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G Hübner

Publications and source records attributed to G Hübner.

At least 19 recordsLinked to original sources
PubMed1998

Activation of thiamin diphosphate in enzymes.

Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

Allosteric Regulation↗
PubMed1998

Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum.

The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in pyruvate oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-pyruvate oxidase ternary complex requires a second step, which was detected at low temperature.

Catalysis↗
PubMed1998

High-energy phosphates in the frontal lobe correlate with Wisconsin Card Sort Test performance in controls, not in schizophrenics: a 31phosphorus magnetic resonance spectroscopic and neuropsychological investigation.

In recent years, a number of 31phosphorus magnetic resonance spectroscopy (P-MRS) studies on the frontal lobe of schizophrenics have been performed, reporting alterations of phospholipids and high-energy phosphates. Deicken et al. (1994b) recently found positive correlations between left frontal phosphomonoester% (PME%) levels and the performance of a specific frontal lobe task, the Wisconsin Card Sorting Test (WCST), in schizophrenics. In the present paper, the correlations between phospholipids and high-energy phosphates in the frontal lobe of 26 schizophrenics and 23 controls measured with a volume-selective P-MRS method were investigated. Overall, we could not find any correlations between WCST results and phospholipid levels, but in controls phosphocreatine% (PCr%) and PCr/adenenosine triphosphate (ATP) ratios were negatively correlated with test performance. Since PCr behaves as a buffer of ATP, in the sense that when ATP is consumed by neuronal activity PCr is catalysed rapidly to ATP, increased PCr% values and, moreover, increased PCr/ATP ratios point to a decreased ATP consumption. Thus, the correlations found between PCr% and PCr/ATP and test performance in controls point to an association between reduced performance in a specific frontal lobe task and decreased energy demanding processes at rest. This association was not found in schizophrenics, possibly due to the influence of neuroleptic medication or the disease process per se.

Adenosine Triphosphate↗
PubMed1997

Decreased energy demanding processes in the frontal lobes of schizophrenics due to neuroleptics? A 31P-magneto-resonance spectroscopic study.

In the present investigation on 31P-magneto-resonance spectroscopic parameters in the frontal lobe, we found phosphocreatine levels and the ratio phosphocreatine/adenosine triphosphate to be increased (12.62 +/- 1.98% resp. 0.31 +/- 0.06) in 50 neuroleptic-treated schizophrenics, whereas no differences were detected in 10 neuroleptic-free patients (11.66 +/- 2.57% resp. 0.29 +/- 0.08) compared to 36 controls (11.37 +/- 1.45 resp. 0.29 +/- 0.04). This result points to a major role of neuroleptics in the metabolism of high-energy phosphates.

Adenosine Triphosphate↗
PubMed1997

Molecular mechanism of regulation of the pyruvate dehydrogenase complex from E. coli.

The pyruvate dehydrogenase multienzyme complex from E. coli shows a sigmoidal dependency of the reaction rate on the substrate concentration when product formation is followed in the presence of physiological concentrations of the cofactor thiamin diphosphate. To elucidate the molecular mechanism of this regulation, the influence of the substrate pyruvate on the coenzyme-protein interaction has been investigated using several coenzyme analogues. The observed binding constants of all coenzymatically active analogues are increased in the presence of the substrate pyruvate, whereas those of all coenzymatically inactive analogues are not altered in the presence of pyruvate. This points to an increased binding affinity of a reaction-intermediate-coenzyme complex to the protein. Since cofactor binding and dissociation at physiological concentrations of thiamin diphosphate are slow compared to the catalytic reaction, a slow transition to the active state of the enzyme occurs. After lowering the pyruvate concentration, the opposite effect, a dissociation of the thiamin diphosphate from the enzyme is observed. This slow substrate dependent enhancement of cofactor binding enables efficient regulation of the pyruvate dehydrogenase complex by its substrate pyruvate.

Coenzymes↗
PubMed1997

A novel type of glutathione peroxidase: expression and regulation during wound repair.

We have previously identified and cloned a novel keratinocyte growth factor (KGF)-regulated gene in human keratinocytes that encodes the human homologue of a bovine non-selenium glutathione peroxidase (GPx). To gain insight into the regulation of this gene in vivo, we isolated the murine homologue from a mouse skin cDNA library. In vitro transcription/translation demonstrated that the cDNA encodes a 27 kDa protein. Furthermore, we amplified by PCR a partial cDNA that most likely corresponds to a related gene. RNase protection analysis revealed tissue-specific expression of both genes and the occurrence of alternative splicing or RNA editing of at least one of the primary transcripts. Similar to that of KGF, expression of GPx was strongly induced after cutaneous injury, and each isoform displayed unique kinetics of expression during the repair process. In situ hybridization studies demonstrated high levels of GPx mRNA in keratinocytes of the hyperproliferative epithelium at the wound edge. Since these cells express functional KGF receptors, induction of GPx expression by KGF might also occur in vivo. These data suggest a role for GPx in the protection of epithelial cells against oxidative stress, particularly during the inflammatory phase of wound repair.

Amino Acid Sequence↗
PubMed1997

Role of Glu51 for cofactor binding and catalytic activity in pyruvate decarboxylase from yeast studied by site-directed mutagenesis.

We investigated the importance of the interaction between the Nl'-atom of the cofactor thiamine diphosphate and glutamic acid residue 51 in pyruvate decarboxylase (EC 4.1. 1.1). The yeast wild type gene PDCl and the respective mutant genes (E51Q and E51A) were expressed in Escherichia coli. The three enzymes were purified to homogeneity. They comigrated as a single band during silver-stained SDS/PAGE with a molecular mass of 60 000 Da. A molecular mass of 61 200 +/- 200 Da was determined by mass spectrometry for the subunit. The native enzyme is a homotetramer as demonstrated by gel filtration experiments. Near- and far-UV CD spectra showed no significant differences for the apoenzyme of the wild type and the mutants. Slight differences in the rate of thiamine diphosphate binding to the apoprotein component were observed between the wild type and the E51Q PDC by CD spectroscopy. Compared to the wild type enzyme, thiamine diphosphate binding at the E51A mutant apoprotein is very slow. Only 0.04% of the catalytic activity of the wild type enzyme was observed for the E51Q mutant; the E51A mutant has no detectable catalytic activity. The S0.5 value for the substrate pyruvate is increased 33-fold for the E51Q mutant. Substrate activation was observed for both the wild type and the E51Q mutant. The interaction between the N1'-atom of the coenzyme and glutamic acid 51 strongly influences the catalytic activity but only moderately the binding of the cofactor to the apoenzyme and the substrate activation rate.

Binding Sites↗
PubMed1997

How thiamine diphosphate is activated in enzymes.

The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

Allosteric Regulation↗
PubMed1997

Partial inhibition of protein synthesis by Pseudomonas exotoxin A deranges catecholamine sensitivity of cultured rat heart myocytes.

To elucidate cellular mechanisms of myocardial depression in Pseudomonas sepsis the effects of sublethal concentrations of P. aeruginosa exotoxin A--a main virulence factor--were studied in cultured neonatal rat cardiomyocytes. It is known that this toxin exerts its pathogenic effect by inhibition of protein synthesis via ADP-ribosylation and thereby inactivation of elongation factor 2 (EF-2). Within 48 72 h, half maximal inhibition of protein synthesis occurs at 4-10 ng/ml. The toxin prevents the beta-adrenoceptor(AR)-mediated myosin heavy chain isozyme shift (V3/V1), while the T3-induced myosin shift is not suppressed. While beta 1-AR-downregulation by excess of norepinephrine (NE) is not affected, protein synthesis-dependent receptor upregulation in the recover period after removal of NE is completely suppressed by P. aeruginosa exotoxin A. Thus, a non-lethal, partial inhibition of global cellular protein synthesis by P. aeruginosa exotoxin A: (1) completely prevents beta 1-AR-mediated myosin isozyme shift and beta-AR upregulation: (2) sustains the cardiomyocytes in a catecholamine-refractory contractile state in the recovery period after catecholamine desensitization: (3) suggests cellular mechanisms by which P. aeruginosa exotoxin A might impair heart function in Pseudomonas sepsis: and (4) may help reveal the possible influence of endogenous inhibitors of EF-2.

ADP Ribose Transferases↗
PubMed1997

Lack of pharmacokinetic interaction of meloxicam with methotrexate in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the pharmacokinetic interaction of oral meloxicam with intravenous (i.v.) methotrexate (MTX) in patients with rheumatoid arthritis (RA). METHODS: Thirteen patients with RA received MTX 15 mg i.v. in the absence of nonsteroidal antiinflammatory drugs (NSAID) and after one week in the presence of steady state levels of meloxicam. Plasma concentrations of MTX and meloxicam were determined using validated high performance liquid chromatography methods. One patient did not complete the study. The interaction of meloxicam and MTX was examined by equivalence testing. The endpoints AUCMTX, VssMTX, CltotMTX, and CmaxMTX were analyzed parametrically, whereas endpoints MRTMTX, CltotMTX, t1/2MTX, and tmaxMTX were analyzed nonparametrically. RESULTS: The MTX plasma concentrations over time, with and without meloxicam, did not differ significantly. The point estimator for the ratio of log transformed data of the primary endpoint AUCMTX was 108%; the lower 95% confidence limit was 100% and the upper 95% confidence limit was 117%. Clinical laboratory values and adverse events revealed no increased MTX toxicity during concomitant treatment with meloxicam. CONCLUSION: In this short term interaction study there was no statistically significantly effect of meloxicam on the pharmacokinetics of MTX. The combination of MTX and meloxicam did not lead to increased MTX toxicity.

Adolescent↗
PubMed1997

Activin A: a novel player and inflammatory marker in inflammatory bowel disease?

Recently, we demonstrated a strong induction of activin expression after cutaneous injury. We speculated, therefore, that activin may be overexpressed during inflammatory processes in other tissues characterized by mesenchymal/epithelial structure. Herein, we show a strikingly increased expression of the activin beta A-subunit in surgical specimens from the gut of patients suffering from ulcerative colitis and Crohn's disease, whereas no activin beta A mRNA could be detected in the normal human digestive tract. The levels of activin beta A expression showed an outstanding correlation with the degree of inflammation as assessed by histologic analysis of adjacent tissue and expression analysis of the proinflammatory cytokine interleukin-1 beta. In situ hybridization studies revealed the highest levels of activin mRNA in the mucosa and submucosa of highly inflamed areas, particularly where the intestinal epithelium was damaged, but not in control tissue. In contrast, activin beta B mRNA levels in most specimens from inflamed areas were only slightly higher compared to control tissue. The strong overexpression of activin beta A in inflammatory bowel disease suggests a novel and important role of this growth and differentiation factor during inflammatory processes of the gut.

Activins↗
PubMed1996

Serum growth factors and proinflammatory cytokines are potent inducers of activin expression in cultured fibroblasts and keratinocytes.

Recently we demonstrated a large induction of activin expression in fibroblasts and keratinocytes after cutaneous injury in mice. To identify possible mediators of activin induction during skin repair, we have now analyzed the regulation of this factor in cultured keratinocytes and fibroblasts. Here we show that activin A mRNA and protein levels are low in quiescent keratinocytes and fibroblasts but expression is strongly induced upon serum treatment. The stimulatory effect of serum on activin expression is likely to be a combinatorial effect of different growth factors, since platelet-poor plasma serum and several purified serum growth factors also stimulated activin expression, although to a lesser extent than complete serum. Furthermore, we found increased expression of activin in keratinocytes and fibroblasts after addition of the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha. Taken together, our data suggest that serum growth factors which are released upon hemorrhage as well as proinflammatory cytokines derived from neutrophils and macrophages might be responsible for induction of activin expression after injury.

3T3 Cells↗
PubMed1996

Strong induction of activin expression after hippocampal lesion.

We studied the temporal and spatial mRNA expression pattern of activin/inhibin beta A, beta B and alpha subunits after unilateral kainic acid lesions of the hippocampal CA3 region. We found a strikingly increased expression of beta A mRNA in the ipsilateral hippocampus 6-24 h after injury. By contrast, the beta B and alpha mRNAs were expressed at equally low levels in normal and injured hippocampi, suggesting that the beta A transcripts give rise to activin A, but not to activin AB or inhibin. In situ hybridization demonstrated the presence of beta A mRNA in neurones near the site of lesion. Expression of all known types of activin receptors could be demonstrated in normal and injured hippocampi by RT-PCR. These findings suggest a role of activin in brain injury.

Activins↗
PubMed1996

[Intensive post-remission therapy in acute myeloid leukemia. Results of a prospective comparative study by the South Germany Hemoblastosis Group].

BACKGROUND: To study intensive postremission therapy in adult patients with acute myeloid leukemia myeloablative therapy followed by allogeneic or unpurged autologous bone marrow transplantation (BMT) was compared with high-dose cytosine-arabinoside/daunorubicin (HDAC) consolidation. PATIENTS AND METHODS: 148 de novo AML patients of maximum 50 years (median 36 years, range 16 to 50) were enrolled in the trial. Following induction and early consolidation chemotherapy consisting of daunorubicin, cytosine-arabinoside and VP-16 (DAV), patients with an HLA-identical sibling underwent allogeneic BMT. The other patients received (by randomization or patient's decision) either HDAC or high-dose busulfan plus cyclophosphamide followed by autologous BMT. RESULTS: Hundred and five 105 (70.9%) patients achieved a complete remission. The event-free survival rates after intensive postremission therapy after 72 months were: after BMT (24 patients) 62% (95% confidence interval +/- 19%), after HDAC (44 patients) 36 +/- 16% and after autologous BMT (12 patients) 18 +/- 22%. Thus allogeneic BMT was superior to autologous BMT (p = 0.04), as was HDAC compared to autologous BMT, although not significantly so (p = 0.15). Patients receiving 2 cycles of HDAC had a better 6-year event-free survival rate (47%) and a lower relapse rate (50%) than patients who received only 1 course (29% and 70% respectively). CONCLUSIONS: High-dose busulfan/cyclophosphamide followed by unpurged autologous BMT early after achieving CR had no advantage over high-dose ara-c/daunorubicin. Two cycles of HDAC yielded better results than 1 cycle. The highest event-free survival rate was reached with myeloablative therapy followed by allogeneic BMT.

Adolescent↗
PubMed1996

[10 years transplantation of bone marrow and hematopoietic stem cells in adults at the Hannover Medical School].

PATIENTS AND METHODS: From January 1986 until August 1995 230 adult patients received an allogeneic or autologous transplantation of bone marrow or hematopoietic blood stem cells. The conditioning and myeloablative treatment regimens were chosen according to the underlying disease and type of transplant. RESULTS: The observation period comprises 1 to 115 months after transplantation. After allogeneic transplantation from HLA-identical family donors, the probabilities of disease-free survival were for acute myeloid leukemia in first complete remission (CR) (n = 35) 77%, for acute lymphoid leukemia in 1st CR (n = 7) 72% and in 2nd CR (n = 10) 40%, in first chronic phase of chronic myeloid leukemia (n = 34) 50% and in severe aplastic anemia (n = 7) 100%. Following myeloablative therapy and autologous transplantation the probabilities of disease-free survival were 47% in relapsed Hodgkin's disease (n = 22) and 42% for relapsed high-grade non-Hodgkin's lymphoma (n = 12). Eight of 10 patients with acute myeloid and 7 of 8 with acute lymphoid leukemia suffered a leukemic relapse after autologous bone marrow transplantation. Three of 8 patients with relapsed testicular cancer survived relapse-free. Treatment failures were due to more advanced acute graft versus host disease after allogeneic transplantation and caused by relapse after autologous transplantation. Current protocols evaluate the allogeneic transplantation of enriched CD34+ blood stem cells. In chronic myeloid leukemia the autologous transplantation of blood stem cells after myeloablative therapy is being studied.

Adolescent↗
PubMed1996

Strong induction of activin expression after injury suggests an important role of activin in wound repair.

Activins are members of the transforming growth factor beta (TGF beta) superfamily, which comprises a growing group of dimeric proteins. TGF beta and several other members of this superfamily are known to play an important role in wound healing. However, expression of activin during wound healing has not been demonstrated so far. In this study we have analyzed the expression pattern of activin and activin receptors in normal and wounded skin. We found a large induction of activin A and a minor induction of activin B mRNA expression 1 day after skin injury and high expression levels of activin A and B were found within the first 7 days after wounding. At 13 days after injury, expression of activin A mRNA had returned to the basal level, whereas high levels of activin B persisted. In situ hybridization studies revealed expression of activin A in the granulation tissue below the wound and activin B in the hyperproliferative epithelium at the wound edge and in the migrating epithelial tongue. All known types of activin receptors as well as the activin binding protein follistatin were expressed in normal and wounded skin. However, no significant induction of receptor gene expression was seen during the repair process. The distribution of activins and activin receptors in the wound suggests multiple autocrine and paracrine activities of the ligands during wound healing. Our data provide evidence for a novel function of activin and indicate that--besides TGF beta s themselves--other members of this superfamily might also play an important role in tissue repair.

Activin Receptors↗