Biomedical subjects

W H Müller

Publications and source records attributed to W H Müller.

At least 19 recordsLinked to original sources

PubMed2002

Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation.

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.

Carboxylic Ester Hydrolases↗

PubMed2000

Automated electron tomography of the septal pore cap in Rhizoctonia solani.

Dolipore septa and septal pore caps (SPCs) in filamentous basidiomycetes may play an important role in maintaining the integrity of hyphal cells. We have investigated the ultrastructure of the dolipore septum and the SPC in Rhizoctonia solani hyphal cells after high-pressure freezing, freeze substitution, and Spurr embedding. We visualized the SPC with associated cell ultrastructures in three dimensions by automated electron tomography of thick-sectioned cells, followed by 3D tomographic reconstructions. Using these methods we were able to document the passage of mitochondria through the SPC, small tubular membranous structures at the entrance of the septal pore channel, filamentous structures connecting the inner side of the SPC with pore-plugging material, thin filaments anchoring the pore-plugging material with the plasma membrane, small vesicles attached to the plugging material, and tubular endoplasmic reticulum continuous with the base of the SPC. We hypothesize that the SPC, the filamentous structures, the plugging material, and the endoplasmic reticulum act in a coordinated fashion to maintain cellular integrity, intercellular communication, and the transport of solutes and cell organelles in the filamentous fungus R. solani.

Automation↗

PubMed2000

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants.

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Aminoglycosides↗

PubMed2000

Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts.

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V(HH) antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

Animals↗

PubMed1999

Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae.

Beta1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, beta1,3-glucan, and chitin. It has been postulated that the synthesis of beta1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of beta1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular beta1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of beta1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of beta1, 6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.

Amino Acid Sequence↗

PubMed1998

Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers.

For scarce antigens or antigens which are embedded in a dense macromolecular structure, on-section labeling, the first method of choice, is not always successful. Often, the antigen can be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method. Most of these methods lead to loss of ultrastructural details and, hence, labeling at electron microscope resolution does not add essential information. The scope of this paper is to compare five permeabilization methods for pre-embedding labelling for electron microscopy. We aim for a method that is easy to use and suitable for routine investigations. For our ongoing work, special attention is given to labeling of the cell nucleus. Accessibility of cytoplasmic and nuclear antigens is monitored with a set of different marker antibodies. From this investigation, we suggest that prefixation with formaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure before using a detergent (Triton X-100 or Brij 58) to permeabilize or remove the membranes. The experimental conditions for labeling should be checked first with fluorescence or fluorescence-gold markers by fluorescence microscopy. Then either ultrasmall gold particles (with or without fluorochrome) with silver enhancement or, if the ultrasmall gold particles are obstructed, peroxidase markers are advised. The most promising technique to localize scarce antigens with good contrast is the combination of a pre-embedding peroxidase/tyramide-FITC or -biotin labeling followed by an on-section colloidal gold detection.

Cell Nucleus↗

PubMed1998

Impaired secretion of a hydrophobic cutinase by Saccharomyces cerevisiae correlates with an increased association with immunoglobulin heavy-chain binding protein (BiP).

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.

Blotting, Western↗

PubMed1997

Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins.

We previously reported that the defects in the Saccharomyces cerevisiae cwh6 Calcofluor white-hypersensitive cell wall mutant are caused by a mutation in SPT14/GPI3, a gene involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Here we describe the effect of cwh6/spt14/gpi3 on the biogenesis of cell wall proteins. It was found that the release of precursors of cell wall proteins from the endoplasmic reticulum (ER) was retarded. This was accompanied by proliferation of ER structures. The majority of the cell wall protein precursors that eventually left the ER were not covalently incorporated into the cell wall but were secreted into the growth medium. Despite the inefficient incorporation of cell wall proteins, there was no net effect on the protein level in the cell wall. It is postulated that the availability of GPI-dependent cell wall proteins determines the rate of cell wall construction and limits growth rate.

Amino Acid Sequence↗

PubMed1995

Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.

A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10,000 x g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14,000 x g pellet. After 2 h, 45,000 x g isopycnic centrifugation of the 14,000 x g pellet on a continuous 20-60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.

Cell Fractionation↗

PubMed1992

Involvement of microbodies in penicillin biosynthesis.

Penicillium chrysogenum strains were constructed which express a mutant acyltransferase lacking the putative targeting signal for microbody proteins. The mutated enzyme was located in vacuoles and in neighbouring cytoplasm. Although acyltransferase was expressed in vivo and was active in vitro, the mutants did not produce penicillin. The results demonstrate the involvement of microbodies in penicillin production.

Acyltransferases↗

PubMed1991

Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.

Acyltransferases↗

PubMed1991

A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno-electron microscopical studies.

A combination of cryofixation without pre-treatment, freeze-substitution and low-temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P.chrysogenum colonies were grown between dissected-dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze-substitution media, but it was reduced after embedding in Epon 812.

Acyltransferases↗

PubMed1990

[Magnet systems for magnetic resonance tomography].

In the past fifteen years, Magnetic Resonance Tomography (MRT) has become a valuable tool for medical diagnostics. At the same time, Magnetic Resonance Spectroscopy (MRS) has attracted more and more attention in medical and other related areas of investigations. For both methods it is necessary to provide a magnetic field, which can be generated by resistive magnets, by permanent magnets as well as by superconducting magnets. These three concepts are described and compared together with the advantages of each design and the considerations that must be addressed regarding the planned use and the availability of a suitable site. In addition to the main magnet important additions like gradient and shim coils, aspects of site planning and future problems are discussed.

Equipment Design↗

PubMed1989

[Neurologic and psychologic disorders in folic acid deficiency].

Folic acid deficiency is the most common of all vitamin deficiencies in North America and in Western Europe, usually in association with other vitamin deficiencies. Alcoholism is considered to be the most frequent cause of folic acid deficiency, as far as the Western world is concerned. Severe neurological and mental disturbances can develop in case of folate deficiency probably long before the manifestation of macrocythaemia. Among the neurological sequelae of folic acid deficiency the main phenomena that are discussed as likely are polyneuropathy, funicular disease of the spine and restless legs. The psychic or mental symptoms that can be ascribed to folic acid deficiency are non-specific and correspond with the symptoms of a psychosis that can have physical causes. Administration of folic acid has repeatedly resulted in significant improvements of neurological and mental symptoms in folic acid deficiency. The exact mechanisms involved in a damage to the nervous system are not sufficiently clarified for folic acid deficiency. Diagnosis is effected mainly by determining the folic acid concentration in the serum. Daily administration of approx. 1.25-15 mg folic acid (oral and/or parenteral) is recommended. Mild side effects such as tiredness and irritability have been noted only with high folic acid dosage levels.

Folic Acid Deficiency↗

PubMed1985

[Induction of gamma-glutamyltransferase in cultured brain cells].

gamma-Glutamyltransferase is a membrane-bound enzyme widely distributed in animal tissues. This enzyme is involved in glutathione metabolism, but its exact biological function is still an open question. In rat brain cells in culture a depletion of L-cystine and L-glutamine or the addition of diethyl maleate to the culture medium leads to a decrease of intracellular glutathione concentration and to an increase of the specific activity of gamma-glutamyltransferase. The induction of the enzyme is inhibited by the addition of cycloheximide or actinomycin D to the culture medium.

Animals↗

PubMed1985

[Induction of alkaline phosphatase by retinoic acid].

Treatment of mammalian cells in culture with retinoic acid causes a time- and concentration-dependent increase of the specific activity of alkaline phosphatase. The increase reaches a factor of 15 and more and begins at a concentration of 10(-8)M retinoic acid. The induction is inhibited by cycloheximide or actinomycin D. The same isoenzyme of alkaline phosphatase is expressed in control and in retinoic acid-treated cells as demonstrated by the inhibitions by amino acids and peptides. The enzyme induction occurs in rat heart, skeletal muscle, brain, lung cells and HeLa cells. No induction was found in two lines of human melanoma cells. After treatment of cells with tunicamycin, the induction of alkaline phosphatase is detectable only in the homogenate and no longer detectable by histochemical methods. This shows that the glycosylation of the protein is an important step in the insertion of this enzyme into the plasma membrane.

Alkaline Phosphatase↗

PubMed1984

[Effect of procaine, nicotinoylprocaine and butacaine on mammalian cells in culture].

The tertiary amines, procaine and nicotinoylprocaine, cause an increase in the specific activities of two glycohydrolases, alpha-fucosidase and beta-N-acetylhexosaminidase, which are involved in membrane metabolism. The specific activity of alkaline phosphatase, a plasma membrane enzyme, is lowered in muscle cells after addition of procaine or nicotinoylprocaine to the culture medium. The specific activities of two transferases, aspartate-amino-transferase and creatine phosphokinase, are increased by 10(-5) mol/l butacaine. A combined addition of butacaine and nicotinoylprocaine causes less effects on the transferases. The specific activities of neutral alpha-glucosidase and beta-N-acetylhexosaminidase are scarcely influenced by butacaine alone. Only butacaine and nicotinoylprocaine together lead to an increase of the activities of these hydrolases. These results suggest two different mechanism of action at least concerning these substances: 1. a specific binding of tertiary amines and 2. a coordinated mechanism on the membrane fluidity.

4-Aminobenzoic Acid↗

PubMed1984

Phototherapy in bile duct-ligated Gunn rats.

Davis and co-workers reported on the failure of phototherapy to reduce plasma bilirubin levels in the bile duct-ligated Gunn rat. We cannot confirm this conclusion. We found that the effect was reduced by a factor of 4-5. With higher effective irradiances distinct decreases of the serum bilirubin concentration can be achieved and the excretion of bilirubin photoproducts in the urine markedly will rise.

Animals↗