Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Recombinant adeno-associated viral (rAAV) vectors hold promise for delivering genes for heart diseases, but cardiac-specific expression by the use of rAAV has not been demonstrated. To achieve this goal rAAV vectors were generated expressing marker or potentially therapeutic genes under the control of the cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter. The rAAV-MHC vectors expressed in primary cardiomyocytes with similar kinetics to rAAV-CMV; however, expression by the rAAV-MHC vectors was restricted to cardiomyocytes. rAAV vectors have low cytotoxicity, and it is demonstrated here that rAAV fails to induce apoptosis in cardiomyocytes compared with a recombinant adenoviral vector. rAAV-MHC or rAAV-CMV vectors were administered to mice to determine the specificity of expression in vivo. The rAAV-MHC vectors expressed specifically in cardiomyocytes, whereas the control rAAV-CMV vector expressed in heart, skeletal muscle, and brain. rAAV-MHC transduction resulted in long term (16 weeks) expression of human growth hormone following intracardiac, yet not intramuscular, injection. Finally, we defined the minimal MHC enhancer/promoter sequences required for specific and robust in vivo expression in the context of a rAAV vector. For the first time we describe a panel of rAAV vectors capable of long term cardiac specific expression of intracellular and secreted proteins.     

Scaling up towards international targets for AIDS, tuberculosis, and malaria: contribution of global fund-supported programs in 2011-2015

Objective

The paper projects the contribution to 2011–2015 international targets of three major pandemics by programs in 140 countries funded by the Global Fund to Fight AIDS, Tuberculosis and Malaria, the largest external financier of tuberculosis and malaria programs and a major external funder of HIV programs in low and middle income countries.

Design

Estimates, using past trends, for the period 2011–2015 of the number of persons receiving antiretroviral (ARV) treatment, tuberculosis case detection using the internationally approved DOTS strategy, and insecticide-treated nets (ITNs) to be delivered by programs in low and middle income countries supported by the Global Fund compared to international targets established by UNAIDS, Stop TB Partnership, Roll Back Malaria Partnership and the World Health Organisation.

Results

Global Fund-supported programs are projected to provide ARV treatment to 5.5–5.8 million people, providing 30%–31% of the 2015 international target. Investments in tuberculosis and malaria control will enable reaching in 2015 60%–63% of the international target for tuberculosis case detection and 30%–35% of the ITN distribution target in sub-Saharan Africa.

Conclusion

Global Fund investments will substantially contribute to the achievement by 2015 of international targets for HIV, TB and malaria. However, additional large scale international and domestic financing is needed if these targets are to be reached by 2015.     

A functional polymorphism in the promoter/enhancer region of the<Emphasis Type="Italic"> FOXP3/Scurfin</Emphasis> gene associated with type 1 diabetes

FOXP3/Scurfin, a member of forkhead/winged-helix proteins, is involved in the regulation of T-cell activation, and essential for normal immune homeostasis. The FOXP3/Scurfin gene is located on chromosome Xp11.23, which includes one of the type 1 diabetes susceptible loci. Therefore, we investigated whether the human FOXP3/Scurfin gene might be a new candidate gene for type 1 diabetes. We first screened the human FOXP3/Scurfin gene for microsatellite and single nucleotide polymorphisms. Next, we performed an association study between the FOXP3/Scurfin gene and type 1 diabetes. Then, the evaluation of promoter/enhancer activity of the intron with (GT)(n) polymorphism was performed by dual luciferase reporter assay. We demonstrated two regions contained microsatellite polymorphisms; one was (GT)(n), located on intron zero and the other (TC)(n) on intron 5, which were under linkage-disequilibrium. The (GT)(15) allele showed a significantly higher frequency in patients with type 1 diabetes than in controls (43.1% vs 32.6%, P=0.0027). The genotype frequencies of (GT)(15)/(GT)(15) in female patients and of (GT)(15) in male patients tended to be higher than those in female ( P=0.064) and male ( P=0.061) controls, respectively. A significant difference in the enhancer activity between (GT)(15) and (GT)(16) dinucleotide repeats was detected. In conclusion, the FOXP3/Scurfin gene appears to confer a significant susceptibility to type 1 diabetes in the Japanese population.     

Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.     

Plant stem cell research is uncovering the secrets of longevity and persistent growth

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project ‘Principles of pluripotent stem cells underlying plant vitality’ was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.     

Analysis of gene expression profile in p130(Cas)-deficient fibroblasts

p130(Cas) (Cas) is a docking protein that becomes tyrosine phosphorylated in v-Src- or v-Crk-transformed cells and in integrin-stimulated cells. Cas -/- fibroblasts show defects in stress fiber formation, cell spreading, cell migration, and transformation by activated Src. To further characterize the role of Cas in signaling, we compared the expression profile in Cas -/- fibroblasts with that in Cas-re-expressing fibroblasts using the microarray methods. In Cas -/- fibroblasts, the expression of heme oxygenase 1 and caveolin-1 was reduced, but the expression of procollagen 1 alpha 1, procollagen 3 alpha 1, procollagen 11 alpha 1, elastin, periostin, TSC-36, and MARCKS was enhanced. The domains in Cas necessary for the change varied among these genes. Activated Src reduced the expression of most of these genes both in Cas -/- and in Cas +/+ fibroblasts. These results suggest the existence of signaling pathways that emanate from Cas to gene expression.     

Development of saxitoxin-conjugated affinity gels

Saxitoxin (STX) and its analogues accumulated in bivalves cause food poisoning through the blockade of sodium channels in the nervous system. In the current studies, STX-conjugated agarose gels as affinity chromatography reagents were prepared for investigation of the fate of the toxins in natural environments and in the human body. A carboxyl moiety was introduced through positions C11 and C13 to leave the most characteristic part of the molecule intact. Two types of synthesized derivatives, 11-(2-carboxyethylthio)saxitoxin and 13-O-hemisuccinyldecarbamoylsaxitoxin, were successfully conjugated to Sepharose 4B in high yield. Affinity gels containing 500 nmol of STX or decarbamoylsaxitoxin per milliliter of gel were accomplished by masking the residual amino groups by acetylation. Finally, the STX-conjugated affinity gel was effective for concentrating STX-binding proteins from pufferfish and bullfrog plasma.     

The Influence of Measurement Parameters on the Specific Rotation of Amino Acids

The effects of solute and hydrochloric acid concentrations on optical rotation were studied using 20 naturally occuring amino acids.

There appeared to be no common factor among the amino acids as far as the inclination of optical rotation was concerned. Lutz-Jirgenson’s rule could be applied to few amino acids in the cationic form. Therefore, in the determination of the optical rotation, the concentration of the solute, nature of solvent and temperature must be rigorously controlled. The optical conditions of measurement and the specific rotation of 20 amino acids were recommended based on this work.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.