Quantitative fluorescence in situ hybridization for investigation of telomere length dynamics in the pituitary gland using samples from 128 autopsied patients.

In order to investigate the population dynamics of telomere status, we measured the telomere lengths of glandular cells in the adenohypophysis (AH) and pituicytes, a type of glial cell, in the neurohypophysis (NH) of 128 autopsied humans (65 men, 63 women, 0 and 102 years) using our original quantitative fluorescence in situ hybridization (Q-FISH) method. Telomeres in the AH shortened with aging in both men and women, but those of pituicytes did not. Pituicyte telomeres were significantly longer in women than in men.

Arm-specific telomere dynamics of each individual chromosome in induced pluripotent stem cells revealed by quantitative fluorescence in situ hybridization.

We have reported that telomere fluorescence units (TFUs) of established induced pluripotent stem cells (iPSCs) derived from human amnion (hAM933) and fetal lung fibroblasts (MRC-5) were significantly longer than those of the parental cells, and that the telomere extension rates varied quite significantly among clones without chromosomal instability, although the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased along with chromosomal abnormalities from an early stage. In the present study we attempted to clarify telomere dynamics in each individual chromosomal arm of parental cells and their derived clonal human iPSCs at different numbers of passages using quantitative fluorescence in situ hybridization (Q-FISH). Although no specific arm of any particular chromosome appeared to be consistently shorter or longer than most of the other chromosomes in any of the cell strains, telomere elongation in each chromosome of an iPSC appeared to be random and stochastic.

Association of telomere shortening in myocardium with heart weight gain and cause of death.

We attempted to clarify myocardial telomere dynamics using samples from 530 autopsied patients using Southern blot analysis. Overall regression analysis demonstrated yearly telomere reduction rate of 20 base pairs in the myocardium. There was a significant correlation between myocardial telomere and aging.

Investigation of telomere length dynamics in induced pluripotent stem cells using quantitative fluorescence in situ hybridization.

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell.

Telomere shortening in the esophagus of Japanese alcoholics: relationships with chromoendoscopic findings, ALDH2 and ADH1B genotypes and smoking history.

Chromoendoscopy with Lugol iodine staining provides important information on the development of squamous cell carcinoma (SCC). In particular, distinct iodine-unstained lesions (DIULs) larger than 10 mm show a high prevalence in high-grade intraepithelial neoplasia. It has also been reported that inactive ALDH2*1/*2 and less-active ADH1B*1/*1, and smoking, are risk factors for esophageal SCC.

Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy.

Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I (Ib) molecule, which plays an important role in immunosuppression. In this study, we investigated the immunomodulating effect of HLA-E in a xenogeneic system, using human placental artery-derived endothelial (hPAE) cells expressing HLA-E in a mouse model. In vitro cell lysis analysis by primed lymphocytes in combination with siRNA transfection showed that HLA-E is necessary for inhibition of the immune response.

CCN3 and bone marrow cells.

CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells.

A role of Wnt/beta-catenin signals in hepatic fate specification of human umbilical cord blood-derived mesenchymal stem cells.

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.

Chromosomal instability in human mesenchymal stem cells immortalized with human papilloma virus E6, E7, and hTERT genes.

Human mesenchymal stem cells (hMSCs) are expected to be an enormous potential source for future cell therapy, because of their self-renewing divisions and also because of their multiple-lineage differentiation. The finite lifespan of these cells, however, is a hurdle for clinical application. Recently, several hMSC lines have been established by immortalized human telomerase reverse transcriptase gene (hTERT) alone or with hTERT in combination with human papillomavirus type 16 E6/E7 genes (E6/E7) and human proto-oncogene, Bmi-1, but have not so much been characterized their karyotypic stability in detail during extended lifespan under in vitro conditions.

The significant cardiomyogenic potential of human umbilical cord blood-derived mesenchymal stem cells in vitro.

We tested the cardiomyogenic potential of the human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs). Both the number and function of stem cells may be depressed in senile patients with severe coronary risk factors. Therefore, stem cells obtained from such patients may not function well.

Menstrual blood-derived cells confer human dystrophin expression in the murine model of Duchenne muscular dystrophy via cell fusion and myogenic transdifferentiation.

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.

Diversifying selection in human papillomavirus type 16 lineages based on complete genome analyses.

Human papillomavirus type 16 (HPV16) is the primary etiological agent of cervical cancer, the second most common cancer in women worldwide. Complete genomes of 12 isolates representing the major lineages of HPV16 were cloned and sequenced from cervicovaginal cells. The sequence variations within the open reading frames (ORFs) and noncoding regions were identified and compared with the HPV16R reference sequence.

[Improvement of musculoskeletal function by cell-based therapy using mesenchymal stem cells with a prolonged life span].

Cell transplantation has recently been attempted to improve musculoskeletal function. Many types of cells, such as embryonic stem cells, fetal cardiomyocytes, myoblasts, bone marrow hematopoietic cells, and mesenchymal stem cells (MSCs), have been transplanted to functionally restore damaged or diseased tissue in animal models, and marrow-derived mononuclear cells have been injected into ischemic limb clinically. MSCs can be a useful source of cell transplantation for several reasons:they have the ability to proliferate and differentiate into mesodermal tissues, including myocytes, they entail no ethical or immunological problems, and bone marrow aspiration is an established routine procedure.

Overlapping reading frames in closely related human papillomaviruses result in modular rates of selection within E2.

A core group of four open reading frames (ORFs) is present in all known papillomaviruses (PVs): the E1 and E2 replication/transcription proteins and the L1 and L2 structural proteins. Because they are involved in processes that are essential to PV propagation, the sequences of these proteins are well-conserved. However, sequencing of novel subtypes for human papillomaviruses (HPV) 54 (AE9) and 82 (AE2/IS39), coupled to analysis of four other closely related genital HPV pairs, indicated that E2 has a higher dN/dS ratio than E1, L1 or L2.

Immortalization of human fetal cells: the life span of umbilical cord blood-derived cells can be prolonged without manipulating p16INK4a/RB braking pathway.

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.

Codetection of a mixed population of candHPV62 containing wild-type and disrupted E1 open-reading frame in a 45-year-old woman with normal cytology.

We have cloned, sequenced, and characterized the complete genome of a novel human papillomavirus (HPV), candHPV62. During cloning, 2 candHPV62 viral isolates were recovered from a single cervical sample; 1 had all anticipated HPV open-reading frames (ORFs) intact, whereas the other exhibited an E1 frame-shift mutation. Further experiments indicated that the 2 strains were equivalent in abundance.

Low prevalence of HPV infection and its natural history in normal oral mucosa among volunteers on Miyako Island, Japan.

Objective: To investigate the prevalence of human papillomavirus (HPV) infection in normal oral mucosa, and to observe the natural history in the oral cavity in oral swab samples collected from healthy volunteers on Miyako Island, Okinawa, Japan.

Study Design: The prevalence of HPV infection in oral buccal mucosa cell scrapes collected between 2000 and 2002 from a cohort of 668 healthy volunteers was determined. HPV DNA was detected by consensus polymerase chain reaction (PCR) using MY09/MY11 primers followed by direct cycle sequencing.

Lack of the canonical pRB-binding domain in the E7 ORF of artiodactyl papillomaviruses is associated with the development of fibropapillomas.

The L-X-C-X-E pRB-binding motif of papillomavirus (PV) E7 proteins has been implicated in the immortalization and transformation of the host cell. However, sequencing of the complete genomes of bovine papillomavirus type 3 (BPV-3), bovine papillomavirus type 5 (BPV-5), equine papillomavirus (EQPV) and reindeer (Rangifer tarandus) papillomavirus (RPV) supports the notion that the pRB-binding motif is not ubiquitous among E7 proteins in the PV proteome. Key among the animal groups that lack the pRB-binding domain are the artiodactyl PVs, including European elk PV (EEPV), deer PV (DPV), reindeer PV (RPV), ovine PVs types 1 and 2 (OvPV-1 and -2) and bovine PVs 1, 2 and 5 (BPV-1, -2 and -5).

Distribution of human papillomavirus types 16 and 18 variants in squamous cell carcinomas and adenocarcinomas of the cervix.

The distributions of human papillomavirus (HPV) types detected in cervical adenocarcinomas and squamous cell tumors differ. However, whether the distributions of intratypic HPV variants seen in these two histological forms of cervical disease differ is unknown. Our objective was to compare the distribution of HPV intratypic variants observed in squamous cell carcinomas (SCC) and cervical tumors of glandular origin (e.

Lack of canonical E6 and E7 open reading frames in bird papillomaviruses: Fringilla coelebs papillomavirus and Psittacus erithacus timneh papillomavirus.

Determination and analyses of the complete sequence of Fringilla coelebs papillomavirus and Psittacus erithacus timneh papillomavirus indicate that they represent a distinct and distant lineage of papillomaviruses. The lack of canonical E6-E7 open reading frames suggests that they serve adaptive functions during papillomavirus evolution.

Felis domesticus papillomavirus, isolated from a skin lesion, is related to canine oral papillomavirus and contains a 1.3 kb non-coding region between the E2 and L2 open reading frames.

We have characterized the complete genome (8300 bp) of an isolate of Felis domesticus papillomavirus (FdPV) from a domestic cat with cutaneous papillomatosis. A BLAST homology search using the nucleotide sequence of the L1 open reading frame demonstrated that the FdPV genome was most closely related to canine oral papillomavirus (COPV). A 384 bp non-coding region (NCR) was found between the end of L1 and the beginning of E6, and a 1.

Identification and characterization of 3 novel genital human papillomaviruses by overlapping polymerase chain reaction: candHPV89, candHPV90, and candHPV91.

Three novel human papillomaviruses (HPVs), candHPV89, candHPV90, and candHPV91, that were previously identified from short polymerase chain reaction (PCR) fragments AE6/CP6108, JC9710, and JC9813, respectively, were cloned and characterized from cervicovaginal cells by use of an overlapping PCR method. The complete nucleotide sequences of candHPV89 (8078 bp), candHPV90 (8033 bp), and candHPV91 (7966 bp) were determined by sequence walking. candHPV89 and candHPV91 were closely related to HPV83 and HPV7 and were placed in the HPV genome homology groups A3 and A8, respectively, by phylogenetic analyses.

Characterization of a novel genital human papillomavirus by overlapping PCR: candHPV86 identified in cervicovaginal cells of a woman with cervical neoplasia.

A novel human papillomavirus (HPV), candHPV86, was cloned and characterized from cervicovaginal cells obtained from a 37-year-old Hispanic woman with cervical intraepithelial neoplasia grade 1 (CIN1) using an overlapping PCR technique. Primers were designed by phylogenetic alignment of closely related HPV genomes using the L1 fragment sequence amplified by GP5+/6+. The 7983 bp complete nucleotide sequence of the HPV genome was determined by sequence walking.