Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Acid-sensing ion channels (ASICs): therapeutic targets for neurological diseases and their regulation

Extracellular acidification occurs not only in pathological conditions such as inflammation and brain ischemia, but also in normal physiological conditions such as synaptic transmission. Acid-sensing ion channels (ASICs) can detect a broad range of physiological pH changes during pathological and synaptic cellular activities. ASICs are voltage-independent, proton-gated cation channels widely expressed throughout the central and peripheral nervous system. Activation of ASICs is involved in pain perception, synaptic plasticity, learning and memory, fear, ischemic neuronal injury, seizure termination, neuronal degeneration, and mechanosensation. Therefore, ASICs emerge as potential therapeutic targets for manipulating pain and neurological diseases. The activity of these channels can be regulated by many factors such as lactate, Zn²⁺, and Phe-Met-Arg-Phe amide (FMRFamide)-like neuropeptides by interacting with the channel’s large extracellular loop. ASICs are also modulated by G protein-coupled receptors such as CB₁ cannabinoid receptors and 5-HT₂. This review focuses on the physiological roles of ASICs and the molecular mechanisms by which these channels are regulated. [BMB Reports 2013; 46(6): 295-304]     

Crif1 Deficiency Reduces Adipose OXPHOS Capacity and Triggers Inflammation and Insulin Resistance in Mice

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS–deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA–encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.     

Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules

Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.     

Five hTRPA1 Agonists Found in Indigenous Korean Mint,Agastache rugosa

Transient receptor potential ankyrin1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) are members of the TRP superfamily of structurally related, nonselective cation channels and mediators of several signaling pathways. Previously, we identified methyl syringate as an hTRPA1 agonist with efficacy against gastric emptying. The aim of this study was to find hTRPA1 and/or hTRPV1 activators in Agastache rugosa (Fisch. et Meyer) O. Kuntze (A.rugosa), commonly known as Korean mint to improve hTRPA1-related phenomena. An extract of the stem and leaves of A.rugosa (Labiatae) selectively activated hTRPA1 and hTRPV1. We next investigated the effects of commercially available compounds found in A.rugosa (acacetin, 4-allylanisole, p-anisaldehyde, apigenin 7-glucoside, L-carveol, β-caryophyllene, trans-p-methoxycinnamaldehyde, methyl eugenol, pachypodol, and rosmarinic acid) on cultured hTRPA1- and hTRPV1-expressing cells. Of the ten compounds, L-carveol, trans-p-methoxycinnamaldehyde, methyl eugenol, 4-allylanisole, and p-anisaldehyde selectively activated hTRPA1, with EC50 values of 189.1±26.8, 29.8±14.9, 160.2±21.9, 1535±315.7, and 546.5±73.0 μM, respectively. The activities of these compounds were effectively inhibited by the hTRPA1 antagonists, ruthenium red and HC-030031. Although the five active compounds showed weaker calcium responses than allyl isothiocyanate (EC₅₀=7.2±1.4 μM), our results suggest that these compounds from the stem and leaves of A.rugosa are specific and selective agonists of hTRPA1.     

Among γ-secretase substrates Notch1 alone is sufficient to block neurogenesis but does not confer self-renewal properties to neural stem cells

Notch signaling pathway enhances neural stem cell characters and regulates cell fate decisions during neural development. Interestingly, besides Notch, other γ-secretase substrates such as APP, LRP2, and ErbB4 have also proven to have biological functions in neural development. We designed a unique experimental setting, combining gain-of- (expression of Notch intracellular domain, NICD) and loss-of-function (γ-secretase inhibition) methods, and were able to examine the function of Notch alone by excluding the activity of other γ-secretase substrates. Here, we show that the frequency and size of neurospheres generated from embryonic neural stem cells (NSCs) significantly decreased by 62.7% and 37.2%, respectively, in the presence of γ-secretase inhibitor even when NICD was expressed. Under the condition of differentiation, however, the γ-secretase inhibitor treatment did not influence the promotion of astrogenesis at the expense of neurogenesis by NICD. These results indicate that other γ-secretase substrate(s) along with Notch are important in the maintenance of the stemness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other γ-secretase substrates during differentiation.     

Production of xylitol by recombinant Saccharomyces cerevisiae containing xylose reductase gene in repeated fed-batch and cell-recycle fermentations

Xylitol is a well-known sugar substitute with low-calorie and anti-cariogenic characteristics. An effort of biological production of xylitol from xylose was made in repeated fed-batch and cell-recycle fermentations of recombinant Saccharomyces cerevisiae BJ3505/δXR harboring the xylose reductase gene from Pichia stipitis. Batch fermentation with 20 g/l xylose and 18 g/l glucose resulted in 9.52 g/l dry cell mass, 20.1 g/l xylitol concentration and approximately 100% conversion yield. Repeated fed-batch operation to remove 10% of culture broth and to supplement an equal volume of 200 g/l xylose was designed to improve xylitol production. In spite of a sudden drop of cell concentration, an increase in dry cell mass led to high accumulation of xylitol at 48.7 g/l. To overcome loss of xylitol-producing biocatalysts in repeated fed-batch fermentation, cell-recycle equipment of hollow fiber membrane was implemented into a xylitol production system. Cell-recycle operation maintained concentration of the recombinant cells high inside a bioreactor. Final dry cell mass of 22.0 g/l, 116 g/l xylitol concentration, 2.34 g/l h overall xylitol productivity were obtained in cell-recycle fermentation supplemented with xylose and yeast extract solution, which were equivalent to 2.3-, 5.8- and 3.8-fold increases compared with the corresponding values of batch-type xylitol production parameters.