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Biomedical subjects

M Stolz

Publications and source records attributed to M Stolz.

At least 19 recordsLinked to original sources
PubMed2004

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide.

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.

Aluminum Silicates↗
PubMed2004

[Aspects of expertise which are jointly valid for German sociomedicine and statutory health care].

A project group of the Medical Advisory Board of the German Federal Rehabilitation Council (BAR) developed fundamental joint principles on experts' opinions according to the social law code no. IX (SGB IX). The principles aim at medical experts working in different social organisations and statutory health care insurance. It was intended to create a "sociomedical language" which should be used as jointly as possible by experts in rehabilitation and social medicine and which is based on the ICF (International Classification of Functioning, Disability and Health, WHO 2001). Its stringent application will increase the utility of medical expertise across different institutions. The authors recommend to evaluate whether this model could provide a tool in the communication and cooperation between different sectors of the health system. Part I describes the theoretical model, Part II its application to a virtual individual case history.

Delivery of Health Care↗
PubMed2003

Hepatitis C virus-polymerase chain reaction minipool testing: 3 years in the largest Swiss blood transfusion service.

BACKGROUND AND OBJECTIVES: Hepatitis C virus-polymerase chain reaction (HCV-PCR) minipool testing can improve the safety of labile blood products owing to a reduction in the diagnostic preseroconversion window period. In Switzerland, HCV-PCR minipool testing for the release of labile blood components became mandatory in September 1999. In the largest Swiss blood transfusion centre, HCV-PCR minipool testing began in January 1999. This report analyses the performance of the test during a 3-year period: 1 January 1999 to 31 December 2001. MATERIALS AND METHODS: EDTA-blood was collected in either standard tubes or plasma preparation (PPT) tubes from 10 blood transfusion services in Switzerland and then sent to the Blood Transfusion Service SRC Berne. Up to 48 donor samples were pooled overnight using Tecan Genesis RSP 200/8 pipettors. Viral RNA was extracted by using the Qiagen QIAamp 96 viral RNA BioRobot kit on a BioRobot 9604. For PCR amplification and detection of HCV or internal control (IC) sequences, the Roche Cobas Amplicor v2.0 test kit was used. Data management, pool resolution and identification of positive samples were performed using the PMS Software from Tecan. RESULTS: In the 3-year period from 1 January 1999 to 31 December 2001, 839056 blood donor samples were tested in minipools of up to 48 samples. Thirty-five HCV-PCR-positive donations were identified. Thirty-four samples had antibodies against HCV and were therefore also detected by screening for antibody to HCV (anti-HCV). In October 2001, one seronegative (but PCR-positive) donor was detected. CONCLUSIONS: HCV-PCR minipool testing was successfully introduced in the largest Swiss blood transfusion service. It was shown that the release of HCV-PCR minipool results can be accomplished concurrently with the results of serological analysis. The challenge with a seronegative, but PCR-positive, donor demonstrates that the minipool testing strategy adds additional safety to blood products.

Blood Preservation↗
PubMed2003

[Aspects of expertising which are jointly valid for German sociomedicine and statutory health care].

A project group of the Medical Advisory Board of the German Federal Rehabilitation Council (BAR) developed fundamental joint principles on experts' opinions according to the social law code no. IX (SGB IX). The principles aim at medical experts working in different social organisations and statutory health care insurances. It was intended to create a "sociomedical language" which should be used as jointly as possible by experts in rehabilitation and social medicine and which is based on the ICF (International Classification of Functioning, Disability and Health, WHO 2001). Its stringent application will increase the utility of medical expertise across different institutions. The authors recommend to evaluate whether this model could provide a tool in the communication and cooperation between different sectors of the health system. Part I describes the theoretical model, Part II its application to a virtual individual case history.

Delivery of Health Care↗
PubMed2000

A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity.

Creatine kinase catalyzes the reversible transphosphorylation of creatine by MgATP. From the sequence homology and the molecular structure of creatine kinase isoenzymes, we have identified several highly conserved residues with a potential function in the active site: a negatively charged cluster (Glu(226), Glu(227), Asp(228)) and a serine (Ser(280)). Mutant proteins E226Q, E226L, E227Q, E227L, D228N, and S280A/S280D of human sarcomeric mitochondrial creatine kinase were generated by in vitro mutagenesis, expressed in Escherichia coli, and purified to homogeneity. Their overall structural integrity was confirmed by CD spectroscopy and gel filtration chromatography. The enzymatic activity of all proteins mutated in the negatively charged cluster was extremely low (0.002-0.4% of wild type) and showed apparent Michaelis constants (K(m)) similar to wild type, suggesting that most of the residual activity may be attributed to wild-type revertants. Mutations of Ser(280) led to higher residual activities and altered K(m) values; S280A showed an increase of K(m) for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease of K(m) for creatine (6-fold). These results, together with the transition state structure of the homologous arginine kinase (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449-8454), strongly suggest a critical role of Glu(226), Glu(227), and Asp(228) in substrate binding and catalysis and point to Glu(227) as a catalytic base.

Binding Sites↗
PubMed2000

Isoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps.

Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of lysine residues that are highly conserved in M-CK but are not present in B-CK. The role of these lysines in mediating M-line interaction was tested with a set of M-CK and B-CK point mutants and chimeras. We found that all four lysine residues are involved in the isoenzyme-specific M-line interaction, acting pair-wise as strong (K104/K115) and weak interaction sites (K8/K24). An exchange of these lysines in MM-CK led to a loss of M-line binding, whereas the introduction of the very same lysines into BB-CK led to a gain of function by transforming BB-CK into a fully competent M-line-binding protein. The role of the four lysines in MM-CK is discussed within the context of the recently solved x-ray structures of MM-CK and BB-CK.

Amino Acid Sequence↗
PubMed2000

Monitoring biomolecular interactions by time-lapse atomic force microscopy.

The atomic force microscope (AFM) is a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effectors and pharmacological stimuli. Direct correlation can therefore be made between structural and functional states of individual biomolecules in an aqueous environment. This review explores how time-lapse AFM has been used to learn more about normal and disease-associated biological processes. Three specific examples have been chosen to illustrate the capabilities of this technique. In the cell, actin polymerizes into filaments, depolymerizes, and undergoes interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli. Such events are critical for the function and maintenance of the molecular machinery of muscle contraction and the dynamic organization of the cytoskeleton. One goal is to use time-lapse AFM to examine and manipulate some of these events in vitro, in order to learn more about how these processes occur in the cell. Aberrant protein polymerization into amyloid fibrils occurs in a multitude of diseases, including Alzheimer's and type 2 diabetes. Local amyloid deposits may cause organ dysfunction and cell death; hence, it is of interest to learn how to interfere with fibril formation. One application of time-lapse AFM in this area has been the direct visualization of amyloid fibril growth in vitro. This experimental approach holds promise for the future testing of potential therapeutic drugs, for example, by directly visualizing at which level of fibril assembly (i.e., nucleation, elongation, branching, or lateral association of protofibrils) a given active compound will interfere. Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the nuclear envelope. Transport of ions, small molecules, proteins, RNAs, and RNP particles in and out of the nucleus occurs via NPCs. Time-lapse AFM has been used to structurally visualize the response of individual NPC particles to various chemical and physical effectors known to interfere with nucleocytoplasmic transport. Taken together, such time-lapse AFM studies could provide novel insights into the molecular mechanisms of fundamental biological processes under both normal and pathological conditions at the single molecule level.

Actins↗
PubMed2000

Vacuolar morphology and cell cycle distribution are modified by leucine limitation in auxotrophic Saccharomyces cerevisiae.

Yeast vacuoles are highly dynamic and flexible organelles. In a previous paper, we have shown that subtle, often unrecognised amino acid limitations lead to much lower final cell densities in cultures of different commonly used auxotrophic Saccharomyces cerevisiae strains (Cakar et al., Biotechnol. Lett. 21 (1999) 611). Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the number and morphology of vacuoles, and that these changes are correlated with the cell cycle in batch cultures in a similar way as is known from synchronized cultures. Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze-substitution techniques for sample preparation that so far have been barely successful in yeast. Cells of leucine-limited cultures had single, large vacuoles with a hexagonal tonoplast pattern and were partially arrested in G1 phase. To relieve leucine-limitation, additional leucine was supplied extracellularly via the medium or intracellularly via enhanced leucine biosynthesis due to plasmid-based expression of a leucine marker gene. Such cultures reached more than two-fold higher final optical densities in stationary phase. Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast pattern and by a smaller proportion of cells in G1 phase. These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and cell cycle distribution present a note of caution for morphological and cell cycle studies in yeast.

Animals↗
PubMed2000

Coupling of creatine kinase to glycolytic enzymes at the sarcomeric I-band of skeletal muscle: a biochemical study in situ.

The specific interaction of muscle type creatine-kinase (MM-CK) with the myofibrillar M-line was demonstrated by exchanging endogenous MM-CK with an excess of fluorescently labeled MM-CK in situ, using chemically skinned skeletal muscle fibers and confocal microscopy. No binding of labeled MM-CK was noticed at the I-band of skinned fibers, where the enzyme is additionally located in vivo, as shown earlier by immunofluorescence staining of cryosections of intact muscle. However, when rhodamine-labeled MM-CK was diffused into skinned fibers that had been preincubated with phosphofructokinase (PFK), a glycolytic enzyme known to bind to actin, a striking in vivo-like interaction of Rh-MM-CK with the I-band was found, presumably mediated by binding of Rh-MM-CK to the glycolytic enzyme. Aldolase, another actin-binding glycolytic enzyme was also able to bind Rh-MM-CK to the I-band, but formation of the complex occurred preferably at long sarcomere length (> 3.0 microm). Neither pyruvate kinase, although known for its binding to actin, nor phosphoglycerate kinase (PGK), not directly interacting with the I-band itself, did mediate I-band targeting of MM-CK. Anchoring of MM-CK to the I-band via PFK, but not so via aldolase, was strongly pH-dependent and occurred below pH 7.0. Labeling performed at different sarcomere length indicated that the PFK/MM-CK complex bound to thin filaments of the I-band, but not within the actomyosin overlap zones. The physiological consequences of the structural interaction of MM-CK with PFK at the I-band is discussed with respect to functional coupling of MM-CK to glycolysis, metabolic regulation and channeling in multi-enzyme complexes. The in situ binding assay with skinned skeletal muscle fibers described here represents a useful method for further studies of specific protein-protein interactions in a structurally intact contractile system under various precisely controlled conditions.

Animals↗
PubMed1998

The isoenzyme-diagnostic regions of muscle-type creatine kinase, the M-260 and M-300 box, are not responsible for its binding to the myofibrillar M-band.

Muscle-type creatine kinase is known for its unique interaction with the myofibrillar M-band, but the molecular origin for this structural relationship is not well understood. A systematic sequence comparison between the highly homologous cytosolic isoforms, muscle-type and brain-type creatine kinase, yielded two isoenzyme-specific regions in the muscle-type creatine kinases, the M-260 box (residues 258-270) and the M-300 box (residues 300-315). These particular regions were conspicuous for the specific interaction of this CK isoenzyme, but not of brain-type creatine kinase, with the sarcomeric M-band. In situ diffusion assays with fluorescently labeled native, as well as mutated muscle-type creatine kinase variants, were used to study by laser confocal microscopy their association with the M-band of chemically skinned muscle fibers. Neither a set of charge mutants of the M-260 box and/or the M-300 box nor a hybrid construct of both isoforms with the entire C-terminal region derived from the brain-type isoform showed any significant alteration in the in situ M-band-binding properties when compared to the wild-type form of muscle-type creatine kinase. This indicates that in the intact protein of muscle type creatine kinase, these C-terminal isoenzyme-specific regions are not important for M-band interaction and that the actual M-band interaction domain(s) lay mostly within the N-terminal half of the molecule. The highly conserved motives (M-260 box and M-300 box) may serve an isoenzyme-specific purpose yet to be identified.

Amino Acid Sequence↗
PubMed1998

Myofibrillar interaction of cytosolic creatine kinase (CK) isoenzymes: allocation of N-terminal binding epitope in MM-CK and BB-CK.

The molecular origin of the isoenzyme-specific interaction of cytosolic creatine kinase isoenzymes, muscle-type creatine kinase and brain-type creatine kinase, with myofibrillar structures has been studied by confocal microscopy in an functional in situ binding assay with chemically skinned, unfixed skeletal muscle fibers using wild-type and chimeric creatine kinase isoproteins. The specific interaction of both wild-type isoforms with the sarcomeric structure resulted in a stable, isoform-characteristic labeling pattern with muscle-type creatine kinase bound exclusively and tightly to the sarcomeric M-band while brain-type creatine kinase was confined to the I-band region. Chimeric proteins of both muscle-type and brain-type creatine kinases were constructed to localize the corresponding binding domain(s). Exchanged domains included the N-terminal part (residues 1-234), the region containing an isoenzyme 'diagnostic box' (residues 235-285) and the C-terminal part (residues 286-380). The purified recombinant proteins were all fully intact and enzymatically active. All chimeric proteins containing the N-terminal region (amino acid 1-234) of muscle-type or brain-type creatine kinase were always specifically targeted to the sarcomeric M-band or I-band, respectively. We therefore propose that the relevant epitope(s), determining the isoenzyme-specific targeting in skeletal muscle, are entirely located within the N-terminal regions of both cytosolic creatine kinase isoforms.

Animals↗
PubMed1997

Regulation of energy metabolism by neurotransmitters in astrocytes in primary culture and in an immortalized cell line.

Evidence suggests that astrocytes might play an important role in cerebral energy metabolism. A recently developed cell line, called DI TNC1, displays several characteristic features of astrocytes. Thus, we have investigated in these cells a number of parameters related to energy metabolism. First, glycogen, the major energy reserve in the brain, is present in these cells and its levels are influenced by the glucose content of the growth medium and the presence of serum. Second, several neurotransmitters including noradrenaline and vasoactive intestinal peptide (VIP) induce a glycogenolytic response. Their effect on glycogen is paralleled by a similar effect on the formation of cyclic AMP, which is presumably the second messenger involved. Third, noradrenaline stimulates glucose utilization (as reflected by 2-deoxyglucose uptake) in DI TNC1 cells, an effect which is mimicked by the second messenger arachidonate. Interestingly, two actions of neurotransmitters, which are well characterized in primary astrocytes, are absent in DI TNC1 cells. These are the noradrenaline- and VIP-induced resynthesis of glycogen and the glutamate-stimulated glycolysis. In summary, the observations reported here lend further support to the concept that astrocytes are important for the control of brain energy metabolism. In addition, DI TNC1 cells might represent an interesting preparation to help decipher some of the astrocytic functions related to energy metabolism.

Animals↗
PubMed1997

The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop.

A histidine residue with a pKa of 7 has been inferred to act as a general acid-base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochondrial isoenzyme Mib-CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small-angle X-ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild-type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters K(m) or Vmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.

Animals↗
PubMed1996

The structure of the prostaglandin EP4 receptor gene and related pseudogenes.

The EP4 prostaglandin receptor (EP4R) is a member of the seven transmembrane receptor superfamily. We have obtained the human EP4 receptor gene sequence and determined its structure relative to EP4R cDNA synthesized from peripheral blood lymphocytes. The EP4R gene spans approximately 22 kb and consists of three exons separated by two introns. The first exon (530 bp) is noncoding. After an intron of 472 bp, the second exon contains a short (43 bp) 5' sequence before a 289-amino-acid open reading frame (ORF). An 11.5-kb intron is found at the end of transmembrane 6, and the rest of the ORF is in exon 3. The gene structure is analogous to those of the thromboxane, PGI, and PGD receptors. The deduced initiation site does not contain a conventional TATA box but is 70% GC-rich and contains CCAAT boxes, SP1 and AP2 motifs, and motifs consistent with activation by proinflammatory cytokines. Southern blot analysis of human genomic DNA shows two genes with homology to the EP4R gene. Both appear to be pseudogenes with 70% amino acid identity to the EP4R up to the "ERY" sequence at the end of transmembrane 3, where an Alu-like repetitive sequence element was found. The ORF sequence is also interrupted by a stop codon. The pseudogenes differ in that one contains a second "repetitive element" (a line 1 repeat) in the 5' end of the ORF. Northern blot analysis of human mRNA using a pseudogene probe showed hybridization only to the EP4 receptor transcript. PCR also failed to detect expression of either pseudogene. This study defines the gene structure of EP4R and suggests the existence of two related pseudogenes.

Amino Acid Sequence↗
PubMed1996

Purification, metal cofactor, N-terminal sequence and subunit composition of a 5-aminolevulinic acid dehydratase from the unicellular green alga Scenedesmus obliquus, mutant C-2A'.

5-Aminolevulinic acid dehydratase was purified to apparent homogeneity from Scenedesmus obliquus, mutant C-2A', starting with serial affinity chromatography according to Wang et al., followed by separation on DEAE-Cellulose DE 52, TSKgel Toyopearl HW-55 and FPLC on Mono Q. The enzyme was purified 117-fold compared with the initial crude soluble enzyme preparation and showed a final specific activity of 9.17 microkat/kg protein at pH 8.2 at a total recovery of 7%. Mg2+ was determined to be the metal cofactor of the enzyme. It can, to a certain extent, be substituted by other divalent cations. From the purified enzyme the first 15 amino acids of the N-terminus could be determined, showing a moderate similarity to 5-aminolevulinic acid dehydratases from spinach, pea, Escherichia coli and yeast. The molecular mass of the native protein was determined by gel filtration to be 282+/-5 kDa. 42+/-1 kDa were ascertained for the subunit size by SDS/PAGE. These investigations, supported by electron microscopy, revealed that the enzyme from Scenedesmus consists of six subunits arranged in a six-membered ring. Additionally, there is some evidence that two of the rings form a sandwich-like complex.

Amino Acid Sequence↗