Quantification of the Pseudomonas population in New Zealand soils by fluorogenic PCR assay and culturing techniques

The genus Pseudomonas contains fast-growing nutritionally versatile bacteria that are able to utilize a wide variety of carbon sources. The ubiquity of the genus has been highlighted by conventional microbiology and the genus is well represented in collections of cultured bacteria. Here we evaluate the Pseudomonas population in New Zealand soils by comparing a culture-independent (real-time PCR combined with fluorescent TaqMan technology) with a culture-dependent (Gould's S1) population estimate. We show that cultivated fluorescent pseudomonads are not numerically dominant and represent a small proportion of <1% of the total Pseudomonas population, and that the total Pseudomonas population itself represents only a small proportion of <1% of the total bacterial population.     

Neisserial pilin genes display extensive interspecies diversity

All Neisseria live in association with host cells, however, little is known about the genetic potential of nonpathogenic Neisseria species to express attachment factors such as pili. In this study, we demonstrate that type IV pilin-encoding genes are present in a wide range of Neisseria species. N. sicca, N. subflava, and N. elongata each contain two putative pilE genes arranged in tandem, while single genes were identified in N. polysaccharea, N. mucosa, and N. denitrificans. Neisserial pilE genes are highly diverse and display features consistent with a history of horizontal gene transfer.     

Kinetic analysis of the L-ornithine transcarbamoylase from Pseudomonas savastanoi pv. phaseolicola that is resistant to the transition state analogue (R)-N delta-(N'-sulfodiaminophosphinyl)-L-ornithine

(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).     

A two-stage normalization method for partially degraded mRNA microarray data

MOTIVATION: The goal of the study is to obtain genetic information from exfoliated colonocytes in the fecal stream rather than directly from mucosa cells within the colon. The latter is obtained through invasive procedures. The difficulties encountered by this procedure are that certain probe information may be compromised due to partially degraded mRNA. Proper normalization is essential to obtaining useful information from these fecal array data. RESULTS: We propose a new two-stage semiparametric normalization method motivated by the features observed in fecal microarray data. A location-scale transformation and a robust inclusion step were used to roughly align arrays within the same treatment. A non-parametric estimated non-linear transformation was then used to remove the potential intensity-based biases. We compared the performance of the new method in analyzing a fecal microarray dataset with those achieved by two existing normalization approaches: global median transformation and quantile normalization. The new method favorably compared with the global median and quantile normalization methods. AVAILABILITY: The R codes implementing the two-stage method may be obtained from the corresponding author.     

Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads

There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.     

Synaptic behaviour of hexaploid wheat haploids with different effectiveness of the diploidizing mechanism

Haploids of three cultivars of Triticum aestivum (Thatcher, Chris, and Chinese Spring) were obtained from crosses with Zea mays. The level of chromosome pairing at metaphase I and the synaptic behaviour at prophase I was studied. There were differences in the meiotic behaviour of the haploids from different cultivars. Thatcher and Chris haploids had significantly higher levels of pairing at metaphase I than Chinese Spring haploids. This metaphase I pairing was correlated with higher levels of synapsis achieved in the Thatcher and Chris prophase I nuclei than in the Chinese Spring nuclei. Variation in the effectiveness of the diploidizing mechanism among cultivars of wheat is proposed to have a genetic origin and the role of the Ph1 locus in the different haploids is discussed.     

Synthesis of novel DNA cross-linking antitumour agents based on polyazamacrocycles

We are seeking to develop more effective alkylating agents as antitumour agents. In previous work conformationally restricted nitrogen mustards were synthesised containing piperidine or pyrrolidine rings. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation and the effects of varying the distances between the two alkylating sites were studied. Some efficient cross-linkers of naked DNA were prepared but few of these compounds exhibited significant cytotoxicity in human tumour cells in vitro. We have extended this work by making tri- and tetra-azamacrocyclic compounds containing two to four potential alkylating sites. Most of these compounds were powerful DNA alkylating agents and showed cytotoxicity (IC(50) values 6-100microM) comparable with chlorambucil (45microM) and melphalan (8.5microM). In particular the cyclen derivative 2a was more than 10(4) times more effective at cross-linking DNA (2a XL(50)<10nM) than chlorambucil (XL(50) 100microM), and showed significant cytotoxicity in human tumour cells in vitro.     

IRREGULAR TRICHOME BRANCH1 in Arabidopsis encodes a plant homolog of the actin-related protein2/3 complex activator Scar/WAVE that regulates actin and microtubule organization

The dynamic actin cytoskeleton is important for a myriad of cellular functions, including intracellular transport, cell division, and cell shape. An important regulator of actin polymerization is the actin-related protein2/3 (Arp2/3) complex, which nucleates the polymerization of new actin filaments. In animals, Scar/WAVE family members activate Arp2/3 complex-dependent actin nucleation through interactions with Abi1, Nap1, PIR121, and HSCP300. Mutations in the Arabidopsis thaliana genes encoding homologs of Arp2/3 complex subunits PIR121 and NAP1 all show distorted trichomes as well as additional epidermal cell expansion defects, suggesting that a Scar/WAVE homolog functions in association with PIR121 and NAP1 to activate the Arp2/3 complex in Arabidopsis. In a screen for trichome branching defects, we isolated a mutant that showed irregularities in trichome branch positioning and expansion. We named this gene IRREGULAR TRICHOME BRANCH1 (ITB1). Positional cloning of the ITB1 gene showed that it encodes SCAR2, an Arabidopsis protein related to Scar/WAVE. Here, we show that itb1 mutants display cell expansion defects similar to those reported for the distorted class of trichome mutants, including disruption of actin and microtubule organization. In addition, we show that the scar homology domain (SHD) of ITB1/SCAR2 is necessary and sufficient for in vitro binding to Arabidopsis BRK1, the plant homolog of HSPC300. Overexpression of the SHD in transgenic plants causes a dominant negative phenotype. Our results extend the evidence that the Scar/WAVE pathway of Arp2/3 complex regulation exists in plants and plays an important role in regulating cell expansion.