Mannoproteins from Saccharomyces cerevisiae stimulate angiogenesis by promoting the akt-eNOS signaling pathway in endothelial cells.

Mannoproteins (MPs) are a major component of yeast cell walls and consist of high levels of mannose in covalent complexes with proteins. MPs complexly enhance the immune system. We previously isolated a mutant yeast, K48L3, with a higher yield of MP from its cell wall than wild-type Saccharomyces cerevisiae, YPH499. We determined that K48L3 induces the release of nitric oxide in macrophage cells. The present study reports nitric-oxide-mediated angiogenesis by MP from K48L3 and the induction of the Akt/eNOS signal pathway. Western blotting and RT-PCR were used to demonstrate that MP treatment resulted in the upregulation of p-Akt, p-eNOS, and angiogenesis-mediated gene expression. Moreover, the angiogenesis activity of the MPs was demonstrated using three angiogenesis assays, namely, a cell migration assay, a tube-forming assay, and an ex vivo aorta ring assay. Thus, this study demonstrates for the first time that MPs from S. cerevisiae K48L3 induce angiogenesis in HUVECs via the Akt-eNOS-dependent signaling pathway.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

Shikonin Induces Apoptotic Cell Death via Regulation of p53 and Nrf2 in AGS Human Stomach Carcinoma Cells.

Shikonin, which derives from Lithospermum erythrorhizon, has been traditionally used against a variety of diseases, including cancer, in Eastern Asia. Here we determined that shikonin inhibits proliferation of gastric cancer cells by inducing apoptosis. Shikonin's biological activity was validated by observing cell viability, caspase 3 activity, reactive oxygen species (ROS) generation, and apoptotic marker expressions in AGS stomach cancer cells. The concentration range of shikonin was 35-250 nM with the incubation time of 6 h. Protein levels of Nrf2 and p53 were evaluated by western blotting and confirmed by real-time PCR. Our results revealed that shikonin induced the generation of ROS as well as caspase 3-dependent apoptosis. c-Jun-N-terminal kinases (JNK) activity was significantly elevated in shikonin-treated cells, thereby linking JNK to apoptosis. Furthermore, our results revealed that shikonin induced p53 expression but repressed Nrf2 expression. Moreover, our results suggested that there may be a co-regulation between p53 and Nrf2, in which transfection with siNrf2 induced the p53 expression. We demonstrated for the first time that shikonin activated cell apoptosis in AGS cells via caspase 3- and JNK-dependent pathways, as well as through the p53-Nrf2 mediated signal pathway. Our study validates in partly the contribution of shikonin as a new therapeutic approaches/ agent for cancer chemotherapy.

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1.

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system.

Genomic annotation for the temperate phage EFC-1, isolated from Enterococcus faecalis KBL101.

The temperate phage EFC-1 was newly isolated from a mitomycin-C-induced lysate of Enterococcus faecalis KBL101. EFC-1 has an isometric head and a long tail. The phage belongs to the family Siphoviridae according to its genomic structure and morphology. The phage EFC-1 has 40,286 base pairs of double-stranded DNA and a G+C content of 35.05 %. Bioinformatic analysis of the phage revealed 60 putative open reading frames (ORFs). The genome of the temperate phage EFC-1 was not significantly similar to that of previously reported bacteriophages from E. faecalis.

Comparative genomic analysis of Lactococcus garvieae phage WP-2, a new member of Picovirinae subfamily of Podoviridae.

To date, a few numbers of bacteriophages that infect Lactococcus garvieae have been identified, but their complete genome sequences have not yet been investigated. For the first time, herein, the complete DNA sequence of a new phage of L. garvieae (phage WP-2) is reported and analyzed. The morphological characteristics indicated that the phage had a small isometric head along with a short and non-contractile tail, suggesting that WP-2 belongs to the family Podoviridae. Bioinformatic analysis revealed that phage WP-2 can be classified as a new member of Ahjdlikevirus in the Picovirinae subfamily because it had a small dsDNA of 18,899 bp with 24 open reading frames and a protein-primed DNA polymerase. The phage nucleotide sequence and predicted protein products have been identified to share very limited evidence of homology with complete genome and proteome of other phages. To our knowledge, this is the first Ahjdlikevirus bacteriophage which can infect a member of the Lactococcus genus.

Insights into new bacteriophages of Lactococcus garvieae belonging to the family Podoviridae.

Lactococcus garvieae is an emerging pathogen responsible for lactococcosis, a serious disease in trout aquaculture. The identification of new bacteriophages against L. garvieae strains may be an effective way to fight this disease and to study the pathogen's biology. Three L. garvieae phages, termed WP-1, WWP-2 and SP-2, were isolated from different environments, and their morphological features, genome restriction profiles and structural protein patterns were studied. Random cloning of HindIII-cut fragments was performed, and the fragments were partially sequenced for each phage. Although slight differences were observed by transmission electron microscopy, all of the phages had hexagonal heads and short non-contractile tails and were classified as members of the family Podoviridae. Restriction digestion analysis of the nucleic acids of the different phages revealed that the HindIII and AseI digests produced similar DNA fragment patterns. Additionally, SDS-PAGE analysis indicated that the isolated phages have similar structural proteins. The sequence BLAST results did not show any significant similarity with other previously identified phages. To the best of our knowledge, this study provides the first molecular characterization of L. garvieae phages.

Complete genome sequence of a novel phage, vB_MoxS-ISF9, infecting methylotrophic Microbacterium: first report of a virulent Microbacterium phage.

Here, we report the first genome sequence of a new virulent phage of Microbacterium oxydans, termed vB_MoxS-ISF9, which was isolated from sewage. Transmission electron microscopy showed that the isolated phage, which has a hexagonal head of about 80 nm in diameter and a long non-contractile tail of about 240 nm, belongs to the family Siphoviridae. The vB_MoxS-ISF9 DNA was completely sequenced and found to be 59,254 bp in length, with a G+C content of 62.76% and 120 putative open reading frames (ORFs). The predicted protein products of the ORFs were identified, and their sequences were analyzed. In a comparison with all available phage genomes, vB_MoxS-ISF9 did not show any significant similarity to other previously reported bacteriophages. To the beast of our knowledge, this is the first report of the isolation and complete genomic sequencing of a virulent phage against a member of the genus Microbacterium.

The immune-enhancing effect of the Cronobacter sakazakii ES2 phage results in the activation of nuclear factor-κB and dendritic cell maturation via the activation of IL-12p40 in the mouse bone marrow.

The bacteriophage ES2 is a virus for bacterial host cells. Unlike other phages that are known for their therapeutic effects, the ES2 phage has never been clearly examined as a therapeutic agent. To systematically and conclusively evaluate its therapeutic efficacy, the expression of the surface markers CD86, CD40, and MHCII, the production of the proinflammatory cytokines IL-6, IL-1α, IL-1ß, and TNF-α, and the underlying NF-κB signaling pathway in murine bone marrow-derived dendritic cells (BM-DCs) in response to ES2 phage infection were examined. The bacteriophage ES2, which was isolated from swine fecal samples an antigen, affected the expression of the cell surface molecules and proinflammatory cytokines that are associated with the DC maturation processes. Treatment with ES2 phage also led to NF-κBp65 activation and translocation to the nucleus, which indicates the activation of NF-κB signaling. Furthermore, the ES2 phage induced the promoter activity of IL-12p40. Our chromatin immunoprecipitation assay revealed that p65 was enriched at the IL12-p40 promoter as a direct target of chromatin. The present study demonstrates that the ES2 phage potently induces DC maturation via immune-enhancement processes.

Development of a random genomic DNA microarray for the detection and identification of Listeria monocytogenes in milk.

We developed a DNA microarray that contains random genomic DNA fragments of Listeria monocytogenes, validated its diagnostic abilities using cells grown in laboratory media and milk, and established enrichment conditions for detection of a low population of L. monocytogenes in milk. Genomic DNA of L. monocytogenes strain ATCC 19111 was fractionated by agarose gel electrophoresis after being cleaved using several different pairs of restriction enzymes. Sixty DNA fragments of different sizes were randomly selected and spotted onto an amine-coated glass slide. To validate diagnostic ability, probes on the DNA microarray were hybridized with genomic DNA extracted from L. monocytogenes, other Listeria spp., and foodborne pathogenic bacteria belonging to other genera grown in laboratory media. The DNA microarray showed 98-100% positive hybridization signals for the 16 strains of L. monocytogenes tested, 7-85% positive signals for 9 strains of other Listeria spp., and 0-32% positive signals for 13 strains of other types of foodborne pathogens. In milk, the detection limit of the DNA microarray was approximately 8 log CFU/mL. When milk contained L. monocytogenes (3-4 log CFU/mL) with other types of bacteria (Bacillus spp., B. cereus, Salmonella Montevideo, Peudomonas aeruginosa, and Yersinia enterocolitica; ca. 3 log CFU/mL each), L. monocytogenes enriched in UVM modified Listeria enrichment broth at 37°C for 24h was successfully detected by the DNA microarray. Results indicate that the DNA microarray can detect L. monocytogenes and distinguish it from other Listeria spp. and other foodborne pathogens in laboratory media and milk. This platform will be useful when developing a DNA microarray to rapidly and simultaneously detect and identify various foodborne pathogens in foods.

Genomic analysis of bacteriophage ESP2949-1, which is virulent for Cronobacter sakazakii.

Virulent phage ESP2949-1, which was isolated from sewage, has an icosahedral head, a contractile tail, and a double-stranded DNA genome with a length of 49,116 bp with 50.09% G+C content. Phage ESP2949-1 showed 3% similarity to enterobacteria phage TLS. Bioinformatics analysis of the phage genome revealed 43 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Based on its morphology, phage ESP2949-1 can be classified as a member of the family Myoviridae. To our knowledge, this is the first report of the genomic sequence and characterization of phage ESP2949-1 isolated from sewage.

Genomic sequence analysis of virulent Cronobacter sakazakii bacteriophage ES2.

Virulent Cronobacter sakazakii bacteriophage ES2 was isolated from swine fecal samples, and the genome sequence by was determined GS-Flx. Bacteriophage ES2 had a double-stranded DNA genome with a length of 22,162 bp and a G+C content of 50.08%. The morphological characteristics under a transmission electron microscope indicated that bacteriophage ES2 belongs to the family Myoviridae. The structural proteins, including the phage coat protein, were separated by SDS-PAGE and identified by Q-TOF. Bioinformatics analysis of the bacteriophage genome revealed 30 putative open reading frames (ORFs). The predicted protein products of the ORFs were determined and described. To our knowledge, the genome of the newly isolated bacteriophage ES2 was not significantly similar to that of any previously reported bacteriophages of members of the family Enterobacteriaceae.

Sequence analysis of the Lactobacillus temperate phage Sha1.

Bacteriophage Sha1, a newly isolated temperate phage from a mitomycin-C-induced lysate of Lactobacillus plantarum isolated from Kimchi, has an isometric head (58 nm × 60 nm) and a long tail (259 nm × 11 nm). The double-strand DNA genome of the phage Sha1 was 41,726 base pairs (bp) long, with a G+C content of 40.61%. Bioinformatic analysis of Sha1 shows that this phage contains 58 putative open reading frames (ORFs). Sha1 can be classified as a member of the large family Siphoviridae by genomic structure and morphology. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage Sha1 from wild-type L. plantarum isolated from kimchi in Korea.

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

Genomic sequence of temperate phage TEM126 isolated from wild type S. aureus.

Bacteriophage TEM126, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild-type Staphylococcus aureus isolated from food, has an isometric head, a noncontractile tail, and a double-stranded DNA genome with a length of 33,540 bp and a G+C content of 33.94%. Bioinformatics analysis of the phage genome revealed 44 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Temperate phage TEM126 can be classified as a member of the family Siphoviridae by morphology and genome structure. Temperate phage TEM126 showed 84% similarity with Staphylococcus phage phiNM1. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage TEM126 from a wild-type S. aureus isolated from foods in Korea.

Complete genomic sequence of virulent Cronobacter sakazakii phage ESSI-2 isolated from swine feces.

A newly identified virulent Cronobacter sakazakii phage, ESSI-2, was isolated from fecal samples from swine. The morphological characteristics evident under a transmission electron microscope indicated that phage ESSI-2 belonged to the family Myoviridae. The genome of phage ESSI-2 comprised a double-stranded DNA of 28,765 bp with a G+C content of 55.17%. Bioinformatic analysis of the phage genome identified 36 putative open reading frames (ORFs). The genome of phage ESSI-2 was not significantly similar to that of a previously reported bacteriophage of the members of Enterobacteriaceae. A lysogeny module was found within the genome of this virulent phage.

A subtractively optimized DNA microarray using non-sequenced genomic probes for the detection of food-borne pathogens.

In this study, we present the successful detection of food-borne pathogens using randomly selected non-sequenced genomic DNA probes-based DNA microarray chips. Three food-borne pathogens, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), and Bacillus cereus, were subjected for the preparation of the DNA microarray probes. Initially, about 50 DNA probes selected randomly from non-sequenced genomic DNA of each pathogen were prepared by using a set of restriction enzyme pairs. The proto-type of DNA microarray chip for detecting three different pathogens simultaneously was fabricated by using those DNA probes prepared for each pathogen. This proto-type DNA microarray has been tested with three target pathogens and additional seven bacteria, and successfully verified with a few cross-hybridized probes. After this primary verification of the DNA microarray hybridization, this proto-type DNA microarray chip was redesigned and successfully optimized by eliminating a few cross-hybridized probes. The specificity of this redesigned DNA microarray chip to each pathogen was confirmed without any serious cross-hybridizations, and its multiplexing capability in its pathogen detection was found to be possible. This randomly selected non-sequenced genomic DNA probes-based DNA microarray was successfully proved to be the high-throughput simultaneous detection chip for the detection of food-borne pathogens, without knowing the exact sequence information of the target bacteria. This could be the first fabrication of DNA microarray chip for the simultaneous detection of different kinds of food-borne pathogens.

Complete genome sequence of ΦMH1, a Leuconostoc temperate phage.

Bacteriophage ΦMH1, a newly isolated temperate phage from a UV-induced lysate of Leuconostoc pseudomesenteroides, has an isometric head, a noncontractile tail, and a double-stranded DNA genome with a length of 38709 bp. Bioinformatic analysis of the phage genome revealed 65 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. ΦMH1 can be classified as a member of the family Siphoviridae by morphology and genome structure. The phage did not show any significant similarity to other previously reported bacteriophages of Leuconostoc species. To our knowledge, this is the first report of genomic sequencing and characterization of a L. pseudomesenteroides temperate phage.

Elevated O-linked N-acetylglucosamine correlated with reduced Sp1 cooperative DNA binding with its collaborating factors in vivo.

O-Linked N-acetylglucosamine (O-GlcNAc), a single GlcNAc modification of proteins, is abundant in nucleocytoplasmic proteins of eukaryotes. Most nuclear transcriptional regulator proteins carry O-GlcNAc, implicating O-GlcNAc in gene regulation. This study suggested the possibility that O-GlcNAc regulates cooperative binding of Sp1 and its collaborating transcription factors, Oct1 and Elf-1, onto DNA templates in vivo. Chromatin immunoprecipitation assays on cells in which O-GlcNAc was modulated pharmacologically revealed that Sp1-Oct1- and Sp1-Elf-1-paired occupancies of previously known target promoter regions were suppressed by elevated O-GlcNAc modification. Since these pairs of transcription factors bind the target promoters cooperatively and DNA binding of Sp1 alone is not affected by O-GlcNAc, our results imply that O-GlcNAc weakens the DNA binding of Sp1 and its cooperative binding partners by inhibiting stable interaction on DNA templates.

Identification and classification of Cronobacter spp. isolated from powdered food in Korea.

Cronobacter is a major food-borne pathogen in powdered infant formula and can lead to serious developmental aftereffect and death to infants. The contamination of Cronobacter may be a high risk for the powdered foods. To isolate and identify Korean Cronobacter from the powdered foods such as powdered infant formula and Saengsik, conventional culture method, rapid identification system, PCR, 16S rDNA sequencing were performed. As the results of isolation, seven Cronobacter were isolated from seven out of 102 powdered infant formulas and 41 Cronobacter were isolated from 41 out of 86 Saengsiks. Forty eight Cronobacter isolates were identified into C.sakazakii and C.dublenisis by 16s rDNA sequence analysis. Almost the isolates were C.sakazakii and 13% of the isolates were C. dublinesis. One fourth of the C.sakazakii showed different biochemical characteristics of negative nitrate reduction and non-motility activities with the other strains reported previously.