Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex,a sequence-selective N3-adenine methylating agent

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < deltamag1< deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.     

Transforming growth factor-beta stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways

Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.     

Photoreceptor death: Spatiotemporal patterns arising from one-hit death kinetics and a diffusible cell death factor

Retinitis pigmentosa (RP) is an inherited disease affecting approximately 1: 4000 individuals in North America. It is characterized clinically by the gradual apoptotic death of photoreceptor cells that occurs nonuniformly across the surface of the retina. Recently, it has been demonstrated that the time of death of many individual photoreceptors is random, a fact that must be reconciled with the spatiotemporal patterns of photoreceptor degeneration that are observed in patients with RP. One possible explanation is that a diffusible toxic factor is released by dying photoreceptors and induces adjacent cells to likewise undergo apoptosis. To determine if such a mechanism can result in patchy distributions of photoreceptor death, as frequently observed in RP patients, we studied cell attrition produced by a bistable biochemical switch in an idealized one-dimensional retina. We found that with a reasonable choice of parameter values, our model was able to produce patterns of cell death resembling those observed in RP. In the context of this model, patches on the order of histologically observable size could develop from a single release event, but their rates of formation were independent of the concentration of toxic factor released. Instead, factor concentration affected the overall rate of cell death, the number of degenerating patches, and their distribution across the retina.     

Reptin and pontin antagonistically regulate heart growth in zebrafish embryos

Organ size is precisely regulated during development, but the control mechanisms remain obscure. We have isolated a mutation in zebrafish, liebeskummer (lik), which causes development of hyperplastic embryonic hearts. lik encodes Reptin, a component of a DNA-stimulated ATPase complex. The mutation activates ATPase activity of Reptin complexes and causes a cell-autonomous proliferation of cardiomyocytes to begin well after progenitors have fashioned the primitive heart tube. With regard to heart growth, beta-catenin and Pontin, a DNA-stimulated ATPase that is often part of complexes with Reptin, are in the same genetic pathways. Pontin reduction phenocopies the cardiac hyperplasia of the lik mutation. Thus, the Reptin/Pontin ratio serves to regulate heart growth during development, at least in part via the beta-catenin pathway.     

Identification and action of juvenile hormone III from sexually mature alate females of the red imported fire ant,Solenopsis invicta

Analysis of extracts of hemolymph obtained from sexually mature alate females of Solenopsis invicta from monogyne colonies resulted in identification of juvenile hormone III (JH III). The average amount of JH III was 0.32±0.04 pmol/μmolof hemolymph. Topical application of 0.038 pmol of JH III was sufficient to stimulate alates to shed their wings in the presence of the queen. The time in which alates were induced to dealate decreased linearly with increasing concentrations of JH III from 0.038 to 3.8 pmol. However, higher JH III concentrations deviated from linearity and did not reach dealation times comparable with those that occur after mating flights. Thus, it appears that the mechanism of dealation that occurs when female alates are out of the influence of their queen is different from the one associated with mating flights. Application of 0.42 μmol of precocene II inhibited dealation of alates in queenless colonies. However, this inhibition was reversed after applying 38 pmol JH III to precocene-treated alates. The sizes of corpora allata (CA) from sexuals treated with JH III did not differ from those of controls. However, the sizes of CA were reduced in alates treated with precocene II. The results indicated that JH was important to dealation.     

In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC). Within the embryonic avian gut, vagal NCC migrate in a rostrocaudal direction to form the majority of neurons and glia along the entire length of the gastrointestinal tract, whereas sacral NCC migrate in an opposing caudorostral direction, initially forming the nerve of Remak, and contribute a smaller number of ENS cells primarily to the distal hindgut. In this study, we have investigated the ability of vagal NCC, transplanted to the sacral region of the neuraxis, to colonise the chick hindgut and form the ENS in an experimentally generated hypoganglionic hindgut in ovo model. Results showed that when the vagal NC was transplanted into the sacral region of the neuraxis, vagal-derived ENS precursors immediately migrated away from the neural tube along characteristic pathways, with numerous cells colonising the gut mesenchyme by embryonic day (E) 4. By E7, the colorectum was extensively colonised by transplanted vagal NCC and the migration front had advanced caudorostrally to the level of the umbilicus. By E10, the stage at which sacral NCC begin to colonise the hindgut in large numbers, myenteric and submucosal plexuses in the hindgut almost entirely composed of transplanted vagal NCC, while the migration front had progressed into the pre-umbilical intestine, midway between the stomach and umbilicus. Immunohistochemical staining with the pan-neuronal marker, ANNA-1, revealed that the transplanted vagal NCC differentiated into enteric neurons, and whole-mount staining with NADPH-diaphorase showed that myenteric and submucosal ganglia formed interconnecting plexuses, similar to control animals. Furthermore, using an anti-RET antibody, widespread immunostaining was observed throughout the ENS, within a subpopulation of sacral NC-derived ENS precursors, and in the majority of transplanted vagal-to-sacral NCC. Our results demonstrate that: (1) a cell autonomous difference exists between the migration/signalling mechanisms used by sacral and vagal NCC, as transplanted vagal cells migrated along pathways normally followed by sacral cells, but did so in much larger numbers, earlier in development; (2) vagal NCC transplanted into the sacral neuraxis extensively colonised the hindgut, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses; (3) RET immunostaining occurred in vagal crest-derived ENS cells, the nerve of Remak and a subpopulation of sacral NCC within hindgut enteric ganglia.     

Enrichment versus biofilm culture: a functional and phylogenetic comparison of polycyclic aromatic hydrocarbon-degrading microbial communities

The effect that culture methods have on the diversity of degradative microbial communities is not well understood. We compared conventional batch enrichment with a biofilm culture method for the isolation of polycyclic aromatic hydrocarbon (PAH)-degrading microbial communities from a PAH-contaminated soil. The two methods were assessed by comparing: (i) the diversity of culturable bacteria; (ii) the diversity of PAH-catabolic genes in isolated bacteria; (iii) the inter- and intraspecific diversity of active PAH-catabolic gene classes; (iv) the diversity of bacteria present in 16S rRNA gene libraries generated from RNA extracted from the two communities and soil; and (v) the estimated diversity of active bacteria in the soil and culture systems. Single-strand conformation polymorphism analysis showed that the biofilm culture yielded 36 bacterial and two fungal species compared with 12 bacterial species from the enrichment culture. Application of accumulation and non-parametric estimators to clone libraries generated from 16S rRNA confirmed that the biofilm community contained greater diversity. Sequencing of clones showed that only species from the Proteobacteria were active in the enrichment culture, and that these species were expressing an identical nahAc-like naphthalene dioxygenase. 16S rRNA clones generated from the biofilm community indicated that species from the Cytophaga/Flavobacterium, high G+C bacteria and Proteobacteria were active at the time of sampling, expressing cndA-, nahAc- and phnAc-like naphthalene dioxygenases. The diversity of active species in the biofilm culture system closely matched that in the PAH-contaminated source soil. The results of this study showed that biofilm culture methods are more appropriate for the study of community-level interactions in PAH-degrading microbial communities. The study also indicated that cultivation of microbial communities on solid media might be the primary source of bias in the recovery of diverse species.     

Separation methods applicable to the evaluation of enzyme-inhibitor and enzyme-substrate interactions

Enzymes catalyze a rich variety of metabolic transformations, and do so with very high catalytic rates under mild conditions, and with high reaction regioselectivity and stereospecificity. These characteristics make biocatalysis highly attractive from the perspectives of biotechnology, analytical chemistry, and organic synthesis. This review, containing 128 references, focuses on the use of separation techniques in the elucidation of enzyme-inhibitor and enzyme-substrate interactions. While coverage of the literature is selective, a broad perspective is maintained. Topics considered include chromatographic methods with soluble or immobilized enzymes, capillary electrophoresis, biomolecular interaction analysis tandem mass spectrometry (BIA-MS), phage and ribosomal display, and immobilized enzyme reactors (IMERs). Examples were selected to demonstrate the relevance and application of these methods for determining enzyme kinetic parameters, ranking of enzyme inhibitors, and stereoselective synthesis and separation of chiral entities.