Fitness cost of parasitoid avoidance behavior in the arrowhead scale, Unaspis yanonensis Kuwana

We examined trade-offs between fitness components and the parasitoid avoidance behavior of the arrowhead scale, Unaspis yanonensis Kuwana (Homoptera: Diaspididae). We examined the performance of two settling modes: singleton and double settling with the burrower under the burrowee. Each of these modes differs in vulnerability to parasitism by Aphytis yanonensis DeBach et Rosen and Coccobius fulvus Compere et Annecke (Hymenoptera: Aphelinidae), and performance. Field and laboratory observations revealed smaller body size and lower parasitism of burrower scales, while both singletons and burrowees showed the opposite tendency (larger body size and a greater parasitism rate). Laboratory observations under parasitoid-free conditions revealed greater mortality and reduced growth rate for simulated burrowers than for singletons during the 40-day nymphal period. This suggests that competition for food resources reduces performance. The results also suggest trade-offs between parasitoid avoidance and resource acquisition ability in U. yanonensis. The trade-offs also explain our previous findings, that introduction of the parasitoids increased the proportion of burrowers, but that within seven years equilibrium was reached at fairly low levels (ca. 5%) in the field, even under intense parasitism.     

Kunitz-type protease inhibitor bikunin disrupts phorbol ester-induced oligomerization of CD44 variant isoforms containing epitope v9 and subsequently suppresses expression of urokinase-type plasminogen activator in human chondrosarcoma cells.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses phorbol ester (PMA)-stimulated expression of urokinase-type plasminogen activator (uPA). In the present study, we tried to answer this mechanism using human chondrosarcoma HCS-2/8 cells. Our results showed the following novel findings: (a) the standard form of CD44 (CD44s; 85 kDa) is expressed in both unstimulated and PMA-stimulated cells, while CD44v isoforms containing epitope v9 (110 kDa) are strongly up-regulated in response to treatment with PMA; (b) CD44v isoforms containing epitope v9 present on the same cell exclusively form aggregates in stimulated cells; (c) induction of uPA mRNA expression could be achieved by using a second cross-linker antibody to cross-link Fab monomers of anti-CD44; (d) co-treatment of stimulated cells with anti-CD44 mAb alone or anti-CD44v9 mAb alone suppresses PMA-induced clustering of CD44, which results in inhibition of uPA overexpression; (e) bikunin efficiently disrupts PMA-induced clustering of CD44, but does not prevent PMA-induced up-regulation of CD44v isoforms containing epitope v9; and (f) after exposure to bik, approximately 150-kDa band is mainly detected with immunoprecipitation and this band is shown to be a heterodimer composed of the 110-kDa v9-containing CD44v isoforms and a 45-kDa bik receptor (bik-R). In conclusion, we provide, for the first time, evidence that the bik-R can physically interact with the CD44v isoforms containing epitope v9 and function as a repressor to down-regulate PMA-stimulated uPA expression, at least in part, by preventing clustering of CD44v isoforms containing epitope v9.     

Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone Cdc37

CK2-dependent phosphorylation of a kinase-specific Hsp90 co-chaperone Cdc37 on a conserved serine residue (Ser13) is essential for the function of Cdc37 [Bandhakavi S. et al. J. Biol. Chem. 278:2829-2836, 2003; Shao J. et al. J. Biol. Chem. 278:38117-38220, 2003; Miyata Y., & Nishida E. Mol. Cell. Biol. 24:4065-4074, 2004]. We have recently produced an anti-[pSer13]-Cdc37 antibody which specifically recognizes Cdc37 that is phosphorylated on Ser 13 [Miyata Y. & Nishida E. FEBS J. 274:5690-5703, 2007]. Here we investigated CK2 activity both in vitro and in cultured cells by using anti-[pSer13]-Cdc37 antibody. Immunoblotting with this antibody showed that heparin and 4,5,6,7-tetrabromobenzotriazole (TBB), known CK2 inhibitors, inhibited in vitro phosphorylation of Cdc37 on Ser13 by CK2 holoenzyme or CK2alpha, confirming the specificity of the antibody to detect CK2 activity. Treatment of cells with TBB resulted in the decrease in the phosphorylation level of endogenous Cdc37 on Ser13, as revealed by anti-[pSer13]-Cdc37, and overexpression of either CK2alpha or CK2beta subunit enhanced the Cdc37 phosphorylation level. While CK2 is suggested to be involved in cell proliferation, mitogenic stimulation of starved cells by fresh serum or insulin-like growth factor-I did not enhance phosphorylation of Cdc37 on Ser13. CK2 inhibitors are known to induce cell apoptosis, suggesting a reverse correlation between cell apoptosis and CK2 activity. However, cellular apoptotic stresses, such as anisomycin treatment and UV irradiation, were found to rather modestly increase phosphorylation of Cdc37 on Ser13. These results show that the anti-[pSer13]-Cdc37 antibody can be a promising new tool to evaluate in vivo CK2 activity.     

Development and characterization of 1C6-203, a new monoclonal antibody specific to human thymidine phosphorylase.

Thymidine phosphorylase (dThdPase) is an essential enzyme for activation of the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, to 5-fluorouracil in tumors. Methods to estimate dThdPase expression in tumor tissue might be useful to predict the efficacy of capecitabine and doxifluridine in cancer patients. We established a new monoclonal antibody (MAb), 1C6-203, applicable for dThdPase immunohistochemistry and compared its staining characteristics with those of a previously established MAb, 654-1. In 4% paraformaldehyde-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 19/30 and 9/30 patients, respectively. In 10% formalin-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 18/30 and 6/30 patients, respectively. In negative 10% formalin-fixed tissues, microwave treatment improved the positivity of 654-1-stained cancer cells. These results suggest that an epitope recognized by 1C6-203 is resistant to epitope masking by formaldehyde fixation, whereas that for MAb 654-1 is sensitive. Therefore, MAb 1C6-203 might be more suitable than MAb 654-1 for evaluating dThdPase expression in colorectal carcinoma.     

Retroposition of the AFC Family of SINEs (Short Interspersed Repetitive Elements) Before and During the Adaptive Radiation of Cichlid Fishes in Lake Malawi and Related Inferences About Phylogeny

Lake Malawi is home to more than 450 species of endemic cichlids, which provide a spectacular example of adaptive radiation. To clarify the phylogenetic relationships among these fish, we examined the presence and absence of SINEs (short interspersed repetitive elements) at orthologous loci. We identified six loci at which a SINE sequence had apparently been specifically inserted by retroposition in the common ancestor of all the investigated species of endemic cichlids in Lake Malawi. At another locus, unique sharing of a SINE sequence was evident among all the investigated species of endemic non-Mbuna cichlids with the exception of Rhamphochromis sp. The relationships were in good agreement with those deduced in previous studies with various different markers, demonstrating that the SINE method is useful for the elucidation of phylogenetic relationships among cichlids in Lake Malawi. We also characterized a locus that exhibited transspecies polymorphism with respect to the presence or absence of the SINE sequence among non-Mbuna species. This result suggests that incomplete lineage sorting and/or interspecific hybridization might have occurred or be occurring among the species in this group, which might potentially cause misinterpretation of phylogenetic data, in particular when a single-locus marker, such as a sequence in the mitochondrial DNA, is used for analysis. Received: 15 December 2000 / Accepted: 30 January 2001     

Localization of mitochondrial large ribosomal RNA in the myoplasm of the early ascidian embryo

Mitochondrial large ribosomal RNA (mtlrRNA) is transferred out of mitochondria and associates with germinal granules in Drosophila and Xenopus embryos. It has been revealed that mtlrRNA outside of mitochondria is required for formation of the germ-line progenitor, or pole cells in Drosophila. In the present study, the distribution of mtlrRNA was examined in embryos of the ascidian, Halocynthia roretzi, during cleavage stages by whole-mount in situ hybridization. Until the 4-cell stage, the distribution of mtlrRNA coincided with that of mitochondria. which are localized to the cortical cytoplasm in the posterior region of the embryos. Both mitochondria and mtlrRNA were preferentially partitioned into muscle-lineage blastomeres during cleavage stages. After the 8-cell stage, a discrepancy in intracellular localization of mitochondria and mtlrRNA became evident. Mitochondria translocated into central yolkless cytoplasm, while mtlrRNA remained in the posterior cortex in the posterior muscle-lineage b astomeres. The significance of the cortical localization of mtlrRNA in muscle precursor cells in ascidian embryos is obscure. However, the results suggest that mtlrRNA is also transferred out of mitochondria in early ascidian embryos and may play some roles in developmental processes.     

Btk29A-Mediated Tyrosine Phosphorylation of Armadillo/β-Catenin Promotes Ring Canal Growth in Drosophila Oogenesis

Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29A^ficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29A^ficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29A^ficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.     

Inhibitory activities of the heterotrimers formed from two α-type phospholipase A2 inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation

α-type phospholipase A₂ inhibitory protein (PLIα) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIα, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIα-LP, is homologous to GbPLIα, but it does not show any inhibitory activity against PLA₂s. When a mixture of denaturant-treated monomeric forms of GbPLIα and EqPLIα-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIα and EqPLIα-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIα and the recombinant chimeric EqPLIα-LP, Eq13Gb37Eq, in which the residues 13–36 were replaced by those of GbPLIα, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIα-LP and the recombinant chimeric GbPLIα, Gb13Eq37Gb, in which the residues 13–36 were replaced by those of EqPLIα-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLIα were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLIα with a single amino acid substitution, GbPLIα(K28E).     

Synthesis and reactivities of binuclear iron(III) complexes with ligands composed of two tridentate chelating groups

Several new binuclear iron(III) complexes were prepared using the binucleating ligands in which two molecules of N,N-bis(2-benzimidazolymethyl)amine are linked by a polyatomic chain, such as-(CH₂)₄(L-4), -(CH₂)₆- (L-6), and -CH₂CH(OH)CH₂H(L-3), etc., and were characterized in terms of the magnetic measurements and cyclic voltammetry. The complexes, Fe₂(L-3′0(NO₃)₅ showed subnormal magnetic moments, 4.10 BM at 299 K and 2.22 BM at 87 K, respectively, suggesting the presence of an alkoxo-bridge structure. This complex showed much higher catalytic activity for the O₂ oxidation of N, N, N′,N′-tetramethyl-1-1,4-diaminobenzene than those of the relevant mononuclear complexes and other binuclear complexes studied here which have no alkoxo-bridge. The complex reacts with catechol and hydrogen peroxide to form 1:1 adducts.