A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens

Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674^T and S. equorum subsp. linens DSM 15097^T, respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA–DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.     

Senescent endothelial cells are prone to TNF-α-induced cell death due to expression of FAS receptor

The senescent endothelial cells show various phenotypes which can increase the incidence of inflammatory cardiovascular diseases, but the fundamental basis for such phenotypic changes of senescing cells remains to be elucidated. This study was undertaken to find transmembrane receptors that might be highly expressed in senescent endothelial cells and play a key role in cell death signal transduction. Comparison of mRNA expression in young and senescent human umbilical vein endothelial cells, using a cDNA microarray method, provided a list of transmembrane receptors including the FAS receptor (tumor necrosis factor receptor superfamily member 6) whose expression levels were significantly increased by cellular senescence. Additional studies focused on FAS demonstrated that a high expression of FAS receptor in senescent endothelial cells is responsible for the susceptibility to apoptotic cell death, as the siRNA-mediated suppression of FAS expression in senescent cells prevented the cell death, and overexpression of exogenous FAS in young cells increased cell death. We also verified that FAS expression level was closely associated with the activation of caspase-3 and caspase-9 involved in apoptosis. The senescence-induced transmembrane receptors including the FAS receptor may provide novel therapeutic targets to prevent cardiovascular diseases.     

Molecular Approaches to Taenia asiatica

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.     

Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation‐induced genotoxicity was investigated via gain‐of‐functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast‐targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild‐type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV‐treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV‐C radiation, the dark‐grown E. coli RecA‐overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation‐induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV‐C radiation‐induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA‐overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.     

The JNK/NFκB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava

Background

The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-κB (NFκB) to incite immune responses.

Methods

To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cultured with SP and its inhibitors. Assays included ³H-thymidine incorporation, flow cytometry, real time polymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay, Western blot, and electrophoretic mobility shift assay (EMSA).

Results

SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3⁺ mature T cells and CD45R/B220⁺ pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG_1a, and IgG_2b compared to that in untreated cells. The subsequent application of JNK (SP600125), NFκB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IκB, the activation of nuclear NFκB p65, and binding of NFκB p65 DNA. Moreover, co-application of both JNK and NFκB inhibitors completely blocked the proliferation of lymphocytes even in the presence of SP.

Conclusion

These results suggest that SP induced T and B cell responses via both JNK and NFκB pathways.

General significance

The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism.     

Deacetylated αβ-tubulin acts as a positive regulator of Rheb GTPase through increasing its GTP-loading

Ras homolog enriched in brain (Rheb) regulates diverse cellular functions by modulating its nucleotide-bound status. Although Rheb contains a high basal GTP level, the regulatory mechanism of Rheb is not well understood. In this study, we propose soluble αβ-tubulin acts as a constitutively active Rheb activator, which may explain the reason why Rheb has a high basal GTP levels. We found that soluble αβ-tubulin is a direct Rheb-binding protein and that its deacetylated form has a high binding affinity for Rheb. Modulation of both soluble and acetylated αβ-tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated αβ-tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated αβ-tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble αβ-tubulin is a novel type of positive regulator of Rheb and may play a role as a temporal regulator for Rheb during the cell cycle.     

Involvement of small GTPase RhoA in the regulation of superoxide production in BV2 cells in response to fibrillar Aβ peptides

Fibrillar amyloid-beta (fAβ) peptide causes neuronal cell death, which is known as Alzheimer's disease. One of the mechanisms for neuronal cell death is the activation of microglia which releases toxic compounds like reactive oxygen species (ROS) in response to fAβ. We observed that fAβ rather than soluble form blocked BV2 cell proliferation of microglial cell line BV2, while N-acetyl-l-cysteine (NAC), a scavenger of superoxide, prevented the cells from death, suggesting that cell death is induced by ROS. Indeed, both fAβ_1–42 and fAβ_25–35 induced superoxide production in BV2 cells. fAβ_25–35 produced superoxide, although fAβ_25–35 is not phagocytosed into BV2 cells. Thus, superoxide production by fAβ does not seem to be dependent on phagocytosis of fAβ. Herein we studied how fAβ produces superoxide in BV2. Transfection of dominant negative (DN) RhoA (N19) cDNA plasmid, small hairpin (sh)-RhoA forming plasmid, and Y27632, an inhibitor of Rho-kinase, abrogated the superoxide formation in BV2 cells stimulated by fAβ. Furthermore, fAβ elevated GTP-RhoA level as well as Rac1 and Cdc42. Tat-C3 toxin, sh-RhoA, and Y27632 inhibited the phosphorylation of p47^PHOX. Moreover, peritoneal macrophages from p47^PHOX (?/?) knockout mouse could not produce superoxide in response to fAβ. These results suggest that RhoA closely engages in the regulation of superoxide production induced by fAβ through phosphorylation of p47^PHOX in microglial BV2 cells.     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

Pyrosequencing-based analysis of bacterial community and metabolites profiles in Korean traditional seafood fermentation: a flatfish-fermented seafood

Bacterial community and metabolites were analyzed in a flatfish jeotgal, a Korean fermented seafood. Inverse relationship of pH and 16S rRNA gene copy number was identified during fermentation. Lactobacillus was the predominant bacterial genus. Increase of Firmicutes was a common characteristic shared by other fermented seafood. Fructose, glucose, and maltose were the major metabolites.     

Loss‐of‐function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa)

STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T–DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild‐type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8‐mediated PCPP in PSII repair.