Phylogeny of the gudgeons (Teleostei: Cyprinidae: Gobioninae)

The members of the cyprinid subfamily Gobioninae, commonly called gudgeons, form one of the most well-established assemblages in the family Cyprinidae. The subfamily is a species-rich group of fishes, these fishes display diverse life histories, appearances, and behavior. The phylogenetic relationships of Gobioninae are examined using sequence data from four loci: cytochrome b, cytochrome c oxidase I, opsin, and recombination activating gene 1. This investigation produced a data matrix of 4114 bp for 162 taxa that was analyzed using parsimony, maximum likelihood, and Bayesian inference methods. The phylogenies our analyses recovered corroborate recent studies on the group. The subfamily Gobioninae is monophyletic and composed of three major lineages. We find evidence for a Hemibarbus-Squalidus group, and the tribes Gobionini and Sarcocheilichthyini, with the Hemibarbus-Squalidus group sister to a clade of Gobionini-Sarcocheilichthyini. The Hemibarbus-Squalidus group includes those two genera; the tribe Sarcocheilichthyini includes Coreius, Coreoleuciscus, Gnathopogon, Gobiocypris, Ladislavia, Paracanthobrama, Pseudorasbora, Pseudopungtungia, Pungtungia, Rhinogobio, and Sarcocheilichthys; the tribe Gobionini includes Abbottina, Biwia, Gobio, Gobiobotia, Huigobio, Microphysogobio, Platysmacheilus, Pseudogobio, Romanogobio, Saurogobio, and Xenophysogobio. The monotypic Acanthogobio is placed into the synonymy of Gobio. We tentatively assign Belligobio to the Hemibarbus-Squalidus group and Mesogobio to Gobionini; Paraleucogobio and Parasqualidus remain incertae sedis. Based on the topologies presented, the evolution of swim bladder specializations, a distinctive feature among cyprinids, has occurred more than once within the subfamily.     

The effect of PKC activity on the TTX-R sodium currents from rat nodose ganglion neurons

To determine how protein kinase C (PKC) activity influences properties of the tetrodotoxin-resistant sodium current (TTX-R I(Na)) in neonatal rat nodose ganglion (NG) neurons, we assessed the effects of phorbol,-12-myristate,13-acetate (PMA), one of the PKC activators, and staurosporine, one of the PKC inhibitors, on the current. PMA (30 and 100 nM) induced an increase in the peak current amplitude of normalized current-voltage curves, a leftward shift in the potential for half activation (V(1/2)) of normalized conductance-voltage curves and a leftward shift of V(1/2) potential for steady-state inactivation curves. The effects of staurosporine (0.1 and 1 muM) on the peak current amplitude and the V(1/2) potential for activation were opposite compared with those seen after PMA application. Staurosporine (1 muM) antagonized PMA (100 nM)-induced modification of TTX-R I(Na). These results suggest that the basal TTX-R I(Na) obtained from neonatal NG neurons is controlled by the level of PKC activity.     

Frequent retention of heterozygosity for point mutations in p53 and Ikaros in N-ethyl-N-nitrosourea-induced mouse thymic lymphomas

In agreement with Knudson's two-hit theory, recent findings indicate that the inactivation of tumor suppressor genes is not only mediated by the loss of function but also by the dominant-negative or gain-of-function activity. The former generally accompanies loss of a wild-type allele whereas in the latter a wild-type allele is retained. N-Ethyl-N-nitrosourea (ENU), which efficiently induces point mutations, reportedly leads to the development of tumors by activating ras oncogenes. Little is known about how ENU affects tumor suppressor genes and, therefore, we examined ENU-induced mutations of p53 and Ikaros in thymic lymphomas and compared these with mutations of Kras. In addition, loss of heterozygosity was examined for chromosome 11 to which both p53 and Ikaros were mapped. The frequency of point mutations in p53 and Ikaros was 30% (8/27) and 19% (5/27), respectively, comparable to that observed in Kras (33%: 9/27). In total, 14 of the 27 thymic lymphomas examined (52%) harbored mutations in at least one of these genes. One Ikaros mutation was located at the splice donor site, generating a novel splice isoform lacking zinc finger 3, Ik (F3del). Interestingly, 90% (10/11) of the tumors with point mutations retained wild-type alleles of p53 and Ikaros. Sequence analysis revealed that the most common nucleic acid substitutions were T>A (4/8) in p53, T>C (4/5) in Ikaros and G>A/T (8/9) in Kras, suggesting that the spectrum of mutations was gene dependent. These results suggest that point mutations in tumor suppressor genes without loss of the wild-type allele play an important role in ENU-induced lymphomagenesis.     

PsaK2 subunit in photosystem I is involved in state transition under high light condition in the cyanobacterium Synechocystis sp. PCC 6803

To avoid the photodamage, cyanobacteria regulate the distribution of light energy absorbed by phycobilisome antenna either to photosystem II or to photosystem I (PSI) upon high light acclimation by the process so-called state transition. We found that an alternative PSI subunit, PsaK2 (sll0629 gene product), is involved in this process in the cyanobacterium Synechocystis sp. PCC 6803. An examination of the subunit composition of the purified PSI reaction center complexes revealed that PsaK2 subunit was absent in the PSI complexes under low light condition, but was incorporated into the complexes during acclimation to high light. The growth of the psaK2 mutant on solid medium was inhibited under high light condition. We determined the photosynthetic characteristics of the wild type strain and the two mutants, the psaK1 (ssr0390) mutant and the psaK2 mutant, using pulse amplitude modulation fluorometer. Non-photochemical quenching, which reflects the energy transfer from phycobilisome to PSI in cyanobacteria, was higher in high light grown cells than in low light grown cells, both in the wild type and the psaK1 mutant. However, this change of non-photochemical quenching during acclimation to high light was not observed in the psaK2 mutant. Thus, PsaK2 subunit is involved in the energy transfer from phycobilisome to PSI under high light condition. The role of PsaK2 in state transition under high light condition was also confirmed by chlorophyll fluorescence emission spectra determined at 77 K. The results suggest that PsaK2-dependent state transition is essential for the growth of this cyanobacterium under high light condition.     

Rho localization in cells and tissues

Rho family small GTPases regulate cytoskeletal organization. Although their spatiotemporal activities appear to be important for cellular morphogenesis, there has been little characterization of the localization of Rho family GTPases in cells and tissues. Here we show precise localization of Rho subfamily proteins in mammalian cultured cells and tissues through evaluation of anti-Rho antibodies and fixation protocols. Although Rho is not a structural protein but functions as a switching molecule, it often localizes at several distinct domains or structures of cells. In cultured epithelial cells, Rho was highly accumulated at lateral membranes. However, in fibroblastic cells, Rho appeared to be distributed evenly in the cytoplasm. Rho concentration at the cleavage furrow at cytokinesis was generally observed. In A431 cells, Rho translocation from the cytoplasm to elongating microvilli at the apical membrane within 30 s after EGF stimulation was clearly demonstrated. Also, Myc- or GFP-tagged RhoA did not always reflect the localization of endogenous Rho, indicating a drawback of protein-tagging methods for localization research. In mouse tissues, Rho localization differed depending on cell type, probably reflecting the functional differences of each cell type.     

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.     

Population genetic structure of Sorex unguiculatus and Sorex caecutiens (Soricidae, Mammalia) in Hokkaido, based on microsatellite DNA polymorphism

We investigated the genetic structure of Sorex unguiculatus and Sorex caecutiens populations in Hokkaido, Japan, using hypervariable microsatellite DNA markers. We used five microsatellite loci to type 475 S. unguiculatus individuals from 20 localities on the Hokkaido mainland and four localities from each of four offshore islands (and 11 shrews from one locality in southern Sakhalin for a particular analysis). We used six microsatellite loci to type 240 S. caecutiens individuals from 13 localities on the Hokkaido mainland. Genetic variation was high in mainland populations of both species and low in the island populations of S. unguiculatus. Allelic richness and island size were positively correlated for S. unguiculatus, suggesting that genetic drift occurred on those islands due to small population size. In addition, four insular populations of S. unguiculatus were genetically differentiated from the mainland populations, although clear phylogeographic clustering was not confirmed among populations on the Hokkaido mainland for either S. unguiculatus or S. caecutiens. Heterozygosity excess was observed in more than half of the populations including the mainland populations of the two species, suggesting recent bottleneck events in these populations. Population dynamics of the shrews might be explained by a metapopulation scheme. According to autocorrelation analysis, the extent of non-random spatial genetic structure was approximately 100 km. Isolation by distance was observed in S. unguiculatus, but not in S. caecutiens although there is a positive trend. The lack of correlation for S. caecutiens might have been due to small sample size. Thus, no obvious differences in population genetic structure were found between the two species on the Hokkaido mainland in the present study, while previous investigations using mitochondrial DNA sequences inferred that these two species might have rather different biogeographic histories.     

Effects of dietary myo-inositol, D-chiro-inositol and L-chiro-inositol on hepatic lipids with reference to the hepatic myo-inositol status in rats fed on 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane

The effects of dietary 0.2% inositol stereoisomers on the hepatic lipids and myo-inositol (MI) status in rats fed with 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) were investigated. Dietary MI reduced the hepatic lipids in the rats fed with DDT. Dietary D-chiro-inositol (DCI) and L-chiro-inositol (LCI) both had a promoting effect on the increase in hepatic lipids due to DDT feeding. Dietary MI enhanced the hepatic free MI level and the phosphatidylinositol/phosphatidylcholine ratio, but dietary DCI reduced the level and ratio.