Effect of Diets Containing Sucrose vs. D‐tagatose in Hypercholesterolemic Mice

Effects of functional sweeteners on the development of the metabolic syndrome and atherosclerosis are unknown. The objective was to compare the effect of dietary carbohydrate in the form of sucrose (SUCR) to D‐tagatose (TAG; an isomer of fructose currently used as a low‐calorie sweetener) on body weight, blood cholesterol concentrations, hyperglycemia, and atherosclerosis in low‐density lipoprotein receptor deficient (LDLr^−/−) mice. LDLr^−/− male and female mice were fed either standard murine diet or a diet enriched with TAG or SUCR as carbohydrate sources for 16 weeks. TAG and SUCR diets contained equivalent amounts (g/kg) of protein, fat, and carbohydrate. We measured food intake, body weight, adipocyte diameter, serum cholesterol and lipoprotein concentrations, and aortic atherosclerosis. Macrophage immunostaining and collagen content were examined in aortic root lesions. CONTROL and TAG‐fed mice exhibited similar energy intake, body weights and blood glucose and insulin concentrations, but SUCR‐fed mice exhibited increased energy intake and became obese and hyperglycemic. Adipocyte diameter increased in female SUCR‐fed mice compared to TAG and CONTROL. Male and female SUCR‐fed mice had increased serum cholesterol and triglyceride concentrations compared to TAG and CONTROL. Atherosclerosis was increased in SUCR‐fed mice of both genders compared to TAG and CONTROL. Lesions from SUCR‐fed mice exhibited pronounced macrophage immunostaining and reductions in collagen content compared to TAG and CONTROL mice. These results demonstrate that in comparison to sucrose, equivalent substitution of TAG as dietary carbohydrate does not result in the same extent of obesity, hyperglycemia, hyperlipidemia, and atherosclerosis.     

Crystal structure of D‐glycero‐Β‐D‐manno‐heptose‐1‐phosphate adenylyltransferase from Burkholderia pseudomallei

The crystal structure of HldC from B. pseudomallei (BpHldC), the fourth enzyme of the heptose biosynthesis pathway, has been determined. BpHldC converts ATP and d ‐glycero‐β‐d ‐manno‐heptose‐1‐phosphate into ADP‐d ‐glycero‐β‐d ‐manno‐heptose and pyrophosphate. The crystal structure of BpHldC belongs to the nucleotidyltransferase α/β phosphodiesterase superfamily sharing a common Rossmann‐like α/β fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues. Intriguingly, a reorientation of the C‐terminal helix seems to guide open and close states of the active site for the catalytic reaction.     

Regulation of adipose‐tissue‐derived stromal cell orientation and motility in 2D‐ and 3D‐cultures by direct‐current electrical field

Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane‐protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose‐tissue‐derived stromal cells (ADSCs) in 2D‐culture on plastic culture dishes and in 3D‐culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L‐lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological‐strength EFs in a homemade EF‐bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time‐lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D‐culture aligned vertically to EF vector and kept a good cell survival rate. In 3D‐culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D‐culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors.     

Efficient racemization of N‐phenylacetyl‐D‐glufosinate for L‐glufosinate production

Most amino acids contain chiral centres and exist as both D‐enantiomer and L‐enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L‐glufosinate (L‐PPT) was obtained by coupling amidase‐mediated hydrolysis of N‐phenylacetyl‐D,L‐glufosinate with racemization of N‐phenylacetyl‐D‐glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D‐enantiomer. To improve the yield of L‐PPT, the racemization reaction conditions were optimized, and through single‐factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L‐PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L‐PPT to 97.02%.     

Structural analysis and reaction mechanism of the disproportionating enzyme (D‐enzyme) from potato

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D‐enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α‐1, 4‐glucan. D‐enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D‐enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26‐meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D‐enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D‐enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D‐enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.     

3D‐Printed Stretchable Micro‐Supercapacitor with Remarkable Areal Performance

While stretchable micro‐supercapacitors (MSCs) have been realized, they have suffered from limited areal electrochemical performance, thus greatly restricting their practical electronic application. Herein, a facile strategy of 3D printing and unidirectional freezing of a pseudoplastic nanocomposite gel composed of Ti₃C₂T_x MXene nanosheets, manganese dioxide nanowire, silver nanowires, and fullerene to construct intrinsically stretchable MSCs with thick and honeycomb‐like porous interdigitated electrodes is introduced. The unique architecture utilizes thick electrodes and a 3D porous conductive scaffold in conjunction with interacting material properties to achieve higher loading of active materials, larger interfacial area, and faster ion transport for significantly improved areal energy and power density. Moreover, the oriented cellular scaffold with fullerene‐induced slippage cell wall structure prompts the printed electrode to withstand large deformations without breaking or exhibiting obvious performance degradation. When imbued with a polymer gel electrolyte, the 3D‐printed MSC achieves an unprecedented areal capacitance of 216.2 mF cm^?2 at a scan rate of 10 mV s^?1, and remains stable when stretched up to 50% and after 1000 stretch/release cycles. This intrinsically stretchable MSC also exhibits high rate capability and outstanding areal energy density of 19.2 µWh cm^?2 and power density of 58.3 mW cm^?2, outperforming all reported stretchable MSCs.     

Bioactive Tyrosine‐Derived Cytochalasins from Fungus Eutypella sp. D‐1

Three new cytochalasins Z₂₄, Z₂₅, Z₂₆ ( 1 – 3 , resp.) and one known compound, scoparasin B ( 4 ), were isolated from the fungus Eutypella sp. D‐1 isolated from the soil of high latitude of the Arctic. The structures of 1 – 3 were elucidated from spectroscopic data (NMR, MS). These compounds were evaluated for cytotoxic activities against several human tumor cell lines. Among them, compound 1 exhibited moderate cytotoxicity toward human breast cancer MCF‐7 cell line with IC₅₀ of 9.33 μM .     

Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D‐tyrosine using random and site directed mutagenesis approaches

The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2‐fold improvement in k_cat, 5.2‐fold lower K_m and 16‐fold improvement in catalytic efficiency for D ‐tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased k_cat for D ‐tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher k_cat and K_m value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D ‐tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9‐fold) improvement in k_cat and a 2.4‐fold increase in K_m compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the K_m up to 2.6‐fold for D ‐tyrosine but one variant 145_V153A increased the K_m 2.4‐fold compared to the WT. Homology based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an α‐helix providing one of the conserved histidine residues of the active site. The k_cat and K_m values for L ‐tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1‐fold high catalytic efficiency compared to the WT which is a 7.6‐fold lower improvement compared to D ‐tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D ‐tyrosine:D ‐DOPA and a 1.4‐fold higher L ‐tyrosine:L ‐DOPA activity ratio compared to the WT. Biotechnol. Bioeng. 2013; 110: 1849–1857. © 2013 Wiley Periodicals, Inc.     

Structure of 6‐diazo‐5‐oxo‐norleucine‐bound human gamma‐glutamyl transpeptidase 1, a novel mechanism of inactivation

Intense efforts are underway to identify inhibitors of the enzyme gamma‐glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma‐glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6‐diazo‐5‐oxo‐norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM ^?1 min^?1 and the K _i was 2.7 ± 0.7 mM . The crystal structure of DON‐inactivated hGGT1 contained a molecule of DON without the diazo‐nitrogen atoms in the active site. The overall structure of the hGGT1‐DON complex resembled the structure of the apo‐enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1‐DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α‐amine of Thr381. The structure of DON‐bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.     

Identification of a naturally‐occurring 8‐[α‐D‐glucopyranosyl‐(1→6)‐β‐D‐glucopyranosyl]daidzein from cultivated kudzu root

Introduction – Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one‐year‐old kudzu root cultivated in Vietnam. Objective – To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI⁺ TOF MS‐MS, ¹H‐, ¹³C‐NMR and enzymatic hydrolysis. Methodology – Samples were prepared by extraction of one‐year‐old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV‐light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the ¹³C and ¹H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. Results – ESI⁺ TOF MS‐MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by ¹H‐, ¹³C‐NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an α‐1,6‐glycosidic bond. This compound is the first naturally‐occurring 8‐[α‐D ‐glucopyranosyl‐(1→6)‐β‐D ‐glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl‐α‐1,6‐puerarin in kudzu root was 2.3 mg/g as determined by HPLC. Conclusion – The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water‐soluble glycosylated puerarin. Copyright © 2009 John Wiley & Sons, Ltd.