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Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family, members of which play essential roles in diverse cellular processes during carcinogenesis, including cell proliferation, differentiation, migration, and invasion. Unlike other MAPKs, ERK3 is an unstable protein with a short half-life. Although deubiquitination of ERK3 has been suggested to regulate the activity, its ubiquitination has not been described in the literature. Here, we report that FBXW7 (F-box and WD repeat domain-containing 7) acts as a ubiquitination E3 ligase for ERK3. Mammalian two-hybrid assay and immunoprecipitation results demonstrated that ERK3 is a novel binding partner of FBXW7. Furthermore, complex formation between ERK3 and the S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) E3 ligase resulted in the destabilization of ERK3 via a ubiquitination-mediated proteasomal degradation pathway, and FBXW7 depletion restored ERK3 protein levels by inhibiting this ubiquitination. The interaction between ERK3 and FBXW7 was driven by binding between the C34D of ERK3, especially at Thr417 and Thr421, and the WD40 domain of FBXW7. A double mutant of ERK3 (Thr417 and Thr421 to alanine) abrogated FBXW7-mediated ubiquitination. Importantly, ERK3 knockdown inhibited the proliferation of lung cancer cells by regulating the G1/S-phase transition of the cell cycle. These results show that FBXW7-mediated ERK3 destabilization suppresses lung cancer cell proliferation in vitro.

Diabetes mellitus is a complex and heterogeneous disease, which has β-cell dysfunction at its core. Glucotoxicity affects pancreatic islets, causing β-cell apoptosis. However, the role of JNK/β-catenin signaling in glucotoxic β-cell apoptosis is not well understood. Recently, we identified tetraspanin-2 (TSPAN2) protein as a proapoptotic β-cell factor induced by glucose, suggesting that TSPAN2 might contribute to pancreatic β-cell glucotoxicity. To investigate the effects of glucose concentration on TSPAN2 expression and apoptosis, we used reverted immortalized RNAKT-15 human pancreatic β cells. High TSPAN2 levels up-regulated phosphorylated (p) JNK and induced apoptosis. p-JNK enhanced the phosphorylation of β-catenin and Dickkopf-1 (Dkk1). Dkk1 knockdown by small interfering (si)RNA up-regulated nuclear β-catenin, suggesting that it is a JNK/β-catenin-dependent pathway. siRNA-mediated TSPAN2 depletion in RNAKT-15 cells increased nuclear β-catenin. This decreased BCL2-associated X protein (Bax) activation, leading to marked protection against high glucose-induced apoptosis. Bax subfamily proteins induced apoptosis through caspase-3. Thus, TSPAN2 might have induced Bax translocation and caspase-3 activation in pancreatic β cells, thereby promoting the apoptosis of RNAKT-15 cells by regulating the JNK/β-catenin pathway in response to high glucose concentrations. Targeting TSPAN2 could be a potential therapeutic strategy to treat glucose toxicity-induced β-cell failure.-Hwang, I.-H., Park, J., Kim, J. M., Kim, S. I., Choi, J.-S., Lee, K.-B., Yun, S. H., Lee, M.-G., Park, S. J., Jang, I.-S. Tetraspanin-2 promotes glucotoxic apoptosis by regulating the JNK/β-catenin signaling pathway in human pancreatic β cells.

Postpartum depression is a severe emotional and mental disorder that involves maternal care defects and psychiatric illness. Postpartum depression is closely associated with a combination of physical changes and physiological stress during pregnancy or after parturition in stress-sensitive women. Although postpartum depression is relatively well known to have deleterious effects on the developing fetus, the influence of genetic risk factors on the development of postpartum depression remains unclear. In this study, we discovered a novel function of T cell death-associated gene 51 (TDAG51/PHLDA1) in the regulation of maternal and depressive-like behavior. After parturition, TDAG51-deficient dams showed impaired maternal behavior in pup retrieving, nursing and nest building tests. In contrast to the normal dams, the TDAG51-deficient dams also exhibited more sensitive depressive-like behaviors after parturition. Furthermore, changes in the expression levels of various maternal and depressive-like behavior-associated genes regulating neuroendocrine factor and monoamine neurotransmitter levels were observed in TDAG51-deficient postpartum brain tissues. These findings indicate that TDAG51 plays a protective role against maternal care defects and depressive-like behavior after parturition. Thus, TDAG51 is a maternal care-associated gene that functions as a crucial regulator of maternal and depressive-like behavior after parturition., Author summary Postpartum depression is a severe emotional and mental disease that can affect women typically after parturition. However, the genetic risk factors associated with the development of postpartum depression are still largely unknown. We discovered a novel function of T cell death-associated gene 51 (TDAG51) in the regulation of maternal behavior and postpartum depression. We report that TDAG51 deficiency induces depressive-like and abnormal maternal behavior after parturition. The loss of TDAG51 in postpartum brain tissues induces changes in the expression levels of various maternal and depressive-like behavior-associated genes that regulate the levels of neuroendocrine factors and monoamine neurotransmitters. TDAG51 is a maternal care-associated gene that functions as a crucial regulator of maternal and depressive-like behavior after parturition.

Summary Phenyl‐2‐pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near‐senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose‐ and time‐dependent manner and resulted in senescence‐associated β‐galactosidase (SA‐β‐gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence‐associated proteins, such as phosphorylated ERK1/2, caveolin‐1, p53, p16ink4a, and p21waf1, were elevated in PPKO‐treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N‐acetylcysteine, 2,2,6,6‐tetramethylpiperidinyloxy, and L‐buthionine‐(S,R)‐sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L‐NG‐nitroarginine methyl ester and L‐NG‐monomethylarginine, PPKO‐induced transient NO production and SA‐β‐gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence‐associated proteins.

The effect of curcumin pretreatment (15-240 μM) in fathead minnow cells infected with viral hemorrhagic septicemia virus (VHSV) was evaluated. Cell viability, apoptosis and viral copy number were analyzed using Cell Counting Kit-8 assay, Annexin V staining, and reverse transcription-PCR, respectively. Pretreatment with 120 μM curcumin showed an increase in viability (>90% of mock) of VHSV-infected cells and reduction in the copy number (0.2-log reduction in VHSV N gene expression), reactive oxygen species and apoptosis in the cells without cytotoxic effects. To understand the mechanisms underlaying the antiviral effects of curcumin pretreatment, a comparative proteomic analysis was performed in four samples (M, mock; C, curcumin-treated; V, VHSV-infected; and CV, curcumin-treated VHSV-infected) in triplicate. In total, 185 proteins were detected. The analysis showed that three proteins, including heat shock cognate 71 (HSC71), actin, alpha cardiac muscle (ACTC1) and elongation factor 1 (EEF1) were differentially expressed between V and CV samples. Network analysis performed by Ingenuity Pathways Analysis (IPA) showed that HSC71 was the primary protein interacting with fibronectin (FN) 1, actins (ACTB, ACTG, F-actin) and gelsolin (GSN) in both V and CV samples and thus is a strong target candidate for the protection from VHSV infection at the viral entry stage. Our proteomics data suggest that curcumin pretreatment inhibits entry of VHSV in cells by downregulating FN1 or upregulating F-actin. For both proteins, HSC71 acts as a binding protein that modulates their functions. Furthermore, consistent with the effect of a heat shock protein inhibitor (KNK437), curcumin downregulated HSC71 expression with increasing viability of VHSV-infected cells and inhibited VHSV replication, suggesting that the downregulation of HSC71 could be responsible for the antiviral activity of curcumin. In conclusion, this study indicates that the suppression of viral entry by rearrangement of the F-actin/G-actin ratio via downregulating HSC71 is a plausible mechanism by which curcumin pretreatment controls the early stages of VHSV infection.

Background: TNF receptor-associated factor 2 (TRAF2) is a key adaptor molecule in the TNF receptor (TNFR) signaling pathway. Results: TRAF-interacting protein (TRIP) inhibits Lys63-linked TRAF2 ubiquitination by blocking the binding of the cofactor sphingosine 1-phosphate (S1P) to the TRAF2 RING domain. Conclusion: TRIP negatively regulates the TRAF2 ubiquitin-dependent pathway by modulating the TRAF2-S1P interaction. Significance: TRIP is an important cellular regulator of the TNFR-mediated inflammatory response. The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys63-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections., Author summary Viral particles must transport their genome into the cytoplasm or the nucleus of host cells to initiate successful infection. Knowledge of how viruses may pirate host cell signaling cascades or molecules to promote their own replication can facilitate the development of antiviral drugs. Group A rotavirus (RVA) is a major etiological agent of acute gastroenteritis in young children and the young of various mammals. RVA enters cells by a complex multistep process. However, the cellular signaling cascades or molecules that facilitate these processes are incompletely understood. Here, we demonstrate that infection with late-penetration RVA strains results in phosphorylation of PI3K, Akt, and ERK signaling molecules at an early stage of infection, a process mediated by the multistep binding of RVAs outer capsid proteins. Specific inhibitors for PI3K/Akt and MEK/ERK signaling pathways trap the viral particles in late endosome, and acidic replenishment restores and releases them. Moreover, the RVA-induced phosphorylated PI3K, Akt, and ERK directly interact with the subunit E of the V-ATPase proton pump, required for endosomal acidification and RVA uncoating. Understanding how RVA-induced early activation of cellular signaling molecules mediates the V-ATPase-dependent endosomal acidification required for uncoating of viral particles opens up opportunities for targeted interventions against rotavirus entry.

The identification of disulfide bonds provides critical information regarding the structure and function of a protein and is a key aspect in understanding signaling cascades in biological systems. Recent proteomic approaches using digestion enzymes have facilitated the characterization of disulfide-bonds and/or oxidized products from cysteine residues, although these methods have limitations in the application of MS/MS. For example, protein digestion to obtain the native form of disulfide bonds results in short lengths of amino acids, which can cause ambiguous MS/MS analysis due to false positive identifications. In this study we propose a new approach, termed planned digestion, to obtain sufficient amino acid lengths after cleavage for proteomic approaches. Application of the DBond software to planned digestion of specific proteins accurately identified disulfide-linked peptides. RNase A was used as a model protein in this study because the disulfide bonds of this protein have been well characterized. Application of this approach to peptides digested with Asp-N/C (chemical digestion) and trypsin under acid hydrolysis conditions identified the four native disulfide bonds of RNase A. Missed cleavages introduced by trypsin treatment for only 3 hours generated sufficient lengths of amino acids for identification of the disulfide bonds. Analysis using MS/MS successfully showed additional fragmentation patterns that are cleavage products of S-S and C-S bonds of disulfide-linkage peptides. These fragmentation patterns generate thioaldehydes, persulfide, and dehydroalanine. This approach of planned digestion with missed cleavages using the DBond algorithm could be applied to other proteins to determine their disulfide linkage and the oxidation patterns of cysteine residues.

Genetic mutations in osteoclastogenic genes are closely associated with osteopetrotic bone diseases. Genetic defects in OSTM1 (osteopetrosis-associated transmembrane protein 1) cause autosomal recessive osteopetrosis in humans. In particular, OSTM1 mutations that exclude the transmembrane domain might lead to the production of a secreted form of truncated OSTM1. However, the precise role of the secreted form of truncated OSTM1 remains unknown. In this study, we analyzed the functional role of truncated OSTM1 in osteoclastogenesis. Here, we showed that a secreted form of truncated OSTM1 binds to the cell surface of osteoclast (OC) precursors and inhibits the formation of multinucleated OCs through the reduction of cell fusion and survival. Truncated OSTM1 significantly inhibited the expression of OC marker genes through the down-regulation of the BLIMP1 (B lymphocyte-induced maturation protein 1)-NFATc1 (nuclear factor of activated T cells c1) axis. Finally, we demonstrated that truncated OSTM1 reduces lipopolysaccharide-induced bone destruction in vivo. Thus, these findings suggest that autosomal recessive osteopetrosis patients with an OSTM1 gene mutation lacking the transmembrane domain produce a secreted form of truncated OSTM1 that inhibits osteoclastogenesis.

Mitochondria are important organelles for cellular respiration within the eukaryotic cell and have many important functions including vitamin synthesis, amino acid metabolism and photorespiration. To investigate the mitochondrial proteome of the roots of wheat seedlings, a systematic and targeted analysis were carried out on the mitochondrial proteome from 15 day-old wheat seedling root material. Mitochondria were isolated by Percoll gradient centrifugation; and extracted proteins were disassociated and analyzed by Tricine SDS-PAGE couple to LTQ-FTICR mass spectrometry. From the isolated the sample, 184 proteins were identified which is composed of 140 proteins as mitochondria and 44 proteins as other subcellular proteins that are predicted by the freeware sub-cellular predictor. The identified proteins in mitochondria were functionally classified into 12 classes using the ProtFun 2.2 servers based on biological processes. Proteins were shown to be involved in amino acid biosynthesis (17.1%), biosynthesis of cofactors (6.4%), cell envelope (11.4%), central intermediary metabolism (10%), energy metabolism (20%), fatty acid metabolism (0.7%), purines and pyrimidines (5.7%), regulatory functions (0.7%), replication and transcription (1.4%), translation (22.1%), transport and binding (1.4%), and unknown (2.8%). These results indicate that many of the protein components present and functions of identifying proteins are common to other profiles of mitochondrial proteins performed to date. These results are provided the extensive and noble clues, to our knowledge, of mitochondrial proteins from wheat roots.

Hepatitis A virus (HAV) infection leads to acute liver failure and death through the intake of contaminated food. The polymerase chain reaction (PCR) has been used to detect HAV in food samples. HAV detection takes a long time, however, due to the virus concentration step required before PCR assay. In this study, a rapid method of detecting the HAVs present in lettuce using immunomagnetic separation combined with quantum dots (IMS-QDs) assay was developed. The detection limit of IMS-QDs for HAV was 10 TCID50/mL, similar to the result that was obtained using RT-PCR combined with PEG or IMS. The application of IMS-QDs assay completed the viral detection within one hour, but this was not possible using PEG combined with RT-PCR. In conclusion, IMS-QDs assay is a rapid and efficient method for detecting HAV at a low concentration in agricultural products.

A halophilic archaeal strain, designated CBA1105(T), was isolated from non-purified solar salt. Strain CBA1105(T) was found to have three 16S rRNA genes, rrnA, rrnB and rrnC; similarities between the 16S rRNA gene sequences were 99.5-99.7 %. The phylogenetic analysis of the 16S rRNA gene sequences showed that strain CBA1105(T) forms a distinct clade with the strains of the closely related genera, Halorientalis and Halorhabdus, with similarities of 94.2 % and 93.9-94.2 %, respectively. Multilocus sequence analysis confirmed that strain CBA1105(T) is closely related to the genus Halorhabdus or Halorientalis. Growth of the strain was observed in 15-30 % NaCl (w/v; optimum 20 %), at 30-45 °C (optimum 37 °C) and pH 7.0-8.0 (optimum pH 7.0) and with 0-0.5 M MgCl2·6H2O (optimum 0.05-0.2 M). The cells of the strain were observed to be Gram-stain negative and pleomorphic with coccoid or ovoid-shape. The cells lysed in distilled water. Tweens 20, 40 and 80 were found to be hydrolysed but starch, casein and gelatine were not. The cells were unable to reduce nitrate under aerobic conditions. Assays for indole formation and urease activity were negative and no growth was observed under anaerobic conditions. Cells were found to be able to utilize L-glutamate, D-glucose, L-maltose, D-mannose and sucrose as sole carbon sources. The polar lipids were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, unidentified glycolipids and an unidentified phospholipid. The G+C content of strain CBA1105(T) was determined to be 66.0 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strain represents a novel species of a novel genus within the family Halobacteriaceae, for which the name Halapricum salinum is proposed with CBA1105(T) (= KCTC 4202(T) = JCM 19729(T)) as the type strain.

Two Gram-stain negative, rod-shaped and motile extreme halophiles, designated CBA1107(T) and CBA1108, were isolated from non-purified solar salt. Based on the phylogenetic analysis, strains CBA1107(T) and CBA1108 were shown to belong to the genus Halolamina, with similarities for the 16S rRNA gene sequences between strains CBA1107(T) and Halolamina pelagica TBN21(T) , Halolamina salina WSY15-H3(T) and Halolamina salifodinae WSY15-H1(T) of 98.3, 97.6 and 97.3 %, respectively; the similarities for the rpoB' gene sequences between the same strains were 96.0, 95.3 and 94.6 %, respectively. The colonies of both strains were observed to be red pigmented on growth medium. Strain CBA1107(T) was observed to grow at 20-50 °C, in the presence of 15-30 % NaCl, at pH 6.0-9.0, and with 0.005-0.5 M Mg(2+). The cells of both strains lysed in distilled water. The DNA-DNA hybridization experiments showed that strain CBA1107(T) shared 97 % relatedness with CBA1108 and50 % relatedness with H. pelagica JCM 16809(T), H. salina JCM 18549(T) and H. salifodinae JCM 18548(T). The genomic DNA G+C content of strain CBA1107(T) was determined to be 65.1 mol%. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and glycolipids including sulfated mannosyl glucosyl diether and mannosyl glucosyl diether. Based on the polyphasic taxonomic analyses, the strains are considered to represent a new taxon for which the name Halolamina rubra sp. nov. is proposed, with the type strain CBA1107(T) (=CECT 8421(T) =JCM 19436(T)).

The accuracy and sensitivity of PCR-based methods for detection of hepatitis A virus (HAV) are dependent on the methods used to separate and concentrate the HAV from the infected cells. The pH and ionic strength affect the binding affinity of the virus to cells. In this study, we initially investigated the effects of pH (4.0–10.0) and metal ions (Fe2+, Co2+, Cu2+, Mg2+, K+, and Ca2+) on the binding of HAV to oyster digestive cells. The lowest relative binding (RB) of HAV to the cells was found at pH 4.0 and in FeSO4 solution (64.6% and 68.1%, respectively). To develop an alternative to antibody-dependent immunomagnetic separation prior to detection of HAV using RT-PCR, the binding of HAV to five lectins, peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Ulex europaeus agglutinin (UEA-1) and soybean agglutinin (SBA), was evaluated using ELISAs. SBA showed significantly higher RB to HAV than the other lectins tested. In addition, HAV could be concentrated within 30 min using SBA-linked magnetic bead separation (SMS) prior to the RT-PCR assay. Our findings demonstrate the feasibility of using SMS combined with RT-PCR to detect HAV at dilutions ranging from 10−1–10−4 of a HAV stock (titer: 104 TCID50/mL).

A novel, red-pigmented, pleomorphic and short rod-shaped haloarchaeon, designated B8(T), was isolated from a salt-fermented seafood. Strain B8(T) was found to be able to grow at 20-45 °C, in the presence of 15-30 % (w/v) NaCl and at pH 7.0-9.0. The optimum requirements were found to be a temperature range of 35-40 °C, pH 8.0 and the presence of 25 % NaCl. The cells of strain B8(T) were observed to be Gram-staining negative and lysed in distilled water. Anaerobic growth did not occur in the presence of nitrate, L-arginine, dimethyl sulfoxide or trimethylamine N-oxide. The catalase and oxidase activities were found to be positive and nitrate was reduced in aerobic conditions. Tween 20, 40 and 80 were found to be hydrolyzed, whereas casein, gelatin and starch were not hydrolyzed. Indole or H2S was not formed and urease activity was not detected. A phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain B8(T) is most closely related to members of the genus Halorubrum in the family Halobacteriaceae. Strain B8(T) was found to have three 16S rRNA genes, rrnA, rrnB and rrnC; similarities between the 16S rRNA gene sequences are 99.0-99.8 %. Strain B8(T) shared 99.0 % 16S rRNA gene sequence similarity with Halorubrum (Hrr.) lipolyticum JCM 13559(T) and Hrr. saccharovorum DSM 1137(T), 98.8 % with Hrr. kocurii JCM 14978(T), 98.3 % with Hrr. lacusprofundi DSM 5036(T), 98.0 % with Hrr. arcis JCM 13916(T), 97.7 % with Hrr. aidingense JCM 13560(T) and 97.0 % with Hrr. aquaticum JCM 14031(T), as well as 93.7-96.5 % with other type strains in the genus Halorubrum. The RNA polymerase subunit B' gene sequence similarity of strain B8(T) with Hrr. kocurii JCM 14978(T) is 97.2 % and lower with other members of the genus Halorubrum. DNA-DNA hybridization experiments showed that strain B8(T) shared equal or lower than 50 % relatedness with reference species in the genus Halorubrum. The genomic DNA G+C content of strain B8(T) was determined to be 64.6 mol%. The major isoprenoid quinone of strain B8(T) was identified as menaquinone-8 and the major polar lipids as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl glucosyl diether and an unidentified phospholipid. Based on this polyphasic taxonomic study, strain B8(T) is considered to represent a new species in the genus Halorubrum, for which the name Hrr. halophilum sp. nov. is proposed. The type strain is B8(T) (=JCM 18963(T) = CECT 8278(T)).

The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding l -alanine dehydrogenase in Mycobacterium smegmatis . The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of l -alanine. The purified AldR protein exists as a homodimer in the absence of l -alanine, while it adopts the quaternary structure of a homohexamer in the presence of l -alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by l -alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N 2 -ATC-N 2 -TC and one putative AldR binding site with the sequence GA-N 2 -GTT-N 2 -TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of l -alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

Current molecular methods that include PCR have been used to detect norovirus in many food samples. However, the protocols require removing PCR inhibitors and incorporate time-consuming concentration steps to separate virus from analyte for rapid and sensitive detection of norovirus. We developed an immunomagnetic separation (IMS) and a quantum dots (QDs) assay to detect norovirus eluted from fresh lettuce with Tris buffer containing 1% beef extract (pH 9.5). IMS facilitated viral precipitation with a 10-min incubation, whereas virus concentration using polyethylene glycol (PEG) requires more than 3 h and an additional high-speed centrifugation step to precipitate virus before reverse transcription PCR (RT-PCR) analysis. The fluorescence intensity of QDs was detected qualitatively on norovirus dilutions of 10(-1) to 10(-3) in a stool suspension (100 RT-PCR units/ml). The results suggest that a fluorescence assay based on IMS and QDs is valid for detecting norovirus qualitatively according to fluorescent signal intensity within the same virus detection limit produced by IMS-RT-PCR and PEG-RT-PCR.

A novel halophilic archaeon designated strain CBA1114T was isolated from solar salt in the Republic of Korea. Strain CBA1114T, cells of which were coccoid and Gram-stain-negative, grew in the presence of 15–30 % (w/v) NaCl (optimum, 20 %) and at 20–50 °C (optimum, 40 °C) and pH 7.0–9.0 (optimum, pH 8.0). Strain CBA1114T required Mg2+ for growth. Strain CBA1114T had three 16S rRNA genes, rrnA, rrnB and rrnC; levels of similarity between the sequences were 99.7–99.9 %. The 16S rRNA gene sequence of strain CBA1114T showed 91.7 % similarity to that of Haloterrigena thermotolerans PR5T. In multilocus sequence analysis (MLSA), five housekeeping genes, atpB, EF-2, radA, rpoB′ and secY, were found to be closely related to those of the members of the genera Halorientalis (89.7 % similarity of the atpB gene sequence), Halomicroarcula (91.9 %, EF-2), Haloterrigena (85.4 %, radA), Natronoarchaeum (89.2 %, rpoB′) and Natrinema (75.7 %, secY). A phylogenetic tree generated from the results of MLSA of the five housekeeping genes showed that strain CBA1114T was closely related to species of the genus Halorientalis in the family Halobacteriaceae . The major polar lipids were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and unidentified lipids. The G+C content of the genomic DNA of strain CBA1114T was 68.1 mol%. According to the results of phylogenetic, phenotypic and chemotaxonomic analyses, we designate strain CBA1114T (=JCM 30111T=KCTC 4206T) as the type strain of Halostella salina gen. nov., sp. nov., a novel species of a new genus within the family Halobacteriaceae .

Leaves are the final site of salinity perception through the roots. To better understand how wheat chloroplasts proteins respond to salt stress, the study aimed to the physiochemical and comparative proteomics analysis. Seedlings (12-days-old) were exposed to 150 mM NaCl for 1, 2, or 3 days. Na(+) ions were rapid and excessively increase in roots, stems and leaves. Photosynthesis and transpiration rate, stomatal conductance, and relative water content decreased whereas the level of proline increased. Statistically significant positive correlations were found among the content of hydrogen peroxide, activity of catalase, and superoxide dismutase under salt stress in wheat. Protein abundance within the chloroplasts was examined by two-dimensional electrophoresis. More than 100 protein spots were reproducibly detected on each gel, 21 protein spots were differentially expressed during salt treatment. Using linear quadruple trap-Fourier transform ion cyclotron resonance (LTQ-FTICR) hybrid mass spectrometry, 65 unique proteins assigned in the differentially abundant spots. Most proteins were up-regulated at 2 and 3 days after being down-regulated at 1 day. Others showed only slight responses after 3 days of treatment, including Rubisco, glutamate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, photosystem I, and pyridoxal biosynthesis protein PDX1.2 and PDX1.3. The ATP synthase (α, β, and γ) and V-type proton ATPase subunits were down-regulated resulting showed negative impact by Na(+) on the photosynthetic machinery. This ephemeral increase and subsequent decrease in protein contents may demonstrate a counterbalancing influence of identified proteins. Several proteins such as cytochrome b6-f (Cyt b6-f), germin-like-protein, the γ-subunit of ATP synthase, glutamine synthetase, fructose-bisphosphate aldolase, S-adenosylmethionine synthase, carbonic anhydrase were gradually up-regulated during the period of treatment, which can be identified as marker proteins.

A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon-gamma knockout (Ifng-KO) mice. Five upregulated and five downregulated proteins were identified with 2D gels and MALDI-TOF/TOF MS analyses in Ifng-KO mouse brain. Of the identified proteins, we focused on creatine kinase brain (CKB) and triose phosphate isomerase 1 (TPI1). Consistent with the proteomic data, reverse transcriptase-mediated PCR, immunoblotting, and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.

Induced pluripotent stem cells (iPSCs) are somatic cells that have been reprogrammed to a pluripotent state via introduction of defined transcription factors. iPSCs are a valuable resource for regenerative medicine, but whether iPSCs are identical to embryonic stem cells (ESCs) remains unclear. In this study, we performed comparative proteomic analyses of human somatic cells [human newborn foreskin fibroblasts (hFFs)], human iPSCs (hiPSCs) derived from hFFs, and H9 human ESCs (hESCs). We reprogrammed hFFs to a pluripotent state using 4 core transcription factors: Oct4 (O), Sox2 (S), Klf4 (K), and c-Myc (M). The proteome of hiPSCs induced by 4 core transcription factors was relatively similar to that of hESCs. However, several proteins, including dUTPase, GAPDH, and FUSE binding protein 3, were differentially expressed between hESCs and hiPSCs, implying that hiPSCs are not identical to hESCs at the proteomic level. The proteomes of iPSCs induced by introducing 3, 5, or 6 transcription factors were also analyzed. Our proteomic profiles provide valuable insight into the factors that contribute to the similarities and differences between hESCs and hiPSCs and the mechanisms of reprogramming.

The extremely halophilic archaeon Halobellus rufus type strain CBA1103(T) (CECT 8423(T) and JCM 19434(T)) was isolated from non-purified solar salt and characterized as an agarase producer. The draft genome sequence contains 3852 303 bp with a G + C content of 64.1% and includes genomic information on various carbohydrate-active enzymes. This is the first sequenced genome of the genus Halobellus, and is expected to provide general sequence information for halophilic carbohydrate-active enzymes and opportunities for biotechnological applications of novel halophilic enzymes.

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.

We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, “ischemia-responsive protein 94 kDa” (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation.

Using silkworm Bombyx mori Bm5 cells, we established a stable cell line expressing the human keratinocyte growth factor (hKGF), named by the Bm5-hKGF cell, in which the protein hKGF is synthesized in the cell and secreted in the cell culture supernatant (CCS) at approximately 15-20 ng/㎖. When the Bm5-hKGF cell was co-expressed with B. mori protein disulfide isomerase (bPDI) cDNA, its secretion increased by about two times the original amount. Through wound healing migration assay, it was demonstrated that the secreted hKGF included in the CCS has a very powerful biological activity of keratinocyte proliferation. We expect to produce useful human recombinant proteins from silkworm cultured cells in large quantities at low prices.

In the field of drug discovery and development, the increasing use of cell-based assays has resulted in an increased demand for novel cellular bioassays. Such bioassays are expected to detect a wide variety of functional molecules in live cells. Fluorescence-based imaging techniques such as fluorescence resonance energy transfer (FRET) and biomolecular fluorescence complementation (BiFC) have been developed to analyze protein–protein interactions (PPIs) and inhibition of PPIs (iPPIs) in live mammalian cells. Although these techniques have been useful, they require a variety of fusion constructs to determine the relative locations of fluorophores and binding pairs for optimal performance as well as appropriate linker domains. Alternatively, translocation-based cellular assays (redistribution approaches), which are cell-based assay techniques utilizing protein translocation as the primary readout, have been used to study the PPIs between specific proteins and other intracellular events. These methods use a bait (target) molecule fused to a protein that changes its localization within the cell following a stimulus. Such assays can be formatted as agonist or antagonist assays, in which compounds are tested for their ability to promote or inhibit, respectively, protein translocation caused by a known agonist. Translocation-based cellular assays do not require much construct optimization and boast a high signal-to-noise ratio. These assays are robust, fast, and flexible; thus, these systems have been considered as an ideal assay for high-contentscreening approaches to drug discovery. Despite these advantages, few experimental applications of translocationbased cellular assays have been reported. Most of these have been based on regulated transport between the cell nucleus and the cytoplasm using a combination of nuclear localization signals and/or nuclear export signals. Several technologies are already commercially available. Recently, the groups of Schultz and Heo independently reported that PPIs can be visualized by cotranslocation of a target protein from the cytoplasm to the plasma membrane and to the endosome, respectively. Schultz et al. demonstrated the direct cotranslocation of a protein complex through the Ca-induced translocation of a bait protein fused to Annexin A4, a phospholipidand Ca-binding protein. Heo and colleagues showed that Rab5, an endosome-localized protein, recruited an interacting protein to the endosome through an FKBP–rapamycin–FRB complex intermediate. These studies were focused on the visual detection of PPIs so that new conceptual and novel applications of redistribution approaches have vastly expanded what can be explored in live cells. Herein we demonstrate that the inhibition of protein– protein interactions (iPPI) using a small molecular inhibitor can be monitored directly by a redistribution approach. Protein kinase C (PKC) is known to translocate from the cytoplasm to the plasma membrane in response to physiological stimuli, as well as exogenous ligands such as phorbol esters. In a study using PKC tagged with green fluorescent protein (GFP) the dynamics of PKC translocation in response to different stimuli was monitored in real time in live cells. PKCd has a C1 domain that binds diacylglycerol, but an impaired C2 domain that does not bind Ca ions. Thus, PKCd responds to an increase in phorbol esters in the cell but not Ca ions. Therefore we hypothesized that a PKCd-fused bait protein would guide cotranslocation with the target protein, and a chemical inhibitor would interrupts PPI, making it possible to monitor iPPI (Scheme 1). To verify our approach, we examined iPPI using the p53 (tumor suppressor)/MDM2 (negative regulator of the p53) protein pair and Nutlin-3 (see the Supporting Information for experimental details). The small molecular inhibitor Nutlin-3 is a cis-imidazoline analogue commonly used in anticancer studies that inhibits the interaction between p53 and MDM2; this inhibitor resulted from the optimization of a lead structure identified by the screening of a chemical library. We prepared the C-terminal fusion constructs PKCd/monomeric red fluorescent protein (mRFP)/p53 (bait) and enhanced GFP (eGFP)/MDM2 (target). Both the pmRFP plasmid encoding PKCd–mRFP–p53 and the peGFP plasmid encoding eGFP–MDM2 were transiently cotransfected into HEK-293T cells. When the exogenous ligand phorbol 12-myristate 13-acetate (PMA) was added, both p53 [*] Dr. K.-B. Lee, J. M. Hwang, Dr. J.-S. Choi, Dr. G.-H. Kim, Dr. S. I. Kim, Dr. S. Kim, Dr. Z.-W. Lee Division of Life Science, Korea Basic Science Institute (KBSI) Daejeon 305-333 (Korea) Fax: (+82)42-865-3419 E-mail: shkim@kbsi.re.kr ezone@kbsi.re.kr J. M. Hwang, Prof. Dr. J. Rho Department of Bioscience and Biotechnology Chungnam National University, Daejeon 305-764 (Korea)

We investigated the wavelength dependence and photon-fluence rate response relationship for phototaxis of wild-type and a cyanobacterial phytochrome 2 (cph2) mutant in cyanobacterium Synechocystis sp. PCC 6803. Compared to wild-type, the cph2 mutant exhibited maximal activity for positive phototaxis at the near-UV spectral range. Two cysteine to serine substitutions in two chromophore-binding domains showed a similar cph2 mutant phenotype under UV-A. Epistasis of a pixJ mutation over a cph2 mutation implied that pixJ gene acts downstream of the cph2 gene with respect to UV-A-induced positive phototaxis. Therefore, we suggest that Cph2 is essential for the inhibition of positive phototaxis toward UV-A.

An important factor in evaluating porcine meat quality is the pH at post-mortem 24 h (pH 24 ). In the current work, we conducted a GeneFishing polymerase chain reaction (PCR)–based screen to identify differentially expressed genes in Berkshire loins with high versus low pH 24 . Total RNAs were extracted from Berkshire loins with high and low pH 24 and subjected to GeneFishing PCR. Four genes were found to be differentially expressed in the high and low pH 24 groups. Semi-quantitative reverse transcription (RT)-PCR confirmed that MYH1 encoding myosin heavy chain 1 was specifically expressed in high pH 24 groups. These results are very useful in establishing the genetic relationship between the composition of muscle fiber type and pH 24 in the Berkshire loin. Key words: GeneFishing TM polymerase chain reaction, myosin heavy chain 1, pork meat quality, post-mortem pH value, pale, soft and exudative.

Sericin is a type of high molecular weight water-soluble glycoprotein surrounding fibroin (silk protein) that has been used as a cell culture supplement and accelerates cell proliferation in various serum-free media. The purpose of this study was to investigate the enhancing effect of thyroglobulin (Tg) secretion by sericin in thyrocytes, FRTL-5 cells. While Tg-mRNA expression was not enhanced, a secreted form of Tg was obviously increased by sericin. In this status, expression of both endoplasmic reticulum (ER) molecular chaperones (Bip & calreticulin) and ER membrane proteins (IRE1, PERK & ATF6) was enhanced. The proximal step of IRE1, XBP1 mRNA splicing was slightly detected however, the proximal step of PERK, phosphorylation of eIF2α, was changeless. In addition, sericin enhanced cell viability by the MTT assay. The above results showing the ability of sericin to promote protein production demonstrated its potential usefulness as a new biomaterial.

We applied proteomics analysis to generate a map of the wild relatives of wheat grain proteins. These differentially expressed proteins are potentially involved in metabolism, stress responses, and other biological activities. Using two-dimensional electrophoresis, we detected 119, 134, and 193 reproducible spots on gels loaded with protein samples extracted from the A, B, and D genomes, respectively, of the mature grain. In all, 89, 53, and 54 distinct proteins, respectively, were found among these genomes through MALDI-TOF mass spectrometry. Of these, 26% (n = 52) proteins were considered distinct. They included 18.89% (n = 17) in the A, 28.30% (n = 15) in the B, and 37.04% (n = 20) in the D genome, all functioning in disease and defense roles. For example, the ABA-inducible protein PHVA1 can be induced by drought, cold, heat, and salinity, while the basic endochitinase confers protection against chitin-containing fungal pathogens. The diverse functional categories found here suggest different biological processes, such as disease/defense, energy metabolism, protein synthesis and storage, cellular organization, signal transduction, transcription, and the facilitation of transport. Our findings demonstrate that these functional proteins have important roles in stress tolerance and the maintenance of quality in mature grains. The interacting effects of genetics and environment on differential protein production may be partially mediated by a regulatory mechanism in those grains.

Porcine pancreas development is not well studied at the molecular level despite being a therapeutic resource for diabetic patients. In this study, we investigated expression of lineage markers and performed proteomic analysis. Expression of the early lineage markers Pdx1 and Ptf1a was developmentally conserved between mice and pigs, whereas expression of the islet differentiation marker Pax4 was delayed in porcine compared with murine pancreas development. Proteomic analysis found that expression levels of chymotrypsinogen were down-regulated during porcine pancreas development while those of digestive enzymes like lipases, elastase and serine protease were up-regulated. In addition, specific isoforms of protein folding assistants such as protein disulfide isomerase and prefoldin were expressed at specific stages during the maturation of digestive enzymes. Taken together, these results show that development of the porcine pancreas is regulated by a concerted interplay of pancreas lineage marker proteins and other specified proteins, resulting in a functional endocrine and exocrine organ.

The unicellular cyanobacterium Synechocystis sp. PCC 6803 glides toward a light source through the interplay of positive phototaxis genes and proteins. In genetic analysis, the complete disruption of the hybrid sensory kinase sll0043 produced negative phototaxis. Furthermore, Sll0043 was found to be a hub protein by in silico prediction of protein-protein interaction, in which Sll0043 was predominantly linked to seven two-component proteins with high confidence. To understand the regulation and networking of positive phototaxis proteins, the proteomic profile of the sll0043 mutant was compared to that of wild-type. In the sll0043 mutant, 18 spots corresponding to 15 unique proteins were altered by 1.3 to 59 fold; the spots were identified by 2-DE/MALDI-MS analysis. Down-regulated proteins in the sll0043 null-mutant included chaperonins, superoxide dismutase, and phycocyanin beta-subunit. In contrast, nine proteins involved in photosynthesis, translation, regulatory function, and other functions were up-regulated. In particular, a twitching motility protein (PilT1) was induced over 2-fold in sll0043 mutant. Moreover, semi-quantitative and quantitative RT-PCR analysis revealed that pilin (pilA1), pili motor (pilT1), and pili switch gene (pilT2) were significantly increased in sll0043 mutant. These results suggest that the hybrid kinase Sll0043 regulates positive phototaxis by suppressing the expression of pili biosynthesis and regulatory genes and through the interplay with positive phototaxis/motility two-component proteins.

We cloned a plant gene, Ntcyc07, conferring arsenite tolerance by expressing a tobacco expression library in WT yeast (Y800). Expression of Ntcyc07 increased the tolerance to As(III) and decreased its accumulation, suggesting that the enhanced As(III) tolerance resulted from a reduction of the intracellular arsenic level. Interestingly, expression of Ntcyc07 increased the expression of the As(III) export carrier ACR3, but repressed that of As(III) uptake channel FPS1. Ntcyc07p interacted with Acr1p, which is the transcriptional activator of ACR3, but not with the ACR3 promoter. Taken together, the data indicated that Ntcyc07p promoted As(III) tolerance by decreasing the intracellular level of As(III) via increasing the expression of ACR3 and reducing that of FPS1.

The use of aluminum sulfate [alum; Al2(SO3)4·14H2O] as top dressing to poultry litter has been proven in reducing ammonia (NH3) volatilization under both laboratory and field tests; however, there has been no information of alum application in mixing methods from poultry litter or rice hulls. The aim of the experiment was to evaluate the effects of alum top dressed or mixed with rice hulls as litter management methods on pH and NH3 emissions. A total of 180 broiler chickens were randomly allocated to 12 pens to a density of 0.07 m2/bird for 5 weeks, creating 4 replicates of 3 experimental treatments with 15 birds per experimental unit as a completely randomized design. The treatments included an untreated control, 100 g of alum (top dressing) and 100 g of alum (mixed)/kg of rice hull. In addition, alum treatment was usually applied by top dressing onto the rice hulls or fully mixed with the rice hulls. During the experimental period, pH and NH3 emissions were significantly reduced by the two different methods of alum amendments (P < 0.05) in the litter over time compared with the controls except for NH3 emissions at 1 through 3 weeks. However, no significant differences (P > 0.05) in pH and NH3 emission were observed between the two different methods with alum for 5 weeks. The reduction in NH3 emission from 100 g of alum top-dressed and 100 g of alum fully mixed with kg of rice hull at 5 weeks was 50 and 51%, respectively. In summary, these results indicate that “mixing” methods of alum as well as top dressing would serve as a suitable method for decreasing NH3 emission, which resulted in lower pH.Key words: Alum, top dressing, mixing, pH, ammonia.

We investigated the effects of rosemary extract (RE), α-tocopherol (AT) and chitosan (CH) added individually or in combination as compared with butylated hydroxyanisole (BHA) on microbiological parameters [total viable count (TVC), lactic acid bacteria (LAB), enterobacteria (ENB), pseudomonas bacteria (PSY)], pH and lipid oxidation of emulsion-type sausages stored for 28 days at 4°C. TVC, LAB, ENB, and PSY counts were significantly increased (P0.05) in all microbial counts between AT and control groups during the whole storage period. Overall storage had a significant effect on lowering pH, but no influence of additives on pH values was detected, except for 2and 28 days of storage. During refrigerated storage, CH and its combination, or BHA in emulsion-type sausages was more effective in delaying lipid oxidation compared to RE and AT (P

We investigated potential protein markers of post-mortem interval (PMI) using rat kidney and psoas muscle. Tissue samples were taken at 12 h intervals for up to 96 h after death by suffocation. Expression levels of eight soluble proteins were analyzed by Western blotting. Degradation patterns of selected proteins were clearly divided into three groups: short-term, mid-term, and long-term PMI markers based on the half maximum intensity of intact protein expression. In kidney, glycogen synthase (GS) and glycogen synthase kinase-3 beta were degraded completely within 48 h making them short-term PMI markers. AMP-activated protein kinase alpha, caspase 3 and GS were short-term PMI markers in psoas muscle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a mid-term PMI marker in both tissues. Expression levels of the typical long-term PMI markers, p53 and beta-catenin, were constant for at least 96 h post-mortem in both tissues. The degradation patterns of GS and caspase-3 were verified by immunohistochemistry in both tissues. GAPDH was chosen as a test PMI protein to perform a lateral flow assay (LFA). The presence of recombinant GAPDH was clearly detected in LFA and quantified in a concentration-dependent manner. These results suggest that LFA might be used to estimate PMI at a crime scene.

A gel-free and label-free quantitative proteomic approach based on a spectral counting strategy was performed to discover prolificacy-related proteins. Soluble proteins of porcine placenta from small litter size group (SLSG) and large litter size group (LLSG) were extracted and subsequently applied to in-solution tryptic digestion followed by liquid chromatography–tandem mass spectrometry analysis. Six and thirteen proteins were highly expressed in SLSG and LLSG, respectively. Of the dominantly expressed proteins, we chose prolificacy-related proteins such as puromycin-sensitive aminopeptidase (PSA) and retinol-binding protein 4 (RBP4). Western blot analysis confirmed that the processed form (70 kDa) of PSA was more expressed and RBP4 (23 kDa) was dominantly expressed in LLSG. These results indicate that PSA and RBP4 are representative proteins involved in porcine fertility traits, and this finding may help to increase litter size of pigs.

Tanshinone IIA is a diterpene quinone isolated from the roots of Salvia miltiorrhiza bunge that has traditionally been used in China for the treatment of cardiovascular and cerebrovascular disorders. Although there is recent evidence showing that tanshinone IIA has an anti-obesity effect, its underlying mechanism of anti-obesity effect is poorly understood. Here, we investigated the effect of tanshinone IIA on lipid accumulation in 3T3-L1 preadipocytes and zebrafish. Notably, tanshinone IIA at 10 μM concentration greatly reduced lipid accumulation and triglyceride (TG) contents during 3T3-L1 preadipocyte differentiation, suggesting its anti-adipogenic effect. On mechanistic levels, tanshinone IIA reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3/5 (STAT-3/5) in differentiating 3T3-L1 cells. In addition, tanshinone IIA strongly inhibited leptin and resistin mRNA expression in differentiating 3T3-L1 cells. Importantly, the tanshinone IIA’s lipid-reducing effect was also seen in zebrafish. In sum, these findings demonstrate that tanshinone IIA has anti-adipogenic effects on 3T3-L1 cells and zebrafish, and its anti-adipogenic effect on 3T3-L1 cells is largely attributable to the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3/5.

Burkholderia sp. K24 is an aniline-degrading soil bacterium that utilizes aniline and its analogues as sole carbon and nitrogen sources. Here, we report the draft genome sequence of this strain that consists of 8,344,181 bp, with a G+C content of 61.7%.

Secretory clusterin (sCLU), an anti-apoptotic protein, is overexpressed in many tumors and enhances tumorigenesis and chemo-resistance. However, the regulation mechanism controlling the sCLU maturation process or activity remains undetermined. In this study, we found PACAP as a negative regulator of CLU. Overexpression of the PACAP gene in cervical cancer cell lines lacking PACAP expression significantly inhibited cell growth and induced apoptosis. We further demonstrated that interaction of PACAP with CLU significantly downregulated CLU expression and secretion, inhibited the Akt–Raf–ERK pathway, and suppressed the growth of human tumor xenografts in nude mice. This novel inhibitory function of PACAP may be applicable for developing novel molecular therapies for tumors with increased sCLU expression.

Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a-7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases' inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of these meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation., Author Summary Although enteropathogenic sapoviruses and noroviruses are leading causes of acute gastroenteritis in both humans and animals, the study of viral pathogenesis and immunity of these ubiquitous pathogens has been hampered due to the lack of a fully permissive cell culture system. Porcine sapovirus Cowden strain provides a suitable system that can be used to identify the molecular mechanisms of viral pathogenesis. Previous studies have shown that carbohydrates and glycolipids play important roles in the attachment of members of the Caliciviridae; histo-blood group antigens (HBGAs) are used by Norovirus genogroups I to IV, as well as members of the Lagovirus, and Recovirus genera, whereas terminal sialic acid is recognized as a receptor for feline calicivirus and murine norovirus. To date, however, the role of carbohydrates in the life cycle of sapoviruses has remained largely unknown. We found that porcine sapovirus binds to susceptible host cells through both α2,3- and α2,6-linked terminal sialic acids which are attached to O-linked glycoproteins. These efforts, findings and insights will significantly contribute to a better understanding of the sapovirus life cycle.

We generated random Tn5 mutations in Synechocystis sp. PCC 6803 in search for genes involved in the signal transduction cascade for the cyanobacterial gliding motility. One of the non-gliding Tn5 mutants, S1-105, had an insertional inactivation in the slr1044 gene encoding a putative methyl-accepting chemotaxis protein. Interposon mutation on the slr1044 (named ctr1) in the bacterium also eliminated gliding motility. In the interposon mutant, the expression of pilA1 was 5-fold decreased compared with that of wild-type and thick pili, that are believed to be the motor for gliding, could not be observed by an electron microscope. Therefore, we suggest that the Ctr1 protein functions as a transducer that regulates the expression of pilA1, and thus is required for the biogenesis of thick pili.

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.

Our previous works have demonstrated that fast atom bombardment tandem mass spectrometry can be a valuable tool in determining the complete structure of glycoglycerolipids and glycerophospholipids. Collision-induced dissociation of sodium-adducted molecular ions ([M + Na] + or [M − H + 2Na] + ) generates diverse product ions informative on the double-bond position in fatty acyl groups as well as the polar head group and fatty acid composition. Here we report that this direct and rapid method can be applied to the structural determination of individual molecular species of each glycerolipid class purified from the total lipid extract of cyanobacterium Synechocystis sp. PCC 6803. Especially, based on the preference for the loss of the fatty acyl group positioned at the sn -2, it was proved that all of the molecular species of diacylglycerolipids contained a palmitoyl group exclusively at the sn -2 position. Additionally, lysoglycerolipids, monoacyl forms of four major membrane diacylglycerolipids, were first isolated together from a fresh extract. Using fast atom bombardment mass spectrometry and tandem mass spectrometry, it was found that each lysoglycerolipid had a molecular species with only palmitic acid as a fatty acyl group. Thus, these compounds could be synthesized by specific enzyme-catalyzed hydrolysis of the sn -1 fatty acyl group of the corresponding diacylglycerolipids.

Lectins found in fish tissues play an important role in the innate immune response against viral infection. A fucose-binding type lectin, RbFTL-3, from rock bream (Oplegnathus fasciatus) was identified using expressed sequence tag (EST) analysis. The expression of RbFTL-3 mRNA was higher in intestine than other tissues of rock bream. To determine the function of RbFTL-3, VHSV-susceptible fathead minnow (FHM) cells were transfected with pcDNA3.1(+) or pcDNA3.1(+)-RbFTL-3 and further infected with VHSV. The results show that the viability of FHM cells transfected with pcDNA3.1(+)-RbFTL-3 is higher than that of cells transfected with pcDNA3.1(+) (relative cell viability: 28.9% vs 56.2%). A comparative proteomic analysis, performed to explore the proteins related to the protective effect of RbFTL-3 in the cells during VHSV infection, identified 90 proteins differentially expressed in VHSV-infected FHM cells transfected with pcDNA3.1(+) or pcDNA3.1(+)-RbFTL-3. The expression of RbFTL-3 inhibits a vascular-sorting protein (SNF8) and diminishes the loss of prothrombin, which are closely associated with controlling viral budding and hemorrhage in fish cells, respectively. Subsequent Ingenuity Pathways Analysis enabled prediction of their biofunctional groupings and interaction networks. The results suggest RbFTL-3 modulates the expression of proteins related to viral budding (SNF8, CCT5 and TUBB) and thrombin signaling (F2) to increase the viability of VHSV infected cells.

Pseudomonas taeanensis MS-3 T , isolated from a crude oil-contaminated seashore in South Korea, is capable of degrading petroleum oils, such as gasoline, diesel, and kerosene. Here, we report the draft genome sequence of this strain, which consists of 5,477,045 bp, with a G+C content of 60.72%.

A halophilic archaeon, designed strain CBA1103(T), was isolated from non-purified solar salt. The cells of strain CBA1103(T) were observed to be Gram-stain negative and pleomorphic, and the colonies appear red. Strain CBA1103(T) was observed to grow between 20 and 55 °C (optimum 37 °C), and in NaCl concentrations of 10-30 % (w/v) (optimum 15 %) with 0-0.5 M MgSO4·7H2O (optimum 0.1 M) and at pH 6.0-9.0 (optimum pH 7.0). Additionally, the cells lyse in distilled water. The major polar lipids of strain CBA1103(T) are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and two glycolipids chromatographically identical to sulfated mannosyl glucosyl diether and manosyl glucosyl diether. Strain CBA1103(T) is shown to belong to the Halobellus genus and exhibits similarity to related taxa; the 16S rRNA gene sequence similarity between strain CBA1103(T) and Halobellus rarus 18362(T), Hbs. limi 16811(T), Hbs. litoreus JCM 17118(T), Hbs. inordinatus YC20(T), Hbs. clavatus TNN18(T) and Hbs. salinus CSW2.24.4(T) is 97.3, 96.5, 96.5, 94.5, 94.5 and 93.7 %, respectively. The RNA polymerase subunit B gene sequence of strain CBA1103(T) shows 93.7 % similarity with the sequence of Hbs. litoreus JCM 17118(T); the similarity is lower with sequences from the type strains of other species of Halobellus. The genomic DNA G+C content of strain CBA1103(T) was determined to be 67.0 mol% a value which is in the range of the genomic DNA G+C content of members of the genus Halobellus (61.5-69.2 mol%). These results suggest that strain CBA1103(T) should be considered to represent a new taxon for which the name Halobellus rufus sp. nov. is proposed, with the type strain CBA1103(T) (=CECT 8423(T) =JCM 19434(T)).

Accumulative evidence suggests ginseng extract and/or its major components, ginsenosides and compound K, a metabolized ginseng saponin, have anti-cancer effects. In the present study, the effects of a ginseng butanolic extract (GBX) and an enzymatically fortified ginseng extract (FGX), with enriched ginsenosides and compound K, on the growth of KATO3 human gastric cancer cells were investigated using a cell viability assay. While treatment with GBX at 31.25-125 mg/ml for 24 h did not affect the proliferation of KATO3 cells, FGX under the same conditions inhibited cell proliferation in a concentration-dependent manner. Furthermore, Annexin V/PI-staining and flow cytometric analysis demonstrated that the population of apoptotic KATO3 cells was increased following treatment with FGX, which was greater than in the GBX-treated cells, suggesting that FGX had a stronger apoptotic effect than GBX. To investigate the underlying mechanism of the cytostatic and cytotoxic effects of the ginseng extracts, apoptosis-associated proteins were assessed using western blot analysis. The data revealed higher expression levels of B-cell lymphoma 2-associated X protein (Bax), lower expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) and reduced phosphorylation of mammalian target of rapamycin (mTOR) and protein kinase B (PKB) in the FGX-treated KATO3 cells than in the GBX-treated cells. Collectively, these results demonstrated for the first time, to the best of our knowledge, that FGX had stronger anti-proliferative and pro-apoptotic effects on KATO3 cells than GBX. The anti-proliferative and/or pro-apoptotic effects of FGX appeared to be mediated via the upregulation of Bax, IκBα proteolysis (activation of nuclear factor-κB) and the blocking of mTOR and PKB signals.