The Bcl-2 Homology Domain 3 (BH3)-only Proteins Bim and Bid Are Functionally Active and Restrained by Anti-apoptotic Bcl-2 Family Proteins in Healthy Liver

An intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family proteins. We previously reported that a fine rheostatic balance between the anti- and pro-apoptotic multidomain Bcl-2 family proteins controls hepatocyte apoptosis in the healthy liver. The Bcl-2 homology domain 3 (BH3)-only proteins set this rheostatic balance toward apoptosis upon activation in the diseased liver. However, their involvement in healthy Bcl-2 rheostasis remains unknown. In the present study, we focused on two BH3-only proteins, Bim and Bid, and we clarified the Bcl-2 network that governs hepatocyte life and death in the healthy liver. We generated hepatocyte-specific Bcl-xL- or Mcl-1-knock-out mice, with or without disrupting Bim and/or Bid, and we examined hepatocyte apoptosis under physiological conditions. We also examined the effect of both Bid and Bim disruption on the hepatocyte apoptosis caused by the inhibition of Bcl-xL and Mcl-1. Spontaneous hepatocyte apoptosis in Bcl-xL- or Mcl-1-knock-out mice was significantly ameliorated by Bim deletion. The disruption of both Bim and Bid completely prevented hepatocyte apoptosis in Bcl-xL-knock-out mice and weakened massive hepatocyte apoptosis via the additional in vivo knockdown of mcl-1 in these mice. Finally, the hepatocyte apoptosis caused by ABT-737, which is a Bcl-xL/Bcl-2/Bcl-w inhibitor, was completely prevented in Bim/Bid double knock-out mice. The BH3-only proteins Bim and Bid are functionally active but are restrained by the anti-apoptotic Bcl-2 family proteins under physiological conditions. Hepatocyte integrity is maintained by the dynamic and well orchestrated Bcl-2 network in the healthy liver.     

Prokaryotic microbiota of recycled paper mills with low or zero effluent

The tendency in the paper industry is to close all water loops to save water. This leads to higher process temperatures and may increase the colloidal and dissolved material in the process circulation. Increase of nutrients in the water circuits may favor microbiological growth and fouling. In this paper the chemical and microbial compositions of water circuits and deposits were studied of two closed cycled paper/board mills, one mill totally closed (0 m(3) waste water t(-1)), and the other low discharging (about 4 m(3 )t(-1)). The zero discharge mill accumulated high amounts (>10 g C L(-1)) of organic carbon in the circulation waters, about 40% of which composed of volatile acids (lactic, acetic, propionic and butyric acid). Water contents of sulfate, chloride, sodium and calcium increased to >1 g L(-1) of each. q-PCR targeted on 16S rRNA genes indicated that the bacteria in water circuits were mainly viable cells. In both mills anaerobic growth (10(6)-10(8) CFU mL(-1)) equalled or exceeded aerobic growth, with odor problem but no actual slime problem. The major part (40%) of all identifiable bacterial sequences were closest but yet distant (<96%) to Enterococcus cecorum and in the 4 m(3 )t(-1) discharging mill also Bacillus thermoamylovorans and Bacillus coagulans. Slimes and deposits from the mills contained high amounts, >/=10(8) g(-1), of archaean, but only the genus Methanothrix was identifiable from the cloned sequences. The findings indicate that closing the water circuits strongly limited diversity of the microbiota but allowed efficient mineralization of the dissolved and suspended matter.     

Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.     

Gene expression profiling identifies a set of transcripts that are up-regulated inhuman testicular seminoma.

Seminoma constitutes one subtype of human testicular germ cell tumors and is uniformly composed of cells that are morphologically similar to the primordial germ cells and/or the cells in the carcinoma in situ. We performed a genome-wide exploration of the genes that are specifically up-regulated in seminoma by oligonucleotide-based microarray analysis. This revealed 106 genes that are significantly and consistently up-regulated in the seminomas compared to the adjacent normal tissues of the testes. The microarray data were validated by semi-quantitative RT-PCR analysis. Of the 106 genes, 42 mapped to a small number of specific chromosomal regions, namely, 1q21, 2p23, 6p21-22, 7p14-15, 12pll, 12p13, 12q13-14 and 22q12-13. This list of up-regulated genes may be useful in identifying the causative oncogene(s) and/or the origin of seminoma. Furthermore, immunohistochemical analysis revealed that the seminoma cells specifically expressed the six gene products that were selected randomly from the list. These proteins include CCND2 and DNMT3A and may be useful as molecular pathological markers of seminoma.     

Negative regulation of NK cell activities by inhibitory receptor CD94/NKG2A leads to altered NK cell-induced modulation of dendritic cell functions in chronic hepatitis C virus infection

NK cells are potent activators of dendritic cells (DCs), but it remains obscure how third-party cells affect the ability of NK cells to modulate DC functions. We show here that NK cells derived from healthy donors (N-NK), when cocultured with human liver epithelial cells, induced maturation as well as activation of DCs, such as increased migratory capacity as well as T cell stimulatory activity. In contrast, NK cells from chronic hepatitis C virus-infected donors (HCV-NK) were not capable of activating DCs under the same conditions. In comparison to N-NK, HCV-NK showed higher expression of CD94/NKG2A and produced IL-10 and TGFbeta when cultured with hepatic cells, most of which express HLA-E, a ligand for CD94/NKG2A. Blockade of NKG2A restored the ability of HCV-NK to activate DCs, which appeared to result from the reduced NK cell production of IL-10 and TGFbeta. The blockade also endowed HCV-NK with an ability to drive DCs to generate Th1-polarized CD4+ T cells. These findings show that NK cell modulation of DCs is regulated by third-party cells through NK receptor and its ligand interaction. Aberrant expression of NK receptors may have an impact on the magnitude and direction of DC activation of T cells under pathological conditions, such as chronic viral infection.     

Morphological discrimination of two genetic groups of a Japanese salamander, Hynobius naevius (Amphibia, Caudata)

Hynobius naevius, distributed on western Honshu, Shikoku, and Kyushu Islands of Japan, includes two genetically distinct groups (Groups A and B) that have never been delimited morphologically. Using specimens from the entire species range, we investigated the possibility of distinguishing these groups morphologically. Multivariate analyses of morphometric characters resulted in recognition of two groups that corresponded well to the two genetic groups. One (Group A) was characterized by larger body, compressed tail, shallower vomerine tooth series, bluish- or reddish-purple ground color, and pale-white lateral markings. In contrast, another (Group B) was characterized by smaller body, cylindrical tail, longer vomerine tooth series, reddish-brown ground color, and white lateral markings. Group A was composed of populations from the Chugoku District of Honshu and northern Kyushu, and could not be divided into subgroups, while Group B encompassed populations from the Chubu and Kinki Districts of Honshu, Shikoku and Kyushu, and was subdivided into three local subgroups that are geographically separated by marine straits. Morphometric differentiation in Group A is presumed to have been less affected by genetic factors than by other factors, such as ecological relationships with other, coexisting species. Differentiation in Group B is assumed to have been enhanced not only by genetic but also by climatological factors.     

Phylogeny and historical demography of Cynops pyrrhogaster (Amphibia: Urodela): Taxonomic relationships and distributional changes associated with climatic oscillations

We investigated the phylogenetic relationships and estimated the historical demography of the Japanese fire-bellied newt, Cynops pyrrhogaster, from Japanese mainlands using 1407-bp sequences of the mitochondrial DNA (NADH6, tRNAglu, cyt b) and 1208-bp sequences of nuclear DNA (Rag-1) genes. Phylogenetic trees based on mitochondrial DNA revealed four major haplotype clades (NORTHERN, CENTRAL, WESTERN, and SOUTHERN clades) within this species. Degree of genetic differentiation among major haplotype clades was very large for intraspecific variation, suggesting this species to be composed of four species lineages that replace each other geographically. Nuclear genetic variation presented no obvious patterns of geographic structure except for the distinctness of populations diagnosed by NORTHERN clade of mitochondrial haplotypes, suggesting results of incomplete lineage sorting. Current distribution and estimated divergence times for the genus Cynops suggest that the common ancestor of two Japanese species (C. pyrrhogaster and C. ensicauda from the Ryukyu Islands) had diverged at the edge of the continent corresponding to the present East China Sea and Central Ryukyus. Subsequent range expansion to Japanese mainland seems to have occurred in the middle Miocene. Population-genetic analyses indicated that all species lineages, except for the SOUTHERN one, experienced geographic population reductions and expansions associated with glacial and postglacial climatic oscillations.     

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.     

Exosome‐mediated extracellular release of polyadenylate‐binding protein 1 in human metastatic duodenal cancer cells

Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis‐ and endocytosis‐like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ‐521) and metastatic (AZ‐P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC‐MS/MS and Western blot analyses, polyadenylate‐binding protein 1 (PABP1) was found to be predominantly abundant in AZ‐P7a exosomes. The amount of exosomal PABP1 in AZ‐P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin‐proteasome pathway (MG‐132 and PYR‐41). Treatment of AZ‐P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1‐immunoreactive products cleaved via a proteolysis‐like process. Taken together, these results suggest that AZ‐P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome‐mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer.