Alterations of Mg<Superscript>2+</Superscript> After Hemorrhagic Shock

The objectives of this study were to measure the concentrations of elements in raw milk by inductively coupled plasma-mass spectrometry (ICP-MS) and evaluate differences in element concentrations among animal species and regions of China. Furthermore, drinking water and feed samples were analyzed to investigate whether the element concentrations in raw milk are correlated with those in water and feed. All samples were analyzed by ICP-MS following microwave-assisted acid digestion. The mean recovery of the elements was 98.7 % from milk, 103.7 % from water, and 93.3 % from a certified reference material (cabbage). Principal component analysis results revealed that element concentrations differed among animal species and regions. Correlation analysis showed that trace elements Mn, Fe, Ni, Ga, Se, Sr, Cs, U in water and Co, Ni, Cu, Se, U in feed were significantly correlated with those in milk (p < 0.05). Toxic and potential toxic elements Cr, As, Cd, Tl, Pb in water and Al, Cr, As, Hg, Tl in feed were significantly correlated with those in milk (p < 0.05). Results of correlation analysis revealed that elements in water and feed might contribute to the elements in milk.     

Complete Genome Sequence Analysis of Leuconostoc kimchii IMSNU 11154

Leuconostoc kimchii IMSNU 11154, isolated from kimchi, a traditional Korean fermented food, is known to be an important antimicrobial lactic acid bacterium with probiotic potential. Here we announce the complete genome sequence of L. kimchii IMSNU 11154 consisting of a 2,101,787-bp chromosome and five plasmids. The strain has genes for dextran formation from sucrose and for mannitol formation from fructose. Antimicrobial and antioxidative functions of L. kimchii IMSNU 11154 could be attributed to a leucosin B-like peptide and multiple enzymes to reduce hydrogen peroxide and oxidized thiols, respectively.Kimchi is a traditional Korean pickled vegetable dish with varied seasonings, and it is known to be an important source of vitamins, minerals, and dietary fiber as well as a good dietary source of lactic acid bacteria (LAB) for humans (2, 3). An exopolysaccharide (EPS)-producing LAB, designated IMSNU 11154, was isolated from kimchi made of cabbage and subsequently classified as a novel species, Leuconostoc kimchii (6). The strain and its culture broth showed antimicrobial activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus cepacia, Candida albicans, and Aspergillus niger. Here we report the genome sequence of Leuconostoc kimchii IMSNU 11154 using a whole-genome shotgun sequencing strategy (4). The complete sequences of one chromosome and five plasmids were obtained. The circular chromosome of strain IMSNU 11154 was 2,101,787 bp in length (37.9% G+C), and the five plasmids were LkipL48 (3,196 bp; 37.1% G+C), LkipL4701 (21,055 bp; 34.3% G+C), LkipL4704 (23,285 bp; 35.6% G+C), LkipL4719 (21,924 bp; 39.1% G+C), and LkipL4726 (29,616 bp; 35.5% G+C). The number of predicted open reading frames (ORFs) is 2,205, covering 89.5% (1,880,952 bp) of the genome. Noncoding RNA regions were identified as 68 tRNAs and 4 copies of rRNA operons. A small gene encoding a leucosin B-like peptide was identified.Strain IMSNU 11154 was missing the fructose 1,6-bisphosphatase enzyme of the Embden-Meyerhof-Parnas pathway and transaldolase of the 6-phosphogluconate/phosphoketolase pathway. Tricarboxylic cycle enzymes were also absent, except for cytochrome oxidase bd-I subunits. The strain metabolized sucrose by invertase, sucrose phosphorylase, and dextransucrase. Formation of EPS and fructose from sucrose by dextransucrase could enhance the probiotic function as well as improve the taste and flavor of kimchi. Strain IMSNU 11154 had a mannitol dehydrogenase gene (mdh) identical to mdh of Leuconostoc mesenteroides (1). By producing mannitol, it maintains its turgor and stabilizes membrane lipids and proteins at low water activity as well as scavenges free reactive oxygen radicals as previously observed for mannitol fermenters (10). Like other Leuconostoc spp., strain IMSNU 11154 does not bear any catalase or superoxide dismutase (SOD) enzymes but has six thioredoxins and four thioredoxin reductases that were shown to be important in coping with acid and oxidative stress (9, 11). Harboring thioredoxin systems on plasmids is a common feature for both L. kimchii IMSNU 11154 and Leuconostoc citreum KM20 (7). Glutathione protected some lactic acid bacteria against oxidative stress (8), but gamma-glutamylcysteine (γ-GC) is the major low-molecular-weight thiol in Leuconostoc spp., including IMSNU 11154 (5). Genes for γ-GC synthesis and reduction are present in strain IMSNU 11154 (5), and there are genes for putative peroxiredoxins that can reduce hydrogen peroxide via small thiols or thioredoxins. Thus, multiple antioxidative systems reduce thiols in strain IMSNU 11154.In conclusion, the genome of Leuconostoc kimchii IMSNU 11154 revealed that its carbohydrate metabolism has adapted to the formation of dextran, fructose, and mannitol from sucrose. The antimicrobial activity of strain IMSNU 11154 could be due to a leucosin B-like peptide, and it contains multiple antioxidative systems to manage acid and oxidative stresses independent of SOD and catalase.     

Hsp70 Interacts with the Retroviral Restriction Factor TRIM5�� and Assists the Folding of TRIM5��

Tripartite motif (TRIM) protein TRIM5α has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5α is not known. In this study, we showed that transiently overexpressed TRIM5α causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5α suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5α-binding partner. The interaction between Hsp70 and TRIM5α was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5α-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5α, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5α-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5α. Studies using a series of TRIM5α deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5α structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5α, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5α in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5α-induced cellular insults.     

Fungicidal effect of prenylated flavonol, papyriflavonol A, isolated from Broussonetia papyrifera (L.) vent. against Candida albicans

Papyriflavonol A (PapA), a prenylated flavonoid (5,7,3',4'-tetrahydroxy-6,5'-di-(r,r-dimethylallyl)-flavonol), was isolated from the root barks of Broussonetia papyriferra. Our previous study showed that PapA has a broad-spectrum antimicrobial activity against pathogenic bacteria and fungi. In this study, the mode of action of PapA against Candida albicans was investigated to evaluate PapA as antifungal agent. The minimal inhibitory concentration (MIC) values were 10~25 microgram/ml for C. albicans and Saccharomyces cerevisiae, gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and gram-positive bacteria (Staphylococcus epidermidis and Staphylococcus aureus). The kinetics of cell growth inhibition, scanning electron microscopy, and measurement of plasma membrane florescence anisotrophy revealed that the antifungal activity of PapA against C. albicans and S. cerevisiae is mediated by its ability to disrupt the cell membrane integrity. Compared with amphotericin B, a cell membrane disrupting polyene antibiotic, the hemolytic toxicity of PapA was negligible. At 10~25 microgram/ml of MIC levels for the tested strains, the hemolysis ratio of human erythrocytes was less than 5%. Our results suggest that PapA could be a therapeutic fungicidal agent having a broad spectrum antimicrobial agent.     

Toxicity and repellency of origanum essential oil and its components against Tribolium castaneum (Coleoptera: Tenebrionidae) adults

The components of Origanum vulgare L. essential oil showing insecticidal activity and repellency against red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae), adults were analysed by GC-MS. All constituents were identified, and the main components were carvacrol (67.2%), p-cymene (16.2%), γ-terpinene (5.5%), thymol (4.9%), and linalool (2.1%). In a vapor phase fumigant assay, the origanum oil was more effective in closed conditions (LD₅₀ = 0.055 mg/cm³) than in open conditions (LD₅₀ > 0.353 mg/cm³). This suggests that toxicity is exerted largely in the vapor phase. Based on 24-h LD₅₀ values, the toxicity of caryophyllene oxide (0.00018 mg/cm³) was comparable with that of dichlorvos (0.00007 mg/cm³). In addition, thymol, camphene, α-pinene, p-cymene, and γ-terpinene showed good insecticidal activity (LD₅₀ = 0.012–0.195 mg/cm³). In repellency tests using 9 constituents of origanum oil, caryophyllene oxide showed complete repellency at 0.03 mg/cm². Hydrogenated monoterpenoids, such as thymol, α-pinene, carvacrol, and myrcene, elicited strong repellency at 0.03 and 0.006 mg/cm². Repellency depended on both time and concentration. These results indicate that origanum oil and its components could be potential candidates as a fumigant and repellent for managing T. castaneum adults.     

Genome-Wide Screening and Identification of Factors Affecting the Biosynthesis of Prodigiosin by Hahella chejuensis,Using Escherichia coli as a Surrogate Host

A marine bacterium, Hahella chejuensis, recently has attracted attention due to its lytic activity against a red-tide dinoflagellate. The algicidal function originates from its red pigment, prodigiosin, which also exhibits immunosuppressive or anticancer activity. Genome sequencing and functional analysis revealed a gene set contained in the hap gene cluster that is responsible for the biosynthesis of prodigiosin. To screen for the factors affecting the prodigiosin biosynthesis, we constructed a plasmid library of the H. chejuensis genomic DNA, introduced it into Escherichia coli strains harboring the hap cluster, and observed changes in production of the red pigment. Among the screened clones, hapXY genes whose products constitute a two-component signal transduction system were elucidated as positive regulators of the pigment production. In addition, an Hfq-dependent, noncoding region located at one end of the hap cluster was confirmed to play roles in regulation. Identification of factors involved in the regulation of prodigiosin biosynthesis should help in understanding how the prodigiosin-biosynthetic pathway is organized and controlled and also aid in modulating the overexpression of prodigiosin in a heterologous host, such as E. coli, or in the natural producer, H. chejuensis.Harmful algal blooms (HABs), commonly called red tide, are a phenomenon in which toxin-producing marine algae rapidly proliferate in the offshore area. The HAB-causing phytoplanktons are reported to interact with other organisms such as bacteria and fungi. Among them, the marine bacteria are known to play important roles in decreasing or developing HABs (3, 5, 14). For instance, Hahella chejuensis, isolated from the coastal area of Marado in South Korea (15), is capable of killing Cochlodinium polykrikoides (12). C. polykrikoides is a major microalga that causes HABs, especially in the Northeast Pacific coastal area (8). The bacterial determinant that kills C. polykrikoides was further characterized as a red pigment referred to as prodigiosin (12). Prodigiosin belongs to a family of tripyrrole antibiotic molecules called prodiginines, which have potential as anticancer agents or immunosuppressants (24). The prodigiosin congener isolated from H. chejuensis also exerts an immunosuppressive effect (11).Through completed genome sequencing of H. chejuensis and its functional analysis, the genomic region involved in biosynthesis of prodigiosin was elucidated (12). This complete set of prodigiosin-biosynthetic genes was named the hap gene cluster. The red pigment prodigiosin was further characterized structurally, and the biosynthetic pathway was proposed by Kim and colleagues (13, 14). Genes of the hap cluster share homology with those in the pig cluster and the red cluster which are involved in prodiginine-biosynthetic intermediates of Serratia marcescens and Streptomyces coelicolor, respectively (7, 23, 25). Enzymes encoded by the genes in the pig and red clusters have been characterized (24). However, gene expression of the hap cluster can be tightly controlled, based on the observation that heterologous expression of the hap cluster alone failed to produce the pigment in Escherichia coli. The recombinant E. coli was able to produce the pigment only when the culture filtrate of H. chejuensis was added to the growth media (12). This result indicates that another regulatory cue is needed for prodigiosin biosynthesis, which prompted us to search for regulatory factors that modulate prodigiosin biosynthesis in H. chejuensis.In this study, regulatory factors for biosynthesis of prodigiosin in H. chejuensis were identified by functional screening. To search for such factors, a plasmid library derived from the genomic DNA of H. chejuensis was constructed and transformed into E. coli strains carrying the hap cluster. In the cases of Serratia marcescens and Streptomyces coelicolor, molecular inputs, such as cell-produced quorum-sensing signal molecules or two-component systems (TCSs) for signal transduction, have been verified as key regulatory signals for prodigiosin biosynthesis so far (4, 9, 10, 20-22). Similarly, some clones of interest uncovered in this study include molecular factors such as those that belong to the TCS. Also, we elucidated that an apparently noncoding region in the hap cluster functions as a key factor of prodigiosin biosynthesis.     

Quantitative proteome analysis of age‐related changes in mouse gastrocnemius muscle using mTRAQ

Aging is associated with a progressive loss of skeletal muscular function that often leads to progressive disability and loss of independence. Although muscle aging is well documented, the molecular mechanisms of this condition still remain unclear. To gain greater insight into the changes associated with aging of skeletal muscle, we performed quantitative proteomic analyses on young (6 months) and aged (27 months) mouse gastrocnemius muscles using mTRAQ stable isotope mass tags. We identified and quantified a total of 4585 peptides corresponding to 236 proteins (protein probability >0.9). Among them, 33 proteins were more than 1.5‐fold upregulated and 20 proteins were more than 1.5‐fold downregulated in aged muscle compared with young muscle. An ontological analysis revealed that differentially expressed proteins belonged to distinct functional groups, including ion homeostasis, energy metabolism, protein turnover, and Ca²⁺ signaling. Identified proteins included aralar1, β‐enolase, fatty acid‐binding protein 3, 3‐hydroxyacyl‐CoA dehydrogenase (Hadh), F‐box protein 22, F‐box, and leucine‐rich repeat protein 18, voltage‐dependent L‐type calcium channel subunit beta‐1, ryanodine receptor (RyR), and calsequestrin. Ectopic expression of calsequestrin in C2C12 myoblast resulted in decreased activity of nuclear factor of activated T‐cells and increased levels of atrogin‐1 and MuRF1 E3 ligase, suggesting that these differentially expressed proteins are involved in muscle aging.     

Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells

Previous studies have demonstrated that a small subset of cancer cells is capable of tumor initiation. The existence of tumor initiating cancer stem cells (CSCs) has several implications in terms of future cancer treatment and therapies. However, recently, several researchers proposed that differentiated cancer cells (non-CSCs) can convert to stem-like cells to maintain equilibrium. These results imply that removing CSCs may prompt non-CSCs in the tumor to convert into stem cells to maintain the equilibrium. Interleukin-6 (IL-6) has been found to play an important role in the inducible formation of CSCs and their dynamic equilibrium with non-stem cells. In this study, we used CSC-like human breast cancer cells and their alternate subset non-CSCs to investigate how IL-6 regulates the conversion of non-CSCs to CSCs. MDA-MB-231 and MDA-MB-453 CSC-like cells formed mammospheres well, whereas most of non-stem cells died by anoikis and only part of the remaining non-stem cells produced viable mammospheres. Similar results were observed in xenograft tumor formation. Data from cytokine array assay show that IL-6 was secreted from non-CSCs when cells were cultured in ultra-low attachment plates. IL-6 regulates CSC-associated OCT-4 gene expression through the IL-6-JAK1-STAT3 signal transduction pathway in non-CSCs. Inhibiting this pathway by treatment with anti-IL-6 antibody (1 μg/ml) or niclosamide (0.5–2 μM)/LLL12 (5–10 μM) effectively prevented OCT-4 gene expression. These results suggest that the IL-6-JAK1-STAT3 signal transduction pathway plays an important role in the conversion of non-CSCs into CSCs through regulation of OCT-4 gene expression.