Direct measurement of oxygen consumption rate on the nematode Caenorhabditis elegans by using an optical technique

It is well known that aging and longevity strongly correlate with energy metabolism. The nematode Caenorhabditis elegans is widely used as an ultimate model of experimental animals. Thus, we developed a novel tool, which is constructed from an optical detector, using an indirect method that can measure simply the energy metabolism of C. elegans. If we measure the oxygen consumption rate using this optical tool, we can easily evaluate the activity of mitochondria as an index in the aging process. However, a direct measurement of the oxygen consumption rate of C. elegans exposed in air is thought to be impossible because of the high concentration of atmospheric oxygen and the small size of the animals. We demonstrate here that we can directly detect the oxygen consumption with a small number of animals (相似文献   

Up-regulation of Na(+),K(+)-ATPase alpha 3-isoform and down-regulation of the alpha1-isoform in human colorectal cancer

We investigated expression levels of Na(+),K(+)-ATPase alpha-isoforms and their ATPase activities in human colorectal cancer tissue and the accompanying normal mucosa. A decrease in expression of the alpha1-isoform protein was observed in all sampled cancer tissues compared with the normal mucosae. The level of ouabain (5 microM)-sensitive Na(+),K(+)-ATPase activity in carcinomas was 81+/-5% that of in the normal mucosae. The mRNA expression of alpha2- and alpha 4-isoforms was decreased in almost all the carcinoma samples. Interestingly, the expression level of the alpha 3-isoform protein in the cancer tissue was higher than that of the normal mucosa. These results indicate that a decrease in the alpha1-isoform expression and an increase in the alpha 3-isoform expression may be associated with colorectal cancer.     

Establishment of stable hFis1 knockdown cells with an siRNA expression vector

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.     

Interaction with protocadherin-gamma regulates the cell surface expression of protocadherin-alpha

The protocadherin-alpha (CNR/Pcdhalpha) and protocadherin-gamma (Pcdhgamma) proteins, members of the cadherin superfamily, are putative cell recognition/adhesion molecules in the brain. Overexpressed cadherins are generally expressed on the cell surface and elicit cell adhesion activity in several cell lines, although hardly any overexpressed CNR/Pcdhalpha proteins are expressed on the cell surface, except on HEK293T cells, which show low expression. We analyzed the expression of CNR/Pcdhalpha and Pcdhgamma in HEK293T cells and found that they formed a protein complex and that Pcdhgamma enhanced the surface expression of CNR/Pcdhalpha. This enhanced surface expression was confirmed by flow cytometry analysis and by marking cell surface proteins with biotin. The enhancement was observed using different combinations of CNR/Pcdhalpha and Pcdhgamma proteins. The surface expression activity was enhanced by the extracellular domains of the proteins, which could bind each other. Their cytoplasmic domains also had binding activity and influenced their localization. Their protein-protein interaction was also detected in extracts of mouse brain and two neuroblastoma cell lines. Thus, interactions between CNR/Pcdhalpha and Pcdhgamma regulate their surface expression and contribute to the combinatorial diversity of cell recognition proteins in the brain.     

Molecular phylogeny of arctoids (Mammalia: Carnivora) with emphasis on phylogenetic and taxonomic positions of the ferret-badgers and skunks

Phylogenetic relationships among the ferret-badger Melogale moschata, the skunk Mephitis mephitis, and 21 other arctoid carnivorans, representing Mustelidae (Mustelinae: Mustela, Martes, Gulo; Lutrinae: Enhydra; Melinae: Meles), Procyonidae (Procyon), and Ursidae (Ursus, Melursus), were evaluated through maximum-parsimony phylogenetic analysis of concatenated partial nucleotide sequences of the nuclear recombination-activating gene 1 (RAG1) and gene encoding interphotoreceptor retinoid-binding protein (IRBP). The analysis strongly supports Melogale as more closely related to a musteline-lutrine clade (containing Mustela and Enhydra) than to Meles or another musteline clade containing Martes and Gulo (causing Melinae and Mustelinae, as traditionally circumscribed, to be nonmonophyletic). This, together with known morphological and karyological evidence for nonmeline affinities of Melogale, justify the exclusion of the ferret-badgers from the monophyletic Melinae. Therefore, we recommend that Melogale be classified in a distinct mustelid subfamily, the monotypic Helictidinae. Our analysis also strongly supports an outgroup position of the skunks to a clade containing Procyonidae and the nonmephitine Mustelidae (causing Mustelidae, as traditionally circumscribed, to be paraphyletic). This position of the skunks agrees with results of most previous genetic studies. However, it is contradicted by known morphological evidence from both living and fossil taxa, as well as genetic evidence from protein electrophoresis. These consistently support the traditional placement of the skunks within the monophyletic Mustelidae (recently in a close relationship to Lutrinae). Therefore, we consider the recent elevation of the skunks to the level of family as premature, and recommend that this clade be left at the subfamily level (Mephitinae) within the family Mustelidae, pending further evidence.     

Inhibition of osteoporosis induced by protein deficient (PD) food intake by active vitamin D(3) and vitamin K(2) in rats

Independent use of K(2) and D(3) and simultaneous application of K(2) and D(3) inhibited the development of osteoporosis caused by PD food intake. The ALP activity of urine as a marker of bone formation osteoporosis did not rise in rats fed PD foods containing D(3), K(2) or both together. Body and womb weights fell in rats fed PD foods with D(3) K(2) and both D(3), K(2). Osteoporosis caused by PD food intake found to be very similar to type II osteoporosis in respects of inhibition by D(3) and K(2) and rising urinary ALP activity.     

Generation of a highly pathogenic avian influenza A virus from an avirulent field isolate by passaging in chickens

Highly virulent avian influenza viruses can arise from avirulent strains maintained in poultry, but evidence to support their generation from viruses in wild birds is lacking. The most likely mechanism for the acquisition of virulence by benign avian viruses is the introduction of mutations by error-prone RNA polymerase, followed by the selection of virulent viruses. To investigate whether this mechanism could apply to wild waterfowl, we studied an avirulent wild-swan virus that replicates poorly in chickens. After 24 consecutive passages by air sac inoculation, followed by five passages in chicken brain, the avirulent virus became highly pathogenic in chickens, producing a 100% mortality rate. Sequence analysis at the hemmaglutinin cleavage site of the original isolate revealed a typical avirulence type of sequence, R-E-T-R, which progressed incrementally to a typical virulence type of sequence, R-R-K-K-R, during repeated passages in chickens. These results demonstrate that avirulent viruses maintained in wild waterfowl in nature and bearing the consensus avirulence type sequence R-E-T-R have the potential to become highly pathogenic while circulating in chickens.     

Expression of vascular endothelial growth factor by granulated metrial gland cells in pregnant murine uteri

Granulated metrial gland (GMG) cells are a characteristic uterine component belonging to a natural killer cell lineage. This study is aimed at revealing their kinetic and spatial relationship with vascular growth during pregnancy and the expression of vascular endothelial growth factor (VEGF). GMG cells and blood vessels were identified by periodic-acid-Schiff-reagent (PAS)-stained granules and positive staining for factor-VIII-related antigen, respectively. GMG cells were widely distributed in the decidua and metrial gland and showed a numerical increase with a peak at day 13 in parallel with the increase of vascular density. Preceding the maximal vascular development at day 13, microvessels with a narrow lumen representative of neovascularization prevailed at days 7-9, and the VEGF content in the decidua/metrial gland was significantly elevated at days 7-13 concurrently with mRNA expression. By immunolight microscopy combined with PAS staining, GMG cells with PAS-stained granules were positive for VEGF. Immunoelectron microscopy demonstrated that immunoreactions were diffuse in the cytoplasm but not localized in the granules. In contrast, fibroblast-like stromal cells were negative. These data indicate that GMG cells express VEGF and may play inducing roles in uterine neovascularization during pregnancy.     

Cu-Zn superoxide dismutase inhibits lactate dehydrogenase release and protects against cell death in murine fibroblasts pretreated with ultraviolet radiation

The effects of adding Cu-Zn superoxide dismutase (Cu-Zn SOD) to culture medium of the murine fibroblast cell line, L-929, pretreated with UV-B (312 nm, 480 mJ/cm(2)) have been investigated. Cell injury was monitored by the release of lactate dehydrogenase (LDH) into the medium, and cell death by the trypan blue exclusion test. UV-B radiation induced cell death by apoptosis, as demonstrated by DNA fragmentation. Over the range 0.1-0.3 microm Cu-Zn SOD, a significant dose-dependent protection against cell death was obtained of the UV-B exposed cells. Cell death correlated with the amount of LDH released into the medium, and Cu-Zn SOD treatment inhibited this. Heat-denatured Cu-Zn SOD did not affect either cell viability or the release of LDH from the cells. Endogenous Cu-Zn SOD activity, monitored by chemiluminescence, decreased by 20% in UV-B-irradiated cells; the addition of 0.3 microm exogenous Cu-Zn SOD to the medium did not affect intracellular Cu-Zn SOD activity. These results establish that Cu-Zn SOD added to extracellular medium can protect cells against injury caused by UV-B exposure.