p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.     

Comparative analysis of virulence genes, genetic diversity, and phylogeny of commensal and enterotoxigenic Escherichia coli isolates from weaned pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

Diversity of Archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. nov

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.     

Studies on the oridonin-loaded poly(D,L-lactic acid) nanoparticles in vitro and in vivo

The purpose of this paper was to investigate the possibility of developing a polymeric nanoparticle delivery system for ORI to increase its solubility, blood circulation time and tissue targeting. Oridonin-loaded poly(D,L-lactic acid) nanoparticles (ORI-PLA-NP) were prepared by the further modified spontaneous emulsion solvent diffusion (MSESD) method. Studies were carried out to characterize and evaluate the produced ORI-PLA-NP both in vitro and in vivo. The experimental results showed that the mean size of the nanoparticles were 137.3 nm, with 87.2% of the nanoparticles distributed between the range of 107 and 195 nm. The entrapment efficiency and actual drug loading of the nanoparticles were 91.88+/-1.83 and 2.32+/-0.05%, respectively. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that ORI existed in the form of amorphous in the nanoparticles. The in vitro release profile of ORI-PLA-NP could be expressed well by the Higuchi equation: Q=8.944t(1/2)+11.246. The results of pharmacokinetics demonstrated that being encapsulated in PLA nanoparticles was remarkably effective for ORI to prolong its blood circulation time. After the i.v. administration of ORI-PLA-NP, we could observe a stable and high concentration of ORI in liver, lung and spleen, while its distribution in heart and kidney decreased.     

Photosynthesis of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> subjected to drought stress is enhanced by choline chloride through alleviating lipid peroxidation and increasing proline accumulation

Rehmannia glutinosa seedlings were pretreated with choline chloride (CC) in concentrations of 0, 0.7, 2.1 and 3.5 mM, and then subjected to drought and rewatering treatment to study the effects of CC on the generation of reactive oxygen species (O₂⁻, H₂O₂), lipid peroxidation, proline accumulation, water status and photosynthesis. The results showed that pretreatment with CC alleviated the inhibition of SOD and APX activity caused by drought stress, and therefore, the rate of O₂⁻ production and H₂O₂ concentration were reduced and lipid peroxidation decreased in pretreated plants. CC pretreatment also accelerated accumulation of proline, maintained higher Ψw and RWC, deferred leaf water loss during drought stress and retarded the drop in proline concentration after rewatering. Consequently, drought-induced decreases in F_m/F₀, F_v/F_m, Φ_PS2, q_P, and A and increase in q_NP were inhibited and the recovery of photosynthesis after rewatering was quicker in pretreated plants. Although differences in F_v/F_m, Φ_PS2 and q_P between treatments were not significant, there was a general trend that the effects of CC increased with the rise of its concentrations. The data suggested that 2.1 mM of CC be suitable for alleviating lipid peroxidation, promoting proline accumulation, retarding leaf water loss and improving photosynthesis of R. glutinosa seedlings under drought stress.     

Phenotypes and fed-batch fermentation of ubiquinone-overproducing fission yeast using ppt1 gene

Ubiquinone (UQ), a component of the electron transfer system in many organisms, has been widely used for pharmaceuticals and cosmetics. In this study, we cloned and overexpressed the full-length ppt1 (MTppt1) gene, which encodes p-hydroxybenzoate:polyprenyltransferase and ERppt1 gene, which was modified to be localized on endoplasmic reticulum in fission yeast. The yeast MTppt1 and ERppt1 transgenic lines showed about 3.7 and 5.1 times increment in UQ content and the recombinant yeasts with a higher UQ level are more resistant to H(2)O(2), Cu(2+) and NaCl, and interestingly their growth was also faster than the wild type at lower temperature. For large-scale cultivation, the direct feedback control of glucose using an on-line ethanol concentration monitor for ubiquinone production of yeast ERppt1 by high-cell-density fermentation was investigated and the fermentation parameters (e.g., dissolved oxygen, pH, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiration quotient) were also discussed. After 90 h cultures, the yeast dry cell weight reached 57 gl(-1) and the ubiquinone yield reached 23 mgl(-1). In addition, plasmid stability was maintained at high level throughout the fermentation.     

Cultivation-dependent and cultivation-independent characterization of the microbial community in acid mine drainage associated with acidic Pb/Zn mine tailings at Lechang, Guangdong, China

Cultivation-based and molecular approaches were used to characterize the phylogenetic composition and structure of the microbial community in an extremely acidic (pH 2.0) acid mine drainage (AMD) associated with Pb/Zn mine tailings that were undergoing vigorous acid generation. Acidophilic bacteria were isolated and enumerated on solid media, and were found to be restricted to isolates related to Acidithiobacillus ferrooxidans and Acidiphilium cryptum. By contrast, cloning and phylogenetic analysis of 16S rRNA genes revealed that, although low in total taxonomically distinct groups, the tailings AMD ecosystem harbored a wide range of phylogenetically diverse microbes. Of the 141 clones examined, 104 were phylogenetically affiliated with the recently discovered, iron-oxidizing Leptospirillum group III within the Nitrospira. It thus appears that iron serves as the major electron donor in this habitat. Thirty clones were affiliated with the Proteobacteria, half of which belonged to organisms related to Alphaproteobacteria species capable of ferric iron reduction. Other clones were grouped with Betaproteobacteria and Gammaproteobacteria (six clones each), and even with Deltaproteobacteria (three clones), a subdivision with anaerobic sulfate or metal (iron) reduction as the predominant physiological trait of its members. Finally, four clones were clustered within the Firmicutes and the Acidobacteria. Approximately half of the sequence types representing the majority of the total clones fell into lineages that are poorly represented by cultured organisms or have thus far been represented by only a few environmental sequences. Thus, the present study extends our knowledge of the biodiversity of microorganisms populating highly acidic AMD environments.     

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.     

Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants

* Using pharmacological and biochemical approaches, the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Treatment with ABA or H(2)O(2) led to significant increases in the concentration of cytosolic Ca(2+) in the protoplasts of mesophyll cells and in the expression of the calmodulin 1 (CaM1) gene and the content of CaM in leaves of maize plants, and enhanced the expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes. The up-regulation of the antioxidant enzymes was almost completely blocked by pretreatments with two CaM antagonists. * Pretreatments with CaM antagonists almost completely inhibited ABA-induced H(2)O(2) production throughout ABA treatment, but pretreatment with an inhibitor or scavenger of reactive oxygen species (ROS) did not affect the initial increase in the contents of CaM induced by ABA. * Our results suggest that Ca(2+)-CaM is involved in ABA-induced antioxidant defense, and that cross-talk between Ca(2+)-CaM and H(2)O(2) plays a pivotal role in ABA signaling.