. These results suggest that versican facilitates chondrogenesis and joint morphogenesis, by localizing TGF-␤ in the extracellular matrix and regulating its signaling.

Mesenchymal cell condensation is an essential step for cartilage development. Versican/PG-M, a large chondroitin sulfate proteoglycan, is one of the major molecules expressed in the extracellular matrix during condensation. However, its role, especially as an environment for cells being condensed, has not been elucidated. Mesenchymal cell condensation is the first step of organogenesis in many tissues. Cartilage formation in limb buds, one of the most typical examples of organogenesis, starts with the condensation of chondrogenic mesenchymal cells (1). This step is thought to be essential for subsequent skeletal development in vertebrates (2) and requires the involvement of not only some cell growth and morphogenetic factors, such as transforming growth factor-␤ (3), growth differentiation factor-5 (4), and bone morphogenetic proteins (BMPs) 4 (5), but also some cellular adhesion and extracellular matrix molecules such as N-cadherin (6), neural cell adhesion molecule (7), fibronectin (8), tenascin (9), versican/PG-M (10), hyaluronan (11), syndecan (12), and perlecan (13).Versican/PG-M was originally isolated as PG-M (medium-sized chondroitin sulfate proteoglycan) from the core (the mesenchymal cell condensation area) of chick embryonic limb bud at stage 23 (10). The cDNA of versican/PG-M was cloned as versican, a fibroblast proteoglycan (14), and also as one of the alternatively spliced forms of the PG-M core protein (15). Versican/PG-M has a molecular mass of more than 1,000 kDa and consists of two globular domains at the N and C termini (G1 and G3 domain, respectively) and the two chondroitin sulfate-attachment domains (CS-␣ and CS-␤) (16, also see Fig. 4A). The G1 and G3 domains are commonly found in proteoglycans belonging to the aggrecan family and are binding sites for hyaluronan and oligosaccharides, respectively (17, 18). The CS-␣ and CS-␤ domains give unique properties to this proteoglycan in that multiple alternative splicing yields the following four variant forms with different numbers of the attached chondroitin sulfate chains: V0 having CS-␣ and CS-␤, V1 having CS-␤, V2 having CS-␣, and V3 having neither of the two (19 in chicken; 15, 16, and 20 in mouse; 14 and 21 in human). An important role for versican/PG-M in the condensation process has been suggested by its unique properties as follows: 1) high transient expression in the mesenchymal cell condensation area during development of cartilage (10,(22)(23)(24), heart (25, 26), hair follicles (25), and kidney (27); 2) specific binding to fibronectin (28), type I collagen (28), hyaluronan (11,29), and tenascin (9), also major matrix molecules in the mesenchymal cell condensation area; and 3) ability to inhibit cell adhesion to a variety of extracellular matrix molecules such as fibronectin or type I collagen through its chondroitin sulfate chains (30 -32). Versican/PG-M interacts with cell surface annexin VI and inhibits the subsequent cellspreading step that needs the rearrangement of cytoskeletal elements (33).Exogenous treatment with versican/PG-...

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Abstract. We have applied a loop-mediated isothermal amplification (LAMP) technique to detect Leishmania donovani DNA. The LAMP technique detected 1 fg of L. donovani DNA, which was 10-fold more sensitive than a conventional polymerase chain reaction (PCR). All nested PCR-positive blood samples from visceral leishmaniasis patients were positive with the LAMP technique, and DNA samples from L. infantum , L. major , L. mexicana , L. tropica , L. braziliensis , Plasmodium falciparum , and healthy humans were negative with the LAMP technique. The advantages of the LAMP method are its shorter reaction time, a lack of requirement of sophisticated equipment, and visual judgment of positivity based on the turbidity of reaction mixture. Our LAMP technique can be a better alternative to a conventional PCR, especially under field conditions.

The influenza virus causes annual epidemics and occasional pandemics and is thus a major public health problem. Development of vaccines and antiviral drugs is essential for controlling influenza virus infection. We previously demonstrated the use of vectored immune-prophylaxis against influenza virus infection. We generated a plasmid encoding neutralizing IgG monoclonal antibodies (mAbs) against A/PR/8/34 influenza virus (IAV) hemagglutinin (HA). We then performed electroporation of the plasmid encoding neutralizing mAbs (EP) in mice muscles and succeeded in inducing the expression of neutralizing antibodies in mouse serum. This therapy has a prophylactic effect against lethal IAV infection in mice. In this study, we established a new method of passive immunotherapy after IAV infection. We performed hydrodynamic injection of the plasmid encoding neutralizing mAbs (HD) involving rapid injection of a large volume of plasmid-DNA solution into mice via the tail vein. HD could induce neutralizing antibodies in the serum and in several mucosal tissues more rapidly than in EP. We also showed that a single HD completely protected the mice even after infection with a lethal dose of IAV. We also established other isotypes of anti-HA antibody (IgA, IgM, IgD, and IgE) and showed that like anti-HA IgG, anti-HA IgA was also effective at combating upper respiratory tract IAV infection. Passive immunotherapy with HD could thus provide a new therapeutic strategy targeting influenza virus infection.

We overexpressed mouse DNA methyltransferase in murine C2C12 myoblast cells and tested the isolated clones for their ability to differentiate. Significant numbers of the clones showed distinct myotubes 24 h after the isolated transformants had been induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point. Transfection of the vacant vector or the plasmid containing the reverse-oriented DNA methyltransferase cDNA did not provide significant numbers of transformants with the accelerated differentiation phenotype, suggesting that the effect is caused by the expression of DNA methyltransferase. The expressions of skeletal muscle myosin and creatine kinase in clones that showed the accelerated differentiation-phenotype were also induced about 24 h earlier and at higher levels relative to the parent C2C12 or the control cells, indicating that the entire process of myogenesis had been accelerated. All the methyltransferase-transfected clones, regardless of their phenotypes, demonstrated about threefold higher DNA methyltransferase activity and higher methylation levels than those of the clones transfected with vector alone or the reverse-oriented plasmid. At the early stage of transfection of the sense-oriented plasmid, high de novo methylation activities were detected. We consider it likely that this high de novo methylation activity is the reason for the high methylation levels and the accelerated myotube formation of the clones transfected with the sense-oriented plasmid. In some transformants which showed the accelerated differentiation phenotype, MyoD1 was already fully expressed under the growth conditions while, in control cells, MyoD1 was expressed at low levels. This elevated level of MyoD1 transcription could account for the accelerated myotube formation observed in the transformants. The methylation state of the HpaII sites in exon 1 through exon 2 of the MyoD1 gene and the expression of the MyoD1 transcript are positively correlated.

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We recently reported the production of the recombinant kinesin-related protein of Leishmania donovani with a molecular weight of 42 kd (rKRP42) and the value of the antigen in serum-based ELISA for the diagnosis of visceral leishmaniasis (VL). In this study, the rKRP42 antigen was validated with ELISA using urine samples (rKRP42 urine ELISA). The urine-based ELISA showed 94% sensitivity (108 positives among 115 VL samples) and 99.6% specificity (239 negatives among 240 non-VL samples). The sensitivity and specificity are almost similar to our previous results by ELISA with acetone-treated L. donovani promastigote antigen and direct agglutination test, both methods being done by use of urine samples. A comparison of the rKRP42 urine ELISA with the commercially available urinary antigen detection kit (KAtex) using 108 VL samples showed much higher sensitivity of the ELISA (96.3%) than KAtex (55.6%). The use of the rKRP42 antigen with urine samples will facilitate epidemiologic studies.