Papillomaviruses are a heterogeneous group of DNA viruses with closed circular double-stranded DNA genomes of about 8 kb in size that contain three general regions. An upstream regulatory region (URR) contains sequences that control transcription and replication, an early region contains genes (e.g., E6, E7, E1, E2, E4, and E5) involved primarily in enzymatic activities, and a structural region produces the L1 capsid protein and L2, which facilitates packaging of the viral DNA. Human papillomaviruses (HPVs) are classified by the sequence similarity of their genomes. A cloned HPV genome whose L1 open reading frame (ORF) displays less than 90% similarity to previously designated types is defined as a novel type. To date, more than 90 different genotypes of the HPV have been fully characterized (21). Intratypic variants and subtypes are defined as HPVs that vary by less than 10% in their L1 DNA sequences (5, 21).HPVs are causally involved in the etiology of cervical cancer and its precursor lesions (16,17,43,49). Of the high-risk HPV types associated with cervical cancer, HPV16 is the most prevalent and is found in approximately half of all cancers (7, 49). Numerous variants of HPV16 have been identified in different geographic locations and ethnic groups (35,42,53,68). Although all HPV16 isolates are closely related, previous studies inferred five distinct phylogenetic branches among HPV16 variants: European (E), Asian (As), Asian-American (As-Am), African-1 (Af-1), and African-2 (Af-2), corresponding to the geographic locations from which the samples were obtained (12,34). Subsequent studies by sequence analyses of the HPV16 variants in other genomic regions (e.g., E6, L2, and L1) expanded and complemented this phylogenetic hypothesis (69).Although HPV16 variants are an important focus of phylogenetic studies and the molecular variants of E2, L2, L1, the URR, and especially the E6 region have been described in detail previously (28), covariation among different ORFs belonging to the same lineage or isolate have not been studied in great detail. HPV16 variants have demonstrable differences in biological properties in vitro which may be responsible, in part, for differences in pathogenicity, carcinogenic risk, and perhaps immunogenicity (28). Furthermore, HPV16 variants are associated with different cervical cancer risks (6,32,65,67). Although the diversifying selection in the HPV16 E6 and E7 oncogenes has been described recently (20), the evolutionary basis of the entire genome, coevolutionary mechanisms among different HPV16 genes, and their underlying biological significance remain unknown. Obtaining whole-genome sequences representative of the major HPV16 variants allowed us to determine with certainty nucleotide and amino acid sequence changes that are of potential evolutionary importance.Comparison of synonymous (silent; d S ) and nonsynonymous

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood-derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood-derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood-derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

ABSTRACT

Human papillomavirus (HPV) infection in the normal oral cavity was studied by the sensitive polymerase chain reaction (PCR) using primers for the L1 region of human papillomavirus DNA and high fidelity amplification system. Cells were scraped from the oral mucosae of 7 (mean age; 42 years) and 30 (mean age; 32 years) volunteers with and without skin warts, respectively. Human papillomavirus DNA was detected in 30/37 (81.1%) specimens and their copy numbers per cell were 10(-1) to 10(-4) (mean, 10(-3)). The human papillomavirus types determined by PCR-based sequencing analysis were HPV-18 (26/30; 86.7%), -61 (18/30; 60%), -59 (7/30; 23.3%), -16 (2/30; 6.7%), -6 (1/30; 3.3%) and an unknown type (HPV-X71) (1/30; 3.3%). Multiple human papillomavirus types were present in 17/30 (56.7%) specimens. HPV-6 was detected in 2 of 7 skin warts and differed from the human papillomavirus types of the corresponding oral specimens. These data suggest that human papillomavirus infection in the oral mucosa occurs much more frequently than previously considered.

To establish a sensitive and specific antibody assay, potent antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied. Fifteen recombinant HHV8-encoded proteins were produced as glutathione S-transferase fusion proteins. The sera from AIDS-associated Kaposi's sarcoma (

Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to serve as an excellent alternative to bone marrow-derived human mesenchymal stem cells. However, it is difficult to study them because of their limited life span. To overcome this problem, we attempted to produce a strain of UCBMSCs with a long life span and to investigate whether the strain could maintain phenotypes in vitro. UCBMSCs were infected with retrovirus carrying the human telomerase reverse transcriptase (hTERT) to prolong their life span. The UCBMSCs underwent 30 population doublings (PDs) and stopped dividing at PD 37. The UCBMSCs newly established with hTERT (UCBTERTs) proliferated for >120 PDs. The p16 INK4a /RB braking pathway leading to senescence can be inhibited by introduction of Bmi-1, a polycomb-group gene, and human papillomavirus type 16 E7, but the extension of the life span of the UCBMSCs with hTERT did not require inhibition of the p16 INK4a /RB pathway. The characteristics of the UCBTERTs remained unchanged during the prolongation of life span. UCBTERTs provide a powerful model for further study of cellular senescence and for future application to cell-based therapy by using umbilical cord blood cells. INTRODUCTIONHuman mesenchymal stem cells (hMSCs) can be a useful source of cells for transplantation for several reasons: they have the ability to proliferate and differentiate into mesodermal tissues, and they entail no ethical or immunological problems (Caplan, 1991;Prockop, 1997;Caplan and Bruder, 2001). hMSCs have been studied extensively over the past 3 decades, and numerous independent research groups have successfully isolated hMSCs from a variety of sources, most commonly, from the bone marrow (Owen, 1988;Umezawa et al., 1992;Jaiswal et al., 1997;Makino et al., 1999;Pittenger et al., 1999;Sekiya et al., 2004). Umbilical cord blood (UCB) contains circulating stem/progenitor cells, and the cells contained in UCB are known to be distinct from those contained in bone marrow and adult peripheral blood (Mayani and Lansdorp, 1998). Isolation, characterization, and differentiation of clonally expanded hMSCs derived from UCB (UCBMSCs) have been reported (Goodwin et al., 2001;Lee et al., 2004), and UCBMSCs have been found to have multipotency, and the immunophenotype of the clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Even now, most UCB is regarded as medical waste in the delivery rooms. Aspirating bone marrow from patients is, however, an invasive procedure, and the proliferation and differentiation capacity of hMSCs decreases with the donor age (D'Ippolito et al., 1999). Therefore, the applications of UCB should be further expanded.UCBMSCs will be useful sources for cell transplantation, however, it is difficult to study and apply them because of their limited life span. One of the reasons for this is that normal human cells undergo a limited number of cell division in culture and then enter a nondividing state called "senescence" (Hayflick, 1976;Campisi...

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Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to be an excellent source of cells for transplantation. In addition, the stem cell plasticity of human UCBMSCs, which can transdifferentiate into hepatocytes, has been reported. However, the mechanisms involved remain to be clarified. To identify the genes and/or signals that are important in specifying the hepatic fate of human UCBMSCs, we analyzed gene expression profiles during the hepatic differentiation of UCBMSCs with human telomerase reverse transcriptase, UCBMSCs immortalized by infection with a retrovirus carrying telomerase reverse transcriptase, but whose differentiation potential remains unchanged. Efficient differentiation was induced by 5-azacytidine (5-aza)/hepatocyte growth factor (HGF)/oncostatin M (OSM)/fibroblast growth factor 2 (FGF2) treatment in terms of function as well as protein expression: 2.5-fold increase in albumin, 4-fold increase in CCAAT enhancer-binding protein α, 1.5-fold increase in cytochrome p450 1A1/2, and 8-fold increase in periodic acid-Schiff staining. Consequently, we found that the expression of Wnt/β-catenin-related genes downregulated, and the translocation of β-catenin was observed along the cell membrane and in the cytoplasm, although some β-catenin was still in the nucleus. Downregulation of Wnt/β-catenin signals in the cells by Fz8-small interference RNA treatment, which was analyzed with a Tcf4 promoter-luciferase assay, resulted in similar hepatic differentiation to that observed with 5-azacytidine/HGF/OSM/FGF2. In addition, the subcellular distribution of β-catenin was similar to that of cells treated with 5-azacytidine/HGF/OSM/FGF2. In conclusion, the suppression of Wnt/β-catenin signaling induced the hepatic differentiation of UCBMSCs, suggesting that Wnt/β-catenin signals play an important role in the hepatic fate specification of human UCBMSCs.