspartate 115 of porcine beta-trypsin promotes crystallization of its complex with bdellastasin.

Bdellastasin is a 59-amino-acid, cysteine-rich, antistasin-type inhibitor of sperm acrosin, plasmin and trypsin, isolated from the medicinal leech Hirudo medicinalis. The complex formed between bdellastasin and porcine beta-trypsin has previously been crystallized in the presence of PEG in a tetragonal crystal form of space group P4(3)2(1)2 and has now been found to crystallize under high-salt conditions in the enantiomorphic space group P4(1)2(1)2. These structures have been solved and refined to 2.8 and 2.7 A resolution, respectively. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure. In the second new crystal form, the flexible N-terminal subdomain is rotated with respect to the C--terminal subdomain by about 90 degrees, fitting into a cavity formed by symmetry-related trypsin molecules. The canonical inhibitor-proteinase interaction is restricted to the primary binding loop comprising residues Leu31-Lys36 of bdellastasin. During the refinement, a bound sodium ion occupying the calcium-binding site of the porcine beta-trypsin component was discovered. This sodium ion is coordinated in a tetragonal-pyramidal manner, with the geometry of the enclosing loop slightly changed compared with the loop in the presence of calcium. In the crystal form of space group P4(3)2(1)2, the electron density for residue 115 of porcine beta-trypsin clearly indicates the presence of a beta-isomerized L-aspartic acid, which is placed in spatial proximity to segment Thr144--Gly148 of a symmetry-related trypsin molecule. This is the first structurally observed example of an L-isoaspartate in beta--trypsin originating from Asn. A comparison with other known crystal structures of porcine beta-trypsin-macromolecular inhibitor complexes suggests that the deamidation, isomerization and racemization of Asn115 is the key step in crystallization.

Animals↗
PubMed2000

Crystallization and preliminary X-ray crystallographic analysis of PdxJ, the pyridoxine 5'-phosphate synthesizing enzyme.

The enzyme PdxJ catalyzes the condensation of 1-deoxy-D-xylulose-5-phosphate (DXP) and 1-amino-3-oxo-4-(phosphohydroxy)propan-2-one to form pyridoxine 5'-phosphate (PNP). The protein from Escherichia coli has been crystallized in several forms under different conditions. The best diffracting crystals were obtained by a combination of the hanging-drop vapour-diffusion and microseeding techniques. Using an in-house image plate, the PdxJ crystals diffracted under cryo-conditions to 2.6 A resolution. The space group has been determined as C222(1), with unit-cell parameters a = 132.5, b = 154. 4, c = 131.4 A, corresponding to four monomers per asymmetric unit. In the search for heavy-atom derivatives, a mercury derivative has been interpreted. The 12 mercury sites located are related by 222 symmetry and, in combination with self-rotation search analyses and gel-filtration experiments, indicate the quaternary assembly of PdxJ into octamers with 422 symmetry.

Bacterial Proteins↗
PubMed2000

Primary structure of a novel subunit in ba3-cytochrome oxidase from Thermus thermophilus.

The bax-type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3-cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).

Amino Acid Sequence↗
PubMed2000

Towards the ecology of hyperthermophiles: biotopes, new isolation strategies and novel metabolic properties.

Ecological studies have shown that water-containing terrestrial, subterranean and submarine high-temperature environments harbor a great diversity of hyperthermophilic prokaryotes, growing fastest at temperatures of 80 degrees C or above. The investigations included cultivation, isolation and detailed analysis of these hyperthermophiles as well as in situ 16S rRNA gene sequence analysis and in situ hybridization studies. For a safe and fast isolation of novel hyperthermophiles from mixed cultures, a new, plating-independent isolation technique was developed, based on the use of a laser microscope ('optical tweezers'). This method, combined with 16S rRNA gene sequence analysis and whole-cell hybridization using fluorescently labelled oligonucleotide probes, even allows the recovery of pure cultures of phylogenetically predicted organisms harboring novel 16S rRNA gene sequences. In their natural habitats, hyperthermophiles form complex food webs, consisting of primary producers and consumers of organic material. Their metabolic potential includes various types of aerobic and anaerobic respiration and different modes of fermentation. In hydrothermal and geothermal environments, hyperthermophiles have important ecological functions in biogeochemical processes. Members of the Sulfolobales are able to mobilize heavy metals from sulfidic ores like pyrite or chalcopyrite. Biomineralization processes of hyperthermophiles include the formation of magnetite from iron or the precipitation of arsenate as realgar, a reaction performed by a novel hyperthermophile that was isolated from Pisciarelli Solfatara, Naples, Italy.

Archaea↗
PubMed2000

Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus.

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

Alcohol Dehydrogenase↗
PubMed2000

Topography of EEG dynamics after sleep deprivation in mice.

Several recent results show that sleep and sleep regulation are not only global phenomena encompassing the entire brain, but have local features. It is well established that slow-wave activity [SWA; mean electroencephalographic (EEG) power density in the 0.75-4.0 Hz band] in non-rapid eye movement (NREM) sleep is a function of the prior history of sleep and wakefulness. SWA is thought to reflect the homeostatic component of the two-process model of sleep regulation. According to this model, originally formulated for the rat and later extended to human sleep, the timing and structure of sleep are determined by the interaction of a homeostatic Process S and a circadian process. Our aim was to investigate the dynamics of SWA in the EEG of two brain regions (frontal and occipital cortex) after sleep deprivation (SD) in two of the mice strains most often used in gene targeting. C57BL/6J (n = 9) and 129/Ola (n = 8) were recorded during a 24-h baseline day, 6-h SD, and 18-h recovery. Both derivations showed a significant increase in SWA in NREM sleep after SD in both strains. In the first hour of recovery, SWA was enhanced more in the frontal derivation than in the occipital derivation and showed a faster decline. This difference resulted in a lower value for the time constant for the decrease of SWA in the frontal derivation (frontal: 10.9 +/- 2.1 and 6.8 +/- 0.9 h in Ola and C57, respectively; occipital: 16.6 +/- 2.1 and 14.1 +/- 1.5 h; P < 0.02; for each of the strains; paired t-test). Neither time constant differed significantly between the strains. The subdivision of SWA into a slower and faster band (0.75-2.5 Hz and 2.75-4.0 Hz) further highlighted regional differences in the effect of SD. The lower frequency band had a higher initial value in the frontal derivation than in the occipital derivation in both strains. Moreover, in the higher frequency band a prominent reversal took place so that power in the frontal derivation fell below the occipital values in both strains. Thus our results indicate that there may be differences in the brain in the effects of SD on SWA in mice, suggesting regional differences in the dynamics of the homeostatic component of sleep regulation. The data support the hypothesis that sleep has local, use- or waking-dependent features that are reflected in the EEG, as has been shown for humans and the laboratory rat.

Animals↗
PubMed2000

Local delivery of platelet-derived growth factor receptor-specific tyrphostin inhibits neointimal formation in rats.

Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) system is involved in the process of postangioplasty restenosis. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the antiproliferative effects of PDGFRbeta-specific tyrphostin AG-1295 in vitro and in vivo. AG-1295 significantly inhibited rat smooth muscle cell growth stimulated by PDGF-BB or FCS. This antiproliferative effect was paralleled by reversible reduction of the total phosphotyrosine level and the degree of PDGFRbeta phosphorylation by the drug in vitro. Local sustained delivery of the drug from perivascularly implanted polymeric matrices resulted in focal AG-1295 levels of 711 and 29.1 ng/mg of dry arterial tissue 1 and 14 days after implantation in rats. AG-1295 delivered from polymeric matrices resulted in a 35% reduction of neointimal formation on day 14 after balloon injury in the rat carotid model. Tyrosine phosphorylation of certain transduction proteins in arterial tissue extracts was significantly upregulated by balloon injury on day 3 but was essentially returned to or below basal levels 14 days after injury. Tyrphostin treatment decreased tyrosine phosphorylation at both time points below the basal levels. Moreover, the enhancement of PDGFRbeta expression 3 and 14 days after arterial injury was strongly inhibited by AG-1295 treatment. It can be concluded that AG-1295 reduces neointimal formation by inhibiting PDGFbeta-triggered tyrosine phosphorylation.

Angioplasty, Balloon↗
PubMed2000

Structural investigations of the highly flexible recombinant ribosomal protein L12 from Thermotoga maritima.

Ribosomal protein L7/L12, the only multicopy component of the ribosome, is involved in translation factor binding and in the ribosomal GTPase center. The gene for L7/L12 from Thermotoga maritima was cloned and the protein expressed at high levels in Escherichia coli. Purification of L7/L12 was achieved under non-denaturing conditions via heat treatment and two chromatographic steps. Circular dichroism melting profiles were monitored at 222 nm, showing the melting temperature of the protein at pH 7.5 around 110 degrees C, compared to approximately 60 degrees C for the highly homologous Escherichia coli protein. The unfolding was reversible and renaturation closely followed the path of the thermal melting. Dynamic light scattering, gel filtration chromatography, and crosslinking experiments suggested that under physiological buffer conditions Thermotoga maritima L7/L12 exists as a tetramer. The protein was crystallized under two conditions, yielding an orthorhombic (C222(1)) and a cubic (12(1)3) space group with an estimated two and three to four L7/L12 molecules per asymmetric unit, respectively. The crystals contained the full-length protein, and cryogenic buffers were developed which improved the mosaic spreads and the resolution limits. For the structure solution isoleucine was mutated to methionine at two separate positions, the mutant forms expressed as selenomethionine variants and crystallized.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the caspase activator human granzyme B, a proteinase highly specific for an Asp-P1 residue.

Granzyme B is the prototypic member of the granzymes, a family of trypsin-like serine proteinases localized in the dense cytoplasmic granules of activated natural killer cells and cytotoxic T lymphocytes. Granzyme B directly triggers apoptosis in target cells by activating the caspase pathway, and has been implicated in the etiology of rheumatoid arthritis. Human granzyme B expressed in a baculovirus system has been crystallized without inhibitor and its structure has been determined to 3.1 A resolution, after considerably improving the diffraction power of the crystals by controlled humidity changes. The granzyme B structure reveals an overall fold similar to that found in cathepsin G and human chymase. The guanidinium group of Arg226, anchored at the back of the S1-specificity pocket, can form a salt bridge with the P1-Asp side chain of a bound peptide substrate. The architecture of the substrate binding site of granzyme B appears to be designed to accommodate and cleave hexapeptides such as the sequence Ile-Glu-Thr-Asp-/Ser-Gly present in the activation site of pro-caspase-3, a proven physiological substrate of granzyme B. These granzyme B crystals, with fully accessible active sites, are well suited for soaking with small synthetic inhibitors that might be used for a treatment of chronic inflammatory disorders.

Amino Acid Sequence↗
PubMed1999

Impaired tolerance to repetitive hypoxia in hippocampal slices of Cu,Zn superoxide dismutase transgenic mice.

Energy metabolism is impaired in the Cu,Zn superoxide dismutase transgenic mouse model of amyotrophic lateral sclerosis. The goal was to investigate tolerance against single and repetitive hypoxia in C57B6SJL-TgN(SOD1-G93A)1GUR mice (G93A mice). Posthypoxic recovery (15 min hypoxia, 45 min recovery) of population spike amplitude in hippocampal region CA1 was 38 +/- 29% (mean +/- SD) in controls and 67 +/- 41% (ns) in G93A mice at day 40. Upon in vivo pretreatment with 20 mg/kg 3-nitropropionate posthypoxic recovery increased to 82 +/- 32% (P < 0.01) in controls and decreased to 35 +/- 33% in G93A mice (P < 0.05 to pretreated controls). Results at day 80 and 110 were similar. We conclude that G93A mice show a long-lasting impairment to sustain repetitive hypoxic episodes whereas tolerance to a single hypoxic episode is comparable to controls.

Amyotrophic Lateral Sclerosis↗
PubMed1999

Crystal structures of the membrane-binding C2 domain of human coagulation factor V.

Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.

Amino Acid Sequence↗
PubMed1999

Pulsed high-frequency electromagnetic field affects human sleep and sleep electroencephalogram.

To investigate whether the electromagnetic field (EMF) emitted by digital radiotelephone handsets affects the brain, healthy, young subjects were exposed during an entire night-time sleep episode to an intermittent radiation schedule (900 MHz; maximum specific absorption rate 1 W/kg) consisting of alternating 15-min on-15-min off intervals. Compared with a control night with sham exposure, the amount of waking after sleep onset was reduced from 18 to 12 min. Spectral power of the electroencephalogram in non-rapid eye movement sleep was increased. The maximum rise occurred in the 10-11 Hz and 13.5-14 Hz bands during the initial part of sleep and then subsided. The results demonstrate that pulsed high-frequency EMF in the range of radiotelephones may promote sleep and modify the sleep EEG.

Adult↗
PubMed1999

Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.

The MunI restriction enzyme recognizes the palindromic hexanucleotide sequence C/AATTG (the '/' indicates the cleavage site). The crystal structure of its active site mutant D83A bound to cognate DNA has been determined at 1.7 A resolution. Base-specific contacts between MunI and DNA occur exclusively in the major groove. While DNA-binding sites of most other restriction enzymes are comprised of discontinuous sequence segments, MunI combines all residues involved in the base-specific contacts within one short stretch (residues R115-R121) located at the N-terminal region of the 3(10)4 helix. The outer CG base pair of the recognition sequence is recognized solely by R115 through hydrogen bonds made by backbone and side chain atoms to both bases. The mechanism of recognition of the central AATT nucleotides by MunI is similar to that of EcoRI, which recognizes the G/AATTC sequence. The local conformation of AATT deviates from the typical B-DNA form and is remarkably similar to EcoRI-DNA. It appears to be essential for specific hydrogen bonding and recognition by MunI and EcoRI.

Binding Sites↗