6,6'-Bieckol inhibits adipocyte differentiation through downregulation of adipogenesis and lipogenesis in 3T3-L1 cells.

BACKGROUND: Brown algae have been used for their nutritional value as well as a source of bioactive compounds with antioxidant, anti-inflammatory, antimicrobial and anti-obesity effects. Obesity is an important condition implicated in various diseases, including diabetes, hypertension, dyslipidemia and coronary heart disease. However, anti-obesity effects of Eisenia bicyclis remain unknown. RESULTS: We investigated the anti-obesity effects of 6,6'-bieckol, 6,8'-bieckol, 8,8'-bieckol, dieckol and phlorofucofuroeckol A isolated from E. bicyclis. Anti-obesity activity was evaluated by examining the inhibition of differentiation of 3T3-L1 adipocytes and the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCATT/enhancer-binding protein α (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c) at the mRNA and protein level. Differentiated 3T3-L1 cells were treated with the purified phlorotannins at concentrations of 10, 25 and 50 µg mL(-1) for 8 days. The results indicated that the purified phlorotannins suppressed the differentiation of 3T3-L1 adipocytes in a dose-dependent manner, without toxic effects. Among the five compounds, 6,6'-bieckol markedly decreased lipid accumulation and expression levels of PPARγ, C/EBPα, SREBP-1c (mRNA and protein), and fatty acid synthase and acyl-coA carboxylase (mRNA). CONCLUSION: These findings suggest that E. bicyclis suppressed differentiation of 3T3-L1 adipocyte through downregulation of adipogenesis and lipogenesis.

Carboxylicivirga gen. nov. in the family Marinilabiliaceae with two novel species, Carboxylicivirga mesophila sp. nov. and Carboxylicivirga taeanensis sp. nov., and reclassification of Cytophaga fermentans as Saccharicrinis fermentans gen. nov., comb. nov.

Two facultatively anaerobic mesophilic bacteria, strains MEBiC 07026(T) and MEBiC 08903(T), were isolated from two different tidal flat sediments and both strains showed approximately 92.2 % 16S rRNA gene sequence similarity with [Cytophaga] fermentans DSM 9555(T). 16S rRNA gene sequence similarity between the two new isolates was 97.5 % but levels of DNA-DNA relatedness between the two were 31.3-31.8 %. Phylogenetic analysis revealed that the two isolates and [Cytophaga] fermentans DSM 9555(T) were affiliated with the family Marinilabiliaceae in the class Bacteroidia. The dominant fatty acids of strains MEBiC 07026(T), MEBiC 08903(T) and [Cytophaga] fermentans DSM 9555(T) were branched-type or hydroxylated C15 : 0, but [Cytophaga] fermentans DSM 9555(T) contained a higher proportion of anteiso-branched fatty acids. The two new isolates contained a markedly higher proportion of monounsaturated fatty acids than other members of the family Marinilabiliaceae. The major respiratory quinone of the strains was MK-7. Strains MEBiC07026(T) and MEBiC08903(T) utilized a wide range of carboxylic acids whereas [Cytophaga] fermentans DSM 9555(T) utilized carbohydrates rather than carboxylic acids. The DNA G+C content of the novel strains was about 44 mol% but that of [Cytophaga] fermentans DSM 9555(T) revealed from the genome sequence was 37.6 mol%. Based on evidence from this polyphasic taxonomic study, a novel genus, Carboxylicivirga gen. nov., is proposed in the family Marinilabiliaceae with two novel species, Carboxylicivirga mesophila sp. nov. with type strain MEBiC 07026(T) ( = KCCM 42978(T) = JCM 18290(T)) and Carboxylicivirga taeanensis sp. nov. with type strain MEBiC 08903(T) ( = KCCM 43024(T) = JCM 19490(T)). Additionally, [Cytophaga] fermentans DSM 9555(T) ( = ATCC 19072(T)) is reclassified as Saccharicrinis fermentans gen. nov., comb. nov.

Enantioselective hydrolysis of racemic styrene oxide and its substituted derivatives using newly-isolated Sphingopyxis sp. exhibiting a novel epoxide hydrolase activity.

(S)-Styrene oxide, (S)-2-chlorostyrene oxide (CSO), (S)-3-CSO and (S)-4-CSO with 99.9 %ee were obtained with a yield of 20.6, 39.3, 28.7 and 26.8 % from 4 mM corresponding racemic substrates using 10 mg cells of a newly-isolated Sphingopyxis sp. at pH 8.0 and 25 °C in 1 ml 100 mM Tris/HCl buffer after 420, 100, 120 and 55 min, respectively. For racemic 2CSO, well-known for one of the racemates that is difficult to obtained in enantiomerically pure form, (S)-2-CSO with 99.9 %ee, 39.3 % yield (theoretical yield 50 %) and enantiomeric ratio of 42.1 was obtained. The newly-isolated strain can thus be used as whole-cell biocatalyst in the production of various (S)-CSO with a chlorine group at different positions.

Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives.

A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.

Cytotoxicity of human recombinant arginase I (Co)-PEG5000 in the presence of supplemental L-citrulline is dependent on decreased argininosuccinate synthetase expression in human cells.

Human recombinant arginase I cobalt [HuArgI (Co)] coupled with polyethylene glycol 5000 [HuArgI (Co)-PEG5000] has shown potent in-vitro depletion of arginine from tissue culture medium. We now show that HuArgI (Co)-PEG5000 is toxic to almost all cancer cell lines and to some normal primary cells examined. In contrast, HuArgI (Co)-PEG5000 in combination with supplemental L-citrulline is selectively cytotoxic to a fraction of human cancer cell lines in tissue culture, including some melanomas, mesotheliomas, acute myeloid leukemias, hepatocellular carcinomas, pancreas adenocarcinomas, prostate adenocarcinomas, lung adenocarcinomas, osteosarcomas, and small cell lung carcinomas. Unfortunately, a subset of normal human tissues is also sensitive to HuArgI (Co)-PEG5000 with L-citrulline supplementation, including umbilical endothelial cells, bronchial epithelium, neurons, and renal epithelial cells. We further show that cell sensitivity is predicted by the level of cellular argininosuccinate synthetase protein expression measured by immunoblots. By comparing a 3-day and 7-day exposure to HuArgI (Co)-PEG5000 with supplemental L-citrulline, some tumor cells sensitive on short-term assay are resistant in the 7-day assay consistent with the induction of argininosuccinate synthetase expression. On the basis of these results, we hypothesize that HuArgI (Co)-PEG5000 in combination with L-citrulline supplementation may be an attractive therapeutic agent for some argininosuccinate synthetase-deficient tumors. These in-vitro findings stimulate further development of this molecule and may aid in the identification of tissue toxicities and better selection of patients who will potentially respond to this combination therapy.

Isolation of a novel strain, Sphingorhabdus sp. YGSMI21 and characterization of its enantioselective epoxide hydrolase activity.

Sphingorhabdus sp. YGSMI21, a novel microbial strain with an enantioselective epoxide hydrolase activity, was isolated from tidal samples contaminated by accidental oil spills subjected to enriched culture with polycyclic aromatic hydrocarbon. This strain was able to optically decompose (R)-styrene oxide (SO) and showed 100% optical purity. In addition, it showed a good enantioselectivity for the derivatives of (S)-SO, (S)-2-chlorostyrene oxide (CSO), (S)-3-CSO and (S)-4-CSO. For (S)-2-CSO, (S)-3-CSO and (S)-4-CSO, 99.9%ee was obtained with the yield of 26.2%, 24.8%, and 11.0%, respectively, when using 10 mg cells of Sphingorhabdus sp. YGSMI21 at pH 8.0 with 4 mM racemic substrates at pH 8.0 and 25°C. The values obtained in this study for (S)-2-CSO, particularly the yield of 26.2%, is noteworthy, considering that obtaining an enantiomerically pure form is difficult. Taken together, Sphingorhabdus sp. YGSMI21 can be regarded as a whole-cell biocatalyst in the production of various (S)-CSO with the chlorine group at a different position.

RLIP76 and Cancer.

RLIP76 is a multifunctional membrane protein that transports glutathione conjugates of electrophilic compounds and other xenobiotics including chemotherapy agents out of cells. The protein is overexpressed in lung carcinomas, ovarian carcinomas, and melanomas. The protein also binds Ral and participates in mitotic spindle function, clathrin-dependent endocytosis, and triggers GTPase-activating protein activity. It is found throughout the cell, in membrane, cytosol, and the nucleus, and is known to shift between these compartments in response to stress. Loss of RLIP76 by antibody or antisense therapy is associated with increased sensitivity to radiation and chemotherapy. Conversely, liposomally delivered RLIP may treat poisoning and wounds.

Cloning and characterization of an epoxide hydrolase from Novosphingobium aromaticivorans.

A gene encoding a putative epoxide hydrolase (EHase) was identified by analyzing an open reading frame of the genome sequence of Novosphingobium aromaticivorans, retaining the conserved catalytic residues such as the catalytic triad (Asp177, Glu328, and His355) and the oxyanion hole. The enantioselective EHase gene (neh) was cloned, and the recombinant EHase could be purified to apparent homogeneity by one step of metal affinity chromatography and further characterized. The purified N. aromaticivorans enantioselective epoxide hydrolase (NEH) showed enantioselective hydrolysis toward styrene oxide, glycidyl phenyl ether, epoxybutane, and epichlorohydrin. The optimal EHase activity toward styrene oxide occurred at pH 6.5 and 45 degrees C. The purified NEH could preferentially hydrolyze (R)-styrene oxide with enantiomeric excess of more than 99% and 11.7% yield after 20-min incubation at an optimal condition. The enantioselective hydrolysis of styrene oxide was also confirmed by the analysis of the vicinal diol, 1-phenyl-1,2-ethanediol. The hydrolyzing rates of the purified NEH toward epoxide substrates were not affected by as high as 100 mM racemic styrene oxide.

Identification of a peptide sequence that improves transport of macromolecules across the intestinal mucosal barrier targeting goblet cells.

In this study, we demonstrated that the CSKSSDYQC-peptide ligand which was identified from a random phage-peptide library through an in vivo phage display technique with rats could prominently improve the transport efficiency of macromolecules, such as large filamentous phage particles (M13 bacteriophage), across the intestinal mucosal barrier. Synthetic CSKSSDYQC-peptide ligands significantly inhibited the binding of phage P1 encoding CSKSSDYQC-peptide ligands to the intestinal mucosal tissue and immunohistochemical analysis showed that the CSKSSDYQC-peptide ligands could be transported across the intestinal mucosal barrier via goblet cells as their specific gateway. Thus, we inferred that CSKSSDYQC-peptide ligand might have a specific receptor on the goblet cells and transported from intestinal lumen to systemic circulation by transcytosis mechanism. These results suggest that CSKSSDYQC-ligand could be a promising tool for development of an efficient oral delivery system for macromolecular therapeutics in the carrier-drug conjugate strategy.

Screening enantioselective epoxide hydrolase activities from marine microorganisms: detection of activities in Erythrobacter spp.

To develop an enantioselective epoxide hydrolase (EHase) from marine microorganisms, marine samples were collected from a variety of marine environments. Strains isolated by the capability of living on styrene oxide (SO) were screened for retaining enantioselective EHase activities toward SO by combining spectrophotometric, GC, and HPLC analysis. Consequently, one strain, JCS358, was selected, and the sequence analysis of 16S rRNA gene showed that the strain belonged to Erythrobacter cluster. Twelve additional Erythrobacter strains from this study or acquired from culture collections were thereby tested for displaying EHase activities, and most of tested strains showed enantioselective hydrolysis toward SO and glycidyl phenyl ether. Kinetic resolution of racemic SO using whole cell of Erythrobacter sp. JCS358 was performed. Enantiopure (S)-SO could be obtained with an enantiomeric excess (ee) higher than 99% after 15 h incubation. The determination of 1-phenyl-1,2-ethanediol configuration derived from racemic SO confirmed the enantioselective hydrolyzing activity of Erythrobacter sp. JCS358.

A cold-adapted epoxide hydrolase from a strict marine bacterium, Sphingophyxis alaskensis.

An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was identified by analyzing the genome sequence of Sphingophyxis alaskensis. The EHase gene (seh) was cloned and expressed in E. coli. To facilitate purification, the gene was fused in-frame to 6x histidine at the C-terminus. The recombinant EHase (rSEH) was highly soluble and could be purified to apparent homogeneity by one step of metal affinity chromatography. The purified SEH displayed hydrolyzing activities toward various epoxides such as styrene oxide, glycidyl phenyl ether, epoxyhexane, epoxybutane, epichlorohydrin, and epifluorohydrin. The optimum activity toward styrene oxide was observed at pH 6.5 and 35 degrees . The purified SEH showed a cold-adapted property, displaying more than 40% of activity at low temperature of 10 degrees compared with the optimum activity. Despite the catalytic efficiency, the purified SEH did not hydrolyze various epoxides enantioselectively. Km and kcat of SEH toward (R)-styrene oxide were calculated as 4+/-0.3 mM and 7.42 s(-1), respectively, whereas Km and kcat of SEH toward (S)-styrene oxide were 5.25+/-0.3 mM and 10.08 s(-1), respectively.

Complete Genome Sequence of Sphingorhabdus sp. YGSMI21, Exhibiting High Enantioselective Epoxide Hydrolase Activity.

Sphingorhabdus sp. YGSMI21 is a novel strain exhibiting high enantioselective hydrolysis activity for styrene oxide. Here, we present its complete genome sequence, consisting of one circular chromosome (3.86 Mb) and one plasmid (0.196 Mb).

Expression and characterization of human growth hormone-Fc fusion proteins for transcytosis induction.

The major obstacle for oral delivery of administered therapeutic proteins is malabsorption in the intestine. This malabsorption could be overcome by induction of neonatal FcRn [Fc (CH2 and CH3 domains of human IgG1 antibody) receptor]-mediated transcytosis in the intestine using recombinant fusion of CH2 and CH3 moieties of human IgG to a therapeutic protein. To this end we developed recombinant hGH (human growth hormone) fused to the N-terminus of Fc moieties [CH2-CH3 or h (hinge)-CH2-CH3] from human IgG1. These recombinant proteins secreted by the methylotrophic yeast Pichia pastoris functionally induced secretion of insulin-like growth factor 1 by HepG2 cells in the response to hGH moiety in the fusion proteins. In a transport study using polarized T84 cells, 3.7% of added dimeric hGH-h-Fc was transported in the apical-to-basolateral direction within 1 h by FcRn-mediated transcytosis of 1 cm(2) monolayers. However, transport of monomeric hGH-Fc (only 0.43%) was much less effective, yet its transport was 2.3 times higher than that of hGH. Finally, we concluded that, upon recombinant fusion, maintenance of dimeric structure of Fc moieties is crucial for the induction of FcRn-mediated transcytosis.

Complete Genome Sequence of Indigo-Producing Bacterium Celeribacter sp. Strain TSPH2.

Celeribacter sp. strain TSPH2, a novel producer of indigo, was isolated from oil-contaminated sediment. We present here its genome sequence consisting of one circular chromosome (4 Mb) and one plasmid (0.15 Mb), with an overall G+C content of 60.9%. This strain contains oxygenase genes involved in indigo synthesis, such as flavin-containing monooxygenase.

Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion.

In a bioreactor culture of genetically engineered Pichia pastoris secreting a bivalent immunotoxin, 64% of the secreted immunotoxin was present in aggregate forms and this resulted in a loss of bioactivity. Biochemical analyses of the secreted immunotoxin and an in vitro aggregation study using purified monomeric immunotoxin suggested that aggregation was primarily an extracellular event. By employing limited methanol feeding at 0.75 mlmin(-1) per 10l initial medium, oxygen consumption was reduced, permitting a lowering of the bioreactor agitation speed from 800 to 400 rpm. By increasing the anti-foam reagent to 0.6 mll(-1), the thickness of the air/liquid interfacial foam layer was reduced by 80%. These steps reduced the immunotoxin aggregates from 64% to 5%. Consequently immunotoxin purification yield was increased from 53.0% to 73.8%. Simultaneously this methodology enhanced immunotoxin secretion to 120 mgl(-1) at 163 h of methanol induction in a toxin resistant production strain. We conclude that minimizing shearing force and reducing the air/liquid interfacial foam area are crucial factors in reducing hydrophobic protein aggregation upon secretory expression in yeast bioreactor cultures.

Therapeutic HPV Cancer Vaccine Targeted to CD40 Elicits Effective CD8+ T-cell Immunity.

Human papillomavirus (HPV), particularly HPV16 and HPV18, can cause cancers in diverse anatomical sites, including the anogenital and oropharyngeal (throat) regions. Therefore, development of safe and clinically effective therapeutic vaccines is an important goal. Herein, we show that a recombinant fusion protein of a humanized antibody to CD40 fused to HPV16.E6/7 (αCD40-HPV16.E6/7) can evoke HPV16.E6/7-specific CD8+ and CD4+ T-cell responses in head-and-neck cancer patients in vitro and in human CD40 transgenic (hCD40Tg) mice in vivo The combination of αCD40-HPV16.E6/7 and poly(I:C) efficiently primed HPV16.E6/7-specific T cells, particularly CD8+ T cells, in hCD40Tg mice. Inclusion of montanide enhanced HPV16.E6/7-specific CD4+, but not CD8+, T-cell responses. Poly(I:C) plus αCD40-HPV16.E6/7 was sufficient to mount both preventative and therapeutic immunity against TC-1 tumors in hCD40Tg mice, significantly increasing the frequency of HPV16-specific CD8+ CTLs in the tumors, but not in peripheral blood. In line with this, tumor volume inversely correlated with the frequency of HPV16.E6/7-specific CD8+ T cells in tumors, but not in blood. These data suggest that CD40-targeting vaccines for HPV-associated malignancies can provide a highly immunogenic platform with a strong likelihood of clinical benefit. Data from this study offer strong support for the development of CD40-targeting vaccines for other cancers in the future. Cancer Immunol Res; 4(10); 823-34. ©2016 AACR.

Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization.

Three thermostable lactose-hydrolases, namely, two beta-glycosidases (bglA and bglB) and one beta-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431 aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70 degrees C, 40 min) and a Ni2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB beta-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 degrees C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 degrees C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.

Separation of bivalent anti-T cell immunotoxin from Pichia pastoris glycoproteins by borate anion exchange.

A major problem encountered in the large-scale purification of the bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), from Pichia pastoris supernatants was the presence of host glycoproteins exhibiting similar charge, size, and hydrophobicity characteristics. We overcame this problem by employing borate anion exchange chromatography. The borate anion has an affinity for carbohydrates and imparts negative charges to these structures. We found that at a concentration of sodium borate between 50 and 100 mM, the nonglycosylated immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but glycoproteins, including aggregates related to the immunotoxin, did. By using this property of the immunotoxin in the presence of sodium borate, we successfully developed a 3-step purification procedure: (i) Butyl-650M hydrophobic interaction chromatography, (ii) Poros 50 HQ anion exchange chromatography in the presence of borate, and (iii) HiTrap Q anion exchange chromatography. The final preparation exhibited a purity of greater than 98% and a yield of greater than 50% from the supernatant. Previously, boronic acid resins have been used to separate glycoproteins from proteins. However, combining borate anion with conventional anion exchange resins accomplishes the separation of the immunotoxin from glycoproteins and eliminates the need to evaluate nonstandard resins with respect to good manufacturing practice guidelines.

C2-ceramide as a cell death inducer in HC11 mouse mammary epithelial cells.

Ceramide is a lipid mediator in cell proliferation, differentiation, and apoptosis in many cell lines. However, the molecular mechanisms for ceramide have not been clarified in HC11 mouse mammary epithelial cells. Under phase contrast microscope, C2-ceramide-treated cells clearly showed morphological changes, which were characteristic features of apoptosis. Treatment with C2-ceramide at 10 microM specifically resulted in the death of 50% of the cells after 48 h as assessed by MTT assay. To further investigate which genes contribute to cell death in C2-ceramide-treated cells, we used the reverse transcription-polymerase chain reaction to assess mRNA levels for five genes in the Bcl-2 family and five genes in the caspases family. The steady-state mRNA levels of Bax, Bad and Bak were not significantly changed for 48 h of C2-ceramide treatment. The increases of mRNA levels of Bcl-2 and Bcl-w were observed for the first 3 h of C2-ceramide treatment and the last 24 h between 24 and 48 h. We also found that in HC11 cells, C2-ceramide increased mRNA levels of the caspases family from 6 to 24 h. These results suggest that in the HC11 cells, C2-ceramide promote cell death by mediating the induction of caspases and that HC11 mouse mammary epithelial cells paradoxically up-regulate the expression of Bcl-2 and Bcl-w to prevent C2-ceramide-mediated cell death.

In-vivo evaluation of human recombinant Co-arginase against A375 melanoma xenografts.

Metastatic melanoma is a deadly form of cancer with few therapeutic options and the cause of more than 9480 deaths annually in the USA alone. Novel treatment options for this disease are urgently needed. Here we test the efficacy of a novel melanoma drug, the human recombinant Co-arginase (CoArgIPEG), against an aggressive A375 melanoma mouse model. CoArgIPEG is a modification of the naturally occurring human enzyme with improved stability, catalytic activity, and potentially lower immunogenicity compared with current amino acid-depleting drugs. Marked tumor growth reductions (mean P=0.0057) with apoptosis induction and proliferation inhibition are noted with CoArgIPEG treatment, both in the presence and in the absence of supplemental citrulline. Further, improved therapeutic efficacy has been noted against A375 xenografts relative to the naturally occurring human recombinant arginase enzyme at lower doses of CoArgIPEG. Unfortunately, after 1 month, half of the relapsing tumors showed argininosuccinate synthase induction, which correlated with Ser62-phosphorylated cMyc. Although argininosuccinate synthase induction could not be induced in vitro, a drug targeting pathway previously demonstrated to be associated with Ser62 cMyc phosphorylation - U0126 - in combination with CoArgIPEG demonstrated an in-vitro synergistic response (combination indices 0.13±0.10 and 0.14±0.10 with or without citrulline, respectively). Overall, favorable efficacy and potential synergy with other antimelanoma drugs support CoArgIPEG as a potent, novel cancer therapeutic.