DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI

Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns. They have also been identified in self-splicing inteins (protein introns). These enzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-specific double-strand break at a target site in an allele that lacks the corresponding mobile intron. The target sites recognized by these small endonucleases are generally long (14-44 base pairs). Four families of homing endonucleases have been identified, including the LAGLIDADG, the His-Cys box, the GIY-YIG and the H-N-H endonucleases. The first identified His-Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 A resolution. I-PpoI displays an elongated fold of dimensions 25 x 35 x 80 A, with mixed alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long alpha-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 A apart. The enzyme possesses a new zinc-bound fold and endonuclease active site. The structure has been determined in both uncleaved substrate and cleaved product complexes.     

Interleukin-1 inhibits renin gene expression in As4.1 cells but not in native juxtaglomerular cells

The assessment of personality disorders is complicated by conceptual and empirical problems. An aspect of the disarray in this area is the general negative connotations associated with these conditions, which is prevalent in the negative language frequently used to describe these disorders. This article presents a simple, straightforward, and objective approach to evaluating and describing the Axis II conditions as an example of how to improve on this problem.     

Epithelial/mesenchymal interactions and branching morphogenesis of the lung

The establishment of the permissible levels for the use of additives in foods must be based on the Acceptable Daily Intake (ADI). A method that may be applied for this purpose is the Danish Budget Method which estimates the maximum amount of the additive that may be added to the food based on the functional properties of the additive, and on the categories of the food in which the additive will be used. Based on the latest information B?r and Würtzen propose some modifications to the original Budget Method, one of which is the addition of a correction factor which takes into account the competition between different food additives with the same functional properties. In the present paper, both the Budget Method and the B?r and Würtzen-modified method were applied to evaluate whether the maximum levels of food colours use exceeded their ADI or not. Applying the original Budget Method, the results showed that the colours Sunset Yellow, Amaranth, Erythrosine, Ponceau 4R and Cochineal possibly exceeded the ADI; while applying the modified method only the colours Erythrosine and Cochineal would exceed the ADI. Brazilian regulatory authorities should be advised to establish maximum limits of use for the following categories of colours: Caramel, Inorganic, Natural and Artificial Colours Identical to the Natural Ones, where ADIs have been evaluated by JECFA.     

Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34+ hematopoietic progenitor cells

PURPOSE: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. METHODS AND MATERIALS: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34+ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34+ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. RESULTS: The radiobiological parameters (D[0] and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34+ cells compared to optimal two-factor combinations. The D[0] values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n=1 for all tested GF(s). DNA repair capacity of CD34+ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34+ cells was similar. CONCLUSION: Our data indicate that increased survival of irradiated CB CD34+ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.     

Determination of fluoxetine and norfluoxetine concentrations in cadaveric allograft skin

Fluoxetine hydrochloride is the sixth most prescribed drug in the United States and is administered to treat major depression. A cadaveric skin donation was obtained from a 46-year-old woman who died as a result of a fluoxetine overdose. Due to the potential penetration of the drug and its major metabolite, norfluoxetine, into skin, the safety of using the skin as an allograft was questioned. Our evaluation showed that mean concentrations in skin were 2304+/-175 and 1353+/-102 ng/g of skin, respectively. The skin:plasma ratio was 0.41. Clinically, the amount of fluoxetine that can be transferred to an allograft recipient depends on many factors. Based on penetration of drug and metabolite into skin, one would have to evaluate carefully the risk:benefit ratio of using allografts from a donor who died from a fluoxetine overdose.     

Angiogenin antagonists prevent tumor growth in vivo

A noncytotoxic neutralizing monoclonal antibody (mAb), 26-2F, to human angiogenin (Ang), a potent inducer of neovascularization, has been reported to prevent or delay the establishment of HT-29 human tumor xenografts in athymic mice. In the present study the tumor model was modified to increase sensitivity to Ang antagonists to facilitate further investigations and comparisons of their capacity to inhibit tumor growth. An increase in the percentage of tumor-free mice from 10-25% to 65% is observed in this modified model after treatment with mAb 26-2F. An additional neutralizing mAb, 36u, that interacts with a different epitope on Ang similarly prevents the appearance of tumors, both alone and in combination with mAb 26-2F. In those tumors that develop in mice treated with these agents, the number of vascular elements is reduced. Actin, an Ang antagonist that unlike the mAbs binds both human and mouse Ang, also prevents the establishment of tumors while exhibiting no toxic effects at daily doses > 50 times the molar amount of circulating mouse Ang. Ang antagonists also inhibit the appearance of tumors derived from two other Ang-secreting human tumor cell lines--i.e., A549 lung adenocarcinoma and HT-1080 fibrosarcoma. These results demonstrate that inhibition of the action of Ang is an effective therapeutic approach for the treatment of malignant disease.