Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235--a dual PI3K/mTOR inhibitor--and RAD001--an mTOR inhibitor--in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n?=?9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n?=?4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.     

Developmental Trajectories of Body Mass Index Among Japanese Children and Impact of Maternal Factors during Pregnancy

Background

The aims of this study were to 1) determine the distinct patterns of body mass index (BMI) trajectories in Japanese children, and 2) elucidate the maternal factors during pregnancy, which contribute to the determination of those patterns.

Methodology/Principal Findings

All of the children (1,644 individuals) born in Koshu City, Japan, between 1991 and 1998 were followed in a longitudinal study exploring the subjects’ BMI. The BMI was calculated 11 times for each child between birth and 12 years of age. Exploratory latent class growth analyses were conducted to identify trajectory patterns of the BMI z-scores. The distribution of BMI trajectories were best characterized by a five-group model for boys and a six-group model for girls. The groups were named “stable thin,” “stable average,” “stable high average,” “progressive overweight,” and “progressive obesity” in both sexes; girls were allocated to an additional group called “progressive average.” Multinomial logistic regression found that maternal weight, smoking, and skipping breakfast during pregnancy were associated with children included in the progressive obesity pattern rather than the stable average pattern. These associations were stronger for boys than for girls.

Conclusions/Significance

Multiple developmental patterns in Japanese boys and girls were identified, some of which have not been identified in Western countries. Maternal BMI and some unfavorable behaviors during early pregnancy may impact a child’s pattern of body mass development. Further studies to explain the gender and regional differences that were identified are warranted, as these may be important for early life prevention of weight-associated health problems.     

Micro-manipulation system with a two-fingered micro-hand and its potential application in bioscience

A micro-manipulation system using a two-fingered micro-hand, an auto-focusing optical microscope, and user interfaces was developed. This micro-hand has 6 degrees of freedom (DOF): 3 DOF for each of the two fingers. These fingers work just like the thumb and forefinger. Thus, this hand can grasp, move, rotate, and release micro-objects, such as biological cells. A human operator can operate this hand using a joystick or a keyboard, while seeing the microscope image displayed on a monitor. The present paper describes two applications of this system to the field of bioscience. The first application involves extraction of cytoplasm from a cell using two, two-fingered micro-hands. One hand holds the cell firmly, while the other hand makes a hole in the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involves measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor based on the Atomic Force Microscope (AFM) principle. The AFM cantilever is placed within the microscopic field. The micro-finger holds a cell and presses it against the cantilever tip. By measuring the pressing force and the deformation of the cell, the cell's force-deformation curve is obtained.     

High Prevalence of Simian T-Lymphotropic Virus Type L in Wild Ethiopian Baboons

Simian T-cell leukemia viruses (STLVs) are the simian counterparts of human T-cell leukemia viruses (HTLVs). A novel, divergent type of STLV (STLV-L) from captive baboons was reported in 1994, but its natural prevalence remained unclear. We investigated the prevalence of STLV-L in 519 blood samples from wild-living nonhuman primates in Ethiopia. Seropositive monkeys having cross-reactive antibodies against HTLV were found among 22 out of 40 hamadryas baboons, 8 of 96 anubis baboons, 24 of 50 baboons that are hybrids between hamadryas and anubis baboons, and 41 of 177 grivet monkeys, but not in 156 gelada baboons. A Western blotting assay showed that sera obtained from seropositive hamadryas and hybrid baboons exhibited STLV-L-like reactivity. A PCR assay successfully amplified STLV sequences, which were subsequently sequenced and confirmed as being closely related to STLV-L. Surprisingly, further PCR showed that nearly half of the hamadryas (20 out of 40) and hybrid (19 out of 50) baboons had STLV-L DNA sequences. In contrast, most of the seropositive anubis baboons and grivet monkeys carried typical STLV-1 but not STLV-L. These observations demonstrate that STLV-L naturally prevails among hamadryas and hybrid baboons at significantly high rates. STLV-1 and -2, the close relative of STLV-L, are believed to have jumped across simian-human barriers, which resulted in widespread infection of HTLV-1 and -2. Further studies are required to know if STLV-L is spreading into human populations.     

Über den Einfluß der Ernährung auf die Darmflora der Fische

Ohne Zusammenfassung     

Highly diversified population structure of the spider <Emphasis Type="Italic">Lycosa ishikariana</Emphasis> inhabiting sandy beach habitats

Sandy beach ecosystems are decreasing worldwide and organisms living there are becoming threatened. The burrowing wolf spider Lycosa ishikariana is one such example. To establish effective conservation strategies under habitat fragmentation, we examined population genetic structure of L. ishikariana from mitochondrial cytochrome oxidase I gene and 6 microsatellite loci. Mitochondrial sequence data revealed 6 population subgroups with very high fixation indices, indicating that L. ishikariana has a clear phylogeographic structure and that the level of differentiation among regions is considerable. In particular, one subgroup in the western Honshu mainland (clade G) has a highly distinct genetic structure, despite having no clear geographic barriers from its parapatric population. Moreover, the distribution ranges of the other two subgroups (clades D and E) were highly restricted, suggesting their vulnerability to local human impacts and highlighting their high conservation priorities. Microsatellite data revealed 10 subgroups that were compatible with the clades identified from the mitochondrial data. Fixation indices among these groups were very high, indicating a limited gene flow induced by male spiders. Based on these results, we proposed six conservation units of L. ishikariana and effective conservation/restoration strategies in the face of ongoing coastal armoring.     

Analysis of Vibrio seventh pandemic island II and novel genomic islands in relation to attachment sequences among a wide variety of Vibrio cholerae strains

Vibrio cholerae O1 El Tor, the pathogen responsible for the current cholera pandemic, became pathogenic by acquiring virulent factors including Vibrio seventh pandemic islands (VSP)‐I and ?II. Diversity of VSP‐II is well recognized; however, studies addressing attachment sequence left (attL) sequences of VSP‐II are few. In this report, a wide variety of V. cholerae strains were analyzed for the structure and distribution of VSP‐II in relation to their attachment sequences. Of 188 V. cholerae strains analyzed, 81% (153/188) strains carried VSP‐II; of these, typical VSP‐II, and a short variant was found in 36% (55/153), and 63% (96/153), respectively. A novel VSP‐II was found in two V. cholerae non‐O1/non‐O139 strains. In addition to the typical 14‐bp attL, six new attL‐like sequences were identified. The 14‐bp attL was associated with VSP‐II in 91% (139/153), whereas the remaining six types were found in 9.2% (14/153) of V. cholerae strains. Of note, six distinct types of the attL‐like sequence were found in the seventh pandemic wave 1 strains; however, only one or two types were found in the wave 2 or 3 strains. Interestingly, 86% (24/28) of V. cholerae seventh pandemic strains harboring a 13‐bp attL‐like sequence were devoid of VSP‐II. Six novel genomic islands using two unique insertion sites to those of VSP‐II were identified in 11 V. cholerae strains in this study. Four of those shared similar gene clusters with VSP‐II, except integrase gene.

     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Occurrence of theobromine synthase genes in purine alkaloid-free species of <Emphasis Type="Italic">Camellia</Emphasis> plants

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-¹⁴C]adenine and [8-¹⁴C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession numbers AB297451 (CjCS1), AB362882 (CgCS1), AB362883 (CgCS2), AB362884 (CkCS1), AB362885 (ClCS1), and AB362886 (CcCS2).