DNA ploidy and proliferative activity of human pulmonary epithelium

DNA ploidy and distribution has been determined in normal and abnormal bronchial, bronchiolar and alveolar epithelium from 22 patients, aged between 0 and 85 years, 9 of whom had received chemotherapy for malignant disease. The DNA ploidy was diploid in all the specimens examined. The S + G2/M fraction was significantly greater in diseased than normal bronchial trees. In the bronchial epithelium, mean values ± the standard deviation (SD) were 5.5 + 2.2% vs 1.1±0.6%, in bronchiolar epithelium 4.6 ± 1.6% vs 1.0 ± 0.9% and in alveolar epithelium 4.6 ± 1.6% vs 0.8 ± 0.5%. The highest S + G2/M value of 8.9% was obtained from inflamed bronchial epithelium. Polyploid cells up to the octaploid range occurred infrequently but their incidence was slightly increased to between 0.16% and 0.9% in diseased lungs and in patients who had received chemotherapeutic drugs. It was concluded that (1) non-cancerous pulmonary epithelium is diploid, that (2) pulmonary epithelium shows steady-state renewal at all ages and polyploid cells are rare under normal conditions and that (3) the S + G2/M fraction increases up to approximately 10% in reactive proliferative states.     

Concordant metaphyseal dysostosis type Schmid in identical twins

Summary Metaphyseal dysostosis type Schmid has been described in identical twin girls. It was concluded that the concordant occurrence of the disease in the twins was the result of a fresh autosomal dominant mutation.     

HuH-7 reference genome profile: complex karyotype composed of massive loss of heterozygosity

Human cell lines represent a valuable resource as in vitro experimental models. A hepatoma cell line, HuH-7 (JCRB0403), has been used extensively in various research fields and a number of studies using this line have been published continuously since it was established in 1982. However, an accurate genome profile, which can be served as a reliable reference, has not been available. In this study, we performed M-FISH, SNP microarray and amplicon sequencing to characterize the cell line. Single cell analysis of metaphases revealed a high level of heterogeneity with a mode of 60 chromosomes. Cytogenetic results demonstrated chromosome abnormalities involving every chromosome in addition to a massive loss of heterozygosity, which accounts for 55.3% of the genome, consistent with the homozygous variants seen in the sequence analysis. We provide empirical data that the HuH-7 cell line is composed of highly heterogeneous cell populations, suggesting that besides cell line authentication, the quality of cell lines needs to be taken into consideration in the future use of tumor cell lines.     

Carboxy-terminal modulator protein induces Akt phosphorylation and activation, thereby enhancing antiapoptotic, glycogen synthetic, and glucose uptake pathways

Carboxy-terminal modulator protein (CTMP) was identified as binding to the carboxy terminus of Akt and inhibiting the phosphorylation and activation of Akt. In contrast to a previous study, we found CTMP overexpression to significantly enhance Akt phosphorylation at both Thr³⁰⁸ and Ser⁴⁷³ as well as the kinase activity of Akt, while phosphatidylinositol 3-kinase (PI3-kinase) activity was unaffected. Translocation of Akt to the membrane fraction was also markedly increased in response to overexpression of CTMP, with no change in the whole cellular content of Akt. Furthermore, the phosphorylations of GSK-3β and Foxo1, well-known substrates of Akt, were increased by CTMP overexpression. On the other hand, suppression of CTMP with small interfering RNA partially but significantly attenuated this Akt phosphorylation. The cellular activities reportedly mediated by Akt activation were also enhanced by CTMP overexpression. UV-B-induced apoptosis of HeLa cells was significantly reversed not only by overexpression of the active mutant of Akt (myr-Akt) but also by that of CTMP. Increases in glucose transport activity and glycogen synthesis were also induced by overexpression of either myr-Akt or CTMP in 3T3-L1 adipocytes. Taking these results into consideration, it can be concluded that CTMP induces translocation of Akt to the membrane and thereby increases the level of Akt phosphorylation. As a result, CTMP enhances various cellular activities that are principally mediated by the PI3-kinase/Akt pathway. phosphatidylinositol 3-kinase     

Species identification of animal cells by nested PCR targeted to mitochondrial DNA

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.     

Epigenetic alteration to activate Bmp2-Smad signaling in Raf-induced senescence

AIM: To investigate epigenomic and gene expression alterations during cellular senescence induced by oncogenic Raf. METHODS: Cellular senescence was induced into mouse embryonic fibroblasts(MEFs) by infecting retrovirus to express oncogenic Raf(RafV 600E). RNA was collected from RafV 600 E cells as well as MEFs without infection and MEFs with mock infection, and a genome-wide gene expression analysis was performed using microarray. The epigenomic status for active H3K4me3 and repressive H3K27me3 histone marks was analyzed by chromatin immunoprecipitation-sequencing for RafV 600 E cells on day 7 and for MEFs without infection. These data for Raf-induced senescence were compared with data for Ras-induced senescence that were obtained in our previous study. Gene knockdown and overexpression were done by retrovirus infection. RESULTS: Although the expression of some genes including secreted factors was specifically altered in either Ras- or Raf-induced senescence, many genes showed similar alteration pattern in Raf- and Ras-induced senescence. A total of 841 commonly upregulated 841 genes and 573 commonly downregulated genes showed a significant enrichment of genes related to signal and secreted proteins, suggesting the importance of alterations in secreted factors. Bmp2, a secreted protein to activate Bmp2-Smad signaling, was highly upregulated with gain of H3K4me3 and loss of H3K27me3 during Raf-induced senescence, as previously detected in Ras-induced senescence, and the knockdown of Bmp2 by sh RNA lead to escape from Raf-induced senescence. Bmp2-Smad inhibitor Smad6 was strongly repressed with H3K4me3 loss in Raf-induced senescence, as detected in Ras-induced senescence, and senescence was also bypassed by Smad6 induction in Raf-activated cells. Different from Ras-induced senescence, however, gain of H3K27me3 did not occur in the Smad6 promoter region during Raf-induced senescence. When comparing genome-wide alteration between Ras- and Raf-induced senescence, genes showing loss of H3K27me3 during senescence significantly overlapped; genes showing H3K4me3 gain, or those showing H3K4me3 loss, also well-overlapped between Ras- and Raf-induced senescence. However, genes with gain of H3K27me3 overlapped significantly rarely, compared with those with H3K27me3 loss, with H3K4me3 gain, or with H3K4me3 loss.CONCLUSION: Although epigenetic alterations are partly different, Bmp2 upregulation and Smad6 repression occur and contribute to Raf-induced senescence, as detected in Ras-induced senescence.     

3-Hydroxykynurenine as a radical scavenger in the blowfly, Aldrichina grahami

3-Hydroxykynurenine (3-HOK) accumulated in may tissues of A. grahami, especially in the Malpighian tubules. The compound was present in the insect during its whole life cycle. Its content in the insect seemed to be more than that needed for the synthesis of ommochrome, and was also 5–10-fold of the concentration of other reductants. It can protect the fat body cells of A. grahami from peroxidation and also suppress the decrease of respiration of the cells due to injury by t-butylhydroperoxide (t-BuOOH). The effects of the compound are superior to those of reduced glutathione and uric acid, and approach those of ascorbic acid. The protection of the respiration against injury from t-BuOOH means that 3-HOK effectively scavenged radicals and the more 3-HOK the tissue contained, the less the fat body was injured by t-BuOOH.     

Integration-free iPS cells engineered using human artificial chromosome vectors

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.     

Recognition of tennis serve performed by a digital player: comparison among polygon, shadow, and stick-figure models

The objective of this study was to assess the cognitive effect of human character models on the observer's ability to extract relevant information from computer graphics animation of tennis serve motions. Three digital human models (polygon, shadow, and stick-figure) were used to display the computationally simulated serve motions, which were perturbed at the racket-arm by modulating the speed (slower or faster) of one of the joint rotations (wrist, elbow, or shoulder). Twenty-one experienced tennis players and 21 novices made discrimination responses about the modulated joint and also specified the perceived swing speeds on a visual analogue scale. The result showed that the discrimination accuracies of the experienced players were both above and below chance level depending on the modulated joint whereas those of the novices mostly remained at chance or guessing levels. As far as the experienced players were concerned, the polygon model decreased the discrimination accuracy as compared with the stick-figure model. This suggests that the complicated pictorial information may have a distracting effect on the recognition of the observed action. On the other hand, the perceived swing speed of the perturbed motion relative to the control was lower for the stick-figure model than for the polygon model regardless of the skill level. This result suggests that the simplified visual information can bias the perception of the motion speed toward slower. It was also shown that the increasing the joint rotation speed increased the perceived swing speed, although the resulting racket velocity had little correlation with this speed sensation. Collectively, observer's recognition of the motion pattern and perception of the motion speed can be affected by the pictorial information of the human model as well as by the perturbation processing applied to the observed motion.