N-hexanoyl-L-homoserine lactone, a mediator of bacterial quorum-sensing regulation, exhibits plant-dependent stability and may be inactivated by germinating Lotus corniculatus seedlings

The half-life of N-hexanoyl-l-homoserine lactone (C6-HSL) was determined under various pH and temperature conditions, and in several plant environments. C6-HSL was sensitive to alkaline pH, a process that was also temperature-dependent. In addition, C6-HSL disappeared from plant environments, i.e. axenic monocot and dicot plants cultivated under gnotobiotic, hydroponic conditions, albeit with variable kinetics. The disappearance was rapid at the root system of legume plants such as clover or Lotus, and slow or non-existent at the root system of monocots such as wheat or corn. These variable kinetics were not dependent upon pH changes that may have affected the growth media of the plants. Furthermore, C6-HSL did not accumulate in the plant, and the plant did not produce inhibitors of the C6-HSL signal. HPLC analyses revealed that C6-HSL disappeared from the media, and hence, Lotus exhibited a natural C6-HSL inactivating ability. This ability was not specific for C6-HSL and allowed the degradation of other N-acyl-homoserine lactones such as 3-oxo-C6-HSL, 3-oxo-octanoyl-HSL and 3-oxo-decanoyl-HSL. Preliminary investigation revealed that the inactivating ability is temperature-dependant and possibly of enzymatic origin.     

Nutrient regulation of PKCepsilon is mediated by leucine, not insulin, in skeletal muscle

Nutrients enhance signaling pathways involved in skeletal muscle growth through an increased rate of protein synthesis. These studies have led to an understanding of the potential role of the mammalian target of rapamycin (mTOR) in this process. However, activation of mTOR cannot account for all the stimulatory effects of nutrients. The purpose of these experiments was to examine the effect of nutrients on the cellular distribution and activation state of novel PKC isoforms (PKCepsilon and PKCdelta) in the gastrocnemius of rats by use of modification state-dependent phosphopeptide-specific antibodies. The phosphorylation of PKCepsilon on the catalytic domain autophosphorylation site (Ser(729)) was elevated during feeding and then returned to basal levels when the feeding period ended. Meal feeding augmented the phosphorylation of the downstream effectors of mTOR, namely S6K1 and 4E-BP1. In contrast, the phosphorylation of PKCdelta on either the catalytic domain autophosphorylation site (Ser(643)) or activation loop site (Thr(505)) was unaffected. Similar results were obtained when animals were given leucine either acutely via gavage or chronically by dietary supplementations. The effect of leucine was not mimicked by injecting animals with insulin but could be induced by gavage with norleucine, a structural analog of leucine that does not increase plasma insulin concentration. Thus rises in insulin secondary to meal intake or leucine gavage are probably not responsible for increased phosphorylation of PKCepsilon in response to meal feeding. Elevating the leucine concentration stimulated the phosphorylation of PKCepsilon in gastrocnemius from perfused hindlimb and caused a shift in the distribution of PKCepsilon from the membrane fraction to the cytosolic fraction. The results indicate that leucine leads to an activation (autophosphorylation) and subcellular redistribution of PKCepsilon, but not PKCdelta, in gastrocnemius both in vivo and in vitro. Furthermore, activation of the mTOR signaling pathway above basal conditions does not appear to be necessary to induce phosphorylation or translocation of PKCepsilon, suggesting that multiple signaling pathways become activated with leucine.     

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.