Memory and modularity in cell-fate decision making

Thomas M. Norman,     Nathan D. Lord,     Johan Paulsson     & Richard Losick

                                                              

Abstract:

Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. We show that the motile state is ‘memoryless’, exhibiting no autonomous control over the time spent in the state. In contrast, the time spent as connected chains of cells is tightly controlled, enforcing coordination among related cells in the multicellular state. We show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.

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Keywords:memory,cell-fate,multicellular

TRPV1 structures in distinct conformations reveal activation mechanisms

Erhu Cao,     Maofu Liao,     Yifan Cheng     & David Julius

                                                                            

Abstract:

Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (consisting of transmembrane segments 1–4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiological modulation exhibited by TRPV1 and other TRP channels.

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Keywords:TRPV1,polymodal,pathophysiological,allosteric,vanilloid,pharmacological probes

Structure of the TRPV1 ion channel determined by electron cryo-microscopy

Maofu Liao,     Erhu Cao,     David Julius     & Yifan Cheng

                                                                       

Abstract:

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5–6 (S5–S6) and the intervening pore loop, which is flanked by S1–S4 voltage-sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with a short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

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keywords:TRPV1,ion channel,electron cryo-microscopy,transmembrane,cytoplasmic

Activation and allosteric modulation of a muscarinic acetylcholine receptor

Andrew C. Kruse,     Aaron M. Ring,     Aashish Manglik,     Jianxin Hu,     Kelly Hu,     Katrin Eitel, Harald Hübner,     Els Pardon,     Celine Valant,     Patrick M. Sexton,     Arthur Christopoulos, Christian C. Felder,     Peter Gmeiner,     Jan Steyaert,     William I. Weis,     K. Christopher Garcia, Jürgen Wess     & Brian K. Kobilka

                                                                            

Abstract:

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors

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Keywords:allosteric,modulation,muscarinic acetylcholine receptor,orthosteric,rhodopsin,G-protein

Fermentation kinetics of production of ubiquinone-10 by Paracoccus dinitrificans NRRL B-3785: Effect of type and concentration of carbon and nitrogen sources

Mahesh V. Bule, Rekha S. Singhal

                                                                              

Abstract
Ubiquinone-10 (CoQ10), a vitamin-like lipophilic component of the membrane-bound electron transport system, has a wide range of therapeutic, neutraceutical, and cosmeceutical applications. The objective of this study was to optimize nutritional requirements for production of CoQ10 by Paracoccus dinitrificans NRRL B-3785 fermentation. Effect of type and concentration of carbon and nitrogen source on fermentation kinetic parameters were analyzed using logistic and Luedeking-Piret equations. In submerged batch fermentation, yield of CoQ10 was 12.22 mg/L when 40 g/L glycerol was used and specific growth rate (0.056/h) as well as growth associated constant (α=0.680 mg/g) were higher as compared to other concentrations. Ammonium nitrate and proteose peptone at 5 (α=0.677 mg/g; β= 0.0072 mg/g·h) and 20 g/L (α=0.806 mg/g; β=0.0074 mg/g·h), respectively, were optimal for CoQ10 production. CoQ10 formation observed to be both growth and nongrowth associated. In optimized medium CoQ10 formation increased considerably from 1.91 to 14.12 mg/L.

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Keywords: ubiquinone-10,araccocus dinitrificans,fermentation kinetics,process modification.

α-Galactosidases production by Debaryomyces hansenii UFV-1

Pollyanna Amaral Viana, Sebastião Tavares de Rezende, Flávia Maria Lopes Passos, Solimar Gonçalves Machado, Gabriela Picollo Maitan, Vinicio Tadeu da Silva Coelho, Valéria Monteze Guimarães

                                                                                       

Abstract
Extracellular and intracellular α-galactosidases were produced by yeast Debaryomyces hansenii UFV-1 grown on different media with several carbon sources. D. hansenii grown in YP-medium (1% yeast extract and 2% peptone) presented maximum cell mass (8.45 mg/mL) after 36 h of cultivation, with lactose as carbon source, followed by sucrose, glucose, raffinose, and galactose. Higher extracellular and intracellular α-galactosidases activities were observed at 48 h of D. hansenii cultivation in YPmedium containing galactose (0.97 and 5.27 U/mL) and lactose (1.28 and 4.88 U/mL), supporting the evidence for the model of induction for the yeast GAL/MEL regulon, such as described in Sacharomyces cereviseae.

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Keywords: Debaryomyces hansenii UFV-1,α-galactosidase,culture media,carbon source,enzyme production.

Effects of γ-irradiation on antioxidant and antimicrobial activities and color ofEcklonia cava

Eu Jin Song, Ah Ram Kim, Koth Bong Woo Ri Kim, So Young Lee, Jin Gyu Park, Jae Hun Kim, Jong Il Choi, Ju Woon Lee, Myung Woo Byun, Dong Hyun Ahn

                                                                                     

Abstract
Ecklonia cava (EC) was irradiated with γ-rays at doses of 3, 7, and 20 kGy. The extraction yields, total polyphenol content (TPC), DPPH radical scavenging activity, antimicrobial activity, and color of the EC extracts were assessed. The results showed that irradiation caused an increase in the extraction yields of ethanol and water. TPC was found to be significantly increased when EC powder was subjected to irradiation, followed by ethanol and water extraction. However, DPPH radical scavenging and antimicrobial activities were stable under all irradiation conditions. In the heat and pH stability tests, the DPPH radical scavenging activities of EC ethanol extracts were not influenced by irradiation. Irradiation caused an increase in the lightness and redness of EC extracts and decreased the yellowness. In conclusion, γ-irradiation increased the extraction yield and TPC and brightened the color, while maintaining the antioxidant and antimicrobial activities.

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Keywords: γ-irradiation,Ecklonia cava,antioxidant,antimicrobial,color.

Antioxidant activity of the differentially processed seeds of Jack bean (Canavalia ensiformis L. DC)

Antioxidant activity of the differentially processed seeds of Jack bean (Canavalia ensiformis L. DC)

                                                              

Abstract
Antioxidant activity of 70% acetone extracts of raw and processed seeds of Jack bean (Canavalia ensiformis L. DC) was evaluated by various in vitro antioxidant assays, including total antioxidant, free radical scavenging, reducing power, metal ion chelating, β-carotene/linoleic acid bleaching, and antihemolytic activities. The total phenolics and tannin contents were higher in the extract of seeds processed by autoclaving with 1% ash solution (3.2 and 1.6 g/100 g extract, respectively). In general, all the extracts of processed seeds exhibited higher activity in various antioxidant systems, when compared to raw seeds but significant differences were noticed between processing methods. The extract of seeds autoclaved with 1% sugar solution showed higher DPPH radical scavenging activity (IC50 10.6 mg/mL). Interestingly, the extract of dry heated seeds registered higher inhibition of hemolysis (76.1%) compared to standards butylated hydroxyanisole (BHA) (66.2%) and α-tocopherol (59.3%) at the concentration of 500 μg/mL

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Keywords: Canavalia ensiformis,total phenolics,legume,antioxidant activity,antihemolytic.

Distribution of newly described enterotoxin-like genes in Staphylococcus aureus isolated from ready-to-eat foods in Korea

Su Kyung Oh, Minseon Koo, Nari Lee, Hyun Jung Kim, Se-Wook Oh, Soon Young Choi

                                                         

Abstract
To investigate the distribution of staphylococcal enterotoxin-like (SEl) genes in Staphylococcus aureus from food, a total of 154 S. aureus isolates from ready-to-eat (RTE) foods in Korea were analyzed by mutiplex PCR for the detection of the following 9 staphylococcal enterotoxin-like genes; sek, sel, sem, sen, seo, sep, seq, ser, and seu. Seventy-nine isolates (51.3%) were found to have at least one of SEl genes. The major SEl genes were sek, sem, sen, and seq. Other SEl genes found in the isolates were seo (21 isolates, 13.6%), seu (12 isolates, 7.8%), sep (8 isolates, 5.2%), sel (7 isolates, 4.5%), and ser (2 isolates, 1.3%). Most (95%) of the isolates with staphylococcal enterotoxin (SE) genes were also carried SEl genes. The genes seg, sei, sem, and sen were all detected in the same isolates. Ninety-seven % of isolates with seg+sei+sem+sen also contained seo or seu. Ninety-four % of isolates with sea+seh were found to coexist with sek+seq. The toxic shock syndrome toxin gene, tst-1, was found in all isolates with egc-2, including seg, sei, sem, sen, and seu. The coexistence of SE and SEl genes in S. aureus isolates from RTE foods can be explained by the mobile genetic elements. Because of the mobile genetic element, SE and SEl genes of S. aureus in foods may be transferable to nontoxigenic S. aureus and other food pathogens. Additional studies must be conducted to prevent spread of pathogenic genes such as enterotoxin gene.

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Keywords: Staphylococcus aureus,staphylococcal enterotoxin-like (SEl) gene,mobile genetic element,ready-to-eat food.

Optimization of the steamed foam cakes prepared with separated-egg-sponge method using response surface methodology

Myung Sook Jang, Jung Eun Park, Sung Ho Kwhak

                                                                                     

Abstract
In preparation of steamed foam cakes by separated-egg-sponge method, effects of whipping time (X1, 4–12 min), quantity of wheat flour (X2, 460–540 g), and number of folding (X3, 110–190 times) of the steamed foam cakes were investigated using response surface method. Measuring R2 of the volume and hardness were 0.9094 and 0.9119, respectively at p<0.05 and gumminess and chewiness were 0.9397 and 0.9429, respectively at p<0.01. According to the result of examining the effect of independent variables on each reaction variable, physical properties was affected most significantly by whipping time. According to the result of examining the response surface graphs and the optimal condition of ordinary points, volume showed a maximal point, hardness a minimal point, and all the other items a saddle point. The experimental results for optimum manufacturing conditions for separated-egg-sponge method, i.e., 7.5 min whipping time, 492 g amount of wheat flour, and 149 times number of folding were empirically proven to fit the predicted levels of physical properties from the final foam cakes.

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Keywords: steamed,foam cake,separated-egg-sponge method,response surface methodology,optimization.

Metabolite fingerprinting of bokbunja (Rubus coreanus Miquel) by UPLC-qTOF-MS

Sujin Heo, Do-Yup Lee, Hyung-Kyoon Choi, Jaehwi Lee, Jung-Hyun Kim, Soo-Muk Cho, Hong Jin Lee, Joong-Hyuck Auh

                                                                    

Abstracts
Metabolite fingerprinting of bokbunja (Rubus coreanus Miquel) using LC-MS according to the cultivation region and the maturity level was performed. The endogenous metabolites were extracted with 80% cold ethanol and the total content of phenolics and anthocyanins compounds was assessed. Accurate mass fingerprinting with chromatographic separation successfully classified bokbunja based on the metabolite profile. Unsupervised classification method (PCA) and supervised prediction model (OPLSDA) provided good capability in categorizing the samples according to the region and the maturity level. Additional tandem-mass spectrometry analysis allowed the identification of some anthocyanin derivatives that showed the most dramatic increase during ripening period (e.g., cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside).

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Keywords : Rubus coreanus Miquel ,metabolite fingerprinting ,LC-MS ,cyanidin.

Expression, purification, and characterization of human intestinal maltase secreted from Pichia pastoris

Hwa-Ja Ryu, Eun-Seong Seo, Hee-Kyoung Kang, Young-Min Kim, Doman Kim

                                                               

Abstract
A gene encoding human intestinal maltase (HMA) was successfully expressed in Pichia pastoris under the control of the methanol-induced alcohol oxidase (AOX1) promoter. The secreted recombinant HMA fused with a His6-tag was produced (150 U/L) and was easily purified from culture supernatants in a 3-step diafiltration, ultrafiltration, and affinity column chromatography protocol. The specific activity of the purified HMA was 16.8 U/mg. Endoglycosidase H digestion of the protein showed that the recombinant HMA was N-glycosylated. The purified HMA was maximally active at pH 6.5 and stable (≥90%) up to 65°C. The kinetic parameters K m and V max were 3.3±0.25 mM maltose and 61.9±2 U/mg, respectively.

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Keywords: α-glucosidase,maltase,diabetes,Pichia pastoris,expression in yeast.

Isolation and identification of cinnamic acid amides as antioxidants from Allium fistulosum L. and their free radical scavenging activity

Gee-Woo Seo, Jeong-Yong Cho, Jae-Hak Moon, Keun-Hyung Park

                                                                             

Abstract
Four compounds obtained from the ethyl acetate-soluble neutral fraction of methanol extracts of the Welsh onion (Allium fistulosum L.) were purified by column chromatography using silica gel, octadecyl silane (ODS), and Sephadex LH-20 with a guided DPPH radical scavenging assay. After purification, the compounds were isolated by ODS-HPLC. The isolated compounds were identified as N-trans-feruloyl-3′-methoxytyramine (1), N-cis-feruloyl-3′-methoxytyramine (2), N-trans-p-coumaroyltyramine (3), and 3,5,7-trihydroxyflavone (kaempferol, 4) based on the spectroscopic data of NMR and MS. To the best of our knowledge, this is the first study to identify compound 1–3 in the Welsh onion. Compound 1 and 2 showed significantly (p<0.05) higher DPPH radical scavenging activities than compound 3.

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Keywords: Allium fistulosum,Welsh onion,cinnamic acid amide,DPPH radical,antioxidant.

Dehydration characteristics of Maesaengi (Capsosiphon fulvescens) in hot-air drying

Seok Min Son, Jong-Whan Rhim, Jun Ho Lee

                                                                      

Abstract
Maesaengi (Capsosiphon fulvescens) was dehydrated in a single layer at drying air temperatures ranging from 50–80°C in a laboratory scale convective dryer with an air velocity of 0.26 m/s. The effect of drying air temperature on the drying kinetic characteristics was determined. Maesaengi was dried to equilibrium moisture content within 200–600 min under these drying conditions. Drying rate curves of maesaengi showed an initial short period of a constant drying rate, followed by a falling drying rate period. The moisture decrease in the sample during dehydration was well described by the zero-order kinetic model. An Arrhenius-type equation was used to test the effect of temperature on drying rate according to an activation energy value of 29.36 kJ/mol

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Keywords: maesaengi,Capsosiphon fulvescens,drying kinetics,modeling,thin-layer drying,hot air.

Antimicrobial and antioxidative effects of onion peel extracted by the subcritical water

Kyoung Ah Lee, Kee-Tae Kim, Seung-Yeol Nah, Myong-Soo Chung, SangWoo Cho, Hyun-Dong Paik

                                                                     

Abstract
The objective of this study was to evaluate the antimicrobial and antioxidative effects of the onion peels extract prepared via the subcritical water extraction (SWE) method. First, the number of cells treated with the extract was reduced by 0.7–1.1 log CFU/mL compared with the control. The SWE extract showed 76.08% scavenging activity and it was more effective than butylated hydroxytoluene (BHT) at 61.3 ppm in lipid peroxidation inhibitory effects. In addition, the antioxidative effect of SWE extract measured via the ferric thiocyanate (FTC) method was 2-fold that of BHT. The results of this study suggest that the onion peels extract prepared via the SWE method may have potential alternative antimicrobial and antioxidative effects as functional substances.

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Keywords: subcritical water extraction,quercetin,onion peel,antimicrobial effect,antioxidative effect.

Novel quantitative method for the degree of branching in dextran

Young-Min Kim, Atsuo Kimura, Doman Kim

                                                                         

Abstract
A novel quantitative method for the determination of degree of branching in Leuconostoc mesenteroides B-512F dextran was developed by using the combination of 3 dextran-degrading enzymes. First, Paenibacillus sp. endo-dextranase was randomly degraded B-512F dextran into linear or branched isomalto-oligosaccharides with various degree of polymerization (2–8). Second, Streptococcus mutans dextran glucosidase hydrolyzed linear or branched isomalto-oligosaccharides into glucose and branched isomalto-penta-saccharides. Third, the branched isomaltopenta-saccharide was degraded into glucose by using Bacteroides thetaimicron α-glucosidase. The number of branching points in B-512F dextran (5.42%) was determined by the difference in the amount of glucose in the reaction digest between BTGase-PDex and DGase-PDex treatments.

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Keywords: dextran,endo-dextranase,exo-dextranase,degree of branching.

Bitter melon (Momordica charantia) extract suppresses cytokineinduced activation of MAPK and NF-κB in pancreatic β-Cells

Kyong Kim, Hye Young Kim

                                                                                       

Abstract
Previously, the aqueous ethanolic extract (AEE) of unripe fruit of bitter melon (BM; Momordica charantia) was demonstrated to inhibit cytokine-induced apoptosis via modulating Bcl-2 family and caspase cascades in MIN6N8 pancreatic β-cells. Here, it was sought to determine whether the anti-apoptotic effect of AEE-BM is mediated by suppressing the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB), major upstream effectors of Bcl-2 family and caspase cascades, in cytokine-treated MIN6N8 cells. The results exhibited that the AEE-BM suppressed the activation of MAPKs including stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and p44/42, and the activity of NF-κB. The findings suggest that BM protects pancreatic β-cells through down-regulation of MAPKs and NF-κB.

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Keywords: bitter melon (Momordica charantia),mitogen-activated protein kinase (MAPK),nuclear factor-κB,apoptosis,β-cell.