The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.     

Reduction in Ames Salmonella mutagenicity of mainstream cigarette smoke condensate by tobacco protein removal

The mutagenic activity of cigarette smoke condensates (CSC) made from tobacco before and after removal of protein was assessed by the Ames Salmonella assay in bacterial strains TA98 and TA100. Removal of protein and peptides from flue-cured tobacco via water extraction followed by protease digestion reduced the mutagenicity of the resultant CSC by 80% in the TA98 strain and 50% in the TA100 strain. Similarly, reductions of 81% in TA98 and 54% in TA100 were seen following water extraction and protease digestion of burley tobacco. The significant reductions in Ames mutagenicity following protein removal suggest that protein pyrolysis products are a principal contributor to the genotoxicity of CSC as measured in this assay.     

Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo

Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/progenitor cells. Exogenously added stromal cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of myeloid progenitor cells in vitro. To further evaluate SDF-1/CXCL12 effects on progenitor cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic myeloid progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and G(alpha)i proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse myeloid progenitors. Consistent with in vitro results, progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased progenitor cell cycling and significant increases in progenitor cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to cell trafficking.     

Urea-triazone N characteristics and uses

Urea-triazone nitrogen (N) is a stable solution resulting from a controlled reaction in aqueous medium of urea, formaldehyde, and ammonia which contains at least 25% total N. This N source contains no more than 40%, nor less than 5%, of total N from unreacted urea and not less that 40% from triazone. All other N shall be derived from water-soluble dissolved reaction products of the above reactants. It is a source of slowly available N. The rate of mineralization of urea-triazone is about 66% that of urea after 8 days when incorporated in a Munjor sandy loam. Ammonia volatilization losses of N applied as urea-triazone were about 41% of those from urea on a Cecil sandy loam in the first week after application. N leaching losses through saturated Yolo loam columns of urea-triazone were about two thirds that of urea or nitrate N. This N source has proven to be a safer and more effective material for direct application on plant foliage. Tomato growth was enhanced with foliar application of urea-triazone relative to that obtained from ammonium nitrate or urea. The stability of this N source from potential losses via ammonia volatilization and nitrate leaching when soil applied is also documented by results from university trials.     

Evidence that a protein-protein interaction 'hot spot' on heterotrimeric G protein betagamma subunits is used for recognition of a subclass of effectors

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.     

The action of soybean lipoxygenase-1 on 12-iodo-cis-9-octadecenoic acid: the importance of C11-H bond breaking

Previous work has demonstrated that the ferric form of soybean lipoxygenase-1 will catalyze an elimination reaction on 12-iodo-cis-9-octadecenoic acid (12-IODE) to produce 9, 11-octadecadienoic acid and iodide ion. Elimination is accompanied by irreversible inactivation of the enzyme on 1 out of 10 turnovers. In the present work, 11,11-dideuterio-12-IODE (D(2)-12-IODE) was synthesized and used to demonstrate that both the elimination reaction and inactivation of the enzyme exhibit very large kinetic isotope effects. The rates with the deuterated compound are so low that the isotope effects are difficult to quantify, but they appear to be comparable to the isotope effects previously observed for the normal reaction catalyzed by lipoxygenase and much larger than can be explained by zero-point energy considerations. ESR spectroscopy was used to demonstrate that 12-IODE can reduce ferric lipoxygenase to the ferrous form, and a large isotope effect on this process was observed with D(2)-12-IODE. It is proposed that the pathway leading to reduction and inactivation by 12-IODE is initiated by homolytic cleavage of the C(11)-H bond. Elimination could be initiated either by homolytic or by heterolytic cleavage of this bond. The results suggest that very large isotope effects may be a general feature of C-H bond cleavages catalyzed by this enzyme.     

Proteolytic cleavage confers nitric oxide synthase inducing activity upon prolactin

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a common post-translational modification that can either activate precursor proteins or confer upon the peptide fragment unique biological actions not exerted by the parent molecule. Recent studies have demonstrated that the 16-kDa N-terminal proteolytic cleavage product of PRL (16K-PRL) acts as a potent inhibitor of angiogenesis. Despite previous demonstrations of 16K-PRL production in vivo, biological functions beyond its antiangiogenic actions remain unknown. Here we show that 16K-PRL, but not full-length PRL, acts to promote the expression of the inducible isoform of nitric oxide synthase (iNOS) and nitric oxide (*NO) production by pulmonary fibroblasts and alveolar type II cells with potency comparable with the proinflammatory cytokines interleukin-1beta, interferon gamma, and tumor necrosis factor alpha. The differential effect of 16K-PRL versus PRL occurs through a receptor distinct from known PRL receptors. Additionally, pulmonary fibroblasts express the PRL gene and endogenously produce 16K-PRL, suggesting that this pathway may serve both autocrine and paracrine roles in the regulation of *NO production. These results reveal that proteolytic cleavage of PRL confers upon this classical hormone potent iNOS inducing activity, suggesting its role in inflammatory/immune processes.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Evaluating distributional shifts in home range estimates

A variety of methods are commonly used to quantify animal home ranges using location data acquired with telemetry. High‐volume location data from global positioning system (GPS) technology provide researchers the opportunity to identify various intensities of use within home ranges, typically quantified through utilization distributions (UDs). However, the wide range of variability evident within UDs constructed with modern home range estimators is often overlooked or ignored during home range comparisons, and challenges may arise when summarizing distributional shifts among multiple UDs. We describe an approach to gain additional insight into home range changes by comparing UDs across isopleths and summarizing comparisons into meaningful results. To demonstrate the efficacy of this approach, we used GPS location data from 16 bighorn sheep (Ovis canadensis) to identify distributional changes before and after habitat alterations, and we discuss advantages in its application when comparing home range size, overlap, and joint‐space use. We found a consistent increase in bighorn sheep home range size when measured across home range levels, but that home range overlap and similarity values decreased when examined at increasing core levels. Our results highlight the benefit of conducting multiscale assessments when comparing distributions, and we encourage researchers to expand comparative home range analyses to gain a more comprehensive evaluation of distributional changes and to evaluate comparisons across home range levels.