Immunohistochemical Localization of Prothoracicotropic Hormone-Producing Neurosecretory Cells in the Brain of Bombyx mori

A monoclonal antibody that recognized the Bombyx prothoracicotropic hormone (PTTH) was produced by immunizing mice with a synthetic pentadecapeptide corresponding to the amino-terminal portion of Bombyx PTTH. The antibody recognized both intact and reduced forms of PTTH. Immunohistochemistry with this antibody has demonstrated that PTTH is produced by two pairs of dorso-lateral neurosecretory cells of the brain and transported to the corpora allata by axons running through the contralateral hemisphere of the brain. Immunoreactive axon terminals in the corpora allata were localized between the glandular cells, suggesting that PTTH is released at the inner part of this organ.     

Effects of space flight on the histological characteristics of the aortic depressor nerve in the adult rat: electron microscopic analysis.

The effects of microgravity on the histological characteristics of the aortic depressor nerve, which is the afferent of the aortic baroreflex arc, were determined in 10 female adult rats. The rats were assigned for nursing neonates in the Space Shuttle Columbia or in the animal facility on the ground (NASA Neurolab, STS-90), and were housed for 16 days under microgravity in space (microg, n=5) or under one force of gravity on Earth (one-g, n=5). In the Schwann cell unit in which the axons of unmyelinated fibers are surrounded by one Schwann cell, the average number of axons per unit in the microg group was 2.1 +/- 1.6 (mean +/- SD, n=312) and significantly less than that in the one-g group (3.0 +/- 2.9, n=397, p<0.05). The proportion of unmyelinated fibers in the aortic depressor nerve in the microg group was 64.5 +/- 4.4% and significantly less than that in the one-g group (74.0 +/- 7.3%, p<0.05). These results show that there is a decrease in the number of high-threshold unmyelinated fibers in the aortic depressor nerve in adult rats flown on the Shuttle Orbiter, suggesting that the aortic baroreflex is depressed under microgravity during space flight.     

PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability

BACKGROUND: PEGylation of adenovirus vectors (Ads) is an attractive strategy in gene therapy. Although many types of PEGylated Ad (PEG-Ads), which exhibit antibody evasion activity and long plasma half-life, have been developed, their entry into cells has been prevented by steric hindrance by polyethylene glycol (PEG) chains. Likewise, sufficient gene expression for medical treatment could not be achieved. METHODS: A set of PEG-Ads, which have different PEG modification rates, was constructed, and gene expression was evaluated using A549 cells. A novel PEGylated Ad (RGD-PEG-Ad), which contained RGD (Arg-Gly-Asp) peptides on the tip of PEG, was developed. We evaluated gene expression both in Coxsackie-adenovirus receptor (CAR)-positive as well as -negative cells, and in vivo gene expression was also determined. Furthermore, the antibody evasion ability and the specificity of infection exhibited by this RGD-PEG-Ad were also evaluated. RESULTS: Whereas PEG-Ads decreased gene expression in CAR-positive cells, RGD-PEG-Ad enhanced gene expression notably, to a level about 200-fold higher than that of PEG-Ads. Moreover, gene expression of RGD-PEG-Ad was almost equal to that of Ad-RGD, which contains an RGD-motif in the fiber and exhibits among the highest gene expression of CAR-positive and -negative cells. Furthermore, although Ad-RGD gene expression decreased remarkably in the presence of anti-Ad antiserum, RGD-PEG-Ad maintained its activity against antibodies. In vivo experiments also demonstrated that the modification of Ads with RGD-PEG induced efficient gene expression. CONCLUSIONS: In the present study, we demonstrated that a new strategy, which combined integrin-targeting the RGD peptide on the tip of PEG and modified the Ad using this material, could enhance gene expression in both CAR-positive and -negative cells. At the same time, this novel PEGylated Ad maintained strong protective activity against antibodies. This strategy could also be easily modified for developing other vectors using other targeting molecules.     

DNA-mediated transformation system in a bipolar basidiomycete, <Emphasis Type="Italic">Pholiota microspora</Emphasis> (<Emphasis Type="Italic">P. nameko</Emphasis>)

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.     

Transforming growth factor-beta1 inhibition of vascular smooth muscle cell activation is mediated via Smad3

Activation of vascular smooth muscle cells (VSMCs) by proinflammatory cytokines is a key feature of atherosclerotic lesion formation. Transforming growth factor (TGF)-beta1 is a pleiotropic growth factor that can modulate the inflammatory response in diverse cell types including VSMCs. However, the mechanisms by which TGF-beta1 is able to mediate these effects remains incompletely understood. We demonstrate here that the ability of TGF-beta1 to inhibit markers of VSMC activation, inducible nitric-oxide synthase (iNOS) and interleukin (IL)-6, is mediated through its downstream effector Smad3. In reporter gene transfection studies, we found that among a panel of Smads, Smad3 could inhibit iNOS induction in an analogous manner as exogenous TGF-beta1. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous iNOS and IL-6. Conversely, TGF-beta1 inhibition of cytokine-mediated induction of iNOS and IL-6 expression was completely blocked in Smad3-deficient VSMCs. Previous studies demonstrate that CCAAT/enhancer-binding protein (C/EBP) and NF-kappaB sites are critical for cytokine induction of both the iNOS and IL-6 promoters. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on C/EBP DNA-protein binding and activity. Smad3 mediates this effect in part by inhibiting C/EBP-beta and C/EBP-delta through distinct mechanisms. Furthermore, we find that Smad3 prevents the cooperative induction of the iNOS promoter by C/EBP and NF-kappaB. These data demonstrate that Smad3 plays an essential role in mediating TGF-beta1 anti-inflammatory response in VSMCs.     

SS-A/Ro52, an autoantigen involved in CD28-mediated IL-2 production

An autoantibody against SS-A/Ro52 (Ro52) is most frequently found in the sera of patients with Sj?gren's syndrome, systemic lupus erythematosus, and congenital heart block from anti-Ro52 Ab-positive mother. However, the physiological function of the autoantigen SS-A/Ro52 has not yet been elucidated. In this study, we describe the role of Ro52 protein in T cell activation. Overexpression of SS-A/Ro52 in Jurkat T cell resulted in enhanced IL-2 production following CD28 stimulation. Furthermore, transfection of anti-Ro52-specific small RNA duplexes partially blocked the expression of native and overexpressed Ro52 in Jurkat T cell, resulting in decreased IL-2 production via CD28 pathway in these cells. Finally, intracellular localization of Ro52 dramatically changed following CD28 stimulation. Our data reveal a novel function of Ro52 in CD28-mediated pathway, which eventually contributes to cytokine production and expression of the T cell biological programs.     

Engineered bio-nanocapsules, the selective vector for drug delivery system

The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.