Quantitative X-ray phase contrast waveguide imaging of bacterial endospores.

Quantitative waveguide-based X-ray phase contrast imaging has been carried out on the level of single, unstained, unsliced and freeze-dried bacterial cells of Bacillus thuringiensis and Bacillus subtilis using hard X-rays of 7.9 keV photon energy. The cells have been prepared in the metabolically dormant state of an endospore. The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations. Additionally, a direct reconstruction based on the contrast transfer function is investigated, and the two approaches are compared. Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 105 Gy. Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.

The isotopic biosignatures of photo- vs. thiotrophic bivalves: are they preserved in fossil shells?

Symbiont-bearing and non-symbiotic marine bivalves were used as model organisms to establish biosignatures for the detection of distinctive symbioses in ancient bivalves. For this purpose, the isotopic composition of lipids (δ13C) and bulk organic shell matrix (δ13C, δ34S, δ15N) from shells of several thiotrophic, phototrophic, or non-symbiotic bivalves were compared (phototrophic: Fragum fragum, Fragum unedo, Tridacna maxima; thiotrophic: Codakia tigerina, Fimbria fimbriata, Anodontia sp.; non-symbiotic: Tapes dorsatus, Vasticardium vertebratum, Scutarcopagia sp.). ∆13C values of bulk organic shell matrices, most likely representing mainly original shell protein/chitin biomass, were depleted in thio- and phototrophic bivalves compared to non-symbiotic bivalves. As the bulk organic shell matrix also showed a major depletion of δ15N (down to -2.2 ‰) for thiotrophic bivalves, combined δ13C and δ15N values are useful to differentiate between thio-, phototrophic, and non-symbiotic lifestyles. However, the use of these isotopic signatures for the study of ancient bivalves is limited by the preservation of the bulk organic shell matrix in fossils. Substantial alteration was clearly shown by detailed microscopic analyses of fossil (late Pleistocene) T. maxima and Trachycardium lacunosum shell, demonstrating a severe loss of quantity and quality of bulk organic shell matrix with time. Likewise, the composition and δ13C-values of lipids from empty shells indicated that a large part of these compounds derived from prokaryotic decomposers. The use of lipids from ancient shells for the reconstruction of the bivalve's life style therefore appears to be restricted.

Characterization and phylogeny of a novel methanotroph, Methyloglobulus morosus gen. nov., spec. nov.

A novel methanotrophic gammaproteobacterium, strain KoM1, was isolated from the profundal sediment of Lake Constance after initial enrichment in opposing gradients of methane and oxygen. Strain KoM1 grows on methane or methanol as its sole source of carbon and energy. It is a Gram-negative methanotroph, often expressing red pigmentation. Cells are short rods and occur sometimes in pairs or short chains. Strain KoM1 grows preferably at reduced oxygen concentrations (pO2=0.05-0.1bar). It can fix nitrogen, and grows at neutral pH and at temperatures between 4 and 30°C. Phylogenetically, the closest relatives are Methylovulum miyakonense and Methylosoma difficile showing 91% 16S rRNA gene sequence identity. The only respiratory quinone is ubiquinone Q8; the main polar lipids are phosphatidyl ethanolamine and phosphatidyl glycerol. The major cellular fatty acids are summed feature 3 (presumably C16:1ω7c) and C16:1ω5c, and the G+C content of the DNA is 47.7mol%. Strain KoM1 is described as the type strain of a novel species within a new genus, Methyloglobulus morosus gen. nov., sp. nov.

Acute peripheral facial palsy in Lyme disease -- a distal neuritis at the infection site.

AIM: Children with acute peripheral facial palsy have often suffered tick bites and/or erythema migrans in the head/neck region on the same side. With respect to the pathogenesis of neuroborreliosis this topographical association was investigated in an animal model. METHODS: A Borrelia garinii strain, isolated from the CSF of a child with acute facial palsy, was injected in 9 rats intracutaneously in the right subauricular region. Infected rats were examined for clinical symptoms of Lyme disease, the spread of the spirochetes was investigated by PCR of necropsies (facial nerves, trigeminus nerves, heart, brain, skin) up to 47 days after infection. The nerve tissues were investigated by histology, immunohistochemistry and electron microscopy. RESULTS: None of the rats developed a facial palsy or other symptoms of Lyme disease. Borrelia DNA was found in the heart after 5 days and in the brain after 7 days of infection up to the end of investigation (47 days), as well as in the ipsilateral peripheral nerves after 7 to 33 days. Borrelia was detected by electron microscopy near endoneural vessels of the facial nerve. Peri-, epi-, and endoneural infiltrations of macrophages, plasma cells and B cells characterized an inflammation of the facial and trigeminus nerves ipsilateral to the infection site. CONCLUSION: An infection with Borrelia garinii in the subauricular region induces an ipsilateral neuritis of peripheral nerves. The particular vulnerability of the human facial nerve may be a result of its long intraosseus course. Thus, an inflammatory edema may injure the nerve in the canalis facialis.

Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy.

Electron microscopy of biofilms and the localization of extracellular polymers at high resolution require the adaptation of conventional electron microscopic preparation and imaging techniques. A method developed for in situ fixation and embedding of biofilms, imaging of unstained thick sections with electron spectroscopic imaging and the application of lectin or antibody-based marker systems allowed interpretation of extracellular polymer distribution at micrometer scale. By this way, it is possible to discriminate in situ between extracellular polymers produced by different organisms.

Temperature optima of enzyme-catalysed reactions in microemulsion systems.

Ternary phase systems (water/surfactant/organic solvent) were utilised to increase and broaden the temperature optima of enzyme-catalysed reactions. Alcohol dehydrogenases from yeast and Thermoanaerobium brockii (EC 1.1.1.1 and EC 1.1.1.2), lactate dehydrogenase from Lactobacillus delbrueckii (EC 1.1.1.28) and the particulate hydrogenase from Ralstonia eutropha (EC 1.18.99.1) were used as model enzymes in microemulsions, consisting of the surfactant Aerosol OT, and various alkane solvent and aqueous phases. All enzymes exhibited, besides an increase in specific activity, an upshift of the temperature optimum of the catalysed reaction. The temperature optimum could be further shifted by variation of the chain length of the solvent used and/or the addition of compatible solutes to the aqueous phase. Under optimised conditions, catalytic reactions of enzymes from mesophilic microorganisms had temperature optima in the range generally obtained with enzymes from thermophilic organisms.

Liposomes for microcompartmentation of enzymes and their influence on catalytic activity.

Modular systems for protein coupling have been applied for anchoring enzyme molecules on liposome surfaces. Two cytoplasmic model enzymes, alpha-amylase from Escherichia coli (EC. 3.2.1.1) and guanylate kinase from Saccharomyces cerevisiae (EC. 2.7.4.8), were directly coupled by a histidine-tag or indirectly via strep-tag and streptavidin or streptactin linker to a liposome membrane. Though the catalytic properties of the enzymes are generally maintained, stability and specific activity of the enzymes are modified after coupling and are especially influenced by the lipid used for the liposome assembly.

Structure and localization of cyclosporin synthetase, the key enzyme of cyclosporin biosynthesis in Tolypocladium inflatum.

The electron microscopic image of native cyclosporin synthetase molecules showed large globular complexes of 25 nm in diameter, built up by smaller interconnected units. Compartmentation of cyclosporin synthetase and the functionally interconnected D-alanine racemase was revealed after sucrose density gradient centrifugation of subcellular fractions and immunoelectron microscopy. A considerable proportion of cyclosporin synthetase and D-alanine racemase was detected at the vacuolar membrane. The product cyclosporin was localized in the fungal vacuole.

Sinorhizobium meliloti strain 1021 bioS and bdhA gene transcriptions are both affected by biotin available in defined medium.

Sinorhizobium meliloti 1021 responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Immunogold labeling and electron microscopy revealed that the BioS protein is located within the S. meliloti cytoplasm. Under biotin-limiting conditions the S. meliloti cell lumen was filled with polyhydroxybutyrate (PHB) granules suggesting that either PHB synthesis or degradation are influenced by biotin. To test this hypothesis a 3-hydroxybutyrate-dehydrogenase-lacZ (bdhA-lacZ) fusion was mobilized into S. meliloti. beta-galactosidase tests revealed an overall 3.6-5.2-fold higher bdhA transcription in the presence of added biotin. Comparison of the bdhA and the bioS promoter regions identified several common motifs.

The aroC gene of Aspergillus nidulans codes for a monofunctional, allosterically regulated chorismate mutase.

The cDNA and the chromosomal locus of the aroC gene of Aspergillus nidulans were cloned and is the first representative of a filamentous fungal gene encoding chorismate mutase (EC 5.4.99.5), the enzyme at the first branch point of aromatic amino acid biosynthesis. The aroC gene complements the Saccharomyces cerevisiae aro7Delta as well as the A. nidulans aroC mutation. The gene consists of three exons interrupted by two short intron sequences. The expressed mRNA is 0.96 kilobases in length and aroC expression is not regulated on the transcriptional level under amino acid starvation conditions. aroC encodes a monofunctional polypeptide of 268 amino acids. Purification of this 30-kDa enzyme allowed determination of its kinetic parameters (k(cat) = 82 s(-1), n(H) = 1. 56, [S](0.5) = 2.3 mM), varying pH dependence of catalytic activity in different regulatory states, and an acidic pI value of 4.7. Tryptophan acts as heterotropic activator and tyrosine as negative acting, heterotropic feedback-inhibitor with a K(i) of 2.8 microM. Immunological data, homology modeling, as well as electron microscopy studies, indicate that this chorismate mutase has a dimeric structure like the S. cerevisiae enzyme. Site-directed mutagenesis of a crucial residue in loop220s (Asp(233)) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity.

Principles of macromolecular organization and cell function in bacteria and archaea.

Structural organization of the cytoplasm by compartmentation is a well established fact for the eukaryotic cell. In prokaryotes, compartmentation is less obvious. Most prokaryotes do not need intracytoplasmic membranes to maintain their vital functions. This review, especially dealing with prokaryotes, will point out that compartmentation in prokaryotes is present, but not only achieved by membranes. Besides membranes, the nucleoid, multienzyme complexes and metabolons, storage granules, and cytoskeletal elements are involved in compartmentation. In this respect, the organization of the cytoplasm of prokaryotes is similar to that in the eukaryotic cell. Compartmentation influences properties of water in cells.

The in situ polymerase chain reaction for detection of chlamydia trachomatis.

The in situ polymerase chain reaction (PCR) is a technique that has important applications in the diagnosis of viral and bacterial diseases. This study investigated an in situ PCR assay established to detect the presence of Chlamydia trachomatis in endocervical swabs. In addition, histological sections of endocervical squamous cell carcinoma were analyzed because previous studies had revealed a significant association with C. trachomatis. A total of 20 cervical neoplasms (squamous cell carcinoma in situ; n = 10; invasive squamous cell carcinoma; n = 10) and endocervical smears taken from five patients with and without inflammatory changes were analyzed by conventional PCR. Chlamydial DNA was found in 10 histological samples (six carcinomas in situ, four invasive carcinomas) and in one endocervical swab from a patient with known C. trachomatis infection. Positive specimens were used for establishing an in situ PCR assay (IS-PCR). After IS-PCR, these samples showed dense cytoplasmic staining of endocervical cells (smears) and non-neoplastic epithelial cells (cervical neoplasms). The other tumor samples and smears did not demonstrate positive PCR reaction. The results indicate that in situ PCR is an effective technique for localizing C. trachomatis in target cells because IS-PCR detection of chlamydial DNA correlated with histological and cytological features.

Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.

Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm.

The F0 complex of the Escherichia coli ATP synthase. Investigation by electron spectroscopic imaging and immunoelectron microscopy.

Cholate-solubilized F0 complexes of the ATP synthase (F0F1) from Escherichia coli were studied by application of conventional transmission electron microscopy and electron spectroscopic imaging (ESI) of negatively stained samples. Using the ESI mode, the structural organization of the F0 complex (diameter of 7.5 +/- 0.5 nm) could be observed in more detail and defined projections could be distinguished. Projection A appears as a deltoid-like structure with bilateral symmetry. Projection B has an overall trapezoidal shape with some similarity in shape to the letter W. Applying the ESI mode to the ac complex dissolved in cholate-containing buffer, an elongated structure consisting of two intensity maxima could be observed. Simulations with models of the F0 and the ac complex revealed that the projections observed can be obtained by tilting and rotating a model in which subunit a and the two copies of subunit b are located outside the subunit c oligomer. This view of structural organization was supported by results obtained with F0 complexes decorated with monoclonal antibodies against subunits a, b or c.

Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg.

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.

[Unusual late complications following total hip endoprosthesis]. / Ungewöhnliche Spätkomplikation nach Totalendoprothese der Hüfte.

A cementless total hip replacement was implanted in a 52-year-old male patient with coxarthrosis. Rapid cranial migration of the cup and ectopic ossification were subsequently observed. Five years after the original operation, revisional arthroplasty had to be performed to replace the cup. At surgery a large amount of granulomatous tissue and the tip of a deep drainage tube were found. Interposed between the polyethylene inlay and the femoral head of the prosthesis, the drain had not been visible on any of the radiographic controls. The X-ray control performed immediately after the first procedure revealed that the drain had been slung around the neck of the prosthesis stem, a procedure preferred by some surgeons. When the drain was removed, the tip must have been caught between the polyethylene inlay and the femoral head of the prosthesis. It is not clear whether the loosening of the cup was ultimately caused by the increased wear due to the presence of the drain tip. However, in light of this finding we no longer sling the drain around the prosthesis shaft, but lay it along the neck of the femoral component in the same direction as it is routed out transcutaneously.

Stability and activity of hydrogenases of Methanobacterium thermoautotrophicum and Alcaligenes eutrophus in reversed micellar systems.

In water-in-oil microemulsion the membrane-associated F420-hydrogenase of Methanobacterium thermoautotrophicum (strain Marburg) and the membrane-bound hydrogenase of Alcaligenes eutrophus H 16 (MBH) showed prolonged activity at elevated temperatures (measured as hydrogen production) as compared to aqueous buffer solution. The temperature optimum of the reactions was about 15 degrees C higher than in aqueous buffer solution. Activity of the almost completely inactivated F420-hydrogenase could be partially recovered by transfer into microemulsion.

Identification of the Methanococcus voltae S-layer structural gene.

We have established that the gene which we had previously identified as encoding the Methanococcus voltae P-type ATPase is, in fact, the structural gene for the M. voltae S-layer protein. This conclusion is based on a comparison of the N-terminal sequence of S-layer protein prepared by two independent methods with that derived from the nucleotide sequence of the cloned gene. This conclusion was further supported by immunocytochemical localization of the antigen directed against the antibodies used in the cloning experiments.

[Results of surgical treatment of posterior knee instability]. / Ergebnisse der operativen Behandlung posteriorer Knieinstabilitäten.

A retrospective study was performed to evaluate the outcome of operative treatment of posterior cruciate ligament (PCL) lesions in 115 patients operated on between 1980 and 1989. Follow-up was possible in 89 patients at 18-124 months postoperatively (average 76 months). In 65 re-examination was possible, while 24 patients returned a questionnaire. The results of patients who were operated on in the acute state were superior to those with chronic instabilities (Lysholm 79.9 +/- 18.5 vs 64.3 +/- 22.1; Tegner 5.7 +/- 2.3 vs 4.2 +/- 2.2; instrumented posterior drawer 5.3 +/- 3.5 mm vs 5.9 +/- 3.8 mm). On the other hand, the preoperative scores of symptomatic patients with chronic instabilities (Lysholm 38.8 +/- 22.0; Tegner 2.1 +/- 1.7) were clearly lower. Extraarticular procedures (Hughston) slightly improved symptoms in posterolateral instabilities. Olecranization of the patella had no influence on the results. Interpretation of the data is difficult as there was no matched group of patients with nonoperative treatment. A reviews of the literature suggests that isolated PCL tears are best treated with conservative management. Only in cases where associated ligamentous injuries require operative treatment should PCL reconstruction be performed. Chronic posterior instabilities should be treated operatively only if the patients are severely symptomatic. However, complete restoration of knee stability was usually not achieved with the techniques presented in this paper.