In vitro and in vivo gene delivery by recombinant baculoviruses

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.     

The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

The cyclic AMP (cAMP) signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C_1a or C₂ domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C_1a or C₂ domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl₂, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC₅₀ were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.     

Mapping of the MRPm5 epitope to the cytosolic region between transmembrane helices 13 and 14 in the drug and organic anion transporter, MRP1 (ABCC1)

Multidrug resistance in human tumour cells is often associated with increased expression of the 190kDa multidrug resistance protein, MRP1, that belongs to the ATP-binding cassette superfamily of transport proteins. MRP1 is also an efficient transporter of many organic anions. In the present study, we have mapped the epitope of the MRP1-specific murine monoclonal antibody (MAb) MRPm5 to the decapeptide (1063)FFERTPSGNL(1072) located in the cytoplasmic loop (CL6) linking transmembrane helices 13 and 14 in the third membrane spanning domain of the protein. Several amino acids in the cytoplasmic loops of MRP1 have been reported to be important for its transport function; nevertheless, MAb MRPm5 does not inhibit vesicular uptake of the high affinity substrate leukotriene C(4). None of the other MRP1-reactive MAbs described to date map to CL6 of MRP1 which in turn enhances the utility of MAb MRPm5 for both clinical and experimental investigations of this transporter.     

Fas-mediated apoptosis is suppressed by calf serum in cultured bovine luteal cells

Calf serum (CS) is a common supplement used in cell culture. It has been suggested that CS contains substances protecting cells against apoptosis. To examine whether a culture system including CS is appropriate for studying apoptosis in bovine luteal cells, we examined the influence of CS on the expression of Fas, bcl-2 and bax gene. Since progesterone (P(4)) is known to be an anti-apoptotic factor in bovine luteal cells, the present study was carried out to examine the P(4) effect on apoptosis. Bovine mid-luteal cells were exposed to Fas ligand (Fas L) in the presence or in the absence of P(4) antagonist (onapristone, OP) in a basal medium (BM) containing 5% CS (BM-CS) or BM containing 0.1% BSA (BM-BSA). Although Fas L alone, OP alone or Fas L plus OP did not show any cytotoxic effect on the cells cultured in BM-CS, administration of OP or OP in combination with Fas L resulted in the killing of 30% and 55% of the cells cultured in BM-BSA medium, respectively (p<0.05). Concomitantly, CS inhibited bax mRNA expression and stimulated bcl-2 expression in the cells (p<0.05). Moreover, in the cells cultured with BM-CS, Fas mRNA expression was smaller than that of cells incubated in BM-BSA medium (p<0.05). The overall results suggest that CS suppressed Fas-mediated cell death in cultured bovine luteal cells by promoting the ratio of bcl-2 to bax expression and by inhibiting Fas expression. Therefore, it may be suggested that CS contains such anti-apoptotic substances (growth factors) amplifying the cell survival pathways in the bovine corpus luteum (CL) in vitro.     

Interaction of Poliovirus with Its Purified Receptor and Conformational Alteration in the Virion

Polypeptides of amino acids 1 to 241 (PVR241) and 1 to 330 (PVR330) of the human poliovirus receptor (hPVR) were produced in a baculovirus expression system. PVR241 contained extracellular domains 1 and 2 of hPVR, and PVR330 contained extracellular domains 1, 2, and 3. These peptides were purified by immunoaffinity column chromatography with an anti-hPVR monoclonal antibody (MAb). After the purification, PVR241 and PVR330 appeared to retain their native conformation as judged by reactivity with an anti-PVR MAb that recognized domain 1 of hPVR in a conformation-dependent manner. The virulent Mahoney strain of poliovirus type 1 was mixed with the purified PVRs in various concentrations. An average of at least 43 PVR330 molecules were able to bind to one virion particle under the conditions used. The equilibrium dissociation constant between the PVR330 molecule and the PVR binding site (canyon) on the virion was determined to be 4.50 ± (0.86) × 10⁻⁸ M at 4°C. Higher rates of conformational change of the virus (160S) to 135S and 80S particles were observed as the concentration of PVR330 was increased. In this in vitro system, the ratio of the amount of the 135S particle to that of the 80S particle seemed to be always constant. After the disappearance of the 160S particle, the amount of the 80S particle was not increased by further incubation at 37°C. These results suggested that the 80S particle was not derived from the 135S particle under the conditions used in this study.     

Molecular Breeding of Transgenic Rice Plants Expressing a Bacterial Chlorocatechol Dioxygenase Gene

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.     

Photosynthetic responses of birch and alder saplings grown in a free air CO2 enrichment system in northern Japan

Though birch and alder are the common pioneer tree species which dominate in northeast Asia, little is known about the effects of the predicted increase in atmospheric CO₂ concentrations ([CO₂]) upon their photosynthesis in field conditions. To investigate this, we grew 2-year-old saplings of three Betulaceae species (Betula platyphylla var. japonica Hara, Betula maximowicziana Regel, and Alnus hirsuta Turcz) for 2 years in a free air CO₂ enrichment system in northern Japan. Since the effect of high [CO₂] is known to depend on soil conditions, we evaluated the responses in two soils which are widely distributed in northern Japan: infertile and immature volcanic ash (VA) soil, and fertile brown forest (BF) soil. For B. platyphylla, photosynthetic down-regulation occurred in both soils, but for B. maximowicziana, down-regulation occurred only in VA soil. The explanation is reduced nitrogen and Rubisco content in the leaf. For A. hirsuta, down-regulation occurred only in BF soil because of the accumulation of starch in foliage, which restricts CO₂ diffusion inside the chloroplast. The higher photosynthetic rate of A. hirsuta in infertile VA soil could be due to the sink for photosynthates in the N₂-fixing symbiont. These three species are all able to down-regulate at high [CO₂]. However, it is possible that A. hirsuta would dominate in VA soil and B. maximowicziana in BF soil in the early stages of forest succession in a CO₂-enhanced world.     

Establishment of Coral–Algal Symbiosis Requires Attraction and Selection

Coral reef ecosystems are based on coral–zooxanthellae symbiosis. During the initiation of symbiosis, majority of corals acquire their own zooxanthellae (specifically from the dinoflagellate genus Symbiodinium) from surrounding environments. The mechanisms underlying the initial establishment of symbiosis have attracted much interest, and numerous field and laboratory experiments have been conducted to elucidate this establishment. However, it is still unclear whether the host corals selectively or randomly acquire their symbionts from surrounding environments. To address this issue, we initially compared genetic compositions of Symbiodinium within naturally settled about 2-week-old Acropora coral juveniles (recruits) and those in the adjacent seawater as the potential symbiont source. We then performed infection tests using several types of Symbiodinium culture strains and apo-symbiotic (does not have Symbiodinium cells yet) Acropora coral larvae. Our field observations indicated apparent preference toward specific Symbiodinium genotypes (A1 and D1-4) within the recruits, despite a rich abundance of other Symbiodinium in the environmental population pool. Laboratory experiments were in accordance with this field observation: Symbiodinium strains of type A1 and D1-4 showed higher infection rates for Acropora larvae than other genotype strains, even when supplied at lower cell densities. Subsequent attraction tests revealed that three Symbiodinium strains were attracted toward Acropora larvae, and within them, only A1 and D1-4 strains were acquired by the larvae. Another three strains did not intrinsically approach to the larvae. These findings suggest the initial establishment of corals–Symbiodinium symbiosis is not random, and the infection mechanism appeared to comprise two steps: initial attraction step and subsequent selective uptake by the coral.     

Synthetic double-stranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection

The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (HA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor 3 and alpha/beta interferons as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.