Effects of fifteen rare-earth metals on Ca2+ influx in tobacco cells

Effects of naturally existing rare-earth metals (REMs; atomic numbers, 39, 57-60, 62-71; Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu), added as chloride salts, on Ca2+ influx induced by two different stimuli, namely hypoosmotic shock and hydrogen peroxide, were examined in a suspension-cultured transgenic cell line of BY-2 tobacco cells expressing aequorin, a Ca(2+)-sensitive luminescent protein in cytosol. Most REM salts used here showed inhibitory effect against Ca2+ influx. Especially NdCl3, SmCl3, EuCl3, GdCl3 and TbCl3 showed the most robust inhibitory action. In contrast, LuCl3, YbCl3, ErCl3 and YCl3 were shown to be poor inhibitors of Ca2+ influx. Since REMs tested here form a sequential range of ionic radii from 86.1 to 103.2 pm and the optimal range of ionic radii required for blocking the flux of Ca2+ was determined for each stimulus. The hydrogen peroxide-induced Ca2+ influx was optimally blocked by REMs with a broad range of ionic radii (93.8-101 pm) which is slightly smaller than or similar to that of Ca2+ (100 pm), while the hypoosmotically induced flux of Ca2+ was inhibited optimally by few REMs with a narrower range of relatively smaller ionic radii around that of Gd3+ (93.8 pm) a well known inhibitor of stretch-activated channels. Possible applications of such series of channel blockers in elucidation of plant signal transduction pathways are encouraged.     

Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio)

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.     

Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins

Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.     

Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.     

Establishment and functional characterization of novel natural killer cell lines derived from a temperature-sensitive SV40 large T antigen transgenic mouse

Natural killer (NK) cells belong to an important lymphocyte population that eliminates transformed cells and invading pathogens without any prior sensitization. NK cells possess not only natural killing activity against non-self and altered-self cells but also exhibit cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC). Despite their important roles in the innate immune system, little is known about the details of NK cell biology. In spite of that several murine NK cell clones have been established, studies have mainly focused on their natural killing activity but not their cytokine production or ADCC. In this study, we established and characterized eight novel, immortalized murine NK cell clones derived from a temperature-sensitive SV40 large-T antigen transgenic mouse. These NK cell lines continuously proliferated for more than 30 months in a culture medium supplemented with interleukin 2. All cell lines contained azurophilic granules in the cytoplasm, and a few clones retained the NK cell functions, such as natural killing activity, cytokine production, and ADCC. In addition, one clone could serve as a host for transient as well as stable gene transfection. Taken together, these findings indicate that the cell lines could constitute useful tools for detailed analysis of murine NK cell biology.     

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.     

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X(35)-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.     

Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation

A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of bone marrow transplantation is presented. Only the expression of HLA-B51, which was a mismatched HLA locus in the graft-versus-host direction, was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. All CTL clones, that were isolated from the recipient's blood when acute graft-versus-host disease developed, recognized the mismatched B(?)51:01 molecule in a peptide-dependent manner. The pre-transplant leukemia blasts were lysed by CTL clones, whereas the post-transplant leukemia blasts were not lysed by any CTL clones. The IFN-γ ELISPOT assay revealed that B(?)51:01-reactive T lymphocytes accounted for the majority of the total alloreactive T lymphocytes in the blood just before leukemia relapse. These data suggest that immune escape of leukemia blasts from CTL pressure toward a certain HLA molecule can lead to clinical relapse after bone marrow transplantation.     

Determination of the cost of worker reproduction via diminished life span in the ant Diacamma sp

Workers of social Hymenoptera can usually produce male offspring, but rarely do so in the presence of a queen despite the potential individual fitness benefit. Various mechanisms have been hypothesized to regulate worker reproduction, including avoiding the colony-level cost of worker reproduction. However, firm quantitative evidence is lacking to support that hypothesis. Here, we accurately quantified this cost by studying an ant species (Diacamma sp.) in which worker reproduction is rare in the presence of the gamergate (the functional queen). A series of experiments to manipulate worker-gamergate contact revealed that short-term brood-production efficiency is not changed by the presence of worker reproduction. However, when workers reproduce, their average life span is reduced to between 74% and 88% of that in the absence of reproduction, indicating a long-term cost to the colony. In theory, this cost can explain the policing of worker reproduction under a queen-single mating system, but the cost does not appear to be high enough to stop worker reproduction. When contact with the gamergate is lost, it is only the nonreproductive workers whose life span was reduced; the reproductive workers lived as long as nonorphaned workers. We suggest that an increased workload can account for the reduction in life span better than a trade-off between reproduction and longevity.