Antioxidant properties of polysaccharide fractions with different molecular mass extracted with hot-water from rice bran

Polysaccharides were extracted with hot-water from rice bran and precipitated with 40% ethanol (PW1), 60% ethanol (PW2) and 80% ethanol (PW3) in turn. PW1 and PW3 were composed of Glu, Man, Gal, Rib and Ara with molar percent of 54.1%, 10.5%, 21.7%, 7.4% and 6.3% for PW1 and 50.7%, 10.1%, 32.7%, 2.4% and 4.1% for PW3. Man was not observed in PW2. The molecular weight distribution of PW1, PW2 and PW3 ranged from 1.2 × 10⁵ to 6.3 × 10⁶ Da, 3.5 × 10⁴ to 7.4 × 10⁴ Da and 5.3 × 10³ to 2.3 × 10⁴ Da, respectively. Antioxidant activity tests revealed that PW1 showed good capability of scavenging superoxide radical, hydroxyl free radical and anti-lipid peroxidation at 1.0 mg/mL. In the meanwhile PW3 exhibited the good potential for reducing power, chelating ferrous ions, scavenging 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonate), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydrogen peroxide effects.     

Identification, mRNA expression and characterization of a novel ANK-like gene from Chinese mitten crab Eriocheir japonica sinensis

Ankyrins are a family of adapter molecules mediating linkages between integral membrane and cytoskeletal proteins. Ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. A novel ANK-like gene of Chinese mitten crab Eriocheir japonica sinensis (denoted as EjsANK) was identified and cloned by expressed sequence tag and rapid amplification of cDNA end approaches. The full-length cDNA of EjsANK is 4375 bp and contains an open reading frame of 1095 bp which encodes a 364 amino acids polypeptide (40.23 kD) bearing seven ankyrin repeats. EjsANK cDNA has a 3073 bp uniquely long 3′ untranslated region with three K-box elements, one GY-like box domain and one Brd-like box domain. Sequence alignment and three-dimensional structural analyses revealed that EjsANK should be a novel cytosolic member of the ankyrin family. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsANK mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that the relative level of EjsANK mRNA expression was significantly higher in the abdomen at the first crab stage. Functional bioinformatics prediction analyses indicated that EjsANK has an analogical effect like IκB which is a key component of IκB/NF-κB complex in mammalian cells playing very important roles in the development process. Results suggest that EjsANK gene is involved in the early developmental regulation of Chinese mitten crab, especially brachyurization regulation.     

Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering

Escherichia coli only maintains a small amount of cellular malonyl-CoA, impeding its utility for overproducing natural products such as polyketides and flavonoids. Here, we report the use of various metabolic engineering strategies to redirect the carbon flux inside E. coli to pathways responsible for the generation of malonyl-CoA. Overexpression of acetyl-CoA carboxylase (Acc) resulted in 3-fold increase in cellular malonyl-CoA concentration. More importantly, overexpression of Acc showed a synergistic effect with increased acetyl-CoA availability, which was achieved by deletion of competing pathways leading to the byproducts acetate and ethanol as well as overexpression of an acetate assimilation enzyme. These engineering efforts led to the creation of an E. coli strain with 15-fold elevated cellular malonyl-CoA level. To demonstrate its utility, this engineered E. coli strain was used to produce an important polyketide, phloroglucinol, and showed near 4-fold higher titer compared with wild-type E. coli, despite the toxicity of phloroglucinol to cell growth. This engineered E. coli strain with elevated cellular malonyl-CoA level should be highly useful for improved production of important natural products where the cellular malonyl-CoA level is rate-limiting.     

Effects of three phthalate esters on the life-table demography of freshwater rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis> Pallas

Phthalate esters (PAEs) are mainly used in the polymer industry as external plasticizers in PVC, and tend to migrate slowly out of the plastic, either into the air by volatilization or into water or other solvents by dissolution. Di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP) and di-2-ethylhexyl phthalate (DEHP) are three members of PAEs, identified as priority controlled hazardous substances by the United States Environmental Protection Agency, and have been shown to have potential for endocrine disrupting effects on vertebrates and humans. The effects of DBP, BBP and DEHP on survival and reproduction of the freshwater rotifer Brachionus calyciflorus were studied using life-table demographic methods. The results showed that all the life-table demographic parameters of B. calyciflorus were markedly affected by DBP and BBP, but not by DEHP. The net reproductive rate representing the output of reproduction was more affected than all the other parameters representing population growth, development or survival of the rotifers. Compared to the solvent control, DBP and BBP, both at 500 μg l⁻¹, significantly increased the net reproductive rate, and prolonged the generation time and the life expectancy at hatching of the rotifers. DBP at 50 μg l⁻¹ markedly decreased the intrinsic rate of population increase of the rotifers, but the reverse was true for BBP at 50 and 500 μg l⁻¹. Among all the parameters, the intrinsic rate of population increase was the most sensitive to DBP and BBP. The levels of PAEs in water from all the studied rivers and lakes in the world did not affect the population growth of rotifers.     

Comparison of antitumor activities of different polysaccharide fractions from the stems of Dendrobium nobile Lindl

The antitumor activities of extracted polysaccharide fractions from the stems of Dendrobium nobile Lindl were investigated. Polysaccharides were sequentially extracted from the stems of D. nobile to obtain three fractions, i.e. water extract fraction (DNP-W), 5% NaOH extract fraction (DNP-OH) and 5% HCl extract fraction (DNP-H). Further the DNP-W was isolated to give six sub-fractions (DNP-W1, DNP-W2, DNP-W3, DNP-W4, DNP-W5 and DNP-W6) by anion-exchange chromatography. The monosaccharide profile, protein content, uronic acid content, total carbohydrate content, viscosity and molecular weight of nine polysaccharide fractions were analyzed. Both the in vivo and in vitro antitumor activities of nine polysaccharide fractions were evaluated and compared. Results indicated that DNP-W1 and DNP-W3 exhibited high antitumor activities against Sarcoma 180 in vivo and HL-60 in vitro. The results suggested that DNP-W1 and DNP-W3 could be considered as an effective natural antitumor source.     

Cholesterol is required for efficient endoplasmic reticulum-to-Golgi transport of secretory membrane proteins

Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3-6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss of mobility of proteins in the ER lumen. This impaired lateral mobility is correlated with impaired ER-to-Golgi transport. These results provide evidence for the first time that cholesterol is required in the ER membrane to maintain mobility of membrane proteins and thus protein secretion.     

Functional characterization of the Bcl-2 gene family in the zebrafish

Members of the Bcl-2 protein family control the intrinsic apoptosis pathway. To evaluate the importance of this family in vertebrate development, we investigated it in the zebrafish (Danio rerio). We found that the zebrafish genome encodes structural and functional homologs of most mammalian Bcl-2 family members, including multi-Bcl-2-homology (BH) domain proteins and BH3-only proteins. Apoptosis induction by gamma-irradiation required zBax1 and zPuma, and could be prevented by overexpression of homologs of prosurvival Bcl-2 family members. Surprisingly, zebrafish Bax2 (zBax2) was homologous to mammalian Bax by sequence and synteny, yet demonstrated functional conservation with human Bak. Morpholino knockdown of both zMcl-1a and zMcl-1b revealed their critical role in early embryonic zebrafish development, and in the modulation of apoptosis activation through the extrinsic pathway. These data indicate substantial functional similarity between zebrafish and mammalian Bcl-2 family members, and establish the zebrafish as a relevant model for studying the intrinsic apoptosis pathway.