Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.     

The analysis of heparin-protein interactions using evanescent wave biosensor with regioselectively desulfated heparins as the ligands

Evanescent wave biosensor has been recently employed as a powerful tool for analyses of macromolecular interactions. In the present study, evanescent wave biosensor analysis was developed to analyze the heparin-protein interaction using as ligands a series of heparin derivatives regioselectively desulfated by chemical methods, particularly to evaluate the effect of each sulfate group of heparin. The method for immobilizing heparin on the cuvette of the evanescent wave biosensor equipment was optimized to obtain the high response required for accurate measurement. The best result was achieved when the amino group introduced at the reducing end of heparin was coupled with carboxymethyl dextran on the surface of the cuvette using glycolchitosan as a multivalent linker. The established system appeared to describe well the interactions of heparin with such proteins as acidic and basic fibroblast growth factors and tissue factor pathway inhibitor.     

(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.     

A human TERT C-terminal polypeptide sensitizes HeLa cells to H2O2-induced senescence without affecting telomerase enzymatic activity

We have constructed a 27-kDa hTERT C-terminal polypeptide (hTERTC27) devoid of domains required for telomerase activity and demonstrated that it is capable of nuclear translocation/telomere-end targeting. Here we showed that expression of a low level of hTERTC27 renders hTERT positive HeLa cells sensitive to H(2)O(2)-induced oxidative stress and subsequent cell senescence. The senescence-associated gene, the cyclin/cdk inhibitor p21(Waf1), was up-regulated. This occurs without changing the expression of endogenous hTERT, causing significant telomere shortening or inhibiting telomerase activity. Results from this study suggest for the first time that in addition to telomerase activity, the C-terminus of hTERT also plays a role in hTERT-mediated cellular resistance to oxidative stress.     

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.     

Effects of bile acids on dog pancreatic duct epithelial cell secretion and monolayer resistance

Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an (125)I(-) efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a (86)Rb(+) efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca(2+) concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger (125)I(-) efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an I(sc) increase from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl(-) gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an I(sc) increase from apically permeabilized PDEC subject to a luminal-to-serosal K(+) gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca(2+)-activated apical Cl(-) channels and basolateral K(+) channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.     

Low-temperature (180 K) crystal structure,electron paramagnetic resonance spectroscopy,and propitious anticonvulsant activities of CuII2(aspirinate)4(DMF)2 and other CuII2(aspirinate)4 chelates

The purpose of this research was to characterize by X-ray crystallography the ternary dimethylformamide (DMF) Cu(II) complex of acetylsalicylic acid (aspirin), in an effort to compare the structure-activity relationships for the anticonvulsant activity of this and other Cu(II)aspirinate chelates. The ternary DMF Cu(II) complex of aspirin was synthesized and crystals grown from a DMF solution were characterized by single crystal X-ray diffraction. This crystalline material was analyzed for anticonvulsant activity in the Maximal Electroshock (MES) Grand Mal and subcutaneous Metrazol (scMET) Petit Mal models of seizure used to detect anticonvulsant activity. The ternary DMF complex was found to be a monomolecular binuclear complex, tetrakis-mu-(acetylsalicylato)bis(dimethylformamido)dicopper(II) [Cu(II)(2)(aspirinate)(4)(DMF)(2)] with the following parameters: monoclinic, space group P2(1)/n, a=12.259 (1), b=10.228 (1), c=16.987 (1) A, beta=92.07 (1) degrees; V=2128.5 (3) A(3); Z=2. The structure was determined at 180 K from 2903 unique reflections (I>1sigma(I)) to the final values of R=0.030 and wR=0.033 using F. This binuclear complex contains four acetylsalicylate bridging ligands which are related to each other in a two by two symmetry center. The four nearest O atoms around each Cu atom form a closely square planar arrangement with the square pyramidal coordination completed by the dimethylformamide oxygen atom occupying an apical position at a distance of 2.154 (1) A. Each Cu atom is displaced towards the DMF ligand by 0.187 A from the plane of the four O atoms. Electron paramagnetic resonance (EPR) spectra of [Cu(II)(2)(aspirinate)(4)(DMF)(2)] crystals show a strong antiferromagnetic coupling of the copper atoms, similar to that observed with other binuclear copper(II)salicylate compounds. Studies used to detect anticonvulsant activity revealed that [Cu(II)(2)(aspirinate)(4)(DMF)(2)] was an effective anticonvulsant in the MES model of seizure but ineffective against scMET-induced seizures. The monomolecular ternary binuclear [Cu(II)(2)(aspirinate)(4)(DMF)(2)] complex is more effective in inhibiting MES-induced seizures than other binuclear or mononuclear Cu(II) chelates of aspirin including: binuclear polymeric [Cu(II)(2)(aspirinate)(4)], [Cu(II)(2)(aspirinate)(4)(H(2)O)], which is anticipated to be less polymeric, and monomolecular ternary [Cu(II)(2)(aspirinate)(4)(DMSO)(2)] and [Cu(II)(aspirinate)(2)(Pyr)(2)]. These and other chelates appear to be more effective in the scMET model of seizure than [Cu(II)(2)(aspirinate)(4)(DMF)(2)]. These structure-activity relationships support the potential efficacy of Cu chelates of aspirin in treating epilepsies.     

High-level expression of a truncated wall-associated protein A from the dental cariogenic Streptococcus mutans

Streptococcus mutans plays a primary role in the formation of dental caries. Previously, in our laboratory, an S. mutans genomic library was prepared, and the wapA gene was cloned into the shuttle vector, pSA4/4B2. To generate overexpression of wapA and to facilitate efficient purification of the WapA protein for use as an immunogen, an expression vector with the strong tac promoter was used. In order to answer questions regarding the optimization of solubility and expression based on gene size or the hydrophobicity of the protein product, 12 truncated constructs of the wapA gene were prepared using PCR. The truncated products were subcloned into the pGEX-6P-1 glutathione S-transferase (GST) fusion vector and expressed in E. coli BL21. The fusion proteins were analyzed by SDS-PAGE and confirmed by analysis with anti-GST and anti-WapA antibodies. Our study suggests that abrogation of the wapA promoter is necessary for expression of this gene in this expression system. Deletion of the signal peptide and the hydrophobic C terminus of WapA increased expression compared with the full-length construct, and truncation at the protease cleavage site of the C-terminal region greatly increased the stability of the protein without a loss in reactivity with the anti-WapA antibody. Western immunoblot analysis with anti-WapA antiserum clearly showed that the majority of the epitopes of the GST-WapA fusions are located in the N-terminal region of WapA. The immunogenicity of the various WapA fusion products is being examined in mice and rats to further map the immunologically dominant regions of the protein. This method effectively increased the expression of WapA and should contribute to the further understanding of gene expression of E. coli, as well as aid in the characterization of this protein for future immunologic evaluation.     

柑桔原生质体辐射诱变筛选抗寒再生植株

用１１６１Ｃ／ｋｇ软Ｘ射线辐射“Ｐａｇｅ”桔柚（ＣｉｔｒｕｓｒｅｔｉｃｕｌａｔａＢｌａｎｃｏ×Ｃ．ｇｒａｎｄｉｓＯｓｂ．ｃｖ．Ｐａｇｅ）胚性愈伤组织分离的原生质体,经－１１℃低温选择后,将存活的原生质体培养再生植株。研究表明,“Ｐａｇｅ”桔柚胚性愈伤组织原生质体在４１２８Ｃ／ｋｇ辐射剂量处理后几乎不能恢复分裂;２０６４Ｃ／ｋｇ辐射时分裂频率低,即使分裂也难以发育为胚状体;而１１６１Ｃ／ｋｇ辐射后,经－１１℃低温处理仍有１０２％～１６９％的分裂频率,且部分细胞团能发育为胚状体。辐射的原生质体培养成胚状体后,经低温筛选,在改良的生芽培养基和生根培养基中萌发成为植株。再生植株中检测出２株的叶片原生质体的低温半致死温度（ＬＴ５０）分别比对照低１．９６℃和１６８℃。     

糖类在植物组织培养中的效应

糖类是影响植物组织培养成功与否的关键之一。迄今为止,已用于植物组织培养的糖类有５０多种。在植物体细胞组织培养中,蔗糖一直作为标准碳源,然而其他糖类物质如：葡萄糖、麦芽糖和山梨醇对植物体细胞培养也产生了一定的影响。在植物花药培养中,蔗糖较好,但应注意麦芽糖对花药培养的促进作用。