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Biomedical subjects

S A Müller

Publications and source records attributed to S A Müller.

At least 19 recordsLinked to original sources

Immunosuppressive standards in simultaneous kidney-pancreas transplantation.

Simultaneous pancreas-kidney transplantation is an established procedure for patients with type I diabetes and end-stage renal disease. Continuous advances in the operation techniques with consequent reduction of perioperative morbidity and mortality and the introduction of modern immunosuppressive agents improved not only patients but also graft survival and significantly decreased rejection episodes of both kidney and pancreas grafts. Availability of a variety of new immunosuppressants in the clinical routine and increasing experience of the transplant specialists allowed further developments of therapeutic schemes with application of induction and maintenance immunosuppressive protocols. In this article, we summarize the current status of immunosuppressive regimens in simultaneous pancreas and kidney transplantation.

Graft Rejection↗

Deadly carousel or difficult interpretation of new diagnostic tools for Whipple's disease: case report and review of the literature.

Whipple's disease is a rare systemic disorder classically presenting with weight loss, arthralgias, and diarrhea, which was first described in 1907. The causative bacterium Tropheryma whipplei, is a fastidious organism not growing on conventional media. Before the introduction of polymerase chain reaction (PCR)-based methods, the diagnostic gold standard was histological detection of diastase-resistant periodic acid Schiff (PAS)-positive macrophages or electron microscopy. As in the present case, contradictory results between the former and new diagnostic methods may obscure the correct diagnosis. We critically summarize the performance of the different diagnostic methods and discuss their impact on the clinical management of patients with suspected Whipple's disease.

Aged↗

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy.

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.

Bacillus subtilis↗

Experimental evaluation of phospholipids and icodextrin in re-formation of peritoneal adhesions.

BACKGROUND: The formation of further adhesions after adhesiolysis is a major problem. Not all agents that are supposed to reduce de novo adhesions can reduce further adhesion formation. The efficacy and safety of intraperitoneally applied phospholipids has been shown previously. This study evaluated the influence of phospholipids and icodextrin on adhesion re-formation. METHODS: In an experimental study on 60 Chinchilla rabbits adhesions were induced by standardized abrasion of defined areas of the ventral abdominal wall, the caecum and the ileum. Open adhesiolysis was performed 10 days later. The animals randomly received either 1.5 per cent phospholipids, 4 per cent icodextrin or Ringer's lactate intraperitoneally. Half of the animals in each group were killed after a further 10 days and the remainder at 20 days. Adhesion formation was assessed by computer-aided planimetry and histopathological examination. RESULTS: Phospholipids (10 days: P = 0.001; 20 days: P = 0.012) and icodextrin (10 days: P = 0.006; 20 days: P = 0.055) reduced adhesion re-formation after both intervals compared with Ringer's lactate controls. CONCLUSION: Phospholipids and icodextrin were effective in preventing re-formation of adhesions.

Animals↗

[Material and structure of drainages].

The long history of surgical drainages stands in contradiction to available and reliable data. As of today, closed systems made of silicone are preferable. Taking possible complications into consideration and the good results of routine operations without drainages, indication for use should be critically evaluated. Interdisciplinary research,however, offers the chance of optimizing material and surface characteristics, biostability, biocompatibility, and flow rates. New drainages with improved performance and reduced side effects may counteract the current tendency to do without.

Animals↗

Transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils.

Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo.

Amyloid Neuropathies, Familial↗

[Sigmoid diverticulitis. Extent of resection and choice of procedure].

Diverticular disease of the colon ranks in prevalence equal to diabetes or coronary heart disease. Surgical treatment of colonic diverticulitis is in general very successful. Therapeutic options have broadened in recent years: while improved diagnostic modalities allow a pre-surgical containment of the disease process by percutaneous techniques, minimally-invasive surgical procedures can replace conventional surgery in many cases without a change in the risk of complications. However, there is no proof of a general superiority of the laparoscopic approach. Surgical morbidity and mortality mainly derives from perforations with diffuse peritonitis. As a rule, these cases should be treated by a Hartmann procedure. In selected cases it is also possible to perform a primary anastomosis. This decision should be guided by well-known scoring systems.

Anastomosis, Surgical↗

Modulation of antigenicity related to changes in antibody flexibility upon lyophilization.

Lyophilization is frequently used to increase the shelf-life of biopharmaceuticals containing antibodies. A case in which an anti-idiotypic antibody, MMA 383, substantially lost its in vivo immunogenic properties although the protein was not degraded, is investigated. The scanning transmission electron microscope allowed the MMA 383 Fab and Fc moieties to be resolved. By averaging the single antibodies, the angle between the Fab moieties can be calculated. Non-lyophilized antibodies displayed a wider range of shapes than their reconstituted, lyophilized counterparts. Accordingly, the angle between the two Fab fragments varied more, indicating greater flexibility. The tryptophan steady-state fluorescence intensity, steady-state fluorescence anisotropy and fluorescence lifetime, were smaller for the lyophilized antibodies. These were also more resistant towards thermal denaturation/aggregation. Circular dichroism spectra detected temperature-dependent differences between the two antibody types in the 236 nm region. The subtle but reproducible structural changes induced by lyophilization may be related to the loss of in vivo immunogenic properties.

Antibodies, Anti-Idiotypic↗

The McrBC restriction endonuclease assembles into a ring structure in the presence of G nucleotides.

McrBC from Escherichia coli K-12 is a restriction enzyme that belongs to the family of AAA(+) proteins and cuts DNA containing modified cytosines. Two proteins are expressed from the mcrB gene: a full-length version, McrB(L), and a short version, McrB(S). McrB(L) binds specifically to the methylated recognition site and is, therefore, the DNA-binding moiety of the McrBC endonuclease. McrB(S) is devoid of DNA-binding activity. We observed that the quaternary structure of the endonuclease depends on binding of the cofactors. In gel filtration experiments, McrB(L) and McrB(S) form high molecular weight oligomers in the presence of Mg(2+) and GTP, GDP or GTP-gamma-S. Oligomerization did not require the presence of DNA and was independent of GTP hydrolysis. Electron micrographs of negatively stained McrB(L) and McrB(S) revealed ring-shaped particles with a central channel. Mass analysis by scanning transmission electron microscopy indicates that McrB(L) and McrB(S) form single heptameric rings as well as tetradecamers. In the presence of McrC, a subunit that is essential for DNA cleavage, the tetradecameric species was the major form of the endonuclease.

Bacterial Proteins↗

Molecular mass, stoichiometry, and assembly of 20 S particles.

N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.

Amino Acid Sequence↗

STEM Analysis of Caenorhabditis elegans muscle thick filaments: evidence for microdifferentiated substructures.

In the thick filaments of body muscle in Caenorhabditis elegans, myosin A and myosin B isoforms and a subpopulation of paramyosin, a homologue of myosin heavy chain rods, are organized about a tubular core. As determined by scanning transmission electron microscopy, the thick filaments show a continuous decrease in mass-per-length (MPL) from their central zones to their polar regions. This is consistent with previously reported morphological studies and suggests that both their content and structural organization are microdifferentiated as a function of position. The cores are composed of a second distinct subpopulation of paramyosin in association with the alpha, beta, and gamma-filagenins. MPL measurements suggest that cores are formed from seven subfilaments containing four strands of paramyosin molecules, rather than the two originally proposed. The periodic locations of the filagenins within different regions and the presence of a central zone where myosin A is located, implies that the cores are also microdifferentiated with respect to molecular content and structure. This differentiation may result from a novel "induced strain" assembly mechanism based upon the interaction of the filagenins, paramyosin and myosin A. The cores may then serve as "differentiated templates" for the assembly of myosin B and paramyosin in the tapering, microdifferentiated polar regions of the thick filaments.

Animals↗

Efficacy of adhesion prevention and impact on wound healing of intraperitoneal phospholipids.

BACKGROUND: Postoperative peritoneal adhesions are a major complication of abdominal surgery. Adjuvant therapy is needed to prevent adhesion formation and subsequent sequelae. Previously published data proved the efficacy of phospholipids (PL) for this indication; however, additional information on drug safety was still outstanding. The underlying study was designed to investigate the influence of phospholipids on three different types of healing tissue. MATERIALS AND METHODS: A total of 48 Chinchilla rabbits underwent median laparotomy, standardized abrasion of the visceral and parietal peritoneum, jejunal anastomosis, and an electrocautery incision of the liver. The operation was completed by intraperitoneal administration of 10 ml/kg of either normal saline (NaCl) or phospholipids (12%). RESULTS: After 5 (NaCl 691 mm(2) vs PL 192 mm(2)) and 10 days (NaCl 625 mm(2) vs PL 88 mm(2)) the control group presented with significantly larger adhesion areas (P < 0.05). The bursting pressure of the anastomosis on the 5th (NaCl 16.1 kPa vs PL 18.2 kPa) and 10th (NaCl 19.7 kPa vs PL 18.6 kPa) postoperative days showed no statistically significant difference. The tensile strength of the laparotomy wound measured after intervals of 5 (NaCl 8.5 N cm(-1) vs PL 6.8 N cm(-1)) and 10 days (NaCl 23.0 N cm(-1) vs PL 20.2 N cm(-1)) was not statistically different either. The collagen protein ratio of anastomoses, laparotomy wounds, and liver incisions as well as the inflammatory-reparative response of the different tissues was not affected by PL. CONCLUSIONS: These results confirm the efficacy of phospholipids in adhesion prevention. The findings of uncompromised healing of anastomoses, laparotomy wounds, and liver incisions demonstrate the safety of this agent. Further data may qualify phospholipids for a clinical trial.

Anastomosis, Surgical↗

Influence of intraperitoneal phospholipid dosage on adhesion formation and wound healing at different intervals after surgery.

BACKGROUND: Adjuvant therapy is needed to prevent adhesion formation as a major cause of postoperative morbidity and mortality. The efficacy of phospholipids (PLs) has been proven; however, information on dosage and drug safety are still outstanding. MATERIALS AND METHODS: Forty-eight Chinchilla rabbits underwent median laparotomy, abrasion of the peritoneum, jejunal anastomosis, and an electrocautery incision of the liver. The operation was completed by intraperitoneal administration of PLs in two different concentrations (30 mg/kg and 70 mg/kg body weight). In the control group, the abdomen was closed without additional treatment. Adhesion area, anastomotic bursting pressure, tensile strength of the midline incision, and healing of the liver wound were assessed on days 3, 5, 7, and 10, respectively. RESULTS: The mean areas of adhesions in the control group were slightly larger than in the PL groups after 3 days and 5 days. On day 7 and day 10, both PL groups presented with significantly smaller adhesion areas (P<0.05). In all groups, we measured equal anastomotic bursting pressures on the 3rd, 5th, and 10th postoperative days. After 7 days, the mean value of the PL 70-mg group (17.2 kPa) was significantly lower than in the other groups (control 22.1 kPa, PL 30 mg 20.7 kPa; P<0.05). The tensile strengths of the laparotomy wound measured after intervals of 5 days and 7 days were not statistically different. On day 3, it was reduced after 30 mg PL but enhanced after 70 mg PL, whereas 10 days after surgery the strength increased with the PL dosage. The inflammatory reparative response to hepatic injury, jejunal anastomosis, and midline incision was not affected by PLs as assessed by histological analysis. CONCLUSION: These results prove the efficacy of PLs in adhesion prevention in two concentrations. The findings reveal an unimpeded healing of anastomoses, laparotomy wounds, and liver incisions at different periods after surgery.

Animals↗

Structure and mass analysis by scanning transmission electron microscopy.

In the scanning transmission electron microscope (STEM) an electron beam of a few angstroms diameter is raster scanned over a thin sample and the scattered electrons are sequentially measured for each sample element irradiated. The mass, the elemental composition and the structure of a protein can be simultaneously assessed if all detector systems of the STEM are used. Aspects affecting the accuracy of the mass measurement technique and the demands placed on the instrument's dark-field detector system are outlined. In addition, the influences of some sample preparation techniques are noted and the mass-loss induced at ambient temperatures by the incidence of 80kV electrons on various biological samples is reported. Finally, the importance of the STEM for the structural analysis of proteins is documented by examples.

Chaperonins↗

Structural characterization of two aquaporins isolated from native spinach leaf plasma membranes.

Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.

Amino Acid Sequence↗

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme.

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.

Animals↗

Studies on the in vitro assembly of a beta 1-40: implications for the search for a beta fibril formation inhibitors.

The progressive deposition of the amyloid beta peptide (Abeta) in fibrillar form is a key feature in the development of the pathology in Alzheimer's disease (AD). We have characterized the time course of Abeta fibril formation using a variety of assays and under different experimental conditions. We describe in detail the morphological development of the Abeta polymerization process from pseudo-spherical structures and protofibrils to mature thioflavin-T-positive/Congo red-positive amyloid fibrils. Moreover, we structurally characterize the various polymorphic fibrillar assemblies using transmission electron microscopy and determine their mass using scanning transmission electron microscopy. These results provide the framework for future investigations into how target compounds may interfere with the polymerization process. Such substances might have a therapeutic potential in AD.

Amyloid beta-Peptides↗

Amyloid fibril formation from full-length and fragments of amylin.

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.

Amyloid↗