Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil–DNA–glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms.     

Cognitive behavioural therapy versus supportive therapy for persistent positive symptoms in psychotic disorders: The POSITIVE Study, a multicenter, prospective, single-blind, randomised controlled clinical trial

Background

It has been demonstrated that cognitive behavioural therapy (CBT) has a moderate effect on symptom reduction and on general well being of patients suffering from psychosis. However, questions regarding the specific efficacy of CBT, the treatment safety, the cost-effectiveness, and the moderators and mediators of treatment effects are still a major issue. The major objective of this trial is to investigate whether CBT is specifically efficacious in reducing positive symptoms when compared with non-specific supportive therapy (ST) which does not implement CBT-techniques but provides comparable therapeutic attention.

Methods/Design

The POSITIVE study is a multicenter, prospective, single-blind, parallel group, randomised clinical trial, comparing CBT and ST with respect to the efficacy in reducing positive symptoms in psychotic disorders. CBT as well as ST consist of 20 sessions altogether, 165 participants receiving CBT and 165 participants receiving ST. Major methodological aspects of the study are systematic recruitment, explicit inclusion criteria, reliability checks of assessments with control for rater shift, analysis by intention to treat, data management using remote data entry, measures of quality assurance (e.g. on-site monitoring with source data verification, regular query process), advanced statistical analysis, manualized treatment, checks of adherence and competence of therapists. Research relating the psychotherapy process with outcome, neurobiological research addressing basic questions of delusion formation using fMRI and neuropsychological assessment and treatment research investigating adaptations of CBT for adolescents is combined in this network. Problems of transfer into routine clinical care will be identified and addressed by a project focusing on cost efficiency.

Discussion

This clinical trial is part of efforts to intensify psychotherapy research in the field of psychosis in Germany, to contribute to the international discussion on psychotherapy in psychotic disorders, and to help implement psychotherapy in routine care. Furthermore, the study will allow drawing conclusions about the mediators of treatment effects of CBT of psychotic disorders.

Trial Registration

Current Controlled Trials ISRCTN29242879     

Murine B16 melanomas expressing high levels of the chemokine stromal-derived factor-1/CXCL12 induce tumor-specific T cell chemorepulsion and escape from immune control

The chemokine, stromal-derived factor-1/CXCL12, is expressed by normal and neoplastic tissues and is involved in tumor growth, metastasis, and modulation of tumor immunity. T cell-mediated tumor immunity depends on the migration and colocalization of CTL with tumor cells, a process regulated by chemokines and adhesion molecules. It has been demonstrated that T cells are repelled by high concentrations of the chemokine CXCL12 via a concentration-dependent and CXCR4 receptor-mediated mechanism, termed chemorepulsion or fugetaxis. We proposed that repulsion of tumor Ag-specific T cells from a tumor expressing high levels of CXCL12 allows the tumor to evade immune control. Murine B16/OVA melanoma cells (H2b) were engineered to constitutively express CXCL12. Immunization of C57BL/6 mice with B16/OVA cells lead to destruction of B16/OVA tumors expressing no or low levels of CXCL12 but not tumors expressing high levels of the chemokine. Early recruitment of adoptively transferred OVA-specific CTL into B16/OVA tumors expressing high levels of CXCL12 was significantly reduced in comparison to B16/OVA tumors, and this reduction was reversed when tumor-specific CTLs were pretreated with the specific CXCR4 antagonist, AMD3100. Memory OVA-specific CD8+ T cells demonstrated antitumor activity against B16/OVA tumors but not B16/OVA.CXCL12-high tumors. Expression of high levels of CXCL12 by B16/OVA cells significantly reduced CTL colocalization with and killing of target cells in vitro in a CXCR4-dependent manner. The repulsion of tumor Ag-specific T cells away from melanomas expressing CXCL12 confirms the chemorepellent activity of high concentrations of CXCL12 and may represent a novel mechanism by which certain tumors evade the immune system.     

Synergistic and additive antiproliferative effects on human leukemia cell lines induced by combining acetylenehexacarbonyldicobalt complexes with the tyrosine kinase inhibitor imatinib

The tyrosine kinase inhibitor imatinib is successfully used in the treatment of chronic myeloid leukemia, but the occurrence of resistance phenomena can significantly limit therapeutic impact. Imatinib shows synergistic effects with cisplatin, suggesting that the coadministration of different cytostatics might reestablish the efficacy of treatment. We recently demonstrated that cobalt alkyne (or acetylenehexacarbonyldicobalt) complexes induce antiproliferative activity in human leukemia and lymphoma cells. The present study evaluates the effects of cobalt alkyne compounds containing propargylic acid esters on human acute (HL-60) and chronic myeloid (LAMA-84 and CML-T1) leukemia cell lines. The cell growth inhibitory activities (IC(50) values of 9.5 microM and higher) and induction of apoptosis (maximum 5.5-fold increase of single-stranded DNA at a drug concentration of 50 microM) achieved with the single agents were moderate. Interestingly, suboptimal concentrations of the cobalt complexes (10 microM) together with imatinib (0.1 microM), when coadministered, showed an additive or synergistic effect on cellular proliferation inhibition. The most promising results were obtained with complexes containing ligands derived from the nonsteroidal antiinflammatory drugs acetylsalicylic acid and naproxene.     

Nuclear localization and interaction with COP1 are required for STO/BBX24 function during photomorphogenesis

Arabidopsis (Arabidopsis thaliana) SALT TOLERANCE/B-BOX ZINC FINGER PROTEIN24 (STO/BBX24) is a negative regulator of the light signal transduction that localizes to the nucleus of plant cells and interacts with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in the yeast (Saccharomyces cerevisiae) two-hybrid system. The protein contains two B-box zinc-finger motives at the N terminus and a conserved motif at the C-terminal part required for the interaction with COP1. BBX24 accumulates during deetiolation of young seedlings in the first hours of exposure to light. However, this accumulation is transient and decreases after prolonged light irradiation. Here, we identified the amino acidic residues necessary for the nuclear import of the protein. In addition, we created mutated forms of the protein, and analyzed them by overexpression in the bbx24-1 mutant background. Our results indicate that the degradation of BBX24 occurs, or at least is initiated in the nucleus, and this nuclear localization is a prerequisite to fulfill its function in light signaling. Moreover, mutations in the region responsible for the interaction with COP1 revealed that a physical interaction of the proteins is also required for degradation of BBX24 in the light and for normal photomorphogenesis.     

Visualization of abscess formation in a murine thigh infection model of Staphylococcus aureus by 19F-magnetic resonance imaging (MRI)

Background

During the last years, ¹⁹F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection.

Methodology and Principal Findings

In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation.

Conclusion and Significance

We introduce ¹⁹F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.