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Lactobacillus plantarum 510, previously isolated from a koshu vineyard in Japan, was found to produce a bacteriocin-like inhibitory substance which was purified and characterized. Mass spectrometry analysis showed that the mass of this bacteriocin is 4,296.65 Da. A partial sequence, NH2- SSSLLNTAWRKFG, was obtained by N-terminal amino acid sequence analysis. A BLAST search revealed that this is a unique sequence; this peptide is thus a novel bacteriocin produced by Lactobacillus plantarum 510 and was termed plantaricin Y. Plantaricin Y shows strong inhibitory activity against Listeria monocytogenes BCRC 14845, but no activity against other pathogens tested. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was completely inactivated by protease K. Furthermore, trypsin-digested bacteriocin product fragments retained activity against L. monocytogenes BCRC 14845 and exhibited a different inhibitory spectrum.

Electrospun fiber can be used as a carrier for releasing active ingredients at the target site to achieve the effects of drug treatment. The objectives of this research work were to study suitable conditions for producing electrospun gelatin fiber loaded with crude Curcuma comosa Roxb. extract (CE) and to study antioxidant, anti-tyrosinase and anti-bacterial activities and its freeze&ndash, thaw stability as well. To achieve optimal conditions for producing electrospun gelatin fiber, the concentration of gelatin was adjusted to 30% w/v in a co-solvent system of acetic acid/water (9:1 v/v) with a feed rate of 3 mL/h and an applied voltage of 15 kV. The lowest percent loading of 5% (w/v) CE in gelatin nanofiber exhibited the highest DPPH radical scavenging activity of 94% and the highest inhibition of tyrosinase enzyme of 35%. Moreover, the inhibition zones for antibacterial activities against S. aureus and S. epidermidis were 7.77 &plusmn, 0.21 and 7.73 &plusmn, 0.12 mm, respectively. The freeze&ndash, thaw stability of CE in electrospun gelatin nanofiber was significantly different (p &lt, 0.05) after the 4th cycle as compared to CE. Electrospun gelatin nanofiber containing CE also showed the capacity of the release of bioactive ingredients possessing anti-oxidant properties and, therefore, it could potentially be used for face masks.

Summary Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. This study investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis, on prolonged maintenance and expansion of cord blood CD34+ cells. Our results showed that the presence of NTL or Flt-3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum- and cytokine-free culture for 35 d. The effect of NTL on the ex vivo expansion of CD34+ cells in the presence of stem cell factor, thrombopoietin (TPO) and FL was also investigated. NTL-enhanced expansion of early progenitors (CD34+, CD34+CD38−, mixed colony-forming units and CFU-GEMM) and committed progenitor cells (granulocyte CFU, erythroid burst-forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d-expanded cells to non-obese diabetic severe combined immunodeficient mice, increased engraftment of human CD45+ cells was observed in the bone marrow of animals that received NTL-treated cells. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.

Persistent hepatitis B virus (HBV) infection is a major risk of hepatocellular carcinoma (HCC). One intriguing feature of HBV-related HCC is the male predominance, with a male to female ratio of 5–7:1. This dominance has been attributed to the elevated androgen level and the enhanced androgen receptor (AR)-mediated activity in the host. How HBV infection and AR signaling modulate HCC is unknown. We investigated whether the HBV nonstructural protein, X protein (HBx) could cooperate with the AR signaling pathway to enhance carcinogenesis. We found that HBx increased the anchorage-independent colony-formation potency of AR in a nontransformed mouse hepatocyte cell line. We also found that HBx functioned as a positive transcriptional coregulator to increase AR-mediated transcriptional activity. This transcription enhancement was increased in the presence of androgen in a concentration-responsive manner, thus explaining a more prominent effect in males. HBx did not physically associate with ligand-bound AR in the nucleus, and it likely augmented AR activity by increasing the phosphorylation of AR through HBx-mediated activation of the c-Src kinase signaling pathway. Our study documents HBx as a previously undescribed class of noncellular positive coregulators for AR. The results reveal a mechanism for the vulnerability of males to microbial infections and the subsequent development of cancer.

Nampt/visfatin acts in both intracellular and extracellular compartments to regulate multiple biological roles, including NAD metabolism, cancer, inflammation, and senescence. However, its function in chronic inflammation and carcinogenesis in hepatocellular carcinoma (HCC) has not been well-defined. Here we use Huh-7 hepatoma cells as a model to determine how Nampt/visfatin affects cellular survival under oxidative stress. We found that the transition of Nampt/visfatin from intracellular into extracellular form was induced by H2O2treatment in 293T cells and confirmed that this phenomenon was not due to cell death but through the secretion of Nampt/visfatin. In addition, Nampt/visfatin suppressed cell viability in oxidative treatment in Huh-7 cells and acted on the inhibition of hepatoma cell growth. Oxidative stress also reduced the Nampt-mediated activation of NF-κB gene expression. In this study, we identify a novel feature of Nampt/visfatin which functions as an adipokine that can be secreted upon cellular stress. Our results provide an example to understand how adipokine interacts with chemotherapeutic treatment by oxidative stress in HCC.

A sulphated polysaccharide (SP-2a) from the brown alga Sargassum patens (Kütz.) Agardh (Sargassaceae) was found to significantly inhibit the in vitro replication of both the acyclovir (ACV)-sensitive and -resistant strains of Herpes simplex virus type 1 (HSV-1), in dose-dependent manners, with 50% inhibitions occurring with 1.5-5.3 microg/ml of the polysaccharide. SP-2a exhibited extracellular virucidal activity only against the ACV-sensitive strains, but not the resistant strain, at the concentration of 100 microg/ml. The strongest antiviral activities against the different strains of HSV-1 were observed when this polysaccharide was present during and after adsorption of the virus to host cells. The inhibitory effect of SP-2a on virus adsorption occurred dose-dependently in all the HSV-1 strains tested, and the adsorption of the ACV-resistant DM2.1 strain was reduced by 81.9% (relative to control) with 4 microg/ml of the polysaccharide. This study clearly demonstrated that the antiviral mode of action of SP-2a is mediated mainly by inhibiting virus attachment to host cells, and this sulphated polysaccharide might have different modes of action against the ACV-sensitive and -resistant strains of HSV-1.

Six hot alkali extracts (HAE-1 to HAE-6) of mushroom (1→3)-β-glucans from the sclerotia of Pleurotus tuber-regium having different molecular weight (Mw) ranging from 1×104 to 42×104 were carboxymethylated to give their corresponding water-soluble derivatives (CMHAE-1 to CMHAE-6) with Mw ranged from 2.08×104 to 53.2×104. In general, the CMHAE β-glucans had higher water solubility and higher in vivo (Sarcoma 180 solid tumor implanted on BALB/c mice) as well as in vitro (HL-60 tumor cell culture) anti-tumor activity than the native HAE β-glucans. From ELISA assay of cytokines, CMHAE fractions could increase production of tumor necrosis factor alpha (TNF-α) in mouse plasma stimulated by lipopolysaccharide (LPS). It was postulated that a more expanded flexible chain of these novel CMHAE β-glucans might account for their in vivo and in vitro anti-tumor activities.

A sulphated polysaccharide (SP2) was isolated from the brown alga Sargassum patens. SP2 inhibited the replication of herpes simplex virus type 2 (HSV-2) dose-dependently by 38.5-96.1% of the control level, after incubations with 0.78-12.5 microg/ml of the polysaccharide. SP2 exhibited extracellular virucidal activity only in high concentrations (>/=12.5 microg/ml) but significantly inhibited the virus attachment to its host cells by 45.1%, at concentration as low as 1 microg/ml. All the results from this study suggested that the antiviral mode of action of SP2 could be ascribed to the inhibition of virus adsorption, which is different from that of the current drug of choice acyclovir.

The acute phase response (APR) in liver during inflammation is one of the well known examples for elucidating the signaling pathways that lead to the combinatorial regulation of gene expression. The APR is exemplified by alpha(1)-acid glycoprotein gene (agp) expression. A number of transcription factors, including CCAAT/enhancer-binding protein beta (C/EBPbeta), glucocorticoid receptor, cAMP-response element-binding protein (CREB), and Nopp140, are known to participate in its induction. The underlying mechanism of Nopp140 and other factors for regulating agp expression remains unclear. Here we demonstrate that protein kinase A (PKA)-dependent phosphorylation of Nopp140, together with C/EBPbeta, induces agp gene expression synergistically. The cooperative activation of the agp gene by Nopp140 and forskolin is sensitive to inhibition by PKI. Results from biochemical and functional characterizations of Nopp140 mutants defective in PKA phosphorylation sites suggest that PKA-dependent Nopp140 phosphorylation is important for its role in agp gene activation. Furthermore, maximal activation of the agp gene by PKA-phosphorylated Nopp140 depends on the presence of CREB and C/EBPbeta. The participation of CREB in the activation is, however, independent of its PKA-mediated phosphorylation. In summary, we demonstrate the existence of a novel Nopp140-mediated PKA signaling pathway that leads to the activation of agp, one of the major acute phase response genes.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a state-of-the-art method of structural analysis of peptides/proteins. Here, using activating transcription factor-2 (ATF2) as an example, we report how LC/MS/MS data were processed to generate selected ion tracings for identification of phosphorylated peptides based on their parallel elution behavior with their nonphosphorylated analogs. Via this approach, we verified that amino acid residues Thr-69, Thr-71, and Ser-90 of ATF2 were the in vitro targets for c-Jun kinase. Selected ion tracing method was also used to quantitatively determine phosphorylation states of peptides. We demonstrated that the phosphorylation of Thr-69/Thr-71 was increased in response to ultraviolet irradiation specifically in subconfluent but not in confluent cultures. About 24% of Thr-69/Thr-71-containing segment were singly phosphorylated in subconfluent cultures, while minimal phosphorylation occurred in confluent cultures. In contrast, Ser-112 phosphorylation remained unaffected by cell densities. This strategy could be applied to the studies of a variety of modifications seen in various regulated cellular processes.

A coccal strain isolated from fresh broccoli was initially identified asEnterococcus saccharolyticus; however, molecular identification and phenotypic traits did not support this identification. DNA–DNA hybridization with the type strain ofE. saccharolyticus(76.4 % relatedness), DNA G+C content (35.7 mol%), phylogenetic analysis based on 16S rRNA,pheSandrpoAgene sequences, rep-PCR fingerprinting and profiles of cellular fatty acids, whole-cell proteins and enzyme activities, together with carbohydrate metabolism characteristics, indicated that this strain is distinct and represents a novel subspecies, for which the nameEnterococcus saccharolyticussubsp.taiwanensissubsp. nov. is proposed. The type strain is 812T( = NBRC 109476T = BCRC 80575T). Furthermore, we present an emended description ofEnterococcus saccharolyticusand proposal ofEnterococcus saccharolyticussubsp.saccharolyticussubsp. nov. (type strain ATCC 43076T = CCUG 27643T = CCUG 33311T = CIP 103246T = DSM 20726T = JCM 8734T = LMG 11427T = NBRC 100493T = NCIMB 702594T).

Lactobacillus pobuzihii E100301 T is a novel Lactobacillus species previously isolated from pobuzihi (fermented cummingcordia) in Taiwan. Phylogenetically, this strain is closest to Lactobacillus acidipiscis , but its phenotypic characteristics can be clearly distinguished from those of L. acidipiscis . We present the draft genome sequence of strain L. pobuzihii E100301 T .

Androgen and androgen receptor (AR) have long been implicated in liver carcinogenesis, especially for the male dominance feature. However, whether AR gene could occur in somatic mutations that might contribute to this process has not yet been studied. DNA sequencing and genotyping were conducted for detecting the genetic aberrations of AR gene in 257 primary hepatocellular carcinomas (HCCs) and also the dysplastic nodules (DN) from another 11 patients. Twenty-one AR somatic mutations causing amino acid changes were identified in HCC and even in the precancerous DN. The missense somatic mutations of AR were rare in HCC (2 cases) but the trinucleotide repeat (TNR) changes, both at (CAG)n and (GGC)n, was a more common one (19 cases). Notably, all these mutations occurred in male patients and most TNR changes belonged to the contraction type (15 out of 19 cases, 78.9%), which has been reported to associate with increased AR transcriptional activity. Most samples with TNR changes did not show microsatellite instability, suggesting a different cause for these TNR mutations. Although no significant correlation was identified between AR mutations and the clinicopathologic parameters, we found the (CAG)n length significantly shorter in hepatitis B virus (HBV)(+) HCCs than in HBV(-) HCCs and the (GGC)n length significantly correlates with the overall survival. In conclusion, the mis-sense somatic mutations of AR were rare in HCC but the TNR change was a more common one, which exclusively occurred in males. Moreover, the length of TNR carried clinical significance in special HCC group.

Severe acute respiratory syndrome (SARS)‐CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. To study the humoral responses against SARS‐CoV, we evaluated nucleocapsid (N) and spike (S) proteins‐specific antibodies in patients' sera by Western blotting and enzyme‐linked immunosorbent assay (ELISA). Recombinant N and S proteins of SARS‐CoV were purified from transformed E. coli. Serum specimens from 40 SARS‐CoV‐infected patients in the convalescent phase were analyzed by Western blotting using the purified antigens. Serial serum specimens from 12 RT‐PCR‐confirmed SARS patients were assayed by ELISA using the recombinant N protein as coated antigen. By Western blotting, 97.5% of the SARS patients were positive for N protein‐specific antibodies whereas only 47.5% of the samples were positive for S protein‐specific antibodies. Using N protein‐based ELISA, 10 out of the 12 patients were positive for N protein‐specific antibodies and 6 of them showed seroconversion at mean of 16 days after onset of fever. Immunoblotting was useful for detecting the humoral immune response after SARS‐CoV infection. Antibodies against SARS‐CoV N protein appear at the early stage of infection, therefore, N protein‐based ELISA could serve as a simple, sensitive, and specific test for diagnosing SARS‐CoV infection. J. Med. Virol. 73:338–346, 2004. © 2004 Wiley‐Liss, Inc.

Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.

Ex vivo expansion of hematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. In this study, we investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis on prolonged maintenance and expansion of cord blood CD34+ cells. Enriched CD34+ cells (1 x 105/mL, n=5) or mononuclear cells (1 x 106/mL, n=8) were cultured in X-VIVO-10 medium for 14, 21, 28 and 35 days without supplementary cytokine or medium changing. Our results showed that the presence of NTL (200 ng/mL) or FL-3 ligand (FL, 40 ng/mL) significantly preserved populations of early stem/progenitor cells (total CFU, BFU/CFU-E, CFU-GM, CFU-GEMM) in these cultures, compared with respective controls at various time points. In the ex vivo expansion study (n=16), the presence of stem cell factor (S, 50 ng/mL), thrombopoietin (T, 50 ng/mL), FL (F, 80 ng/mL) effectively expanded total nucleated cells (TNC) at day 8 (116 ± 20.2 fold) and day 12 (424 ± 68.8 fold), as well as all subsets of progenitor cells as demonstrated by flow cytometry and CFU assays. The presence of NTL (200 ng/mL) significantly increased TNC (148 ± 24.5 fold at day 8; 572 ± 91.9 fold at day 12; P < 0.01) and expansion of early progenitor cells (CD34+, CD34+CD38−, CFU-GEMM) and committed CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E) and the megakaryocytic lineage (CFU-MK) (P < 0.01 compared with respective TSF cultures). There was also slight but consistent increase of CD61+CD41+ cells in the presence of NTL (8.58 ± 2.14 x 105 vs. 7.30 ± 1.82 x 105 cells/mL, P < 0.001). Significantly, the increased expansion was not only contributed by the higher TNC, but also by the increase in the proportion of CD34+ cells, CD34+CD38− cells and the density of differential CFU. Six weeks after enriched CD34+ cells at day 0 or expanded cells at day 12 were infused into sub-lethally irradiated NOD/SCID mice, human CD45+ cells were detectable in the BM, spleen and PB of the mice. In the BM, there were engraftments of human hematopoietic cells of the early (CD34+), myeloid (CD33+, CD14+), B-lymphoid (CD19+) and megakaryocytic (CD61+) lineages. In animals that received day 12 expanded cells in the TSF + NTL group, there was a significant increase of human CD45+ cells in the BM (19.3% vs. 11.5%, P = 0.03, n = 15) when compared with those only exposed to TSF, and a trend of increased engraftment in their spleen (P = 0.07, n = 14). Comparison of the complete amino acid sequences of NTL and FRIL (a dicot mannose-binding lectin shown to preserve hematopoietic stem cells, PNAS, 96, 646–650, 1999) showed 10.2% identity and both peptides contain putative functional/structural sites such as those for N-myristoylation, casein kinase II phosphorylation, protein kinase C phosphorylation and N-glycosylation. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.