A Novel Enhanced Green FluorescentProtein-Expressing NOG Mouse for Analyzingthe Microenvironment of Xenograft Tissues

The interaction between transplanted cells and host tissues is important for the growthand maintenance of transplanted cells. To analyze the mechanisms of these interactions, asystemic fluorescent protein-expressing mouse is a useful recipient. In this study, wegenerated a novel NOG strain, which strongly expresses enhanced green fluorescent protein(EGFP; PgkEGFP-NOG), especially in the liver, kidney, gastrointestinal tract, and testis.Because the host tissues expressed EGFP, xenotransplanted human cancer cells were clearlyidentified as EGFP-negative colonies in PgkEGFP-NOG mice. Immunohistochemical analysisrevealed that EGFP-expressing stromal tissues formed a complicated tumor microenvironmentwithin xenograft tissues. Moreover, a similar microenvironment was observed in human iPScell-derived teratomas. Collectively, these results indicated that a suitablemicroenvironment is essential for the growth and maintenance of xenotransplanted cells andthat PgkEGFP-NOG mice represent a useful animal model for analyzing the mechanisms ofmicroenvironment formation.     

Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs

We isolated a rat glia maturation factor-gamma(rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-beta (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs.     

Na(+) action potentials in human photoreceptors

Mammalian photoreceptors are hyperpolarized by a light stimulus and are commonly thought to be nonspiking neurons. We used the whole-cell patch-clamp technique on surgically excised human retina to examine whether human photoreceptors can elicit action potentials. We discovered that human rod photoreceptors express voltage-gated Na(+) channels, and generate Na(+) action potentials, in response to membrane depolarization from membrane potentials of -60 or -70 mV. Na(+) spikes in human rods were elicited at the termination of a light response that hyperpolarized the potential well below -50 mV. This served to amplify the release of a neurotransmitter when a bright light is turned off, and thus selectively amplify the off response to the light signal.     

An insect cell line discrimination method by RAPD-PCR

Summary RAPD-PCR with a tenmar single primer for discrimination of insect cell lines was devised. The base sequence of the primers used were TTCGAGCCAG, CCGCATCTAC, GAACGGACTC, and TGAGTGGGTG (GC contents were 60%). Genome DNA was extracted by modified Landry et al. (1993) method. The reaction mixture consisted of 10 μl buffer, 8 μl dNTP mixture (2.5 mM each), 4 μl primer (50 μM), Taq DNA polymerase (2.5 units), 1 μl template DNA; and the reaction was run at 94° C for 2 min (denaturation), followed by 31 cycles of 94° C for 1 min, 42° C for 1 min (annealing), and 72° C for 2 min (extension) and terminated with 72° C for 7 min. By developing the reaction products with agarose gel electrophoresis, it became evident that DNA fragments were amplified with all the primers used. Among four primers, the second primer was selected as a suitable primer for distinguishing cell lines. With this method, cell lines derived from different species were clearly distinguished.     

Maximum Likelihood Analysis of the Complete Mitochondrial Genomes of Eutherians and a Reevaluation of the Phylogeny of Bats and Insectivores

The complete mitochondrial genomes of two microbats, the horseshoe bat Rhinolophus pumilus, and the Japanese pipistrelle Pipistrellus abramus, and that of an insectivore, the long-clawed shrew Sorex unguiculatus, were sequenced and analyzed phylogenetically by a maximum likelihood method in an effort to enhance our understanding of mammalian evolution. Our analysis suggested that (1) a sister relationship exists between moles and shrews, which form an eulipotyphlan clade; (2) chiropterans have a sister-relationship with eulipotyphlans; and (3) the Eulipotyphla/Chiroptera clade is closely related to fereuungulates (Cetartiodactyla, Perissodactyla and Carnivora). Divergence times on the mammalian tree were estimated from consideration of a relaxed molecular clock, the amino acid sequences of 12 concatenated mitochondrial proteins and multiple reference criteria. Moles and shrews were estimated to have diverged approximately 48 MyrBP, and bats and eulipotyphlans to have diverged 68 MyrBP. Recent phylogenetic controversy over the polyphyly of microbats, the monophyly of rodents, and the position of hedgehogs is also examined. Received: 21 December 2000 / Accepted: 16 February 2001     

A case of renin producing leiomyosarcoma originating in the lung.

A 54-year-old woman was referred to our hospital for the treatment of a tumor of the right chest wall. Clinical examination revealed hypertension, hypokalemia, metabolic alkalosis, hyperaldosteronism and hyperreninemia. Computed tomography and an abdominal echogram indicated a tumor in the right phrenic area and two tumors in the retroperitoneum near the pancreas head. After the surgical resection of these tumors, the primary reninism was diminished. The pathological diagnosis of these tumors was leiomyosarcoma. Plasma active and inactive (trypsin-activated) renin activities (PRA) were 85.7 and 38.9 ng angiotensin I/ml/h, respectively. These PRA did not respond to either postural stimulation or suppression by the volume expansion. Active and inactive renin activities in a right phrenic area tumor were 208 and 32 ng angiotensin I/mg protein /h, respectively. Those of an abdominal tumor were 196 and 30 ng angiotensin I/mg protein/h, respectively. Renin mRNA identical in molecular size to that of the human kidney was identified by northern blot analysis. This is the first case report of renin producing leiomyosarcoma derived from the lung, which is characterized by relatively lower plasma prorenin concentrations.     

Tinocladia sanrikuensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

The new species Tinocladia sanrikuensis sp. nov. H.Kawai, K.Takeuchi & T.Hanyuda (Ectocarpales s.l., Phaeophyceae) is described from the Pacific coast of the Tohoku region, northern Japan based on morphology and DNA sequences. The species is a spring–summer annual growing on lower intertidal to upper subtidal rocks and cobbles on relatively protected sites. T. sanrikuensis has a slimy, cylindrical, multiaxial erect thallus, slightly hollow when fully developed, branching once to twice, and resembles T. crassa in gross morphology. The erect thalli are composed of a dense medullary layer, long subcortical filaments, and assimilatory filaments of 11–35 cells, up to 425 μm long and curved in the upper portion. Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most closely related to T. crassa and has the same basic thallus structures but differs in having thinner and longer assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes support the distinctness of this species.     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.