Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Induction of innate immune gene expression following methyl methanesulfonate-induced DNA damage in sea urchins.

Sea urchins are noted for the absence of neoplastic disease and represent a novel model to investigate cellular and systemic cancer protection mechanisms. Following intracoelomic injection of the DNA alkylating agent methyl methanesulfonate, DNA damage was detected in sea urchin cells and tissues (coelomocytes, muscle, oesophagus, ampullae and gonad) by the alkaline unwinding, fast micromethod. Gene expression analyses of the coelomocytes indicated upregulation of innate immune markers, including genes involved in NF-κB signalling. Results suggest that activation of the innate immune system following DNA damage may contribute to the naturally occurring resistance to neoplastic disease observed in sea urchins.

Influence of ensiling time and inoculation on alteration of the starch-protein matrix in high-moisture corn.

The fates of hydrophobic zein proteins, which encapsulate corn starch to create vitreous endosperm, have not been investigated in high-moisture corn (HMC). To assess influences of ensiling time and inoculation on zein proteins in HMC, quadruplicate samples of 2 random corn hybrids (A and B), containing 25.7 and 29.3% moisture, were ground, inoculated with (I) or without 600,000 cfu/g of Lactobacillus buchneri 40788 (Lallemand Animal Nutrition, Milwaukee, WI), and ensiled for 0, 15, 30, 60, 120, and 240 d. Nutrient composition [crude protein (CP), starch, acid detergent fiber, and neutral detergent fiber], fermentation (pH, lactate, and acetate), and protein degradation markers (buffer-soluble CP, isopropanol-soluble CP, and NH(3)-N) were evaluated. At 0 and 240 d, α, γ, δ, and ß zein subunits were profiled using HPLC. Data were evaluated as a split-split plot using the PROC MIXED procedures of SAS. Ensiling time and inoculation decreased pH, and altered lactate and acetate contents of HMC. Lactate and acetate contents of A, AI, B, and BI at 240 d were 0.40, 0.32, 1.11, 0.73, and 0, 0.35, 0.30, and 0.87% of DM, respectively. Buffer-soluble CP in HMC increased from 1.5 to 2.0% of DM at 0 d to >4.0% of DM at 240 d. Inoculation had no effect on buffer-soluble CP but increased NH(3)-N content of HMC. Corn A contained more isopropanol-soluble CP than did corn B and peak areas for 6 α, and all γ and δ zein regions were greater for corn A. Ensiling (0 vs. 240 d) decreased all zein subunits with the exception of 2 α and 1 δ subunit. Ensiling decreased (42.2-73.2%) γ zeins, which are primarily responsible for cross-linking in the starch-protein matrix. Despite altering lactate and acetate contents, inoculation had no effect on degrading hydrophobic zein proteins in HMC. Data suggest that hydrophobic zein proteins in the starch-protein matrix of HMC are degraded by proteolytic activity over an extended ensiling time.

Serum TRAIL levels in patients with epithelial ovarian cancer or primary peritoneal cancer treated with neoadjuvant chemotherapy. A pilot study.

AIMS: The study attempted to evaluate the kinetics of changes in serum TRAIL levels as a potential predictive and prognostic factor in patients with epithelial ovarian cancer (EOC) or primary peritoneal carcinoma (PPC), eligible for an interval debulking surgery (IDS). MATERIAL AND METHODS: 17 patients with primary inoperable EOC or PPC in FIGO Stage IIIC or IV who underwent an exploratory operation were enrolled to the study. Serum TRAIL levels were determined by ELISA method (DIACLONE, Besancon Cedex, France) before and after two courses of neoadjuvant chemotherapy (NAC) based on paclitaxel and platinum analogue (cisplatin or carboplatin). The control group consisted of six healthy volunteers. The median difference in concentration of TRAIL (dTRAIL) between the initial marking and after two courses of NAC in each patient was 192 pg/ml and it was used for dichotomization of the test group. RESULTS: Suboptimal interval debulking surgery (IDS) was performed in 23.5% (4/17) and optimal IDS in 76.5% (13/17) patients. TRAIL concentration before chemotherapy did not differ significantly between patients with EOC or PPC [1426.96 +/- 321.06 pg/ml (mean +/- SD) (U = 26, p = 0.08)] and the control group [1160.40 +/- 256.39 pg/ml (mean +/- SD. After two courses of NAC serum TRAIL concentration level was 1247.49 +/- 378.46 pg/ml (mean +/- SD). The difference was significant (Z = 2.44, p = 0.0147). Statistical analysis showed that dTRAIL did not significantly influence either extent of IDS (U = 35, p = 0.0962) or time to progression (log-rank test, p = 0.1185), overall survival (log-rank test, p = 0.1973) and response to treatment according to RECIST criteria (U = 35.5, p = 0.9616). CONCLUSIONS: Serum TRAIL concentration levels changed significantly during NAC. However, it seems that the concentration of this protein has no critical value as a predictive or prognostic factor in patients with EOC or PPC.

Marine invertebrates as models for aging research.

The oceans are home to many of the earth's longest lived animals with several species of non-colonial marine invertebrates documented to live for more than 100 years. Many of these animals grow and reproduce throughout their lifespans and there is no apparent functional decline or increase in mortality rate with age. Studying these animals may reveal some exceptionally effective defenses against the destructive process of aging thus providing a valuable alternative model for aging research. The life histories of commercially important marine invertebrates are well studied, but little is known of the molecular or cellular changes that occur with increasing age or the factors that determine lifespan. The objectives of this review are to present data on cellular and molecular aspects of aging in marine invertebrates with a focus on bivalves and sea urchins. This review will serve to evaluate their potential as model systems for aging and provide direction for future research efforts so that we can begin to understand the underlying molecular mechanisms responsible for the tremendous longevity and good health of key species.

Antiproliferative effect of 4-thiouridylate on OCM-1 uveal melanoma cells.

PURPOSE: Brachytherapy is a well-established, effective treatment for uveal melanoma with a failure rate of 15%. The fatal consequence of unsuccessful treatments offers reason for improvement of the method. The authors propose using an apoptosis inducing agent locally, concomitantly with the well-established therapy, to sensitize the tumor cells. The authors propose a new nontoxic moderately active apoptosis inducing agent, 4-thio-uridylate (s4UMP), for this purpose. METHODS: OCM-1 uveal melanoma cells were treated with various concentrations of s4UMP and its effect was monitored by measuring the cell viability (MTT assay). The following apoptosis detecting methods were performed to reveal the mechanism of decreased cell viability: light microscopy, DNA fragmentation assay, determination of caspase 9 activity, and FACS analysis. RESULTS: The viability of uveal melanoma cells was decreased by 32%, 40%, and 9% after 24, 48, and 72 hours of treatment with 10 microg/mL (30 microM) s4UMP. The effect was not dose dependent; it rather followed a saturation-type inhibition and the cells at lower drug concentration recovered after 72 hours. Characteristic apoptotic cell morphology and DNA fragmentation was detected in treated cells. The caspase-9 was activated upon treatment showing maximal activity at 48 hours suggesting the induction of apoptosis. The annexin binding activity further verified the apoptogenic activity of s4UMP. CONCLUSIONS: Uveal melanoma, more than other solid tumors, is resistant to most of the chemotherapeutic protocols as indicated by the high mortality rate of metastatic disease. The authors showed that s4UMP, a naturally occurring nucleotide, could induce apoptosis in uveal melanoma cells, suggesting a potential supplementary therapeutic application of the compound.

Antiproliferative effect of 4-thiouridylate on OCM-1 uveal melanoma cells.

PURPOSE: Brachytherapy is a well-established, effective treatment for uveal melanoma with a failure rate of 15%. The fatal consequence of unsuccessful treatments offers reason for improvement of the method. The authors propose using an apoptosis inducing agent locally, concomitantly with the well-established therapy, to sensitize the tumor cells. The authors propose a new nontoxic moderately active apoptosis inducing agent, 4-thio-uridylate (s4UMP), for this purpose. METHODS: OCM-1 uveal melanoma cells were treated with various concentrations of s4UMP and its effect was monitored by measuring the cell viability (MTT assay). The following apoptosis detecting methods were performed to reveal the mechanism of decreased cell viability: light microscopy, DNA fragmentation assay, determination of caspase 9 activity, and FACS analysis. RESULTS: The viability of uveal melanoma cells was decreased by 32%, 40%, and 9% after 24, 48, and 72 hours of treatment with 10 g/mL (30 M) s4UMP. The effect was not dose dependent; it rather followed a saturation-type inhibition and the cells at lower drug concentration recovered after 72 hours. Characteristic apoptotic cell morphology and DNA fragmentation was detected in treated cells. The caspase-9 was activated upon treatment showing maximal activity at 48 hours suggesting the induction of apoptosis. The annexin binding activity further verified the apoptogenic activity of s4UMP. CONCLUSIONS: Uveal melanoma, more than other solid tumors, is resistant to most of the chemotherapeutic protocols as indicated by the high mortality rate of metastatic disease. The authors showed that s4UMP, a naturally occurring nucleotide, could induce apoptosis in uveal melanoma cells, suggesting a potential supplementary therapeutic application of the compound.

Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells.

Membrane proteins of cytotoxic T cells specifically reorganize to form an immunological synapse (IS) on interaction with their specific target. In this paper, we investigated the redistribution of Kv1.3 channels, which are the dominant voltage-gated potassium channels, in the plasma membrane of allogen-activated human cytotoxic T lymphocytes (CTLs) on interacting with their specific target cells. Kv1.3 channels bearing a FLAG epitope were expressed in the CTLs and the cell-surface distribution of fluorescently labeled ion channels was determined from confocal laser-scanning microscopy images. FLAG epitope-tagged Kv1.3 channels showed a patchy distribution in CTLs not engaged with target cells, whereas the channels were accumulated in the IS formed between CTLs and specific target lymphocytes. Localization of Kv1.3 channels in the IS might open an unrevealed possibility in the regulation of ion channel activity by signaling molecules accumulated in the IS.

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes.

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts.

The glycosylphosphatidylinositol-anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co-receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcgamma-receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co-clustering with Toll-like receptor 4, Fcgamma-RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand-specific cellular responses initiated by CD14.