Characterization of a novel sucrose phosphorylase from Paenibacillus elgii and its use in biosynthesis of α-arbutin.

α-Arbutin, a naturally occurring glycosylated derivative of hydroquinone (HQ), effectively inhibits melanin biosynthesis in epidermal cells. It is widely recognized as a fourth-generation whitening agent within the cosmetic industry. Currently, enzymatic catalysis is universally deemed the safest and most efficient method for α-arbutin synthesis. Sucrose phosphorylase (SPase), one of the most frequently employed glycosyltransferases, has been extensively reported for α-arbutin synthesis. In this study, a previously reported SPase known for its effectiveness in synthesizing α-arbutin, was used as a probe sequence to identify a novel SPase from Paenibacillus elgii (PeSP) in the protein database. The sequence similarity between PeSP and the probe was 39.71%, indicating a degree of novelty. Subsequently, the gene encoding PeSP was coexpressed with the molecular chaperone pG-Tf2 in Escherichia coli, significantly improving PeSP's solubility. Following this, PeSP was characterized and employed for α-arbutin biosynthesis. The specific activity of co-expressed PeSP reached 169.72 U/mg, exhibited optimal activity at 35℃ and pH 7.0, with a half-life of 3.6 h under the condition of 35℃. PeSP demonstrated excellent stability at pH 6.5-8.5 and sensitivity to high concentrations of metal ions. The kinetic parameters Km and kcat/Km were determined to be 14.50 mM and 9.79 min- 1·mM- 1, respectively.The reaction conditions for α-arbutin biosynthesis using recombinant PeSP were optimized, resulting in a maximum α-arbutin concentration of 52.60 g/L and a HQ conversion rate of 60.9%. The optimal conditions were achieved at 30℃ and pH 7.0 with 200 U/mL of PeSP, and by combining sucrose and hydroquinone at a molar ratio of 5:1 for a duration of 25 h.

Phospholipase C inhibits apoptosis of porcine primary granulosa cells cultured in vitro.

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 µM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCß1, protein kinase C ß (PKCß), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calcium-sensitive targets and several downstream genes might take part in the processes.

The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro.

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m-3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m-3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m-3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.

Facile synthesis of prickly platinum-palladium core-shell nanocrystals and their boosted electrocatalytic activity towards polyhydric alcohols oxidation and hydrogen evolution.

Herein, prickly platinum-palladium core-shell nanocrystals (Pt@Pd NCs) were prepared by a facile one-pot aqueous method, only taking sodium pyrrolidone carboxylate (PCA-Na) as the structure director and stabilizing agent. The products were mainly characterized by microscopic analysis, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), followed by discussing the formation mechanism in details. The electrochemical characterizations were examined by cyclic voltammetry (CV), linear sweep voltammetry (LSV) and chronoamperometry (CA). The results revealed that the prepared architectures had the biggest current density (58.4 mA cm-2) for ethylene glycol oxidation, which is 3.5-fold, 1.2-fold, 2.3-fold and 2.4-flod enhancement relative to those of home-made single Pt nanoparticles (NPs) and Pd NPs, commercial Pt black and Pd black catalysts, respectively. Also, the obtained Pt@Pd NCs showed improved catalytic activity and stability towards glycerol oxidation and hydrogen evolution reactions compared to the references.

Effect of FH535 on in vitro maturation of porcine oocytes by inhibiting WNT signaling pathway.

Wingless-int (WNT) signaling pathway is vital to modulate life processes, including cell fate determination, cell differentiation, cell proliferation, cell apoptosis and embryogenesis. To demonstrate the uncertain effect of the canonical WNT signaling pathway on oocyte maturation, immature porcine oocytes were collected and cultured in vitro with the WNT/ß-catenin inhibitor FH535. The concentrations of FH535 were selected as 0.00, 0.01, 0.10, 1.00 and 10.00 µmol/L. The results showed that the optimum concentration of FH535 on oocyte maturation was 1.00 µmol/L. In this concentration, the proportion of MII oocytes increased (P < 0.05). The rate of cleavage was the same with the control (P > 0.05), while the rate of blastocysts in the 1.00 µmol/L FH535 treated group was higher than that of control (P < 0.01). Additionally, the average number of nuclei in blastocysts raised significantly (P < 0.05). The inhibition of WNT could regulate expression of maturation-related genes, including Cdc-2, Bmp-15, Gdf-9 and Mos. In the 1.00 µmol/L FH535 treated group, the messenger RNA level of ß-catenin showed no significant change compared to the control (P > 0.05), but the protein abundance was decreased (P < 0.05). This study revealed that the inhibition of FH535 on the WNT signaling pathway could promote the maturation of porcine oocytes and altered gene expressions in vitro.

One-pot aqueous fabrication of reduced graphene oxide supported porous PtAg alloy nanoflowers to greatly boost catalytic performances for oxygen reduction and hydrogen evolution.

Herein, reduced graphene oxide supported porous PtAg alloy nanoflowers (PtAg NFs/rGO) were synthesized by a simple one-pot aqueous method using pyridinium-based dicationic ionic liquid (1,4-bis(pyridinium)butane dibromide, Bpb-2Br) as the new structure-director and stabilizing agent. The products were characterized by a series of techniques. The obtained nanocomposite had more positive onset potential (1.03 V) for oxygen reduction reaction (ORR) in alkaline electrolyte than those of commercial Pt/C (50 wt%, 0.96 V) and home-made Pt nanoparticles (NPs)/rGO (Pt NPs/rGO, 0.97 V), showing the enhanced catalytic performance for hydrogen evolution reaction (HER) with the positive onset potential (-26 mV) and a small Tafel slope (31 mV dec-1) relative to Pt/C (-18 mV, 31 mV dec-1) and Pt NPs/rGO (-42 mV, 36 mV dec-1) in 0.5 M H2SO4.

Melatonin modulates the functions of porcine granulosa cells via its membrane receptor MT2 in vitro.

Melatonin (N-acetyl-5-methoxytryptamine) is documented as a hormone involved in the circadian regulation of physiological and neuroendocrine function in mammals. Herein, the effects of melatonin on the functions of porcine granulosa cells in vitro were investigated. Porcine granulosa cells were cultivated with variable concentrations of melatonin (0, 0.001, 0.01, 0.1, 1.0, and 10ng/mL) for 48h. Melatonin receptor agonist (IIK7) and antagonist (Luzindole, 4P-PDOT) were used to further examine the action of melatonin. The results showed optimum cell viability and colony-forming efficiency of porcine granulosa cells at 0.01ng/mL melatonin for 48-h incubation period. The percentage of apoptotic granulosa cells was significantly reduced by 0.01 and 0.1ng/mL melatonin within the 48-h incubation period as compared with the rest of the treatments. Estradiol biosynthesis was significantly stimulated by melatonin supplementation and suppressed for the progesterone secretion; the minimum ratio of progesterone to estradiol was 1.82 in 0.01ng/mL melatonin treatment after 48h of cultivation. Moreover, the expression of BCL-2, CYP17A1, CYP19A1, SOD1, and GPX4 were up-regulated by 0.01ng/mL melatonin or combined with IIK7, but decreased for the mRNA levels of BAX, P53, and CASPASE-3, as compared with control or groups treated with Luzindole or 4P-PDOT in the presence of melatonin. In conclusion, the study demonstrated that melatonin mediated proliferation, apoptosis, and steroidogenesis in porcine granulosa cells predominantly through the activation of melatonin receptor MT2 in vitro, which provided evidence of the beneficial role of melatonin as well as its functional mechanism in porcine granulosa cells in vitro.

Effects of melatonin on follicular atresia and granulosa cell apoptosis in the porcine.

The accumulation of reactive oxygen species is detrimental to the health of the ovarian follicle. The protective, antioxidant properties of melatonin, an endogenous component of porcine follicular fluid, on apoptosis of granulosa cells were evaluated in this study. Porcine granulosa cells from medium-sized (3-5 mm), healthy follicles were cultured in serum-free conditions with melatonin (0, 0.01, 0.1, 1.0, 10, and 100 ng/mL) with or without its receptor antagonist, luzindole, followed by evaluation of apoptotic markers in the treated cells. Results revealed that endogenous, intrafollicular melatonin concentration decreased as follicular atresia progressed, whereas the percentage of apoptotic granulosa cells increased. Spontaneous apoptosis of granulosa cells, triggered by serum deprivation in vitro, was remarkably blocked by melatonin (1.0 ng/mL melatonin, 32.7 ± 0.5%, vs. control, 47.0 ± 1.0%; P < 0.05). Treatment with 1.0 ng/mL of melatonin also significantly elevated MT2, SOD1, and GPX4 while lowering FASL, CHOP, and GRP78 mRNA abundance compared to the untreated control. The anti-apoptotic effect and some changes of apoptotic-relevant genes in granulosa cells invoked by melatonin supplementation were markedly blocked by luzindole, suggesting that melatonin could prevent the apoptosis of porcine granulosa cells during follicular atresia via its membrane receptors and its free-radical-scavenging activity. These findings provide new insights into the regulatory mechanism of melatonin in follicular atresia-related functions. Mol. Reprod. Dev. 83: 692-700, 2016 © 2016 Wiley Periodicals, Inc.

Testing Rhodiola sachalinensis saccharide as cryoprotectant for bovine spermatozoa.

Rhodiola sachalinensis saccharide (RSS) was extracted from the rhizome of Herba Rhodiolae and was expected as a novel cryoprotectant. The aim of this study was to test the effects of RSS on motility of bull sperm and the activities of superoxide dismutase (SOD), lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (GOT) in bull sperm during cryopreservation. Rhodiola sachalinensis saccharide was added at the concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10 mg/mL to the extenders, which were used to store bovine semen. It was found that the RSS-added extends resulted in a higher percentage of cryopreserved sperm motility, mitochondrial activity, and membrane and acrosome integrity than those of RSS-free extenders. The SOD, LDH, and GOT activities were all decreased during the process of freezing and thawing. The extenders supplemented with RSS improved the SOD, LDH, and GOT activities after cryopreservation compared with the RSS-free groups. In conclusion, RSS conferred great cryoprotective capacity to the basic extender for bull spermatozoa during the process of freezing-thawing, and the optimal concentration of RSS for the extender was 0.06 mg/mL.

New formation of imines of C19-diterpenoid alkaloids by heating with DMSO.

Treatment of some C(19)-diterpenoid alkaloids (3, 6, 10 and 12) with anhydrous DMSO at 100-170 degrees C for 3-7 h led to the formation of the corresponding imines (4, 7/8, 11, 13/14) in 65-83% yield, respectively. This is a new simpler formation of the imines of the C(19)-diterpenoid alkaloids.