Improving freezing tolerance of transgenic tobacco expressing sucrose: sucrose 1-fructosyltransferase gene from Lactuca sativa

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.     

Synthesis and evaluation of isosteres of N-methyl indolo[3,2-b]-quinoline (cryptolepine) as new antiinfective agents

Isosteres of cryptolepine (1) were synthesized and evaluated for their antiinfective activities. Overall, the sulfur isostere, 5-methyl benzothieno[3,2-b]quinolinium salt (5b), was equipotent to 1 and has shown no cytotoxicity at 23.8 microg/mL. Compound 5b was also found to have a broad spectrum of activity. Both the carbon and oxygen isosteres were less potent than cryptolepine. A limited library of 2-substituted analogs of 5b has been synthesized and evaluated in antifungal screens but did not show increase in potency compared to the unsubstituted 5b. Similarly, evaluation of tricyclic benzothieno[3,2-b]pyridines while showing promise in individual screens did not produce an overall increase in potency. Overall, the evaluation of the activities of 5b compared with standard antifungal/anti-protozoal agents suggests that the benzothienoquinoline scaffold could serve as a lead for optimization.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.     

High performance liquid chromatographic determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel organoselenium compound, in dog plasma using pre-column derivatization and its application in pharmacokinetic study

A novel HPLC-UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05-2 microg/ml (r(2)=0.9995). The lower limit of quantification (LOQ) was 0.05 microg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC(0-t) were 5.72+/-2.42 and 1.35+/-0.41 microg h/ml; t(1/2) were 4.6+/-2.1 and 1.7+/-0.6h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.     

CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.     

A new model of phycobilisome inSpirulina platensis

Phycobilisomes (PBS) were isolated from blue-green algaSpirulina platensis. Scanning tunneling microscope was used to investigate the three-dimensional structure of PBS deposited on freshly cleaved highly oriented pyrolytic graphite (HOPG) in ambient condition at room temperature. The results showed that the rods of PBS radiated from the core to different directions in the space other than arrayed in one plane, which was different from the typical hemi-discoidal model structure. The diameter of PBS was up to 70 nm, and the rod was approximately 50 nm in length. Similar results were observed in Langmuir-Blodgett (LB) film of PBS. The dissociated PBS could reaggregate into rod-like structures and easily form two-dimensional membrane while being absorbed on HOPG, however, no intact PBS was observed. The filling-space model structure of PBS inSpirulina platensis with STM from three-dimensional real space at nanometer scale was found, which showed that this new structural model of PBS surely exists in blue-green algae and red algae. The function of this structural model of PBS was also discussed.     

Studies on the Chemical Constituents of Limonium aureum (L.) Hill

Five different crystals have been obtained for the first time from the aerial parts of Limonium aureum (L.)Hill ex kuntze. They were identified as follows: (Ⅰ) homoeriodictyol, (Ⅱ) naringenin, (Ⅲ) eriodictyol (Ⅳ) myricetin-3-O-β-D-glucoside and (Ⅴ) myricetin-3-O-β-D-galactoside.     

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.     

Expression Patterns and Potential Biological Roles of Dip2a

Disconnected (disco)-interacting protein 2 homolog A is a member of the DIP2 protein family encoded by Dip2a gene. Dip2a expression pattern has never been systematically studied. Functions of Dip2a in embryonic development and adult are not known. To investigate Dip2a gene expression and function in embryo and adult, a Dip2a-LacZ mouse model was generated by insertion of β-Gal cDNA after Dip2a promoter using CRISPR/Cas9 technology. Dip2a-LacZ mouse was designed to be a lacZ reporter mouse as well as a Dip2a knockout mouse. Heterozygous mice were used to study endogenous Dip2a expression and homozygotes to study DIP2A-associated structure and function. LacZ staining indicated that Dip2a is broadly expressed in neuronal, reproductive and vascular tissues, as well as in heart, kidney, liver and lung. Results demonstrate that Dip2a is expressed in ectoderm-derived tissues in developing embryos. Adult tissues showed rich staining in neurons, mesenchymal, endothelial, smooth muscle cells and cardiomyocytes by cell types. The expression pattern highly overlaps with FSTL1 and supports previous report that DIP2A to be potential receptor of FSTL1 and its protective roles of cardiomyocytes. Broad and intense embryonic and adult expression of Dip2a has implied their multiple structural and physiological roles.