Follicle characteristics and follicle developmental related Wnt6 polymorphism in Chinese indigenous Wanxi-white goose

In birds, downy feather quantity mainly affected by the follicles. Wnt6, a secreted cysteine-rich protein, plays a key role in follicular development as an intercellular signaling molecule. The present study was to investigate the follicle development and Wnt6 polymorphism in Wanxi-white geese, a Chinese indigenous breed. In total, 300 fertilized eggs were hatched. At embryonic stage and on early birth goslings, the diameter and density of follicles from different sites were examined after sectioning and staining. The results showed that the diameter of primary feather follicles in thorax, venter, dorsum and flank had no difference at embryonic stage. In contrast, after birth, thorax and ventral feather follicles had greater diameter than those on dorsum and flank. Similarly, the primary feather follicle density was higher in thorax and venter than in dorsum and flank at embryonic stage. The secondary feather follicle diameter in flank was greater than that in other sites examined. The secondary follicle showed lush growth in E27 with thickest in ventral and thorax. Overall, follicle formed consistently in dorsal and flank, and follicle in thorax and ventral formed in another consistent way. The polymorphism study showed 2 single nucleotide polymorphisms of Wnt6 and 3 genotypes identified. Sequencing revealed two nucleotide transitions, T451C and A466G, which were synonymous mutations causing codons for aspartate and lysine to change from GAU to GAC and from AAA to AAG, respectively. This information about follicle development and Wnt6 polymorphisms would provide potential utilization in marker-assisted selection program for down feather selection.     

Ferroportin 1 and hephaestin expression in BeWo cell line with different iron treatment

The process of placental iron transfer is an important physiological process during pregnancy. However, the molecular mechanism of placental iron transport has not been completely elucidated until now. Ferroportin 1 (FPN1) and hephaestin (Heph) have been identified as the important molecules involved in duodenal iron export. However, whether they participate in the placental iron efflux has been undefined until now. In this study, the BeWo cells were treated with desferrioxamine and Holo-transferrin human in different concentrations and harvested at 48 and 72 h. The mRNA expression of FPN1 and Heph was detected with quantitative real-time polymerase chain reaction, and the protein expression was detected with western blots. The results showed an up-regulated FPN1 expression with desferrioxamine treatment and down-regulated expression with Holo-transferrin human supplementation. However, the change of FPN1 expression at protein level was limited. Heph expression enhanced when cells were treated with desferrioxamine although the quantity of Heph expression was low. Heph expression showed no significant change with Holo-transferrin human supplementation. It indicates that FPN1 may participate in placental iron transport, and placental FPN1 expression is obviously not dependent on the iron regular element/iron regular protein regulation. An alternatively spliced FPN1 isoform that lacks an iron regular element may be the predominant expression in BeWo cells. It also demonstrates that Heph is active in placenta but may not play a key role in placental iron transport because it is not the main part of placental copper oxidase.     

Novel highly branched water-soluble heteropolysaccharides as immunopotentiators to inhibit S-180 tumor cell growth in BALB/c mice

The in vitro antitumor activities and in vivo immunomodulatory effects of water-soluble highly branched heteropolysaccharides isolated from Rhizoma panacis Japonici were evaluated by three cell lines and BALB/c mice implanted with Sarcoma 180 (S-180) tumors. The heteropolysaccharides showed potent tumor therapeutic effect by potentiating the animal's immune responses including an increase in white blood cell count and lymphocyte number. The heteropolysaccharides also induced apoptosis in the S-180 cells and alleviate the short-term side effect of cisplatin (DDP) to the animal during the treatment period. The bioactivity of the heteropolysaccharides might be due to their unique structures that had an α-(1 → 4)-d-glucan main chain with side chains containing mannose and galactose residues, a spherical chain conformation, as well as high water solubility, all of which facilitated their interaction with the surface receptors of the immune cells.     

Regulation of enzyme activity of alcohol dehydrogenase through its interactions with pyruvate-ferredoxin oxidoreductase in Thermoanaerobacter tengcongensis

Alcohol dehydrogenases (ADHs) from thermophilic microorganisms are interesting enzymes that have their potential applications in biotechnology and potentially provide insight into the mechanisms of action of thermo-tolerant proteins. The molecular mechanisms of ADHs under thermal stress in vivo have yet to be explored. Herein, we employed a proteomic strategy to survey the possible interactions of secondary-ADH (2-ADH) with other proteins in Thermoanaerobacter tengcongensis (T. tengcongensis) cultured at 75°C and found that 2-ADH, pyruvate-ferredoxin oxidoreductase (PFOR) and several glycolytic enzymes coexisted in a protein complex. Using anion exchange chromatography, the elution profile indicated that the native 2-ADH was present in two forms, PFOR-bound and PFOR-free. Immuno-precipitation and pull down analysis further validated the interactions between 2-ADH and PFOR. The kinetic behaviours of 2-ADH either in the recombinant or native form were evaluated with different substrates. The enzyme activity of 2-ADH was inhibited in a non-competitive mode by PFOR, implying the interaction of 2-ADH and PFOR negatively regulated alcohol formation. In T. tengcongensis, PFOR is an enzyme complex located at the upstream of 2-ADH in the alcohol generation pathway. These findings, therefore, offered a plausible mechanism for how alcohol metabolism is regulated by hetero-interactions between 2-ADH and PFOR, especially in anaerobic thermophiles.     

Synthesis and evaluation of ursolic acid derivatives as potent cytotoxic agents

Structural modification was performed at the C-3 and C-28 positions of ursolic acid (UA). Ten UA derivatives with distinct electrical property were synthesized. They could be divided into two groups according to their charge under physiological conditions: (1) Group I negatively charged and (2) Group II positively charged. The anti-proliferative capability of the derivatives was evaluated against HepG2, AGS, HT-29 and PC-3 cells by the MTT assay. Flow cytometry and Annexin V/PI dual staining assay were carried out to explore the antitumor mechanism. The results showed the cytotoxic capacity of the compounds was: Group I相似文献   

Proteomic analysis of the nucleus accumbens in rhesus monkeys of morphine dependence and withdrawal intervention

It has been known that the reinforcing effects and long-term consequences of morphine are closely associated with nucleus accumbens (NAc) in the brain, a key region of the mesolimbic dopamine pathway. However, the proteins involved in neuroadaptive processes and withdrawal symptom in primates of morphine dependence have not been well explored. In the present study, we performed proteomes in the NAc of rhesus monkeys of morphine dependence and withdrawal intervention with clonidine or methadone. Two-dimensional electrophoresis was used to compare changes in cytosolic protein abundance in the NAc. We found a total of 46 proteins differentially expressed, which were further identified by mass spectrometry analysis. The identified proteins can be classified into 6 classes: metabolism and mitochondrial function, synaptic transmission, cytoskeletal proteins, oxidative stress, signal transduction and protein synthesis and degradation. Importantly, we discovered 14 proteins were significantly but similarly altered after withdrawal therapy with clonidine or methadone, revealing potential pharmacological strategies or targets for the treatment of morphine addiction. Our study provides a comprehensive understanding of the neuropathophysiology associated with morphine addiction and withdrawal therapy in primate, which is helpful for the development of opiate withdrawal pharmacotherapies.     

Tuning the properties of elastin mimetic hybrid copolymers via a modular polymerization method

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)(2) (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)(2)X (AK2-CBD1) and X(AKAAAKA)(2)XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.     

Lysosome-membrane fusion mediated superoxide production in hyperglycaemia-induced endothelial dysfunction

Lysosomal exocytosis and fusion to cellular membrane is critical in the oxidative stress formation of endothelium under apoptotic stimulus. We investigated the role therein of it in hyperglycaemia-induced endothelial dysfunction. The lysosome-membrane fusion was shown by the expression of lamp1, the lysosomal membrane marker, on cellular membrane and the transportation of lysosomal symbolic enzymes into cultural medium. We also examined the ceramide production, lipid rafts (LRs) clustering, colocalization of gp91^phox, a NADPH oxidase subunit (NOX) to LRs clusters, superoxide (O₂ ^{. -}) formation and nitric oxide (NO) content in human umbilical vein endothelial cells (HUVEC) and the endothelium-dependent NO-mediated vasodilation in isolated rat aorta. As compared to normal glucose (5.6 mmol/l, Ctrl) incubation, high glucose (22 mmol/l, HG) exposure facilitated the lysosome-membrane fusion in HUVEC shown by significantly increased quantity of lamp1 protein on cellular membrane and enhanced activity of lysosomal symbolized enzymes in cultural medium. HG incubation also elicited ceramide generation, LRs clustering and gp91^phox colocalization to LRs clusters which were proved to mediate the HG induced O₂ ^{. -} formation and NO depletion in HUVEC. Functionally, the endothelium-dependent NO-mediated vasodilation in aorta was blunted substantially after HG incubation. Moreover, the HG-induced effect including ceramide production, LRs clustering, gp91^phox colocalization to LRs clusters, O₂ ^{. -} formation and endothelial dysfunction could be blocked significantly by the inhibition of lysosome-membrane fusion. We propose that hyperglycaemia-induced endothelial impairment is closely related to the lysosome-membrane fusion and the following LRs clustering, LRs-NOX platforms formation and O₂ ^{. -} production.     

RNT4 3'-UTR insertion/deletion polymorphisms are not associated with atrial septal defect in Chinese Han population: a brief communication

Atrial septal defect (ASD) is a common type of congenital heart disease, which is defined as any communication through atrial septum. Several studies have revealed that genetic factors may influence the susceptibility of ASD. Recent studies have shown that reticulon 4 (RTN4) gene might be involved in some processes relevant to heart development, such as regulation of cell migration and vascular remodeling. This study aimed to evaluate RTN4 gene polymorphisms of CAA and TATC insertion/deletion in relation to the risk of ASD in Chinese Han population. A total of 175 ASD patients and 308 unrelated healthy controls were successfully investigated. The polymorphisms of patients were determined by polymerase chain reaction-polyacrylamide gel electrophoresis. There was no significant difference in the allele frequencies of CAA and TATC insertion/deletion in RNT4 gene between ASD patients and controls. The same results were seen in their genotypes. The present study suggests that CAA and TATC insertion/deletion polymorphisms of RNT4 gene may not be a useful marker to predict the susceptibility of ASD in Chinese Han population.     

Ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production

Yeast flocculation is an important trait in the brewing industry as well as in ethanol production, through which biomass can be recovered by cost-effective sedimentation. However, mass transfer limitation may affect yeast growth and ethanol fermentation if the flocculation occurs earlier before fermentation is completed. In this article, a novel type of cell-cell flocculation induced by trehalose-6-phosphate synthase 1 (TPS1) promoter was presented. The linear cassette HO-P(TPS1)-FLO1(SPSC01)-KanMX4-HO was constructed to transform the non-flocculating industrial yeast S. cerevisiae 4126 by chromosome integration to obtain a new flocculating yeast strain, ZLH01, whose flocculation was induced by ethanol produced during fermentation. The experimental results illustrated that flocculation of ZLH01 was triggered by 3% (v/v) ethanol and enhanced as ethanol concentration increased till complete flocculation was achieved at ethanol concentration of 8% (v/v). Real time PCR analysis confirmed that the expression of FLO1(SPSC01) was dependent on ethanol concentration. The growth and ethanol fermentation of ZLH01 were improved significantly, compared with the constitutive flocculating yeast BHL01 engineered with the same FLO gene but directed by the constitutive 3-phosphoglycerate kinase promoter PGK1, particularly under high temperature conditions. These characteristics make the engineered yeast more suitable for ethanol production from industrial substrates under high gravity and temperature conditions. In addition, this strategy offers advantage in inducing differential expression of other genes for metabolic engineering applications of S. cerevisiae.