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.

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.

Chemopreventive Properties of Genipin on AGS Cell Line via Induction of JNK/Nrf2/ARE Signaling Pathway.

Roles of dietary phytochemicals in cancer chemoprevention via induction of nuclear factor-erythroid-2-related factor 2 (Nrf2)-mediated antioxidant enzymes have been well established in a number of studies. In this study, FACS analysis was used to reveal that the intracellular reactive oxygen species level decreased at 0-25 µM of genipin treatment. Furthermore, immunofluorescence analysis and Western blotting were used to demonstrate that genipin treatment resulted in the upregulation and nuclear translocation of Nrf2, as well as upregulation of gastrointestinal glutathione peroxidase. Finally, we found that C-Jun-NH2-kinase (JNK) was also dose-dependently activated, where depleting JNK by using a biochemical inhibitor indicated that JNK was upstream of Nrf2. Interestingly, the antioxidant effects were limited to the treatment in the lower dosage of genipin, where higher dosage of genipin treatment resulted in the increased reactive oxygen species level and cytotoxicity. Thus, this study demonstrates for the first time that lower dosage of genipin results in the induction of JNK/Nrf2/ARE signaling pathway and protection from cell death.

Induction of apoptosis by genipin inhibits cell proliferation in AGS human gastric cancer cells via Egr1/p21 signaling pathway.

Natural compounds are becoming important candidates in cancer therapy due to their cytotoxic effects on cancer cells by inducing various types of programmed cell deaths. In this study, we investigated whether genipin induces programmed cell deaths and mediates in Egr1/p21 signaling pathways in gastric cancer cells. Effects of genipin in AGS cancer cell lines were observed via evaluation of cell viability, ROS generation, cell cycle arrest, and protein and RNA levels of p21, Egr1, as well as apoptotic marker genes. The cell viability of AGS cells reduced by genipin treatment via induction of the caspase 3-dependent apoptosis. Cell cycle arrest was observed at the G2/M phase along with induction of p21 and p21-dependent cyclins. As an upstream mediator of p21, the transcription factor early growth response-1 (Egr1) upregulated p21 through nuclear translocation and binding to the p21 promoter site. Silencing Egr1 expression inhibited the expression of p21 and downstream molecules involved in apoptosis. We demonstrated that genipin treatment in AGS human gastric cancer cell line induces apoptosis via p53-independent Egr1/p21 signaling pathway in a dose-dependent manner.

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.

Cyanidin is an agonistic ligand for peroxisome proliferator-activated receptor-alpha reducing hepatic lipid.

To investigate the underlying mechanism of targets of cyanidin, a flavonoid, which exhibits potent anti-atherogenic activities in vitro and in vivo, a natural chemical library that identified potent agonistic activity between cyanidin and peroxisome proliferator-activated receptors (PPAR) was performed. Cyanidin induced transactivation activity in all three PPAR subtypes in a reporter gene assay and time-resolved fluorescence energy transfer analyses. Cyanidin also bound directly to all three subtypes, as assessed by surface plasmon resonance experiments, and showed the greatest affinity to PPARα. These effects were confirmed by measuring the expression of unique genes of each PPAR subtype. Cyanidin significantly reduced cellular lipid concentrations in lipid-loaded steatotic hepatocytes. In addition, transcriptome profiling in lipid-loaded primary hepatocytes revealed that the net effects of stimulation with cyanidin on lipid metabolic pathways were similar to those elicited by hypolipidemic drugs. Cyanidin likely acts as a physiological PPARα agonist and potentially for PPARß/δ and γ, and reduces hepatic lipid concentrations by rewiring the expression of genes involved in lipid metabolic pathways.

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.

Effect of anthocyanins on expression of matrix metalloproteinase-2 in naproxen-induced gastric ulcers.

Non-steroidal anti-inflammatory drugs cause gastric ulceration through a number of mechanisms including inhibition of PG synthesis, generation of reactive oxygen species (ROS) and induction of apoptosis. Recently, matrix metalloproteinases (MMP) have been suggested to play a crucial role in these mechanisms. The present study investigated the protective effect of anthocyanins isolated from black rice bran (Heugjinjubyeo) against naproxen-induced gastric mucosal injury in rats. The oral administration of anthocyanins (5, 25 or 50 mg/kg body weight) showed significant protection against naproxen (80 mg/kg body weight)-induced gastric ulcer and inhibited lipid peroxidation in the gastric mucosa. In addition, pretreatment with anthocyanins resulted in a significant increase in the activities of radical-scavenging enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Also biochemical and zymographic analyses suggested that the administration of anthocyanins gives a significant protection against naproxen-induced gastric antral ulcer through scavenging ROS and regulation of matrix metalloproteinase-2 (MMP-2) activity. The results of intracellular radical activation show that anthocyanins suppress the generation of intracellular ROS and attenuate the suppression of MMP-2 activity by naproxen. These results suggest that anthocyanins extracted from black rice may offer potential remedy of gastric antral ulceration.

Complete genomic sequence of the Lactobacillus temperate phage LF1.

Bacteriophage LF1, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild type Lactobacillus fermentum, was found to contain a double-strand DNA of 42,606 base pairs (bp) with a G+C content of 45%. Bioinformatic analysis of the phage genome revealed 57 putative open reading frames (ORFs). The predicted protein products of ORFs were determined and described. According to morphological analysis by transmission electron microscopy (TEM), LF1 has an isometric head and a non-contractile tail, indicating that it belongs to the family Siphoviridae. The temperate phage LF1 has a good genetic mosaic relationship with ΦPYB5 in the packaging module. To our knowledge, this is first report of genomic sequencing and characterization of temperate phage LF1 from wild-type L. fermentum isolated from Kimchi in Korea.

Complete nucleotide sequence of the temperate bacteriophage LBR48, a new member of the family Myoviridae.

The complete genomic sequence of LBR48, a temperate bacteriophage induced from a lysogenic strain of Lactobacillus brevis, was found to be 48,211 nucleotides long and to contain 90 putative open reading frames. Based on structural characteristics obtained from microscopic analysis and nucleic acid sequence determination, phage LBR48 can be classified as a member of the family Myoviridae. Analysis of the genome showed the conserved gene order of previously reported phages of the family Siphoviridae from lactic acid bacteria, despite low nucleotide sequence similarity. Analysis of the attachment sites revealed 15-nucleotide-long core sequences.

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.

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.

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.

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.

O-GlcNAc inhibits interaction between Sp1 and sterol regulatory element binding protein 2.

O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine on the serine and threonine residues of nucleocytoplasmic proteins, is a novel protein modification that is ubiquitous among eukaryotes and implicated in cell regulation. Recent evidence indicates that O-GlcNAc regulates protein-protein interactions. Here we provide evidence that O-GlcNAc interrupts a known interaction between Sp1 and sterol regulatory element binding protein 2 (SREBP2), thereby inhibiting expression of the gene encoding acetyl-CoA synthetase 1, which is involved in lipid synthesis. This study suggests a novel mechanism in which lipid biosynthesis may be regulated by O-GlcNAc.

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.