Lumenal transmission of decapentaplegic in Drosophila imaginal discs

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.     

Negative transcriptional regulation of virulence and oncogenes of the Ti plasmid by Ros bearing a conserved C2H2-zinc finger motif

The chromosomal ros gene in Agrobacterium tumefaciens encodes a repressor of virulence and oncogenes that are located on a resident Ti plasmid. Mutational inactivation of ros de-represses the expression of the virC and virD operons, causing premature processing and accumulation of T-DNA molecules, and the premature expression of the oncogene, ipt, leading to the synthesis of cytokinin in the bacterium rather than in the plant host cell. Ros is a 15.5 kDa protein containing a novel "eukaryotic" C(2)H(2) zinc finger. Amino acid substitutions in the finger result in the loss of binding of Ros to the ros box, a 40 bp sequence within the operator of virC/D and ipt gene promoters; and the loss of binding of a zinc ion. The ros gene is highly conserved in members of the Rhizobiaceae. Evolutionary distance tree analyses revealed distant ties to the Japanese puffer fish, Fugu rupripes rather than to plants. Interestingly, ros homologues were found in microorganisms derived from marine sources, supporting the hypothesis that ros may have originated from a marine rather than a terrestrial organism.     

Expression and endocytosis of VEGF and its receptors in human colonic vascular endothelial cells

Normal human colonic microvascular endothelial cells (HUCMEC) have been isolated from surgical specimens by their adherence to Ulex europaeus agglutinin bound to magnetic dynabeads that bind alpha-L-fucosyl residues on the endothelial cell membrane. Immunocytochemistry demonstrated the presence of a range of endothelial-specific markers on HUCMEC, including the von Willebrand factor, Ulex europaeus agglutinin, and platelet endothelial cell adhesion molecule-1. The growing cells form monolayers with the characteristic cobblestone morphology of endothelial cells and eventually form tube-like structures. HUCMEC produce vascular endothelial growth factor (VEGF) and express the receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase, through which VEGF mediates its actions in the endothelium. VEGF induces the tyrosine phosphorylation of KDR and a proliferative response from HUCMEC comparable to that elicited from human umbilical vein endothelial cells (HUVEC). On binding to HUCMEC or HUVEC, (125)I-labeled VEGF internalizes or dissociates to the medium. Once internalized, (125)I-labeled VEGF is degraded and no evidence of ligand recycling was observed. However, significantly less VEGF is internalized, and more is released to the medium from HUCMEC than HUVEC. Angiogenesis results from the proliferation and migration of microvascular, not large-vessel, endothelial cells. The demonstration that microvascular endothelial cells degrade less and release more VEGF to the medium than large-vessel endothelial cells identifies a mechanism permissive of the role of microvascular cells in angiogenesis.     

The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.     

Polygalacturonase beta-subunit antisense gene expression in tomato plants leads to a progressive enhanced wound response and necrosis in leaves and abscission of developing flowers

Tomato (Lycopersicon esculentum var. Better Boy) plants were transformed with a tomato leaf wound-inducible polygalacturonase (PG) beta-subunit gene in the antisense orientation (PGbetaS-AS) under the control of the cauliflower mosaic virus 35S promoter. The leaves of the transgenic plants exhibited small localized lesions, which eventually enlarged and spread throughout the entire surfaces of the leaves, resulting in cell death. The same lesions were also observed in the peduncle of developing flowers, extending to the whole flower causing abscission, resulting in a sterile phenotype. Leaves of transgenic plants exhibited elevated levels of PG activity, hydrogen peroxide, and enhanced defense signaling in response to wounding and elicitor treatment. The defense signaling increased was accompanied by an increased resistance toward tobacco hornworm (Manduca sexta) larvae. The cumulative results suggest that in the absence of the beta-subunit protein in tomato leaves, an increase in PG activity occurred that led to an enhanced wound response, the formation of lesions leading to severe necrosis, and an abscission of developing flowers.     

Detection of fatty acids from intact microorganisms by molecular beam static secondary ion mass spectrometry

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.     

Expanded divalent metal-ion tolerance of evolved ligase ribozymes

Class I ligases are artificial ribozymes that catalyze the joining of two single-stranded RNAs. These ribozymes are between 120 and 160 nucleotides in length, making them intermediate in size for catalytic RNAs. Previous characterization of the b1-207 ribozyme suggests that it behaves similar to larger ribozymes in terms of divalent metal-ion dependence. This molecule displays a strong preference for magnesium for catalysis, and is inactive in any other metal except manganese, which actually inhibits its operation in magnesium. Here, we sought to examine the metal-ion usages of two ligases that were obtained through continuous evolution in vitro from the b1-207 sequence framework. We found an expanded catalytic range for the E(100)(#3) and B(16)(#19) ribozymes, as they are both catalytically active in calcium and strontium, and less inhibited by manganese. Though not selected for activity in these salts, the evolved ribozymes exhibit several adaptations to in vitro catalysis, and their ability to accommodate metals other than magnesium can be viewed as an example of a molecular exaptation.     

Assessing tillage disturbance on assemblages of ground beetles (Coleoptera: Carabidae) by using a range of ecological indices

Many ecological studies have used diversity indices to assess the impact of environmental disturbance. In particular, ground beetles have been advocated as a good group for assessing disturbance. Most studies on various organisms have used only one or two indices. For our study of the impact of tillage disturbance on carabid beetles in farm fields in southern Ontario, Canada, we used seven different diversity indices (richness, Shannon–Wiener, Berger–Parker, Q-statistic, Margalef, and evenness). Few studies have used deviation from diversity abundance models as a measure of disturbance; however, we use three that are applicable to our data (geometric, log-normal and log-series). The indices and models were used to test the null hypothesis that there is no change in diversity with increasing tillage disturbance, and that there is no difference in diversity with different crops or years. We were not able to reject the null hypothesis that there is any diversity difference among farms. We also found that there was no single diversity index or model that was better than any other at detecting disturbance. These results are supplemented by a meta-analysis of 45 published data sets for the same taxon but in different habitats. The meta-analysis supports the conclusions from our field research that diversity indices and models are not useful for detecting the possible effect of disturbance on assemblages of carabid beetles.     

Isolation of Functional RNA from Periderm Tissue of Potato Tubers and Sweet Potato Storage Roots

A reliable and efficient protocol is given for the isolation of mRNA from the periderm of potato tubers and sweet potato storage roots. The method relies on a urea-based lysis buffer and lithium chloride to concentrate total RNA away from most of the cytoplasmic components and to prevent oxidation of phenolic complexes. To enhance the physical separation of the RNA from other macromolecular components, the RNA fraction was incubated in the presence of the cationic surfactant Catrimox-14. Poly(A)⁺ mRNA was separated from total RNA and other contaminants by using Promega's MagneSphere technology. The mRNA was suitable for cDNA library construction and RNA fingerprinting.