A novel cardiolipin-remodeling pathway revealed by a gene encoding an endoplasmic reticulum-associated acyl-CoA:lysocardiolipin acyltransferase (ALCAT1) in mouse

Cardiolipin is a major membrane polyglycerophospholipid that is required for the reconstituted activity of a number of key mitochondrial enzymes involved in energy metabolism. Cardiolipin is subjected to remodeling subsequent to its de novo biosynthesis to attain appropriate acyl composition for its biological functions. Yet, the enzyme(s) involved in the remodeling process have not been identified. We report here the identification and characterization of a murine gene that encodes an acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1). Expression of the ALCAT1 cDNA in either insect or mammalian cells led to a significant increase in acyl-CoA:monolysocardiolipin acyltransferase and acyl-CoA: dilysocardiolipin acyltransferase activities that exhibited a dependence upon ALCAT1 enzyme levels. The recombinant ALCAT1 enzyme recognizes both monolysocardiolipin and dilysocardiolipin as substrates with a preference for linoleoyl-CoA and oleoyl-CoA as acyl donors. In contrast, no significant increases in acyltransferase activities by the recombinant ALCAT1 were detected against either glycerol-3-phosphate or a variety of other lysophospholipids as substrates, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine. Immunocytohistochemical analysis showed that the ALCAT1 enzyme is localized in the endoplasmic reticulum, which is supported by a significant ALCAT activity in isolated liver and heart microsomes. Northern blot analysis indicates that the mouse ALCAT1 is widely distributed, with the highest expression in heart and liver. In support of a role for ALCAT1 in maintaining heart function, the ALCAT1 gene is conserved among different species of vertebrates, but not in non-atrium organisms. ALCAT1 represents the first identified cardiolipin-remodeling enzyme from any living organism; its identification implies a novel role for the endoplasmic reticulum in cardiolipin metabolism.     

Heat shock protein 90 and tyrosine kinase regulate eNOS NO* generation but not NO* bioactivity

An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion (O(2)(-*) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME), GA, and RAD. Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO. donor, increased vasodilation of arterioles pretreated with GA, RAD, and L-NAME equally well except at 10(-5) M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with L-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO* release from stimulated endothelial cell cultures and increased O(2)(-*) production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated O(2)(-*) production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.     

Enhanced antibiotic activity of Xenorhabdus nematophila by medium optimization

Nutrition had highly influence on the antibiotic production by Xenorhabdus nematophila YL001. Glucose and peptone were identified as the best carbon and nitrogen sources that significantly affected antibiotic production using one-factor-at-a-time approach. Response surface methodology was applied to optimize the medium constituents (Glucose, peptone and minerals) for antibiotic production by X. nematophila YL001. Higher antibiotic activity (328.9 U/ml) was obtained after optimizing medium components. The optimal levels of medium components were (g/l): glucose 6.13, peptone 21.29, MgSO(4).7H(2)O 1.50, (NH(4))(2)SO(4) 2.46, KH(2)PO(4) 0.86, K(2)HPO(4) 1.11 and Na(2)SO(4) 1.72. An overall 16% and 35% increase in antibiotic activity were obtained as compared with mean observed response (283.7U/ml) at zero level of all variables and YSG medium.     

Structure-based design of selective phosphodiesterase 4B inhibitors based on ginger phenolic compounds

Phosphodiesterase 4 (PDE4) has been established as a drug target for inflammatory diseases of respiratory tract like asthma and chronic obstructive pulmonary disease. The selective inhibitors of PDE4B, a subtype of PDE4, are devoid of adverse effects like nausea and vomiting commonly associated with non-selective PDE4B inhibitors. This makes the development of PDE4B subtype selective inhibitors a desirable research goal. Thus, in the present study, molecular docking, molecular dynamic simulations and binding free energy were performed to explore potential selective PDE4B inhibitors based on ginger phenolic compounds. The results of docking studies indicate that some of the ginger phenolic compounds demonstrate higher selective PDE4B inhibition than existing selective PDE4B inhibitors. Additionally, 6-gingerol showed the highest PDE4B inhibitory activity as well as selectivity. The comparison of binding mode of PDE4B/6-gingerol and PDE4D/6-gingerol complexes revealed that 6-gingerol formed additional hydrogen bond and hydrophobic interactions with active site and control region 3 (CR3) residues in PDE4B, which were primarily responsible for its PDE4B selectivity. The results of binding free energy demonstrated that electrostatic energy is the primary factor in elucidating the mechanism of PDE4B inhibition by 6-gingerol. Dynamic cross-correlation studies also supported the results of docking and molecular dynamics simulation. Finally, a small library of molecules were designed based on the identified structural features, majority of designed molecules showed higher PDE4B selectivity than 6-gingerol. These results provide important structural features for designing new selective PDE4B inhibitors as anti-inflammatory drugs and promising candidates for synthesis and pre-clinical pharmacological investigations.     

VEGF165 induces differentiation of hair follicle stem cells into endothelial cells and plays a role in in vivo angiogenesis

Within the vascular endothelial growth factor (VEGF) family of five subtypes, VEGF165 secreted by endothelial cells has been identified to be the most active and widely distributed factor that plays a vital role in courses of angiogenesis, vascularization and mesenchymal cell differentiation. Hair follicle stem cells (HFSCs) can be harvested from the bulge region of the outer root sheath of the hair follicle and are adult stem cells that have multi‐directional differentiation potential. Although the research on differentiation of stem cells (such as fat stem cells and bone marrow mesenchymal stem cells) to the endothelial cells has been extensive, but the various mechanisms and functional forms are unclear. In particular, study on HFSCs’ directional differentiation into vascular endothelial cells using VEGF165 has not been reported. In this study, VEGF165 was used as induction factor to induce the differentiation from HFSCs into vascular endothelial cells, and the results showed that Notch signalling pathway might affect the differentiation efficiency of vascular endothelial cells. In addition, the in vivo transplantation experiment provided that HFSCs could promote angiogenesis, and the main function is to accelerate host‐derived neovascularization. Therefore, HFSCs could be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischaemic diseases.     

Glycoprotein profiling of stem cells using lectin microarray based on surface plasmon resonance imaging

Lectin microarrays have emerged as a novel platform for glycan analysis during recent years. Here, we have combined surface plasmon resonance imaging (SPRi) with the lectin microarray for rapid and label-free profiling of stem cells. In this direction, 40 lectins from seven different glyco-binding motifs and three different cell lines—mouse embryonic stem cells (mESCs), mouse-induced pluripotent stem cells (miPSCs), and mouse embryonic fibroblast stem cells (MEFs)—were used. Pluripotent mouse stem cells were clearly distinguished from non-pluripotent stem cells. Eight lectins—DBA, MAL, PHA_E, PHA_L, EEL, AAL, PNA, and SNA—generated maximal value to define pluripotency of mouse stem cells in our experiments. The discriminant function based on lectin reactivities was highly accurate for the determination of stem cell pluripotency. These results suggested that glycomic analysis of stem cells leads to a novel comprehensive approach for quality control in cell-based therapy and regenerative medicine.     

Estimation of genetic parameters for disease‐resistance traits in Cynoglossus semilaevis (Günther, 1873)

The objective of this study was to estimate genetic parameters for three traits based on 28 Cynoglossus semilaevis families approximately 6 months of age (at least 5 cm total length), including trait_1 (survival of 26 families, 3434 individuals in total subjected to challenge tests with Edwardsiella tarda), trait_2 (survival of 20 families, 2016 individuals in total subjected to challenge tests with Vibrio anguillarum) and trait_3 (survival of 27 families, 9340 individuals tagged at circa 180 days of age and reared in indoor ponds for circa another 5 months). The result showed that there were large differences in the survival of the families after challenge (11.11–65.31% for E. tarda and 9.18–70.54% for V. anguillarum). Additionally, the survival of families reared in indoor ponds was also different, varying from 21.00% to 73.67%. Heritabilities of the three traits varied from 0.14 to 0.26, as estimated by the linear model (LM) and the threshold model (TM). The trait_1 heritabilities (0.26 and 0.19 estimated by LM and TM) were higher than those of the others (0.20 and 0.23 estimated by LM, 0.14 and 0.19 estimated by TM). The estimates of heritabilities using LM were consistently higher than those of TM in this study. There were significant medium genetic correlations of 0.44 and 0.42 between trait_1 and trait_2 obtained from LM and TM (P < 0.05). However, very low and non‐significant genetic correlations of trait_1 and trait_3 (?0.10 for LM, ?0.05 for TM), as well as those of trait_2 and trait_3 (0.05 for LM, 0.04 for TM) were obtained. Therefore, indirect selection for trait_1 and trait_2 was effective, but almost ineffectual for trait_1 and trait_3 as well as trait_2 and trait_3. Otherwise, there was no significant difference in the predictive abilities of LM and TM. Two families resistant to both Edwardsiella tarda and Vibrio anguillarum were selected plus one family resistant to both Vibrio anguillarum and naturally infected by unknown pathogens through family selection. As there was very low and non‐significant genetic correlation of trait_3 and trait_1 as well as trait_2, superior strains are anticipated with the ability to resist two or more kinds of diseases, through the crossing of families selected for the three traits described above. The results support the hypothesis that genetic variation exists for disease survival, which could be used to design a breeding program for selecting strains of Cynoglossus semilaevis with high disease resistance.     

SHORT-ROOT 1 is critical to cell division and tracheary element development in rice roots

The exocyst is a key factor in vesicle transport and is involved in cell secretion, cell growth, cell division and other cytological processes in eukaryotes. EXO70 is the key exocyst subunit. We obtained a gene, SHORT-ROOT 1 (SR1), through map-based cloning and genetic complementation. SR1 is a conserved protein with an EXO70 domain in plants. SR1 mutation affected the whole root-development process: producing shorter radicles, adventitious roots and lateral roots, and demonstrating abnormal xylem development, resulting in dwarfing and reduced water potential and moisture content. SR1 was largely expressed in the roots, but only in developing root meristems and tracheary elements. The shortness of the sr1 mutant roots was caused by the presence of fewer meristem cells. The in situ histone H4 expression patterns confirmed that cell proliferation during root development was impaired. Tracheary element dysplasia was caused by marked decreases in the inner diameters of and distances between the perforations of adjacent tracheary elements. The membrane transport of sr1 mutants was blocked, affecting cell division in the root apical region and the development of root tracheary elements. The study of SR1 will deepen our understanding of the function of EXO70 genes in Oryza sativa (rice) and guide future studies on the molecular mechanisms involved in plant root development.