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Biomedical subjects

M Wyss

Publications and source records attributed to M Wyss.

At least 19 recordsLinked to original sources
PubMed1999

Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance.

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.

6-Phytase↗
PubMed1999

Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties.

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37 degreesC ranged from 23 to 196 U. (mg of protein)-1, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, alpha- and beta-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.

6-Phytase↗
PubMed1998

[From psychosomatic disorder to autoimmune disease--50 years urticaria and Quincke edema].

While the hereditary angioneurotic edema is a rare but serious disorder 10 to 25% of the population experience urticaria during a lifetime. Urticaria is for the patient a very impressive disease. He therefore has a great desire to know its cause. In acute urticaria this is usually possible since drugs or specific foods are the most common triggers. In chronic urticaria the search for a cause is much more difficult and successful only in 20 to 30% of cases. Over the past years it has been proven that about 30% of patients with chronic urticaria have antibodies against the high affinity Fc-receptor of mast cells. Thus a fraction of patients with chronic urticaria formerly often associated with psychosomatic illness suffer in fact from an autoimmune disorder.

Angioedema↗
PubMed1998

The therapeutic potential of oral creatine supplementation in muscle disease.

The decrease in intracellular creatine concentration observed in a number of muscle diseases may deplete energy homeostasis and may, therefore, be one of the factors determining and/or aggravating muscle weakness and degeneration. Two hypotheses are put forward in the present communication to explain: (i) the mechanisms leading to the disturbances in creatine metabolism found in various muscle diseases; and (ii) the potential of oral creatine supplementation in alleviating the clinical symptoms.

Animals↗
PubMed1998

Histopathologic and ultrastructural study of lupus-like skin lesions in a patient with Bloom syndrome.

The histopathology of the lupus-like skin lesions associated with Bloom syndrome has been sporadically described. Skin biopsies from a 2-year-old boy with the classical features of Bloom syndrome, including lupus-like skin lesions, demonstrated marked interface changes with basal liquefaction degeneration, a moderate superficial mononuclear infiltrate, pigmentary incontinence, and capillary dilation in the papillary dermis. Immunophenotyping of the dermal infiltrate revealed predominance of T-cells. Basement membrane thickening on periodic acid-Schiff examination was not seen. Direct immunofluorescence failed to demonstrate deposits of immunoglobulin other than nonspecific IgM deposition along the basement membrane zone of lesional skin. Ultrastructurally, the most striking findings were disintegration of basal cell cytoplasm and tubuloreticular inclusions in vascular endothelia. Taken together, the histologic and ultrastructural features of lupus-like lesions associated with Bloom syndrome mimic those of cutaneous lupus erythematosus, with the exception of paucity of immune deposits at the dermoepidermal junction.

Basement Membrane↗
PubMed1998

Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger PH 2.5 acid phosphatase.

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90 degrees C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70 degrees C. After heat denaturation at temperatures up to 90 degrees C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90 degrees C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of >/=80 degrees C. In feed pelleting experiments performed at 75 degrees C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85 degrees C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80 degrees C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.

6-Phytase↗
PubMed1997

Activation of sea-urchin sperm motility is accompanied by an increase in the creatine kinase exchange flux.

The kinetics of the creatine kinase (CK) reaction were studied in suspensions of quiescent and active, intact sea-urchin spermatozoa in artificial seawater, using 31P-NMR magnetization transfer. In inactive sperm, no CK-mediated exchange flux was detected, whereas in activated motile sperm, the forward pseudo-first-order rate constant was 0.13+/-0.04 s-1 at 10 degrees C, corresponding to a steady-state CK flux of 3.1+/-0.5 mM.s-1. Intracellular pH shifted from 6.6+/-0.1 to 7.6+/-0.1 upon activation. The phosphocreatine (PCr)/ATP and PCr/Pi ratios were only marginally reduced in activated sperm, whereas the estimated cytosolic free ADP concentration increased remarkably from 9 microM in quiescent, to 114 microM in activated spermatozoa. The elevation of CK flux upon sperm activation is discussed in the light of the proposition that in sea-urchin spermatozoa, which are fuelled entirely by oxidative phosphorylation, high-energy phosphate transport is mediated by a 'CK/PCr shuttle'.

Adenosine Diphosphate↗
PubMed1997

Purification and characterization of human sarcomeric mitochondrial creatine kinase.

In order to set the basis for detailed clinical investigations, mitochondrial creatine kinase (Mi-CK) was purified to homogeneity from human cardiac muscle. Biophysical characterization by SDS-PAGE, gel permeation chromatography and by electron microscopy of negatively stained single molecules demonstrated that, similar to other vertebrate Mi-CKs, human sarcomeric Mi-CK occurs in two different oligomeric forms, dimers and octamers, that are readily interconvertible. The apparent MTs of Mi-CK protomers, dimers and octamers were 43,600 +/- 800, 79,700 +/- 800 and 371,000 +/- 3000, respectively. In addition, isoelectric focussing proved to be a suitable technique for routinely distinguishing Mi-CK from cytosolic MM-CK and gave pl values of 8.30 +/- 0.04 and 7.44 +/- 0.04 for octameric and dimeric human sarcomeric Mi-CK. Circumstantial evidence suggests that both the highly symmetrical structure and the high pI value of Mi-CK octamers are crucial determinants for the physiological functions of this enzyme.

Creatine Kinase↗
PubMed1997

Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus.

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.

6-Phytase↗
PubMed1997

High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders.

High resolution respirometry in combination with the skinned fiber technique offers the possibility to study mitochondrial function routinely in small amounts of human muscle. During a period of 2 years, we investigated mitochondrial function in skeletal muscle tissue of 13 patients (average age = 5.8 years). In all of them, an open muscle biopsy was performed for diagnosis of their neuromuscular disorder. Mitochondrial oxidation rates were measured with a highly sensitive respirometer. Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obtain maximal respiratory rates for seven different substrates (pyruvate/malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine/malate, succinate, durochinol and ascorbate/TMPD). Decreased respiration rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements. In 4 patients with mitochondrial encephalomyopathy (MEM) the respiration pattern indicated a specific mitochondrial enzyme defect, which was confirmed in every patient by measurements of the individual enzymes (one patient with PDHC deficiency, one with complex I deficiency and two patients with combined complex I and IV deficiency). In the 6 patients with spinal muscular atrophy (SMA) oxidation rates were found to be decreased of 23 +/- 5% of controls. The normalized respiration pattern was comparable to that of the controls indicating a decreased content of mitochondria in SMA muscle with normal functional properties. Also in the 3 patients with Duchenne muscular dystrophy (DMD) decreased oxidation rates (42 +/- 5%) were detected. In addition a low RCI (1.2) indicated a loose coupling of oxidative phosphorylation in the mitochondria of these patients. It is concluded that investigation of mitochondrial function in saponin skinned muscle fibers using high resolution respirometry in combination with multiple substrate titration offers a valuable tool for evaluation of mitochondrial alterations in muscle biopsies of children suffering from neuromuscular disorders.

Child↗
PubMed1996

Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments.

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure. In contrast, co-expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi-CK by far- and near-UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C-terminal fragment refolded into a native-like conformation in vitro, whereas the N-terminal fragment was largely unfolded. Prefolded [168-380] interacted in vitro with [1-167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1 = 17 microM) and then isomerize slowly to an active enzyme (k2 = 0.12 min-1; k-2 = 0.03 min-1). Our data suggest that the C-terminal fragment of Mi-CK represents an autonomous folding unit, and that the folding of the C-terminal part might precede the conformational stabilization of the N-terminal moiety in vivo.

Adenosine Triphosphate↗
PubMed1996

Identification of two distinctly localized mitochondrial creatine kinase isoenzymes in spermatozoa.

The creatine kinase (CK) isoenzyme system is essential for motility in rooster and sea urchin sperm. In the present study, biochemical characterization as well as immunofluorescence and confocal laser microscopy with highly specific antibodies against various chicken CK isoenzymes revealed that cytosolic brain-type CK isoenzyme (B-CK) is the only CK isoenzyme in rooster seminal plasma, while three isoenzymes, cytosolic B-CK, sarcomeric mitochondrial CK (Mib-CK), and a variant of ubiquitous Mi-CK ('Mia-CK variant'), are found in rooster spermatozoa. These three isoenzymes are localized in different regions of the sperm cell. B-CK and Mib-CK were localized along the entire sperm tail and in the mitochondria-rich midpiece, respectively. The 'Mia-CK variant', on the other hand, was found predominantly at the head-midpiece boundary, in a non-uniform manner in the midpiece itself and, surprisingly, at the distal end of the sperm tail as well as at the acrosome. Several lines of evidence show that the 'Mia-CK variant' shares some characteristics with purified Mia-CK from chicken brain, but also displays distinctive features. This is the first evidence for two different Mi-CK isoenzymes occurring in one cell and, additionally, for the co-expression of Mib-CK and cytosolic brain-type B-CK in the same cell. The relevance of these findings for sperm physiology and energetics is discussed.

Animals↗
PubMed1995

Re-evaluation of the structure and physiological function of guanidino kinases in fruitfly (Drosophila), sea urchin (Psammechinus miliaris) and man.

Purification and biophysical characterization of mitochondrial creatine kinase (Mi-CK) from sperm of the sea urchin Psammechinus miliaris, as well as gel-permeation chromatography of human heart Mi-CK demonstrate that these two Mi-CK isoenzymes form highly symmetrical octameric molecules with an M(r) of approx. 350,000, a value similar to that found for all other Mi-CK isoenzymes investigated so far. The absolute evolutionary conservation of this oligomeric form from sea urchins to mammals points both to its essentiality for Mi-CK function and to an important role of octameric Mi-CK in the energy metabolism of tissues and cells with high and fluctuating energy demands. To investigate whether a similar physiological principle also operates in an even more distantly related animal phylum, the arginine kinase (ArgK) isoenzyme system of Drosophila flight muscle was investigated with two independent subcellular fractionation procedures and subsequent analysis of the fractions by SDS/PAGE, immunoblotting and native isoenzyme electrophoresis. In contrast with a previous report [Munneke and Collier (1988) Biochem. Genet. 26, 131-141], strong evidence against the occurrence of a Mi-ArgK isoenzyme in Drosophila was obtained. The findings of the present study are discussed in the context of CK and ArgK function in general and of structural and bioenergetic differences between vertebrate striated muscles and arthropod flight muscles.

Animals↗
PubMed1995

Childhood NHL in Switzerland: incidence and survival of 120 study and 42 non-study patients.

Based on the Swiss Pediatric Oncology Group (SPOG) cancer registry data during 1981-1991, a high average incidence of 8 new NHL per million children younger than 15 years per year was found. Of 162 children with NHL registered in 1976-1991, 120 were study patients, i.e., officially registered and treated according to SPOG or Pediatric Oncology Group (POG) protocols, while 42 were non-study patients, i.e., patients not officially enrolled on protocols. Overall, 91 of 120 (76%) study patients remained alive. Seventy-nine study patients were treated according to older SPOG protocols, and 53 (67%) of these survived, while 38 of 41 (93%) study patients treated according to newer POG protocols remained alive (P = 0.0068). Only 22 (52%) of the 42 non-study patients survived (P = 0.0001). There was no improvement if the survival of non-study patients before and since 1986 was compared. Population-based treatment results in Switzerland were similar to those in the United Kingdom. They provided an important base for the development of future treatment strategies.

Adolescent↗
PubMed1995

Incidence of childhood acute lymphoblastic leukemia (ALL) and population-based treatment results in Switzerland: experiences with 507 study and 149 nonstudy patients.

Of 656 patients with ALL (all types) diagnosed in Switzerland during 4 consecutive 4-year periods (1976-1979, 1980-1983, 1984-1987, 1988-1991), 507 were officially registered on protocols ("study" patients) while 149 were not ("nonstudy" patients). The mean incidence of 3.8/100,000 children < 15 years/year is higher than reported for other Western countries. Evidence is presented suggesting that the 656 patients represent only approximately 90% of all children with ALL residing in Switzerland, indicating that the true incidence of ALL might even be higher. The fraction of "nonstudy" patients fell from 40% (1976-1979) to 15% (1984-1987). The rate of survival at 4 years of all patients with ALL ("study" and "nonstudy") increased by 17% during the three consecutive periods 1976-1979, 1980-1983, and 1984-1987. As expected, a higher increase (20%) was observed in "study" patients and a statistically nonsignificant lower one (10%) in "nonstudy" patients.

Adolescent↗
PubMed1995

Lamellar ichthyosis is genetically heterogeneous--cases with normal keratinocyte transglutaminase.

We recently identified mutations of the keratinocyte transglutaminase gene as a cause of lamellar ichthyosis. In this study we analyzed two sporadic cases of lamellar ichthyosis. Transglutaminase activity measured in membrane extracts from cultured differentiating keratinocytes was within the range observed in normal individuals. Western blot and Northern blot analysis revealed normal size and quantities of keratinocyte transglutaminase protein and mRNA. Sequencing of the 15 exons and their flanking regions demonstrated no deviation from the published sequence except for two silent polymorphisms. These results exclude mutations of keratinocyte transglutaminase as a cause for lamellar ichthyosis in these patients, indicating that lamellar ichthyosis is a genetically heterogeneous disorder.

Genetic Heterogeneity↗
PubMed1995

Scalp dermatitis, distinctive hair abnormalities and atopic disease in the ectrodactyly-ectodermal dysplasia-clefting syndrome.

We report a 2-year-old girl with a combination of the ectodermal dysplasia, ectrodactyly, cleft lip/palate (EEC) syndrome, distinctive hair abnormalities, scalp dermatitis and atopic disease. To our knowledge, this is the first report of scalp dermatitis in the EEC syndrome. Distinctive structural hair abnormalities shared by the syndromes of ectodermal dysplasia and clefting are helicotrichia, pili torti et canaliculi, and cuticle defects, all of which were observed in the present case. A high incidence of atopic disease in certain subsets of ectodermal dysplasia has been reported. In our patient this manifested as food-induced exacerbation of atopic dermatitis, associated with positive prick tests and significant levels of circulating specific IgE.

Abnormalities, Multiple↗