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Michael Becker

Publications and source records attributed to Michael Becker.

33 records · Page 2Linked to original sources
PubMed2003

Purification, characterization and functional cloning of inositol oxygenase from Cryptococcus.

The enzyme inositol oxygenase (myo-inositol : oxygen oxidoreductase; E.C. 1.13.99.1) is a monooxygenase that converts inositol into glucuronic acid in the presence of molecular oxygen. This enzyme is integrated into a pathway leading to either degradation and energy production or the biosynthesis of precursors for polysaccharides. The enzyme was purified from the yeast Cryptococcus lactativorus by a five-step chromatography procedure. The purified enzyme shows a molecular mass of 37 kDa on SDS-PAGE, similar to the estimation of the size of the native enzyme determined by size exclusion chromatography. Peptides of the inositol oxygenase protein derived from a tryptic digest were sequenced de novo by nanoelectrospray tandem mass spectrometry. Using degenerate oligonucleotides, the corresponding gene was cloned from first strand cDNA. The open reading frame encodes a 315 amino acid polypeptide with a predicted molecular mass of 36.9 kDa. Inositol oxygenase is a single copy gene in C. lactativorus. It has close homologues in other fungi such as Cryptococcus neoformans and Neurospora crassa. Biochemical characterization of the enzyme showed a pH optimum of 6-6.5 and a temperature optimum of 30 degrees C. Myo-inositol is the only accepted substrate with a Km of ca. 5 mM. The enzyme contains a Fe-centre but the enzyme activity is resistant to KCN.

Amino Acid Sequence↗
PubMed2003

Treatment of chronic graft-versus-host disease with anti-CD20 chimeric monoclonal antibody.

We reviewed the clinical outcome of 8 patients with steroid-refractory chronic graft-versus-host disease (GVHD) who received an anti-CD20 chimeric monoclonal antibody (rituximab). Rituximab was given by intravenous infusion at a weekly dose of 375 mg/m(2) for 4 weeks. All patients had received extensive treatment with various immunosuppressive agents; 6 patients had also received extracorporeal photopheresis. All patients had extensive chronic GVHD with diffuse or localized sclerodermoid GVHD and xerophthalmia. Other extracutaneous involvements included cold agglutinin disease with the Raynaud phenomenon, membranous glomerulonephritis, and restrictive or obstructive lung disease. Four patients responded to treatment with ongoing resolution or improvement ranging from 265 to 846 days after therapy, despite recovery of B cells in 3 patients. Rituximab seems to have significant activity in the treatment of refractory chronic GVHD and should be considered for further study in patients with early disease. This study suggests a participating role of B cells in the pathogenesis of chronic GVHD.

Adult↗
PubMed2003

Emerging therapeutic targets in chronic heart failure: part II.

Chronic heart failure is characterised by functional deficiencies of the myocardium. Structural abnormalities of the left ventricular wall occur in many cases as a consequence of myocardial infarction (MI). The overburdened postMI heart is characterised by an active reorganisation of the remaining myocardium. Increased expression and activity of matrix metalloproteinases lead to altered composition and arrangement of the extracellular matrix, which is accompanied by eccentric hypertrophy of cardiomyocytes. The altered geometry of the heart muscle fosters biomechanical stress, driving the heart into a dead-end situation. Clearly, novel therapeutic concepts must be developed to reverse this process. Part II of the current review will focus on emerging therapeutic targets for small molecule therapeutics in the fields of cardiac remodelling and impaired survival of cardiomyocytes in the diseased heart. Finally, innovative therapeutic concepts for heart gene therapy and replacement options for destroyed post-MI myocardium using embryonic and adult stem cells are described.

Apoptosis↗
PubMed2002

13C-urea breath test threshold calculation and evaluation for the detection of Helicobacter pylori infection in children.

BACKGROUND: The 13C-urea breath test (UBT) is performed in adults and children with epigastric pain for non-invasively diagnosing a suspected H. pylori infection. Criteria for UBT interpretation have not been generally agreed on and test reliability has not been established in children of different ages. This study aimed at identifying reliable UBT thresholds in children by using 251 UBTs in conjunction with reference histology and by analyzing 1232 UBTs. METHODS: At baseline and 30 and 60 minutes after the administration of 75 mg 13C-urea to children and adolescents (0.25 to 18 years of age), the differences (Delta) of 13CO2/12CO2 ratio in exhaled air (delta) were determined by mass spectrometry. UBT Deltadelta value thresholds were calculated in random subgroups and evaluated in complementary subgroups using logistic regressions on reference histology or bimodal distribution analyses of Deltadelta values from UBTs alone. RESULTS: Deltadelta values were higher (median, 15.4 per thousand) in positive (133/251, 53 %) than in negative histology (2.4 per thousand). At 30 minutes, the calculated cut-off was 5.3 per thousand (mean regression determination R2 = 0.91), and sensitivity (0.95), specificity (0.97), positive (0.97) and negative predictive values (0.95) were higher than at 60 minutes (threshold 6.8 per thousand, R2 = 0.85). Similar thresholds resulted from UBTs analysis (5.8 per thousand and 6.2 per thousand) when sensitivity and specificity were maximized (concordance probabilities, 0.99 and 0.99). There was no systematic age effect. CONCLUSIONS: In children, 13C UBT cut-offs were obtained and specially validated, entailing high accuracy of non-invasively testing for gastric H. pylori infection.

Administration, Oral↗
PubMed2002

Validation of targets and drug candidates in an engineered three-dimensional cardiac tissue model.

High-throughput target discovery confronts the biopharmaceutical industry with a plethora of target candidates. The validation of these candidates in disease-specific animal models often lacks the required throughput. Here, we discuss perspectives and limitations of a novel engineered three-dimensional cardiac tissue, which enables the influence of gene and drug intervention to be monitored on a cellular and molecular level under physiological conditions in sufficient throughput. The model is an extremely helpful filter to prioritize multiple development candidates before moving a project into large animal models with higher predictivity.

Animals↗
PubMed2002

Refinements in nerve to muscle neurotization.

A modified surgical technique is introduced, enabling restoration of muscle function with direct muscular neurotization. Reliable clinical outcomes result from this technique. We report on a series of 10 patients in whom the supplying motor nerve had been lost at the level of the neuromuscular junction as the result of trauma or tumor resection. Our modification of the operative technique ensures a wide distribution of nerve fibers throughout the remaining muscle tissue and produces a mean motor recovery of M4 after a period of 1 to 2 years.

Adult↗
PubMed2002

Tropical calcific pancreatitis: strong association with SPINK1 trypsin inhibitor mutations.

BACKGROUND & AIMS: Tropical calcific pancreatitis (TCP) is a chronic pancreatitis unique to developing countries in tropical regions. The cause of TCP is obscure. Whereas environmental factors, such as protein energy malnutrition and ingestion of cassava, have been implicated, a genetic predisposition to the disease also may be important. In the present study we report on mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene in north Indian patients with TCP. METHODS: We studied 66 unrelated TCP patients (44 men, 49 with diabetes, and 6 with family history of TCP), 25 relatives, and 92 healthy control subjects. Samples were analyzed for SPINK1 variants (-53C>T, L14P, N34S, P55S, and 272T>C) and cationic trypsinogen (PRSS1) variants (A16V, K23R, N29I, and R122H) by melting curve analysis. RESULTS: Twenty-nine patients (44%) carried the N34S missense mutation, of whom 9 (14%) were homozygotes. In contrast, only 2 (2.2%) control subjects were N34S heterozygotes (prevalence ratio 20.2; 95% confidence interval 5.0-81.8; P < 0.0001 vs. TCP). The severity of pancreatitis did not differ between TCP patients with or without N34S, or among those heterozygous or homozygous for N34S. Among TCP patients with or without diabetes, the frequency of N34S carriers (43% vs. 47%) and N34S homozygotes (14% vs. 12%) was similar. CONCLUSIONS: TCP is highly associated with the SPINK1 N34S mutation. The high prevalence of N34S in TCP patients with and without diabetes suggests that these 2 subtypes have a similar genetic predisposition. The genetic predisposition to TCP resembles, at least in part, the idiopathic chronic pancreatitis found in industrialized countries.

Adult↗
PubMed2002

Sensory reconstruction of the fingertip using the bilaterally innervated sensory cross-finger flap.

The authors present a series of 15 patients with large soft-tissue defects of the fingertips as a prospective, nonrandomized study. In all cases, reconstruction was achieved using a bilaterally innervated sensory cross-finger flap. This sensory fasciocutaneous flap relies on the dorsal branch of the proper digital nerves, which branch off at the level of the head of the proximal phalanx; sensory supply to the dorsal skin of the middle phalanx is thus ensured. The reconstructive procedure consists of two steps. First, the contralateral dorsal branch of the proper digital nerve is elevated with the flap at proximal interphalangeal joint level. Microsurgical coaptation is performed to the proximal nerve stump of the injured fingertip. After 3 weeks, when the pedicle is dissected, the second nerve is dissected and coapted. Clinical results were evaluated after 12 months. Because the regenerative distance is only 1.5 to 2.5 cm, good sensory regeneration should be expected. In nine of 16 flaps, sensory quality of S2+ (Highet) was present in the flap after 3 weeks. After 12 months, two-point discrimination was present in all patients, the values ranging between 2 and 6 mm (for two-point discrimination), with an average of 3.6 mm. The rate of complications was low. With acceptable additional operative action, a good functional result can be achieved. The indications of this method are discussed in comparison with other methods of fingertip reconstruction.

Adolescent↗
PubMed2002

Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis.

OBJECTIVE: The pathogenesis of chronic pancreatitis (CP) is poorly understood. Genetic studies revealed mutations in the cationic trypsinogen gene and an increased frequency of cystic fibrosis gene mutations in patients with CP. Recently, a point mutation (N34S) in the gene encoding the serine protease inhibitor, Kazal type 1 (SPINK1), was found in approximately 20% of patients with CP. The aim of our study was to determine the frequency of the N34S SPINKI gene mutation in a well-defined patient cohort with idiopathic CP (ICP) and to compare the incidence with healthy controls. In addition, we investigated the impact of this mutation on the long-term course of CP. METHODS: Fourteen patients with early-onset and four patients with late-onset CP of our well-defined pancreatitis cohort were enrolled in the present study, and 397 healthy individuals served as a control population. Coding exonic and the flanking intronic sequences of SPINK1 were investigated by direct DNA sequencing. The mutations found were confirmed by melting curve analysis. In addition, the N34S mutation was detected by analyzing the DNA fragments generated by digestion with restriction enzyme TspR I. Clinical data of patients with the N34S mutation were compared with those without mutations. RESULTS: The N34S mutation was detected in six of 14 (43%) patients with early-onset ICP. One patient was homozygous, and five patients were heterozygous for this mutation. The N34S mutation in a heterozygous state was found in four of 397 healthy controls (1.0%). The different allele frequency observed (seven of 28 vs four of 794) was significant (odds ratio = 66, 95% CI = 18-242, p < 0.0001). The clinical course was similar in patients with a mutation compared with those without a mutation. No other SPINKI mutations were detected. The N34S mutation was not found in patients with late-onset ICP. CONCLUSIONS: Our results indicate that the N34S mutation in the SPINKI gene is strongly associated with ICP, especially with the early-onset type. The natural course is similar in patients with mutations compared with SPINK1 mutation-negative patients. The N34S mutation may easily be screened for by restriction digestion with TspR I.

Adult↗
PubMed2002

Emerging therapeutic targets in chronic heart failure: part I.

Chronic heart failure (CHF) is a life threatening disease with an enormous medical requirement. Approximately 15 million people worldwide suffer from CHF. The prevalence will inevitably increase due to the ageing population. Nevertheless, current treatment options based on angiotensin-converting enzyme inhibitors and beta-adrenergic receptor antagonists merely slow progression of the disease. Novel treatment concepts based on new therapeutic targets must have the capability to reverse the severity of this disease. This review, focusing on the emerging targets in the most promising therapeutic areas for the treatment of CHF, will be divided into two parts. In Part I, disease concepts such as altered calcium handling and ion channel activity, pathophysiological hypertrophy and inefficient cardiac metabolism are discussed. Validation status and potential therapeutic value for new targets in each research field is given by summarising the results of in vitro and in vivo studies.

Animals↗
PubMed1999

Intramolecular Ligand Hydroxylation: Mechanistic High-Pressure Studies on the Reaction of a Dinuclear Copper(I) Complex with Dioxygen.

We provide a mechanistic study of a monooxygenase model system and detail low-temperature stopped-flow kinetics studies in acetone as solvent, employing both the use of rapid-scanning diode-array observation and variable high-pressure (20-100 MPa) techniques. The dicopper(I) complex employed is [Cu(2)(H-XYL-H)](2+) (1), with the H-XYL-H ligand wherein a m-xylyl group links two bis[2-(2-pyridyl)ethyl]amine units. This reacts with O(2) reversibly (k(1)/k(-)(1)) giving a peroxo-dicopper(II) intermediate [Cu(2)(H-XYL-H)(O(2))](2+) (2), which thereupon irreversibly (k(2)) reacts by oxygen atom insertion (i.e., hydroxylation) of the xylyl group, producing [Cu(2)(H-XYL-O(-))(OH)](2+) (3). Activation parameters are as follows: k(1), DeltaH() = 2.1 +/- 0.7 kJ/mol, DeltaS() = -174 +/- 3 J/(K mol); k(-)(1), DeltaH() = 80.3 +/- 0.8 kJ/mol, DeltaS() = 77 +/- 3 J/(K mol); k(2), DeltaH() = 58.2 +/- 0.2 kJ/mol, DeltaS() = -5.8 +/- 0.9 J/(K mol). These values are similar to values obtained in a previous study in dichloromethane. At low temperatures and higher concentrations, the situation in acetone is complicated by a pre-equilibrium of 1 to an isomer form. The present study provides the first determination of activation volumes for individual steps in copper monooxygenase reactions. The data and analysis provide that DeltaV()(k(1)) = -15 +/- 2.5 cm(3)/mol and DeltaV()(k(-)(1)) = +4.4 +/- 0.5 cm(3)/mol for formation and dissociation of 2, respectively, while DeltaV()(k(2)) = -4.1 +/- 0.7 cm(3)/mol; a volume profile for the overall reaction has been constructed. The significance of the findings in the present study is described, and the results are compared to those for other systems.

Journal Article↗
PubMed1998

Intramolecular Ligand Hydroxylation: Mechanistic Studies on the Reaction of a Copper(I) Schiff Base Complex with Dioxygen.

The dinuclear copper(I) complex of 1,3-bis[N-(2-pyridylethyl)formimidoyl]benzene, [Cu(2)(H-BPB-H)(CH(3)CN)(2)](BF(4))(2), as well as the 5-nitro derivative, [Cu(2)(NO(2)-BPB-H)(CH(3)CN)(2)](BF(4))(2), react with dioxygen to form phenolate-bridged complexes as products. In a detailed kinetic study activation parameters of DeltaH() = 47 +/- 9 kJ/mol, DeltaS() = -53 +/- 11 J/(mol K), and DeltaV() = -9.5 +/- 0.5 cm(3)/mol for the reaction of [Cu(2)(H-BPB-H)(CH(3)CN)(2)](BF(4))(2) with dioxygen were obtained which account, together with further kinetic findings, for the occurrence of an intermediate peroxo complex that cannot be observed spectroscopically. The crystal structures of the products of the reaction were determined. Crystal data: complex [Cu(2)(H-BPB-O)-OH(H(2)O)](BF(4))(2), formula C(22)H(24)B(2)Cu(2)F(8)N(4)O(3), monoclinic space group P2(1)/c, Z = 4, a = 10.122(2) Å, b = 28.711(6) Å, c = 9.283(2) Å, alpha = 90 degrees, beta = 100.78(3) degrees, and gamma = 90 degrees; complex [Cu(2)(NO(2)-BPB-O)-OH(H(2)O)(3)](BF(4))(2), formula C(22)H(27)B(2)Cu(2)F(8)N(5)O(7), triclinic space group, P&onemacr;, Z = 2, a = 10.144(2) Å, b = 10.7612(2) Å, and c = 16.000(4) Å.

Journal Article↗
PubMed1996

Mechanistic Studies of the Formation and Decay of Diiron(III) Peroxo Complexes in the Reaction of Diiron(II) Precursors with Dioxygen.

Mechanistic studies of the reactions of three analogous alkoxo-bridged diiron(II) complexes with O(2) have been carried out. The compounds, which differ primarily in the steric accessibility of dioxygen to the diiron(II) center, form metastable &mgr;-peroxo intermediates when studied at low temperature. At ambient temperatures, these intermediates decay to form (&mgr;-oxo)polyiron(III) products. The effect of ligand steric constraints on the O(2) reactivity was investigated. When access to the diiron center was unimpeded, the reaction was first-order with respect to both [Fe(II)(2)] and [O(2)] and the activation parameters for O(2) addition were similar to those for O(2) reacting with the dioxygen transport protein hemerythrin. When the binding site was occluded, however, reduced order with respect to [O(2)] was observed and a two-step mechanism was required to explain the kinetic results. Decay of all three peroxide intermediates involves a bimolecular event, implying the formation of tetranuclear species in the transition state.

Journal Article↗
PubMedDate not supplied

Tacrolimus and steroids versus ciclosporin microemulsion, steroids, and azathioprine in children undergoing liver transplantation: randomised European multicentre trial.

BACKGROUND: Results of studies in adult recipients of liver allograft suggest that tacrolimus is more efficacious than ciclosporin microemulsion in the prevention of acute rejection. We aimed to compare these drugs in children undergoing liver transplantation. METHODS: This 12-month multicentre, open-label, parallel-group, randomised study compared a dual tacrolimus regimen (tacrolimus/corticosteroids, n=93) with a triple ciclosporin microemulsion regimen (ciclosporin microemulsion/corticosteroids/azathioprine, n=92) in children who had had liver transplants (age < or =16 years, bodyweight < or =40 kg). Initial oral daily doses were 0.30 mg/kg for tacrolimus and 10 mg/kg for ciclosporin microemulsion. Primary endpoint was the incidence of and time to first histologically proven acute rejection. We excluded patients from analysis if they did not receive the study drug, or were given incorrect medication. Otherwise patients were analysed in accordance with their random treatment allocation, irrespective of whether they switched medication during the trial. FINDINGS: Median age was 22 months (IQR 9-56) in the tacrolimus group and 17 months (9-54) in the ciclosporin microemulsion group. We noted no difference between treatment groups with respect to patient survival (93.4% vs 92.2%; p=0.77) or graft survival (92.3% vs 85.4%; p=0.16) at month 12 after transplant. The acute rejection free rate at study end (Kaplan-Meier method) was 55.5% for patients on tacrolimus and 40.2% for patients on ciclosporin microemulsion (p=0.0288). The Kaplan-Meier estimate of patients free from corticosteroid-resistant acute rejection at study end was 94.0% for tacrolimus-treated patients and 70.4% for ciclosporin-microemulsion-treated patients (p<0.0001). Overall, incidence of adverse events did not differ between groups. INTERPRETATION: Tacrolimus is a safe and effective treatment for the prevention of rejection after liver transplantation in children.

Administration, Oral↗