Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIM_S, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.     

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na(+) and K(+) concentrations is important for bacterial cells, which, in the absence of Na(+) extrusion systems, cannot grow in the presence of high external Na(+). Likewise, bacteria require K(+) uptake systems when the external K(+) concentration becomes too low to support growth. At present, we have little knowledge of K(+) toxicity and bacterial outward-directed K(+) transport systems. We report here that high external concentrations of K(+) at alkaline pH are toxic and that bacteria require K(+) efflux and/or extrusion systems to avoid excessive K(+) accumulation. We have identified the first example of a bacterial K(+)(specific)/H(+) antiporter, Vp-NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter-1 (CPA1) family, was able to mediate K(+) extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na(+)/H(+) antiporters, Vp-NhaA and Vp-NhaB, which also exhibit a novel ion specificity toward K(+), implying that they work as Na(+)(K(+))/H(+) exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K(+) extrusion against a K(+) chemical gradient, indicating that E. coli also possesses an unidentified K(+) extrusion system(s).     

Automated drug delivery system to control systemic arterial pressure, cardiac output, and left heart filling pressure in acute decompensated heart failure.

Pharmacological support with inotropes and vasodilators to control decompensated hemodynamics requires strict monitoring of patient condition and frequent adjustments of drug infusion rates, which is difficult and time-consuming, especially in hemodynamically unstable patients. To overcome this difficulty, we have developed a novel automated drug delivery system for simultaneous control of systemic arterial pressure (AP), cardiac output (CO), and left atrial pressure (Pla). Previous systems attempted to directly control AP and CO by estimating their responses to drug infusions. This approach is inapplicable because of the difficulties to estimate simultaneous AP, CO, and Pla responses to the infusion of multiple drugs. The circulatory equilibrium framework developed previously (Uemura K, Sugimachi M, Kawada T, Kamiya A, Jin Y, Kashihara K, and Sunagawa K. Am J Physiol Heart Circ Physiol 286: H2376-H2385, 2004) indicates that AP, CO, and Pla are determined by an equilibrium of the pumping ability of the left heart (SL), stressed blood volume (V), and systemic arterial resistance (R). Our system directly controls SL with dobutamine, V with dextran/furosemide, and R with nitroprusside, thereby controlling the three variables. We evaluated the efficacy of our system in 12 anesthetized dogs with acute decompensated heart failure. Once activated, the system restored SL, V, and R within 30 min, resulting in the restoration of normal AP, CO, and Pla. Steady-state deviations from target values were small for AP [4.4 mmHg (SD 2.6)], CO [5.4 ml x min(-1) x kg(-1) (SD 2.4)] and Pla [0.8 mmHg (SD 0.6)]. In conclusion, by directly controlling the mechanical determinants of circulation, our system has enabled simultaneous control of AP, CO, and Pla with good accuracy and stability.     

Vascular Endothelial-Cadherin Stabilizes at Cell–Cell Junctions by Anchoring to Circumferential Actin Bundles through α- and β-Catenins in Cyclic AMP-Epac-Rap1 Signal-activated Endothelial Cells

Vascular endothelial (VE)-cadherin is a cell–cell adhesion molecule involved in endothelial barrier functions. Previously, we reported that cAMP-Epac-Rap1 signal enhances VE-cadherin–dependent cell adhesion. Here, we further scrutinized how cAMP-Epac-Rap1 pathway promotes stabilization of VE-cadherin at the cell–cell contacts. Forskolin induced circumferential actin bundling and accumulation of VE-cadherin fused with green fluorescence protein (VEC-GFP) on the bundled actin filaments. Fluorescence recovery after photobleaching (FRAP) analyses using VEC-GFP revealed that forskolin stabilizes VE-cadherin at cell–cell contacts. These effects of forskolin were mimicked by an activator for Epac but not by that for protein kinase A. Forskolin-induced both accumulation and stabilization of junctional VEC-GFP was impeded by latrunculin A. VE-cadherin, α-catenin, and β-catenin were dispensable for forskolin-induced circumferential actin bundling, indicating that homophilic VE-cadherin association is not the trigger of actin bundling. Requirement of α- and β-catenins for forskolin-induced stabilization of VE-cadherin on the actin bundles was confirmed by FRAP analyses using VEC-GFP mutants, supporting the classical model that α-catenin could potentially link the bundled actin to cadherin. Collectively, circumferential actin bundle formation and subsequent linkage between actin bundles and VE-cadherin through α- and β-catenins are important for the stabilization of VE-cadherin at the cell–cell contacts in cAMP-Epac-Rap1 signal-activated cells.     

Random AT library: autonomously replicating sequence (ARS) activity of chemically synthesized random sequences for transformation of nonconventional yeast species

In a search for sequences that confer on bacterial plasmids the capacity of autonomous replication in yeast cells, we chemically synthesized polynucleotides 80 bp in length from an equimolar mixture of A and T. The random AT-polymer population, W80, was inserted into the plasmid YIp5-Kan1 (which carries the markers URA3 and G418(R), but does not replicate in yeast) and amplified in Escherichia coli. This library, representing 10 000 different AT sequences, was transformed into three species of yeast: Saccharomyces cerevisiae, Kluyveromyces lactis and Torulaspora delbrueckii. The aim was to evaluate the frequency, if any, of autonomously replicating sequences (ARSs) in the random sequences. A large number of transformants were obtained from each species. Many of them showed a stable transformed phenotype. Several W80 sequences were found many times for a given species, suggesting that each species preferred particular sequences for ARS function, although they are diverse in their primary sequence. In view of the high frequency and stability of the replicative plasmids found in the different hosts, this small random AT library may be conveniently used as a source of replicative gene vectors for genetic manipulation of many nonconventional yeast species, in place of searching for species-specific chromosomal ARSs.     

Antioxidant action of sugar-pendant C60 fullerenes

The action of C₆₀ fullerene and its derivatives as a radical-scavenging antioxidant has received much attention, but their reactivity toward free radicals and antioxidant capacity have not been well elucidated yet. In the present study, the reactivity of the two types of water-soluble, sugar-pendant C₆₀ fullerenes, C₆₀-1S and C₆₀-2S, toward peroxyl radical and their effect against human plasma lipid peroxidation were measured. The rate constants for the reaction of C₆₀-1S and C₆₀-2S with peroxyl radicals were obtained from their effect on the bleaching of β-carotene in lipid-SDS micelle system as 4.6 × 10³ and 8.0 × 10³ M^?1 s^?1 at 37 °C, respectively. They inhibited the free radical-induced lipid peroxidation in human plasma in a concentration-dependent manner. These results suggest that the sugar-pendant fullerenes C₆₀-1S and C₆₀-2S act as a radical-scavenging antioxidant with the activity similar to the phenolic antioxidants.     

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of E^rns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.     

猪胰岛素前体在酵母Kluyveromyces lactis中的分泌表达及其转化为人胰岛素

通过将包括猪胰岛素前体（ＰＩＰ）基因在内的表达框架克隆至质粒ｐＫＤ１衍生的两种载体上而在酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓｌａｃｔｉｓ中分泌表达猪胰岛素前体。根据放射免疫测定结果,猪胰岛素前体的表达水平为２０～３０ｍｇ／Ｌ。猪胰岛素前体经过转肽被转变为基因工程人胰岛素。分析结果表明,来自Ｋ．ｌａｃｔｉｓ的人胰岛素,其氨基酸组成、晶体形状和生物活力与天然胰岛素相同。