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H Witt

Publications and source records attributed to H Witt.

At least 37 records · Page 2Linked to original sources
PubMed2001

Rapid detection of the Wilson's disease H1069Q mutation by melting curve analysis with the LightCycler.

Wilson's disease is an inherited autosomal recessive disorder of copper transport characterized by progressive copper accumulation in the liver and the central nervous system. The disease is caused by mutations in the ATP7B gene. Although many different mutations in this gene were described, a substitution of a histidine by a glutamine residue at codon 1069 (H1069Q) accounts for approximately 30-60% of all mutations in Caucasian patients. We describe a DNA-based method using fluorescence resonance energy transfer probes on the LightCycler for rapid determination of the common H1069Q mutation in the ATP7B gene. We screened 53 patients with Wilson's disease for the H1069Q mutation by the melting curve analysis. The reliability and discriminating power of this technique were documented by comparing results of the LightCycler assay with direct DNA sequencing. The protocol allows genotyping of 30 samples in less than 1 hour without a need for restriction enzyme digestion or gel electrophoresis.

Adenosine Triphosphatases↗
PubMed2001

Results of a routine exercise treadmill testing strategy early after percutaneous transluminal coronary angioplasty.

BACKGROUND: Some physicians use routine exercise treadmill testing early after percutaneous transluminal coronary angioplasty (PTCA), yet there have been few prospective studies examining the results of such a functional testing strategy. OBJECTIVES: To examine the results of a routine post-PTCA exercise treadmill testing strategy, and to explore the impact of such a strategy on follow-up functional tests and cardiac procedures. PATIENTS AND METHODS: Functional test results were examined from a cohort of 226 patients who underwent PTCA at a single institution in which a routine functional testing strategy is used. Tests were defined as 'routine' if they were performed solely because the patient had a prior PTCA. Tests were defined as 'selective' if they were done for a clinical indication or if there was no functional testing during the six-month follow-up. RESULTS: A total of 183 patients (81%) underwent at least one functional test during the six-month period after PTCA. Of these patients, 150 (82%) underwent routine functional testing, the majority (73%) of which were exercise treadmill tests. Sixty-seven per cent of patients who had routine functional testing had their first functional test less than seven weeks after their PTCA. Most routine exercise treadmill tests were found to be electrically or clinically indeterminate (46%), but a substantial number were either electrically or clinically positive (28%), or electrically and clinically negative (39%). Of patients who had electrically or clinically positive routine tests, 57% had repeat functional testing and 23% had repeat revascularization. Of patients who had electrically and clinically negative tests, 53% had repeat functional testing and 10% had repeat revascularization. Of the patients who had electrically or clinically indeterminate tests, 53% had repeat functional testing and 14% had repeat revascularization. The rate of clinical events was less than 4%, irrespective of the results of the exercise treadmill tests. CONCLUSIONS: Routine exercise treadmill testing early after PTCA is associated with a low clinical yield. Most routine exercise treadmill testing results in indeterminate and negative tests, which often lead to more functional tests with little difference in clinical outcome. These results call into question the use of a routine post-PTCA functional testing strategy.

Angioplasty, Balloon, Coronary↗
PubMed2000

Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance raman study.

Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process.

Animals↗
PubMed2000

Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis.

Chronic pancreatitis (CP) is a continuing or relapsing inflammatory disease of the pancreas. In approximately one-third of all cases, no aetiological factor can be found, and these patients are classified as having idiopathic disease. Pathophysiologically, autodigestion and inflammation may be caused by either increased proteolytic activity or decreased protease inhibition. Several studies have demonstrated mutations in the cationic trypsinogen gene (PRSS1) in patients with hereditary or idiopathic CP. It is thought that these mutations result in increased trypsin activity within the pancreatic parenchyma. Most patients with idiopathic or hereditary CP, however, do not have mutations in PRSS1 (ref. 4). Here we analysed 96 unrelated children and adolescents with CP for mutations in the gene encoding the serine protease inhibitor, Kazal type 1 (SPINK1), a pancreatic trypsin inhibitor. We found mutations in 23% of the patients. In 18 patients, 6 of whom were homozygous, we detected a missense mutation of codon 34 (N34S). We also found four other sequence variants. Our results indicate that mutations in SPINK1 are associated with chronic pancreatitis.

Adolescent↗
PubMed2000

The incidence and mechanisms of pharyngeal and upper esophageal dysfunction in partially paralyzed humans: pharyngeal videoradiography and simultaneous manometry after atracurium.

BACKGROUND: Residual neuromuscular block caused by vecuronium alters pharyngeal function and impairs airway protection. The primary objectives of this investigation were to radiographically evaluate the swallowing act and to record the incidence of and the mechanism behind pharyngeal dysfunction during partial neuromuscular block. The secondary objective was to evaluate the effect of atracurium on pharyngeal function. METHODS: Twenty healthy volunteers were studied while awake during liquid-contrast bolus swallowing. The incidence of pharyngeal dysfunction was studied by fluoroscopy. The initiation of the swallowing process, the pharyngeal coordination, and the bolus transit time were evaluated. Simultaneous manometry was used to document pressure changes at the tongue base, the pharyngeal constrictor muscles, and the upper esophageal sphincter. After control recordings, an intravenous infusion of atracurium was administered to obtain train-of-four ratios (T4/T1) of 0.60, 0.70, and 0.80, followed by recovery to a train-of-four ratio of more than 0.90. RESULTS: The incidence of pharyngeal dysfunction was 6% during the control recordings and increased (P < 0.05) to 28%, 17%, and 20% at train-of-four ratios 0.60, 0.70, and 0.80, respectively. After recovery to a train-of-four ratio of more than 0.90, the incidence was 13%. Pharyngeal dysfunction occurred in 74 of 444 swallows, the majority (80%) resulting in laryngeal penetration. The initiation of the swallowing reflex was impaired during partial paralysis (P = 0.0081). The pharyngeal coordination was impaired at train-of-four ratios of 0.60 and 0.70 (P < 0.01). A marked reduction in the upper esophageal sphincter resting tone was found, as well as a reduced contraction force in the pharyngeal constrictor muscles. The bolus transit time did not change significantly. CONCLUSION: Partial neuromuscular paralysis caused by atracurium is associated with a four- to fivefold increase in the incidence of misdirected swallowing. The mechanism behind the pharyngeal dysfunction is a delayed initiation of the swallowing reflex, impaired pharyngeal muscle function, and impaired coordination. The majority of misdirected swallows resulted in penetration of bolus to the larynx.

Adult↗
PubMed1999

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase.

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Cloning, Molecular↗
PubMed1999

A signal peptide cleavage site mutation in the cationic trypsinogen gene is strongly associated with chronic pancreatitis.

BACKGROUND & AIMS: In pancreatitis, a key role has been attributed to the inappropriate conversion of trypsinogen to trypsin. Recently, two mutations of the cationic trypsinogen gene were found in families with hereditary pancreatitis. This study was conducted to determine the spectrum and frequency of cationic trypsinogen mutations in unrelated patients with idiopathic or hereditary chronic pancreatitis (CP). METHODS: DNA samples from 44 unrelated children and adolescents with CP (30 patients with idiopathic CP and 14 with hereditary CP) and from 56 family members were investigated. The cationic trypsinogen gene was screened for mutations by single-strand conformation polymorphism analysis and DNA sequencing. RESULTS: A mutation in the cationic trypsinogen gene was detected in 5 patients: in 2 patients with a family history of CP and in 3 patients with idiopathic CP. In 1 patient the formerly described R122H mutation was detected. In 4 patients a hitherto unknown mutation was found at the signal peptide cleavage site leading to an alanine to valine exchange in codon 16. The mutations were inherited in all cases. In 95 unrelated control individuals the A16V mutation was not found. CONCLUSIONS: Heterozygosity for the A16V mutation is strongly associated with CP. These results indicate that a significant percentage of patients with idiopathic CP may have a genetic basis for their disorder; therefore, genetic testing should be included in the diagnostic evaluation of these patients.

Adolescent↗
PubMed1999

HFE gene mutation and transferrin saturation in very low birthweight infants.

AIM: To determine if there is an association between high transferrin saturation and the C282Y HFE gene mutation in very low birthweight (VLBW) infants. METHODS: One hundred and forty three VLBW infants receiving recombinant erythropoietin and 3 to 9 mg/kg/day of enteral iron were studied. Genomic DNA was extracted from filter paper cards. The C282Y mutation was determined by restriction fragment length polymorphism analysis. RESULTS: Six infants were heterozygous for the mutation; none was homozygous. Ten infants had a transferrin saturation above 80% at least once. No infant was positive for both transferrin saturation above 80% and the mutation. CONCLUSIONS: The data strongly suggest that there is no association between high transferrin saturation and the HFE gene mutation in VLBW infants during the first weeks of life.

Female↗
PubMed1998

Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes.

Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I-IV is obtained when the purification procedure is performed in beta-d-dodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I-II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.

Journal Article↗
PubMed1998

Tryptophan 121 of subunit II is the electron entry site to cytochrome-c oxidase in Paracoccus denitrificans. Involvement of a hydrophobic patch in the docking reaction.

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the KM remains largely unchanged. Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.

Copper↗
PubMed1998

Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans.

To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre-steady state conditions was measured using stopped flow spectroscopy. Taken together, the results demonstrate that the initial interaction of cytochrome c and oxidase is mediated through glutamates and aspartates mainly located in subunit II. The crystal structure of oxidase reveals that the participating residues are clustered, creating an extended, negatively charged patch. We propose this clustering to be a decisive factor in the recognition of positively charged patches on the surface of cytochrome c.

Binding Sites↗
PubMed1998

Swallowing disorders after esophageal atresia evaluated with videomanometry.

BACKGROUND: Patients with repaired esophageal atresia often show persistent motility disorders of the esophagus. METHODS: In this study, the authors used a newly developed method, videomanometry, to study intraluminal pressures and function of the pharynx and upper esophagus in such a group of patients and compared the results with those in healthy age-matched controls. RESULTS: A significant difference was found between timing of the pharynx contraction and upper esophageal sphincter (UES) relaxation, indicating a dyscoordination of swallowing in the patient group. Transit time of bolus from the pharynx to the esophagus was also shorter for the patient group. Such dyscoordination may be a risk factor that can cause aspiration and respiratory symptoms. CONCLUSION: UES resting pressure and residual pressure on swallowing, did not differ between the two groups.

Adult↗
PubMed1997

Asp-193 and Glu-218 of subunit II are involved in the Mn2+-binding of Paracoccus denitrificans cytochrome c oxidase.

Cytochrome c oxidase contains a binding site for a non-redox-active metal at the interface of subunits I and II, usually a magnesium ion. In Paracoccus denitrificans oxidase, typically 20% may be replaced by manganese, using standard growth media. Site-directed mutants were constructed in subunit II (D193N and E218Q), and the isolated enzymes analyzed by total-reflection X-ray fluorescence spectrometry and EPR. Both mutants show a strong reduction of the manganese stoichiometry and a diminished electron transfer activity, demonstrating that D193 and E218 are involved in the binding of a manganese/magnesium ion in this site.

Amino Acid Sequence↗
PubMed1997

Isolation, analysis, and deletion of the gene coding for subunit IV of cytochrome c oxidase in Paracoccus denitrificans.

The gene coding for subunit IV of the cytochrome c oxidase in Paracoccus denitrificans has been cloned and sequenced. The derived amino acid sequence shows no significant homology to any known protein. Gene deletion has no consequences for the integrity of the complex and its spectral and enzymatic properties. Complementation of the deletion mutant in trans results in expression of subunit IV; sequence analysis of the 5'-noncoding region leads to the identification of a putative promoter sequence.

Base Sequence↗
PubMed1997

Functional assessment of the pharynx at rest and during swallowing in partially paralyzed humans: simultaneous videomanometry and mechanomyography of awake human volunteers.

BACKGROUND: Functional characteristics of the pharynx and upper esophagus, including aspiration episodes, were investigated in 14 awake volunteers during various levels of partial neuromuscular block. Pharyngeal function was evaluated using videoradiography and computerized pharyngeal manometry during contrast bolus swallowing. METHODS: Measurements of pharyngeal constrictor muscle function (contraction amplitude, duration, and slope), upper esophageal sphincter muscle resting tone, muscle coordination, bolus transit time, and aspiration under fluoroscopic control (laryngeal or tracheal penetration) were made before (control measurements) and during a vecuronium-induced partial neuromuscular paralysis, at fixed intervals of mechanical adductor pollicis muscle train-of-four (TOF) fade; that is, at TOF ratios of 0.60, 0.70, 0.80, and after recovery to a TOF ratio > 0.90. RESULTS: Six volunteers aspirated (laryngeal penetration) at a TOF ratio < 0.90. None of them aspirated at a TOF ratio > 0.90 or during control recording. Pharyngeal constrictor muscle function was not affected at any level of paralysis. The upper esophageal sphincter resting tone was significantly reduced at TOF ratios of 0.60, 0.70, and 0.80 (P < 0.05). This was associated with reduced muscle coordination and shortened bolus transit time at a TOF ratio of 0.60. CONCLUSIONS: Vecuronium-induced partial paralysis cause pharyngeal dysfunction and increased risk for aspiration at mechanical adductor pollicis TOF ratios < 0.90. Pharyngeal function is not normalized until an adductor pollicis TOF ratio of > 0.90 is reached. The upper esophageal sphincter muscle is more sensitive to vecuronium than is the pharyngeal constrictor muscle.

Adult↗
PubMed1997

Microbial growth inside saline-filled breast implants.

In vitro and in vivo experiments were conducted to determine whether intraluminal saline in breast implants can support the growth of common wound-infecting microorganisms over a prolonged period of time. The bacteria tested were Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Corynebacterium jeikeium, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Three fungal species also were tested: Aspergillus fumigatus, Paecilomyces variotii, and Candida albicans. In the in vitro study, four organisms survived in flasks of sterile saline for the 2 weeks in which serial cultures were performed: K. pneumoniae, C. albicans, A. fumigatus, and P. variotii. In the in vivo study, 61 white rabbits (122 implants) received both an experimental implant inoculated with one of the test organisms and a control implant containing only sterile saline. They were sacrificed at 1-, 3-, or 6-month scheduled endpoints. None of the control implants containing sterile saline had positive cultures. In contrast, the intraluminal saline was culture positive for 7 of the 10 inoculated organisms after varying lengths of time: S. epidermidis, E. coli, E. cloacae, K. pneumoniae, P. aeruginosa, A. fumigatus, and P. variotii. Samples of capsular tissue also were cultured. Of the 122 capsular tissue specimens, 21 (17 percent) had positive cultures and surrounded both inoculated and sterile implants. In most instances, capsules that were culture positive contained an organism different from the one that had been inoculated in the group. In only 3 cases was the same organism cultured from both the periprosthetic tissue and the intraluminal saline, and these may represent instances of the inoculated organism migrating through the implants filler valves. The data show that several types of bacteria (particularly gram-negative species) and fungi can grow and reproduce in a restricted saline environment for extended periods of time.

Animals↗
PubMed1995

Site-directed mutagenesis of cytochrome c oxidase reveals two acidic residues involved in the binding of cytochrome c.

Site-directed mutagenesis in subunit II of the cytochrome c oxidase (haem aa3) from Paracoccus denitrificans reveals that two carboxylic residues, Glu-246 and Asp-206 (corresponding to 198 and 158 in the bovine subunit II), are involved in the binding of cytochrome c. Spectrophotometric and polarographic measurements with the isolated enzymes of both mutant strains show a strongly reduced activity compared to wild-type oxidase, with the overall catalytic capacity (kcat/KM) of both mutants decreased about 8-fold. EPR spectra reveal no significant differences between the wild-type and the mutant enzymes, indicating that neither residue contributes significantly to the structure of the CuA centre. We conclude that Glu-246 and Asp-206 constitute an essential part of the binding site for cytochrome c.

Binding Sites↗