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1.  Reliability of a Fully Automated Interpretation of γ-H2AX Foci in Lymphocytes of Moderately Trained Subjects under Resting Conditions 
Background. Analysis of γ-H2AX foci is a promising approach to evaluate exercise-induced DNA damage. However, baseline levels and day-to-day variability of γ-H2AX foci have not been investigated in healthy subjects at rest. Methods. Blood was taken from eight moderately trained healthy males (29 ± 3 yrs, 1.84 ± 0.03 m, and 85 ± 6 kg) at two separate days (M1/M2) after 24-hour exercise cessation. Number of γ-H2AX foci per 100 lymphocytes (N), number of foci per affected lymphocyte (NAL), percentage of affected lymphocytes (PAL), and diameter (D) of γ-H2AX foci were analyzed (mean ± SD). Differences between M1 and M2 were analyzed using paired t-tests (α = 0.05). Day-to-day variability was evaluated by calculating the coefficients of variation (CV%), bias, and limits of agreement (LoA). Results. There were no statistically significant differences between M1 (N: 7.6 ± 4.4, NAL: 1.2 ± 0.2, PAL: 5.9 ± 2.6%, and D: 0.63 ± 0.07) and M2 (N: 8.4 ± 4.6, NAL: 1.3 ± 0.1, PAL: 6.9 ± 4.2%, and D: 0.66 ± 0.06). CV was calculated to be 98.5% (N), 88.9% (PAL), 11.3% (NAL), and 8.0% (D). Bias (LoA) was 0.75 (−15.2/13.7), −0.02 (−0.36/0.33), −1.0 (−11.9/9.9), and −0.04 (−0.16/0.09), respectively. Conclusions. Background level in healthy subjects is approximately 0.07 to 0.09 γ-H2AX foci/cell. NAL and D are reliable measures.
PMCID: PMC4132490  PMID: 25147735
2.  Maintenance treatment with the immunomodulator MGN1703, a Toll-like receptor 9 (TLR9) agonist, in patients with metastatic colorectal carcinoma and disease control after chemotherapy: a randomised, double-blind, placebo-controlled trial 
This phase II study evaluated the synthetic DNA-based immunomodulator and Toll-like receptor 9 agonist MGN1703 as maintenance treatment in metastatic colorectal carcinoma (mCRC).
Fifty-nine patients with mCRC and disease control after standard first-line chemotherapy were randomised to MGN1703 60 mg (N = 43) or placebo (N = 16).
The hazard ratio (HR) for the primary endpoint [progression-free survival (PFS) from the start of maintenance] was 0.56 (95 % CI 0.29–1.08; P = 0.07) and 0.55 (95 % CI 0.3–1.0; P = 0.04) by independent and investigator review, respectively. MGN1703 significantly improved PFS measured from the start of induction therapy versus placebo on independent (HR 0.49; 95 % CI 0.26–0.94; P = 0.03) and investigator review (HR 0.50; 95 % CI 0.31–1.02; P = 0.02). Overall survival (OS) data remain immature (HR 95 %; 95 % CI 0.3–1.5; P = 0.29) with 28/43 patients alive after a medium follow-up of >17 months. Retrospective subgroup analysis showed a significant effect of MGN1703 on PFS versus placebo in patients with greater than median tumour size reduction and normalised carcinoembryonic antigen concentrations following induction therapy, and in patients with elevated activated NKT cells ≥3.08 %. Adverse events were mild to moderate and limited to injection-site reactions or linked to general immune system activation.
MGN1703 maintenance treatment was well tolerated and appears to induce durable and prolonged PFS and disease control in a subgroup of patients with mCRC following induction therapy. Activated NKT cells may be a predictive biomarker for selecting patients likely to benefit more from MGN1703.
Electronic supplementary material
The online version of this article (doi:10.1007/s00432-014-1682-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4131138  PMID: 24816725
MGN1703; Immunomodulator; TLR9 agonist; Colorectal cancer; Maintenance
3.  Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas 
Advanced adult soft-tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs). The goal of this study was to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. The data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers.
Advanced adult soft-tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs); the latter have mainly been isolated from adult bone marrow as plastic-adherent cells with differentiation capacity into mesenchymal tissues. Recently, a panel of antibodies has been established that allows for the prospective isolation of primary MSCs with high selectivity. Similar to cancer stem cells in other malignancies, sarcoma stem cells may bear immunophenotypic similarity with the corresponding precursor, that is, MSCs. We therefore set out to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. In addition, fibroblasts from different sources were examined. The results document a significant amount of MSC markers shared by sarcoma cells. The expression pattern includes uniformly expressed markers, as well as MSC markers that only stained subpopulations of sarcoma cells. Expression of W5C5, W8B2 (tissue nonspecific alkaline phosphatase [TNAP]), CD344 (frizzled-4), and CD271 marked subpopulations displaying increased proliferation potential. Moreover, CD271+ cells displayed in vitro doxorubicin resistance and an increased capacity to form spheres under serum-free conditions. Interestingly, another set of antigens, including the bona fide progenitor cell markers CD117 and CD133, were not expressed. Comparative expression patterns of novel MSC markers in sarcoma cells, as well as fibroblasts and MSCs, are presented. Our data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers.
PMCID: PMC3659743  PMID: 23283492
Mesenchymal stem cell; Sarcoma stem cell; Cancer stem cell; Stem cell marker
5.  Effects of short‐term treatment strategies over 4 weeks in Achilles tendinopathy 
The therapeutic efficacy of non‐surgical treatment strategies in Achilles tendinopathy (AT) has not been well clarified. Time‐consuming and costly combinations of treatment for pain, physiotherapy and biomechanical procedures are often applied.
To analyse the efficacy of single therapeutic regimens commonly used over a short period of 4 weeks.
31 male runners (mileage >32 km/week) with unilateral, untreated AT completed 4 weeks of either physiotherapy (10 treatments: deep‐friction, pulsed ultrasound, ice, sensory motor training; (P)), wearing custom fit semirigid insoles (I) or remained without treatment (control group C). Before and after treatment, all patients underwent a treadmill test and a plantar flexion strength exercise. Subjective pain (Pain Disability Index, Pain Experience Scale), as well as strength performance capacity (peak torque), was analysed (mean, 95% CI, repeated measures analysis of variance, α = 0.05).
Pain was reduced to <50% of the baseline value after physiotherapy or after wearing insoles (p<0.05). Individual pain reduction was >50% (25%) in 89% (100%) of subjects in I and 55% (73%) in P. Higher eccentric plantar flexion peak torques after treatment were observed in I and P.
Most patients with AT experience a reduction in pain after only 4 weeks of differentiated, non‐surgical treatment consisting of physiotherapy or semirigid insoles.
PMCID: PMC2465365  PMID: 17261560
6.  Resistance to Platinum-Containing Chemotherapy in Testicular Germ Cell Tumors Is Associated with Downregulation of the Protein Kinase SRPK11 
Neoplasia (New York, N.Y.)  2004;6(4):297-301.
Male germ cell tumors (GCTs) are extremely sensitive to platinum-containing chemotherapy, with only 10% of patients showing therapy resistance. However, the biological basis of the high curability of disseminated GCTs by chemotherapy is still unknown. Recently, we demonstrated that the mammalian serine/arginine-rich protein-specific kinase 1 (SRPK1) is a cisplatin-sensitive gene, inactivation of which leads to cisplatin resistance. Because, in mammalians, the expression of SRPK1 is preferentially high in testicular tissues, cisplatin responsiveness of male GCTs might be associated with SRPK1 levels. In the present study, we monitored SRPK1 protein expression in a unique series of nonseminomatous GCTs by immunohistochemistry. Randomly selected GCTs (n = 70) and tumors from patients responding to standard chemotherapy (n = 20) generally showed strong SRPK1 staining. In contrast, expression in refractory GCTs (n = 20) as well as in GCTs from poor-prognosis patients responding to high-dose chemotherapy only (n = 11) was significantly lower (two-sided Wilcoxon rank sum test: P < .001). In conclusion, our data suggest that SRPK1 expression might be an important prognostic indicator for the chemoresponsiveness of nonseminomatous GCTs.
PMCID: PMC1502111  PMID: 15256051
Chemotherapy resistance; germ cell tumors; chemotherapy sensitivity; protein kinase SRPK1; immunohistochemistry
7.  Characterization of a Spontaneous Nonmagnetic Mutant of Magnetospirillum gryphiswaldense Reveals a Large Deletion Comprising a Putative Magnetosome Island 
Journal of Bacteriology  2003;185(19):5779-5790.
Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.
PMCID: PMC193972  PMID: 13129949
8.  Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli 
On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo.
PMCID: PMC92822  PMID: 11282658
9.  ComP, a Pilin-Like Protein Essential for Natural Competence in Acinetobacter sp. Strain BD413: Regulation, Modification, and Cellular Localization 
Journal of Bacteriology  2000;182(13):3673-3680.
We recently identified a pilin-like competence factor, ComP, which is essential for natural transformation of the gram-negative soil bacterium Acinetobacter sp. strain BD413. Here we demonstrate that transcription and synthesis of the pilin-like competence factor ComP are maximal in the late stationary growth phase, whereas competence is induced immediately after inoculation of a stationary-phase culture into fresh medium. Western blot analyses revealed three forms of ComP, one with an apparent molecular mass of 15 kDa, which correlates with the molecular mass deduced from the DNA sequence, one 20-kDa form, which was found to be glycosylated, and one 23-kDa form. The glycosylation of ComP was not required for its function in DNA binding and uptake. The 20-kDa form was present in the cytoplasmic membrane, the periplasm, and the outer membrane, whereas the 23-kDa form was located in the outer membrane and might be due to a further modification. Immunological data suggest that ComP is not a subunit of the pilus structures. Possible functions of ComP in the DNA transformation machinery of Acinetobacter sp. strain BD413 are discussed.
PMCID: PMC94537  PMID: 10850981
10.  Purification and Properties of a Thermoactive Glucoamylase from Clostridium thermosaccharolyticum 
A bacterial glucoamylase was purified from the anaerobic thermophilic bacterium Clostridium thermosaccharolyticum and characterized. The enzyme, which was purified 63-fold, with a yield of 36%, consisted of a single subunit with an apparent molecular mass of 75 kDa. The purified enzyme was able to attack α-1,4- and α-1,6-glycosidic linkages in various α-glucans, liberating glucose with a β-anomeric configuration. The purified glucoamylase, which was optimally active at 70°C and pH 5.0, attacked preferentially polysaccharides such as starch, glycogen, amylopectin, and maltodextrin. The velocity of oligosaccharide hydrolysis decreased with a decrease in the size of the substrate. The Km values for starch and maltose were 18 mg/ml and 20 mM, respectively. Enzyme activity was not significantly influenced by Ca2+, EDTA, or α- or β-cyclodextrins.
PMCID: PMC183570  PMID: 16348541
11.  Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy 
Applied and Environmental Microbiology  1987;53(12):2785-2792.
Clostridium thermocellum JW20 and YM4 both synthesize cellulolytic enzyme complexes, cellulosomes, when grown on medium containing cellulose. Electron microscopic studies showed that, in the early stages of growth of strain JW20, clusters of tightly packed cellulosomes, i.e., polycellulosomes, were located on the cell surface and were bound to cellulose. The polycellulosome was estimated to have a particle mass of 50 × 106 to 80 × 106 daltons (Da), while that of the cellulosome was estimated to be 2 × 106 to 2.5 × 106 Da and to contain about 35 polypeptides ranging from 20 to 200 kDa. The cellulosome produced by strain YM4 was found to be somewhat larger, with the estimated particle mass being 3.5 × 106 Da, and the number of polypeptides was counted to be 45 to 50, ranging from 20 to 200 kDa. In the early stages of cultivation, the cellulosomes from both species exist as tightly packed complexes (tight cellulosomes). These subsequently decompose to loosely packed complexes (loose cellulosomes) and ultimately to free polypeptides. Examination of the loose cellulosomal particles showed that they contain rows of equidistantly spaced, similarly sized polypeptide subunits, with an apparently identical orientation arranged parallel to the major axis of the cellulosome. It is postulated that on binding of a cellulose chain alongside such a row of subunits a simultaneous multicutting event occurs that leads to the release of cellooligosaccharides of four cellobiose units in length (C4). Rows of smaller-sized subunits with lower center-to-center distances, which are also present in the cellulosome, subsequently cleave the C4 fragments (or cellulose) to C2 (cellotetraose) or C1 (cellobiose). In this way the cellulosome can catalyze the complete hydrolysis of cellulose.
PMCID: PMC204199  PMID: 16347495
12.  Plain and Complex Flagella of Pseudomonas rhodos: Analysis of Fine Structure and Composition 
Journal of Bacteriology  1974;117(2):844-857.
Cells of Pseudomonas rhodos 9-6 produce two morphologically distinct flagella termed plain and complex, respectively. Fine structure analyses by electron microscopy and optical diffraction showed that plain flagellar filaments are cylinders of 13-nm diameter composed of globular subunits like normal bacterial flagella. The structure comprises nine large-scale helical rows of subunits intersecting four small-scale helices of pitch angle 25°. Complex filaments have a conspicuous helical sheath, 18-nm wide, of three close-fitting helical bands, each about 4.7-nm wide, separated by axial intervals, 4.7 nm wide, running at an angle of 27°. The internal core has similar but not identical substructure to plain filaments. Unlike plain flagella, the complex species is fragile and does not aggregate in bundles. Mutants bearing only one of two types of flagellum were isolated. Cells with plain flagella showed normal translational motion, and cells with complex flagella showed rapid spinning. Isolated plain flagella consist of a 37,000-dalton subunit separable into two isoproteins. Complex filaments consist of a 55,000-dalton protein; a second 43,000-dalton protein was assigned to complex flagellar hooks. The results indicate that plain and complex flagella are entirely different in structure and composition and that the complex type represents a novel flagellar species. Its possible mode of action is discussed.
PMCID: PMC285582  PMID: 4129995
13.  Electron Microscope Study of Length and Partial Denaturation of Rhizobium Bacteriophage DNA 
Journal of Virology  1973;11(6):946-952.
By electron microscopy and melting of the DNA of some bacteriophages from the soil bacterium Rhizobium lupini, it was found that molecular weights range between 27 and 50 Mdaltons and GC contents between 53 and 62%. All DNAs studied are linear; one has exposed single-stranded terminals. Partial heat denaturation of two phage DNA permitted mapping of AT-rich sites within unique nucleotide sequences. The maps are asymmetric with respect to the midpoints of the DNA.
PMCID: PMC355202  PMID: 4712964
14.  Isolation and Characterization of a Bacteriophage Tail-Like Bacteriocin from a Strain of Rhizobium 
Journal of Virology  1972;9(1):160-173.
Bacterial strain 16-3 spontaneously produces a bacteriocin which inhibits the growth of closely related strain 16-2. Both strains were newly isolated from root nodules of lupines and probably belong to the species Rhizobium lupini. Production of infectious progeny of newly isolated virulent phage 16-2-4 in strain 16-2 is inhibited completely if complexes are bacteriocin-treated during the first half of the latent period. Treatment begun during the second half leads to premature lysis of complexes and inactivates only those progeny phages which were not yet fully matured at the moment of the particle-induced lysis. Examination by electron microscope of the bacteriocin enrichment revealed the presence of particles 123 nm in length which resemble the tails of T-even bacteriophages. Since the particles sediment together with the bactericidal activity in the sucrose gradient and adsorb specifically to bacteriocin-sensitive cells, it is concluded that they are identical with the bactericidal agent. The particles are not found attached to phage heads and cannot self-propagate; they are regarded as incomplete and are named INCO particles. INCO particles consist of a core enveloped by a contractile sheath. One end of the sheath is connected to a baseplate to which six fibers, each 32 nm in length, are attached. These connect the baseplate of an adsorbing particle to the cell surface. Since INCo cores are probably empty, it is concluded that specific adsorption of the particles to the bacterial surface is sufficient to inactive sensitive cells irreversibly.
PMCID: PMC356273  PMID: 4110105

Results 1-14 (14)