Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano‐LC–MS/MS following 1D SDS‐PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung‐enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.     

Toward unraveling the structure of Brassica rapa genome

Genomic research in any organism encompasses understanding structure of the target genome and genes, their function, and evolution. Brassica rapa , which is phylogenetically related to Arabidopsis thaliana , is an important species with respect to its uses as vegetable, oil, and fodder. The availability of suitable genetic and genomic resources is a prerequisite to undertake genomic research in B. rapa . We have developed reference mapping populations of Chinese cabbage ( B. rapa ssp. pekinensis ) comprising 78 doubled haploid lines and over 250 recombinant inbred lines. Two Bacterial Artificial Chromosome (BAC) libraries, generated by restriction enzymes Hin dIII (KBrH) and Bam HI (KBrB), comprise 56 592 and 50 688 clones, respectively. We have also constructed 22 cDNA libraries from different plant tissues consisting of 104 914 clones with an average length of 575 bp. Initial BAC-end sequence analysis of 1473 clones of the KBrH library led us to understand the structure of B. rapa genome with respect to extent of genic sequences and their annotation, and relative abundance of different types of repetitive DNAs. Full-length sequence analysis of BAC clones revealed extensive triplication of B. rapa DNA segments coupled with variable gene losses within the segments. The formulation of the 'Multinational Brassica Genome Project' has laid the foundation to sequence the complete genome of B. rapa ssp. pekinensis by the international Brassica research community. It has been proposed to undertake BAC-to-BAC sequencing of genetically mapped seed BACs. In recent years, development of bioinformatics tools in Brassica has given a boost to structural genomics research in Brassica species. The research undertaken with the availability of various genomic resources in the public domain has added to our understanding of the structure of B. rapa .     

Inhibitory role of RhoA on senescence-like growth arrest by a mechanism involving modulation of phosphatase activity

Recently, negative effects of phosphatase in tumorigenesis and metastasis have been suggested in various tumor types. In this study, we showed that RhoA activation modulated phosphatase during senescence-like arrest in human prostate cancer cells. Under senescence-inducing condition, decreased Erk phosphorylation was detected in caRhoA-transfected cells and inactivation of Erk, but not p38, prevented doxorubicin-induced cell senescence. Cells were induced to senescence by inhibition of phosphatase activity (VHR, MKP3, or PP2A) without additional cellular stress. Of interest, caRhoA prevented doxorubicin-induced decrease of phosphatase. Thus, we postulate that RhoA signaling may protect cells against cellular senescence by maintaining phosphatase activity and Erk dephosphorylation.     

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.     

Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Effects of NaCl stress on germination, antioxidant responses, and proline content in two rice cultivars

We investigated the physiological and biochemical bases for salt tolerance in two rice (Oryza sativa L.) cultivars — relatively salt-tolerant ‘Dongjin’ and salt-sensitive ‘Kumnam’. Salinized hydroponic cultures were studied at the germination and seedling stages. NaCI inhibited germination more severely in ‘Kumnam’ than in ‘Dongjin’. Increasing the salt concentration also deterred growth to a larger extent in the former. Moreover, the leaves of ‘Kumnam’ exhibited greater increases in lipid peroxidation and Na⁺ accumulation than those of ‘Dongjin’ under stress. The activities of constitutive and salt-induced superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (AP, EC 1.11.1.11) were also higher in ‘Kumnam’, while only catalase (CAT, EC 1.11.1.6) activity was slightly higher in stressed plants of ‘Dongjin’. The positive correlation between leaf proline levels and NaCI concentration was more evident in ‘Kumnam’. However, ‘Dongjin’ seeds, which had higher germinability in the presence of NaCI, also contained more proline. These results suggest that the higher salt tolerance in ‘Dongjin’ seedlings could be ascribed to their lower NaCI accumulations in the leaves. This presumably is due to reductions in the uptake or transport rates of saline ions to the shoots from the roots. Finally, we believe that the higher germination rate by ‘Dongjin’ is caused by its higher seed proline content.     

Separation of protein and fatty acids from tuna viscera using supercritical carbon dioxide

Supercritical carbon dioxide extraction was investigated as a method for removing lipids and bad flavor from tuna viscera. To find the optimum conditions, different experimental variables, such as pressure, temperature, flow rate of solvent and sample size, were evaluated for the effective removal of lipids and the undesirable smell. Ethanol was used as the entrainer, with a 3% by vol CO₂ flow rate. By increasing the pressure at constant temperature, the efficiency of the lipid removal was improved and the protein was concentrated without denaturalization. The main fatty acids extracted from the tuna viscera were palmitic acid (16∶0), heptadecanoic acid (17∶1), oleic acid (18∶1) and docosahexaenoic acid (22∶6). The major amino acids in the tuna viscera treated by supercritical carbon dioxide were glutamic acid, leucine and lysine, and the free amino acids werel-proline, taurine andl-α-aminoadipic acid.     

<Emphasis Type="Italic">Hymenobacter agri</Emphasis> sp. nov., a novel bacterium isolated from soil

A bacterial isolate was recovered from a soil sample collected in Jeollabuk-do Province, South Korea, and subjected to polyphasic taxonomic assessment. Cells of the isolate, designated strain S1-2-1-2-1^T, were observed to be rod-shaped, pink in color, and Gram-stain negative. The strain was able to grow at temperature range from 10 to 30 °C, with an optimum of 25 °C, and growth occurred at pH 6–8. Comparative 16S rRNA gene sequence analysis showed that strain S1-2-1-2-1^T belongs to the genus Hymenobacter, with closely related type strains being Hymenobacter daeguensis 16F3Y-2^T (95.8% similarity), Hymenobacter rubidus DG7B^T (95.8%), Hymenobacter soli PB^T (95.7%), Hymenobacter terrenus MIMtkLc17^T (95.6%), Hymenobacter terrae DG7A^T (95.3%), and Hymenobacter saemangeumensis GSR0100^T (95.2%). The genomic DNA G+C content of strain S1-2-1-2-1^T was 63.0 mol%. The main polar lipid of this strain was phosphatidylethanolamine, the predominant respiratory quinone was menaquinone-7, and the major fatty acids were C_15:0 iso (27.3%), summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) (16.5%), C_15:0 anteiso (15.3%), and C_16:0 (14.7%), supporting the affiliation of this strain with the genus Hymenobacter. The results of this polyphasic analysis allowed for the genotypic and phenotypic differentiation of strain S1-2-1-2-1^T from recognized Hymenobacter species. On the basis of its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain S1-2-1-2-1^T is considered to represent a novel species of the genus Hymenobacter, for which the name Hymenobacter agri sp. nov. is proposed. The type strain is S1-2-1-2-1^T (=KCTC 52739^T?=?JCM 32194^T).