Genome sequence of strain HIMB55, a novel marine gammaproteobacterium of the OM60/NOR5 clade

Strain HIMB55 is a phylogenetically unique member of the OM60/NOR5 clade of the Gammaproteobacteria isolated from coastal seawater of Kaneohe Bay on the northeastern shore of Oahu, Hawaii, by extinction culturing in seawater-based oligotrophic medium. Here we present the genome sequence of strain HIMB55, including genes for bacteriochlorophyll-based phototrophy.     

Hydroxylation of the mycotoxin zearalenone at aliphatic positions: novel mammalian metabolites

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species and frequently found as a contaminant of food and feed. Earlier studies have disclosed that ZEN is biotransformed in microsomes from human and rat liver to multiple hydroxylated metabolites, two of which have recently been identified as products of aromatic hydroxylation. Here, we report for the first time on the structure elucidation of metabolites arising through hydroxylation of the aliphatic ring of ZEN at various positions. By using reference compounds and ZEN labeled with deuterium at specific positions, evidence was provided for the preferential hydroxylation of ZEN at C-8 and, to a lesser extent, at C-9, C-10, and C-5. In contrast, hydroxylation at C-6 could be ruled out, as could oxidation of the olefinic double bond. These results imply that the phase I metabolism of ZEN in the mammalian organism is more extensive than previously thought, and warrant further studies on the in vivo formation of the novel ZEN metabolites and their biological activities.     

Tropomodulin binds two tropomyosins: a novel model for actin filament capping

Tropomodulin, a tropomyosin-binding protein, caps the slow-growing (pointed) end of the actin filament regulating its dynamics. Tropomodulin, therefore, is important for determining cell morphology, cell movement, and muscle contraction. For the first time we show that one tropomodulin molecule simultaneously binds two tropomyosin molecules in a cooperative manner. On the basis of the tropomodulin solution structure and predicted secondary structure, we introduced a series of point mutations in regions important for tropomyosin binding and actin capping. Capping activity of these mutants was assayed by measuring actin polymerization using pyrene fluorescence. Using direct methods (circular dichroism and native gel electrophoresis) for detecting tropomodulin/tropomyosin binding, we localized the second tropomyosin-binding site to residues 109-144. Despite previous reports that the second binding site is for erythrocyte tropomyosin only, we found that both short nonmuscle and long muscle alpha-tropomyosins bind there as well, though with different affinities. We propose a model for actin capping where one tropomodulin molecule can bind to two tropomyosin molecules at the pointed end.     

Design, synthesis and preliminary biological evaluations of novel amphiphilic drug carriers

The synthesis of a new fluorocarbon amphiphilic drug carrier is described. A polyfunctional amino acid endowed with a fluorocarbon chain and a sugar moiety providing the amphiphilic character constitutes the central element of this structure. A (14)C-radiolabelled acetyl group was grafted onto the third function and the bioavailability of this molecule was specified in mice after IV administration. This amphiphilic drug carrier exhibits a rapid and homogeneous distribution to the whole tissues and slow elimination half-lives (higher than one day) through a biliary excretion without any toxicity (no measured DL 50 for concentrations up to 500 mg/kg).     

Piperazinyl-glutamate-pyrimidines as potent P2Y12 antagonists for inhibition of platelet aggregation

Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y₁₂ antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physicochemical properties were fine-tuned through modifications at the 4-position of the piperidine ring leading to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.     

Piperazinyl-glutamate-pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl-glutamate-pyridine as a P2Y₁₂ antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 22J with good human PRP potency, selectivity, in vivo efficacy and oral bioavailability.     

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

Background

RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation.

Methodology/Principal Findings

Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment.

Conclusions/Significance

A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.     

Polycomb Group Protein Bmi1 Is Required for Growth of RAF Driven Non-Small-Cell Lung Cancer

Background

We have previously described a RAF oncogene driven transgenic mouse model for non small cell lung cancer (NSCLC). Here we examine whether tumor initiation and growth requires the stem cell self-renewal factor Bmi1.

Principal Findings

In order to evaluate Bmi1 function in NSCLC two founder lines that differ in incidence and latency of tumor formation were compared. Ablation of Bmi1 expression in both lines had a dramatically decreased tumor growth. As the line with shorter latency matched the life span of Bmi1 knock out mice, these mice were chosen for further study. The absence of Bmi1 did not decrease the number of tumor initiation in these mice as only the size and not the number of tumors decreased. Reduction in tumor growth resulted from an increase in cell death and decrease in cell cycle progression that corresponded with up-regulation of the p16^INK4a and p19^ARF.

Significance

The data identifies Bmi1 as an important factor for expansion but not initiation of RAF driven NSCLC.     

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.     

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log₁₀ and 6 log₁₀ CFU g⁻¹.Bovines are a reservoir for verotoxigenic Escherichia coli O157:H7 and Campylobacter jejuni, pathogenic microorganisms responsible for severe human gastrointestinal disease (5, 12). Qualitative and quantitative detection of these organisms in bovine feces is essential for evaluating risk to human health. Real-time PCR (quantitative PCR [qPCR]) assays have been developed to detect and quantify both E. coli O157:H7 and C. jejuni bacteria by using DNA directly extracted from animal feces (20, 22). Analysis of DNA extracted from bovine feces can generate a high level of correlation between the actual target cell density and the PCR signal (7, 8). However, the detection of E. coli O157:H7 and C. jejuni by direct DNA extraction is less sensitive and more variable than detection by procedures based on a preliminary enrichment step (e.g., laboratory culture) (7, 9, 16, 20). We explored the potential of lyophilization for improving overall detection by qPCR through increasing the amount of bovine fecal material available for DNA extraction.Four sets of five fresh bovine fecal samples were collected, and each sample was divided into four equal portions. Samples were seeded with either (i) E. coli O157:H7 (strain NZRM 3614) grown for 18 h at 37°C in tryptic soy broth (BD, Sparks, MD) or (ii) C. jejuni (strain NZRM 1958) grown for 48 h at 42°C in Exeter broth (11) to obtain the following concentrations: set 1, 0 CFU g⁻¹ (unseeded) and 3.5 log₁₀, 4.5 log₁₀, and 5.5 log₁₀ CFU of E. coli O157:H7 g⁻¹, and set 2, 0 CFU g⁻¹ (unseeded) to 5.2 log₁₀ CFU of E. coli O157:H7 g⁻¹. Set 3 and 4 concentrations varied from 0 CFU g⁻¹ (unseeded) to 6.4 log₁₀ C. jejuni CFU g⁻¹. DNA was either extracted directly from fresh samples or extracted from samples after lyophilization. Lyophilization involved mixing of prepared fecal samples in phosphate-buffered saline (145 mM NaCl, 59 mM Na₂HPO₄, 8 mM KH₂PO₄, pH 7.5) at a ratio of 1:10 (wt/vol), homogenization with a lab blender model 400 (Seward Medical, London, United Kingdom), cooling to −35°C, and concentration using a 1015GP lyophilizer (Cuddon Ltd., Blenheim, New Zealand). Total DNA was extracted from 0.2 g of a fresh or lyophilized fecal sample by using a QIAamp DNA stool minikit (Qiagen Inc., Mississauga, Canada). DNA was amplified using either a TaqMan E. coli O157:H7 detection kit (Applied Biosystems, Foster City, CA) or mapA primers and a corresponding probe (1). Amplification and fluorescence data were collected with optical-grade 96-well plates by using a TaqMan 7300 PCR system (Applied Biosystems). For each DNA sample, a mean threshold cycle (C_T) value for triplicate qPCR runs was calculated. When no C_T value was obtained, an arbitrary C_T value of 40 was assigned. All data were reported as equivalent concentrations in fresh feces. Significance levels were determined by one-way analysis of variance. The relationship between the log₁₀ numbers of CFU g⁻¹ fresh feces (viable-cell counts) and C_T values was analyzed using GenStat software (version 10.2.0.175; VSN International, Oxford, United Kingdom). Confidence intervals were obtained using the software program Flexi (21).Lyophilized samples were associated with significantly improved sensitivity (P < 0.001) at seeding levels of 4.5 and 5.5 log₁₀ E. coli O157:H7 CFU g⁻¹ (Table ​(Table1).1). At 3.5 log₁₀ CFU g⁻¹, the rate of E. coli O157:H7 detection was also higher, with all lyophilized samples producing a C_T value of <40 (Table ​(Table1).1). Individual C_T values for the three qPCR amplification runs were sufficiently similar to allow averaging (P > 0.05). Regression analysis of the averaged set 2 and 3 data (Fig. ​(Fig.1)1) demonstrated that the detection of both E. coli O157:H7 and C. jejuni was linear for seeding levels ranging from ca. 2 log₁₀ to 6 log₁₀ CFU g⁻¹ fresh feces. The range of concentrations used reflects the reported range of concentrations of these bacteria in feces (i.e., 0 to 6 log₁₀ CFU g⁻¹) as determined by conventional culture (3, 4, 18, 19). The high coefficients of correlation for the relationships between the log₁₀ numbers of CFU g⁻¹ feces and the C_T values indicated the specific amplification of the target DNA. The reproducibility of detection of E. coli O157:H7 was reduced at the lowest seeding concentration (i.e., 2.2 log₁₀ CFU g⁻¹ feces), with 75% of the samples giving a C_T value of <40. The limit for 100% successful detection after lyophilization was 2.9 log₁₀ E. coli O157:H7 CFU g⁻¹. The detection of C. jejuni by qPCR varied between sets. For set 3, 100% reproducibility occurred at 2.2 log₁₀ C. jejuni CFU g⁻¹. For set 4, satisfactory detection was obtained only after dilution of the DNA extract prior to qPCR. Despite this requirement for dilution, C. jejuni was still detected in 80% of the samples of set 4 seeded at a density of 2.2 log₁₀ C. jejuni CFU g⁻¹.Open in a separate windowFIG. 1.Ranges of quantification of E. coli O157:H7 (A) and C. jejuni (B) bacteria obtained from lyophilized fecal samples by real-time PCR. Each point represents the average C_T value for triplicate runs of one fecal sample at one seeding concentration. The hatched areas represent the 95% confidence intervals.

TABLE 1.

Difference in C_T values obtained for real-time PCR detection of E. coli O157:H7 in seeded fecal samples (n = 5) with and without lyophilization