In vitro activity of oxazaphosphorines in childhood acute leukemia: preliminary report

Glufosfamide (beta-D-glucosyl-ifosfamide mustard) is a new agent for cancer chemotherapy. Its pharmacology is similar to commonly used oxazaphosphorines, but it does not require activation by hepatic cytochrome P-450 and preclinically demonstrates lower nephrotoxicity and myelosuppression than ifosfamide. The aim of the study was a comparison of the drug resistance profiles of glufosfamide and other oxazaphosphorines in childhood acute leukemias. Leukemic cells, taken from children with ALL on diagnosis (n = 41), ALL on relapse (n = 12) and AML on diagnosis (n = 13) were analyzed by means of the MTT assay. The following drugs were tested: glufosfamide (GLU), 4-HOO-ifosfamide (IFO), 4-HOO-cyclophosphamide (CYC) and mafosfamide cyclohexylamine salt (MAF). In the group of initial ALL samples median cytotoxicity values for GLU, IFO, CYC and MAF were 15.5, 33.8, 15.7 and 7.8 microM, respectively. In comparison with initial ALL samples, the relative resistance for GLU and IFO in relapsed ALL samples was 1.9 (p = 0.049) and 1.3 (ns), and in initial AML samples 31 (p < 0.001) and 5 (p = 0.001), respectively. All oxazaphosphorines presented highly significant cross-resistance. Glufosfamide presented high activity against lymphoblasts both on diagnosis and on relapse.     

Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide     

The Effect of Farmer Attitudes and Objectives on the Heterogeneity of Farm Attributes and Management in Switzerland

Identifying and interpreting the heterogeneity of farmer behaviour is becoming increasingly important in support of policy- and decision-making goals. This paper explores whether observed differences in farming practices can be interpreted from the heterogeneity of farmer behaviour. Farmer attitudes and objectives were analysed using a combination of principal components and cluster analysis applied to responses to statements in a telephone-based survey. Respondents were classified into four profiles; business-oriented, lifestylers, multifunctionalists and traditionalists. Each profile differed in terms of farm management practices, the amount of land farmers either managed or owned, the existence of successors and the importance placed on household members in providing information. The results suggest that knowledge of farmer behavioural profiles could support more targeted policy development that accounts for alternative farmer goals. However, similarities were also found between the profiles, suggesting that farmer behaviour would be better interpreted as a dynamic set of identities, rather than as static profiles.     

Optimization of a Method for Chromatin Immunoprecipitation Assays in the Marine Invertebrate Chordate Ciona

Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip and ChIP-sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here, we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms.     

Nucleotide-Specific Recognition of Iron-Responsive Elements by Iron Regulatory Protein 1

IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem–loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.     

High production of cold-tolerant chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus Lecanicillium muscarium CCFEE 5003: Bioprocess optimization and characterization of two main enzymes

The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flasks to check its chitinase production on rough shrimp and crab wastes. Production on shrimp shells was much higher than that on crab shells (104.6 ± 9.3 and 48.6 ± 3.1 U/L, respectively). For possible industrial applications, bioprocess optimization was studied on shrimp shells in bioreactor using RSM to state best conditions of pH and substrate concentration. Optimization improved the production by 137% (243.6 ± 17.3). Two chitinolytic enzymes (CHI1 and CHI2) were purified and characterized. CHI1 (MW ca. 61 kDa) showed optima at pH 5.5 and 45 °C while CHI2 (MW ca. 25 kDa) optima were at pH 4.5 and 40 °C. Both enzymes maintained high activity levels at 5 °C and were inhibited by Fe⁺⁺, Hg⁺⁺ and Cu⁺⁺. CHI2 was strongly allosamidin-sensitive. Both proteins were N-acetyl-hexosaminidases (E.C. 3.2.1.52) but showed different roles in chitin hydrolysis: CHI1 could be defined as “chitobiase” while CHI2 revealed a main “eso-chitinase” activity.     

Therapeutic angiogenesis for revascularization in peripheral artery disease

Therapeutic angiogenesis for peripheral artery disease (PAD), achieved by gene and cell therapy, has recently raised a great deal of hope for patients who cannot undergo standard revascularizing treatment. Although pre-clinical studies gave very promising data, still clinical trials of gene therapy have not provided satisfactory results. On the other hand, cell therapy approach, despite several limitations, demonstrated more beneficial effects but initial clinical studies must be constantly validated by larger randomized, multi-center, double-blinded, placebo-controlled trials. This review focuses on previous and recent gene and cell therapy studies for limb ischemia, including both experimental and clinical research, and summarizes some important papers published in this field. Moreover, it provides a short comment on combined gene and cell therapy approach on the example of heme oxygenase-1 overexpressing cells with therapeutic properties.     

Biomarker responses and accumulation of hazardous substances in mussels (Mytilus trossulus) transplanted along a pollution gradient close to an oil terminal in the Gulf of Finland (Baltic Sea)

Baltic Sea blue mussels (Mytilus trossulus) were used as sentinel organisms to detect the biological effects of chemical contamination in the low salinity environment. Mussels naturally adapted to a salinity of ca. 6.0 PSU were caged for 30 days at four sites along an assumed pollution gradient (salinity ca. 4.5 PSU) in the vicinity of Finland's largest oil refinery and harbor Kilpilahti in the Gulf of Finland. Tissue concentrations and accumulation rates of especially organic contaminants (PAHs, PCBs and organotins) were clearly elevated at the innermost coastal stations near the harbor area. Biological effects of contaminant exposure on caged mussels were evaluated by measuring a suite of biomarkers including catalase, glutathione S-transferase, superoxide dismutase, glutathione reductase, lipid peroxidation, acetylcholinesterase activity and lysosomal membrane stability. Mussels transplanted near the harbor area were able to elevate their antioxidant defense in response to environmental contamination. Reduced morphometric condition index and soft tissue growth rate together with increased lipid peroxidation and low lysosomal membrane stability were also observed at the most contaminated site. The results suggest that caging of M. trossulus for four weeks at lower salinity is a feasible method for the detection of environmental pollution also in low salinity areas of the Baltic Sea.