The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair.

OBJECTIVE: Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS: MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS: The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS: Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.

Cloning and sequencing of a novel hemolysis gene of Vibrio cholerae.

A hemolysis gene (hlx) which lyses sheep erythrocytes on blood agar plates when expressed in Escherichia coli was cloned from Vibrio cholerae. The cloned gene is predicted to encode a polypeptide of 92 amino acid residues with a deduced molecular mass of 10,451. E. coli transformed with this gene lysed sheep, goose, horse and chicken erythrocytes but not those of guinea pig and human. The hlx gene was observed in classical- and El Tor-biotype V. cholerae O1, V. cholerae non-O1, and V. mimicus, but not in V. parahaemolyticus.

An automatic bone segmentation method based on anatomical structure for the knee joint in MDCT image.

The purpose of this study is to propose an automatic segmentation about each bone (the femur, the tibia, the patellar, and fibular) of the knee in MDCT image. The proposed method was applied for six patients (Age 33 ± 13, four males/tew females). The proposed method segmented the knee joint into each bone by using anatomical structure for the knee joint. The experiments calculate matching rate of the manual and the proposed method for evaluating it. As a result, The matching rate of the femur, the tibia, the patellar, and fibula were 95.84 ± 0.57%, 94.12 ± 1.01%, 94.49 ± 0.83%, 86.37 ± 4.28%, respectively. This study concluded that the proposed method is enough to segment the knee bones.

Intramolecular chaperone activity of the pro-region of Vibrio cholerae El Tor cytolysin.

Vibrio cholerae synthesizes a toxin named El Tor cytolysin/hemolysin, which lyses erythrocytes and other mammalian cells. This toxin is encoded by the hlyA gene and is synthesized as a precursor form, prepro-HlyA. Prepro-HlyA consists of, from the amino terminus of this protein, a signal peptide, a pro-region, and a mature region. The pro-region is cleaved off extracellularly resulting in activation. To analyze the role of the pro-region, we substituted the native hlyA gene with the pro-region-deleted hlyA gene (hlyA delta pro). The hemolytic activity of the mutant organism was markedly decreased; the product of the hlyA delta pro gene, secreted in the periplasm, was degraded. To compare their abilities to form tertiary structure, the purified mature- and pro-HlyA were denatured and then renatured by reducing the concentration of denaturant; the denatured pro-HlyA recovered almost all activity while the mature-HlyA was not renatured. The sequences of the pro-region and a molecular chaperone, Hsp90, were similar. The pro-region expressed in Escherichia coli containing the hlyA delta pro gene increased the cytolytic activity. The purified pro-region peptide also facilitated renaturation of the denatured mature HlyA. These results suggest that the pro-region possibly guides the folding of the cytolysin similar to a molecular chaperone; the pro-region and molecular chaperones share common function and structure.

Gametocytogenesis induction by Berenil in cultured Plasmodium falciparum.

Berenil, which is known to inhibit synthesis of nucleic acid, DNA and RNA, and polyamine, induced gametocytogenesis in cultured Plasmodium falciparum, although the mechanism by which Berenil induces gametocytogenesis is unknown. Asexual parasites of a strain of P. falciparum that seldom produce gametocytes in in vitro culture began gametocytogenesis after 2-24 hr treatment with RPMI 1640 medium containing Berenil (final concentration, 0.1-10.0 micrograms/ml) with horse serum. Gametocytogenesis was consistently observed from 3 days after the addition of Berenil to culture. After parasites were cultivated in RPMI 1640 medium containing Berenil (final concentration, 3.0-10.0 micrograms/ml) and concanavalin A with horse serum for 2 hr, Berenil and concanavalin A were removed from culture medium by centrifugation. Nevertheless, induction of gametocytogenesis was observed. The addition of concanavalin A (final concentration, 10 micrograms/ml) to RPMI 1640 medium containing Berenil enhanced the induction of gametocytogenesis by Berenil. However, when RPMI 1640 medium with concanavalin A was used without the addition of Berenil, no gametocytogenesis was observed as in RPMI 1640 medium alone.

In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases.

Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei.

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B/GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.