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Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

Up to now large scale mapping (scales >1:10000) is done nearly exclusively with aerial photographs. New digital camera systems will replace these analogue systems in the near future. This paper describes an approach for automated detection of houses, using a data set of the High Resolution Stereo Camera-Airborne (HRSC-A). The system provides multispectral information with a resolution of 15 cm (3000 m flight altitude) and Digital Surface Models with a resolution of 50 cm, depicting elevation in steps of 10 cm. The elevation and spectral information supplied by the sensor was used for this study. New object-oriented approaches allow the interpretation of these high resolution data sets. The combination of a new approach (multiresolution segmentation, hierarchical networks) and the multispectral high resolution data of HRSC-A with its accurate Digital Surface Model shows very promising results.

This study evaluated the dimensional accuracy of various impression materials for monophase elastic impression making. To isolate this parameter, a direct measurement of the impressions was made without taking the model material into consideration.A total of eight materials were tested; six impression materials were addition-curing silicones, and two were polyether impression materials. All materials were processed according to the manufacturers' instructions. A specially developed precision mold made of stainless steel served as basis for measuring the elastomeric impression materials. Using a stereomicroscope at a temperature of 23.0 +/- 1.0 degrees C and with a precision linear adjustment and lathe, sights were set on the marking points of customized posts. The measurement was performed after the earliest time possible for fabricating the model according to the manufacturer (time 1) and after 90 minutes (time 2). In a one-way analysis of variance, multiple average comparisons of dimensional accuracy were made (Por = .05) between the impression materials under investigation.Under the conditions of this study, the impression materials tested demonstrated a very high dimensional accuracy. The arithmetic means of the dimensional changes ranged from -11 to 19 microns for both measuring times.Since as a rule, no significant dimensional changes occurred for the different impression materials between time 1 and time 2, this time interval for fabricating a model can be recommended.

The BMP2-dependent onset of osteo/chondrogenic differentiation in the acknowledged pluripotent murine mesenchymal stem cell line (C3H10T1/2) is accompanied by the immediate upregulation of Fibroblast Growth Factor Receptor 3 (FGFR3) and a delayed response by FGFR2. Forced expression of FGFR3 in C3H10T1/2 is sufficient for chondrogenic differentiation, indicating an important role for FGF-signaling during the manifestation of the chondrogenic lineage in this cell line. Screening for transcription factors exhibiting a chondrogenic capacity in C3H10T1/2 indentified that the T-box containing transcription factor Brachyury is upregulated by FGFR3-mediated signaling. Forced expression of Brachyury in C3H10T1/2 was sufficient for differentiation into the chondrogenic lineage in vitro and in vivo after transplantation into muscle. A dominant-negative variant of Brachyury, consisting of its DNA-binding domain (T-box), interferes with BMP2-mediated cartilage formation. These studies indicate that BMP-initiated FGF-signaling induces a novel type of transcription factor for the onset of chondrogenesis in a mesenchymal stem cell line. A potential role for this T-box factor in skeletogenesis is further delineated from its expression profile in various skeletal elements such as intervertebral disks and the limb bud at late stages (18.5 d.p.c.) of murine embryonic development.

Eschericha coli was genetically engineered to produce recombinant human bone morphogenetic protein-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system. High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 g l−1) were obtained by applying a high-cell-density cultivation procedure. After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to 100 mg l−1 by means of a simple dilution method with yields exceeding 50%. Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer. With the established renaturation and purification protocols, yields of more than 10 mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth. The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells.

Background: Cardiac allograft vascular disease is (CAVD) the most important cause of death following heart transplantation (HTX). Although in the past, researchers focused predominantly on mechanisms of endothelial injury, the possible role of recipient-related and genetically determined factors has not been studied in detail. Methods Stimulated by recent observations in native coronary artery disease, we analyzed the potential impact of angiostensin-converting enzyme (ACE) polymorphism (insertion/deletion [I/D], intron 16) on development and progression of CAVD. We characterized genotype in 146 patients 1 to 12 years after HTX (121 men; mean age, 46.2 ± 11.3 years; observation period, 6.1 ± 3.8 years) and correlated genotype to the onset and progression of CAVD, defined as luminal obstruction > 50%. Results We found allelic frequencies to be 28.8% ( n = 42) for ACE-DD, 49.3% ( n = 72) for ACE-DI, and 21.9% ( n = 32) for ACE-II. Differences in actuarial freedom from vasculopathy were significant 6 years after transplantation, with 84.6% for ACE-II compared with 54.4% for ACE-DD. We observed intermediate results for ACE-DI genotype (77.3%, p = 0.015). Conclusions In this large cohort study, we demonstrated a close relationship between the recipient-related ACE-D genotype and development of advanced CAVD. These observations suggest that gene–environment interactions might be clinically important in coronary vasculopathy after HTX.

Gene transfer is a process by which bacterial populations substantially increase their rates of evolution and adaptation (12, 59). Particularly, plasmid-located genes, which are transferred by conjugation from donor to recipient cells, can disseminate rapidly between even phylogenetically different bacterial groups (17, 36, 41) and microbial communities in different spatial habitats (34, 71). Such microbial genetic networks should be considered in risk assessments of releases of genetically engineered microorganisms into the environment (22, 37, 43). The probability and rate of plasmid transfer from a donor to indigenous microorganisms in a given habitat are influenced by plasmid-borne genes which determine the type of transfer mechanism (self-transmissible or mobilizable) and the host range of autonomous plasmid replication. Additionally, specific physicochemical conditions, such as temperature, water potential, and the availability of energy (substrates) for donor and recipient cells, are important factors influencing gene transfer rates in terrestrial and aquatic environments (23, 53, 64). The spread of plasmid-borne genes is still extremely difficult to predict for terrestrial habitats, since a large variety of microhabitat conditions which are not well characterized exists. In bulk soil under laboratory conditions, conjugative gene transfer from recombinant bacterial donor strains to indigenous soil bacteria has been found only under specific selective conditions or on rare occasions (11, 20, 24, 27, 50, 61). Several studies failed to detect such transfer events, and it was concluded that heterogeneity and low densities of recipient cells, as well as a lack of substrates for microbial metabolism, prevented efficient plasmid transfer in bulk soil (19, 49, 54, 75). Plant exudates increased rates of gene transfer in soil (33, 48), and higher rates of gene transfer were found in rhizospheres than in bulk soil (50, 61). It was assumed that other microsites which favor gene transfer in terrestrial habitats are associated with soil invertebrates (74). However, to date little experimental evidence to prove this assumption is available. Intraspecies transconjugants of added Enterobacter cloacae donor and recipient cells could be isolated from microcosm experiments with the variegated cutworm, Peridroma saucia, and plant material (2). The investigators in that study concluded that gene transfer events happened, most likely, in the digestive tracts or in the feces of the insects. Another recent report demonstrated that a conjugative plasmid was transferred between fed Escherichia coli strains in the guts of Rhabditis nematodes (1). Earthworms mediated transport and enhanced plasmid transfer from added donor cells to added recipients and to indigenous bacteria in soil (14, 15). High rates of intraspecies plasmid transfer, comparable to those obtained in pure broth cultures, were detected with Bacillus thuringiensis in infected lepidopterous larvae (31). Microarthropods (collembolans and mites) are the most abundant invertebrate group in the majority of soils (5) but have not been recognized, so far, for their impact on microbial gene transfer. There are some indications that microarthropods harbor a large variety of microorganisms in their guts and thereby contribute to microbial biodiversity in terrestrial environments (7, 9, 57). In the accompanying paper, we have described the gut of Folsomia candida (Collembola) as a habitat and species-specific vector for microorganisms (67). The gut of this soil-dwelling insect, which has a volume of only several nanoliters, was found to be densely colonized, predominantly by rod-shaped bacterial cells. We were interested to know whether such bacterial cells act as recipients for plasmids and thereby promote gene transfer in microbial communities. F. candida feeds, under natural conditions, on bacteria (3), fungal mycelia (6, 66), and nematodes (35). Here, we report on the results of experiments in which plasmid-bearing E. coli strains were fed to F. candida in microcosms. Self-transferable plasmids, as well as mobilizable plasmids with different host ranges, and a nonmobilizable plasmid were included in this study in order to determine the specific capacities of these different classes of plasmids to spread into indigenous bacterial populations. For detection purposes, all plasmids were engineered by the insertion of the luciferase-encoding marker gene luc or lux (30, 47).

Interaction potentials between soil microarthropods and microorganisms were investigated with Folsomia candida (Insecta, Collembola) in microcosm laboratory experiments. Microscopic analysis revealed that the volumes of the simple, rod-shaped guts of adult specimens varied with their feeding activity, from 0.7 to 11.2 nl. A dense layer of bacterial cells, associated with the peritrophic membrane, was detected in the midgut by scanning electron microscopy. Depending on the molting stage, which occurred at intervals of approximately 4 days, numbers of heterotrophic, aerobic gut bacteria changed from 4.9 × 10 2 to 2.3 × 10 6 CFU per specimen. A total of 11 different taxonomic bacterial groups and the filamentous fungus Acremonium charticola were isolated from the guts of five F. candida specimens. The most abundant isolate was related to Erwinia amylovora (96.2% DNA sequence similarity to its 16S rRNA gene). F. candida preferred to feed on Pseudomonas putida and three indigenous gut isolates rather than eight different type culture strains. When luciferase reporter gene-tagged bacterial strains were pulse fed to F. candida , gut isolates were continuously shed for 8 days to several weeks but Escherichia coli HB101 was shed for only 1 day. Ratios of ingested to released bacterial cells demonstrated that populations of nonindigenous gut bacteria like Sinorhizobium meliloti L33 and E. coli HB101 were reduced by more than 4 orders of magnitude but that the population of gut isolate Alcaligenes faecalis HR4 was reduced only 500-fold. This work demonstrates that F. candida represents a frequently changeable but selective habitat for bacteria in terrestrial environments and that microarthropods have to be considered factors that modify soil microbial communities.

Comparison of the patterns of N-linked oligosaccharides attached to several proteins isolated from human cerebrospinal fluid and serum reveals striking differences (“brain-type” versus “serum-type” glycosylation) which are attributed to the different physiological surroundings of both body fluids. No modification of oligosaccharides was found to occur during passage of proteins across the blood-brain barrier. Glycosylation analysis of CSF-proteins can thus be used to gain information on the site of biosynthesis (i.e. systemic or intrathecal) of specific proteins in question.

The carbohydrate structures of beta-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N-glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N-acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N-acetylglucosamine residues of nearly all structures were found to be alpha 1,6-fucosylated. Peripheral fucose (about 20% alpha 1,3-linked to N-acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N-acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N-acetylglucosamine only were present. At least 20 different N-glycans were identified. Analysis of the site-specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N-acetylglucosamine, as well as terminal N-acetylglucosamine and galactose residues, and significant amounts of N-acetylneuraminic acid in alpha 2,3 linkage), this protein can be classified as "brain-type" glycosylated.

beta-Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23-29 kDa was determined for the polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of beta-trace protein as prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (Thr instead of Ser) was detected at amino acid position 154 of the beta-trace polypeptide chain in the corresponding tryptic peptide. The two N-glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29 and Asn56 bear exclusively complex-type oligosaccharide structures (partially sialylated with alpha 2-3- and/or alpha 2-6-linked N-acetylneuraminic acid) that are almost quantitatively alpha 1-6 fucosylated at the proximal N-acetylglucosamine; approximately 70% of these molecules contain a bisecting N-acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.

Eine amerikanische Studie (Ann Int Med 2006;145:869–879) kam kurzlich zu dem Ergebnis, dass die Wirksamkeit eines Cimicifugaproduktes bei Wechseljahresbeschwerden Placebo nicht uberlegen ist (siehe auch MMW Nr. 3/2007, S. 1). Eine Leserin zweifelt an den von den Autoren gezogenen Schlussfolgerungen und an der ubertragbarkeit der Ergebnisse auf andere Produkte

This review focuses on the molecular aspects of osteogenesis and chondrogenesis as they relate to cellular and developmental levels. We discuss BMP (bone morphogenetic protein)-mediated signaling pathways for the patterning of skeletal elements and mechanisms for the induction of cartilage and bone formation. We emphasize BMP-mediated initiation of chondrogenesis and osteogenesis and consider BMP-dependent signaling cascades in mesenchymal progenitor cells during the development of the axial and appendicular skeleton and during growth plate and joint formation. In particular, we discuss BMP-signaling mediators of the Smad and TAK family and the modulation of these signals by cross-talk and integration with other signaling pathways.

Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible lambda pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to 10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(-) orientation is as efficient as the switch from G(-) to G(+).

Asiolo-transferrin from human cerebrospinal fluid was purified to homogeneity. Investigation of the structural characteristics of its oligosaccharides support our hypothesis of ‘brain-type’ glycosylation of intrathecally synthesized cerebrospinal fluid proteins. For carbohydrate structural analysis, high-pH anion-exchange chromatography, methylation analysis, liquid secondary ion- and matrix-assisted laser desorption/ ionization mass spectrometry of the permethylated derivatives were used. The major structure turned out to be a complex-type agalacto-diantennary oligosaccharide with bisecting N-acetylglucosamine and proximal fucose. Analysis of a second transferrin preparation containing both asialo- and sialo-transferrin revealed another major glycan species derived from the sialylated transferrin variant which is galactosylated and lacks bisecting N-acetylglucosamine and fucose.