Nuclear sex-determining genes cause large sex-ratio variation in the apple snail Pomacea canaliculata

Evolutionary maintenance of genetic sex-ratio variation is enigmatic since genes for biased sex ratios are disadvantageous in finite populations (the "Verner effect"). However, such variation could be maintained if a small number of nuclear sex-determining genes were responsible, although this has not been fully demonstrated experimentally. Brood sex ratios of the freshwater snail Pomacea canaliculata are highly variable among parents, but population sex ratios are near unity. In this study, the effect of each parent on the brood sex ratio was investigated by exchanging partners among mating pairs. There were positive correlations between sex ratios of half-sib broods of the common mother (r = 0.42) or of the common father (r = 0.47). Moreover, the correlation between full-sib broods was very high (r = 0.92). Thus, both parents contributed equally to the sex-ratio variation, which indicates that nuclear genes are involved and their effects are additive. Since the half-sib correlations were much stronger than the parent-offspring regressions previously obtained, the variation was caused by zygotic sex-determining genes rather than by parental sex-ratio genes. The number of relevant genes appears to be small.     

Construction of a human immunodeficiency virus type 1 (HIV-1) library containing random combinations of amino acid substitutions in the HIV-1 protease due to resistance by protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 x 10(5)) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 microM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.     

Occurrence and reproduction of tropical fishes in ocean warming hotspots of Japanese temperate reefs

Reproduction of tropical species beyond their geographic range associated with ocean warming is regarded as the key indicator of a range shift. However, the lack of historical breeding records poses challenges for detecting distinct range shifts of tropical fishes. To obtain baseline data of the current status of the occurrence and breeding activity of tropical pomacentrid and apogonid fishes in ocean warming hotspots of temperate reefs (Kochi and Wakayama, 33°N) of Japan, we conducted a two-year underwater visual survey and synthesized those data with recently published information. By combining data from the present as well as past studies, the results confirmed the occurrence of 52 pomacentrid and 34 apogonid species, whereas breeding activity was confirmed for 19 and 16 species, respectively. Species richness and abundance of recruitment periphery and breeding active species were high at the warmer site adjacent to the Kuroshio Current. Most observed species were found beyond their known geographic range. Some species showing active breeding were widespread tropical fishes (e.g., Amphiprion clarkii, Pomacentrus coelestis and Apogon notatus) and probably have established breeding populations irrespective of recent global warming. The winter sea water temperature around the study sites will continue to rise, increasing by >2 °C by the end of the century; therefore, our results are highly relevant and represent the first step to elucidate the potential range extension of tropical fishes into temperate reefs with climate change.     

Structure-based screening for functional non-coding RNAs in fission yeast identifies a factor repressing untimely initiation of sexual differentiation

Non-coding RNAs (ncRNAs) ubiquitously exist in normal and cancer cells. Despite their prevalent distribution, the functions of most long ncRNAs remain uncharacterized. The fission yeast Schizosaccharomyces pombe expresses >1800 ncRNAs annotated to date, but most unconventional ncRNAs (excluding tRNA, rRNA, snRNA and snoRNA) remain uncharacterized. To discover the functional ncRNAs, here we performed a combinatory screening of computational and biological tests. First, all S. pombe ncRNAs were screened in silico for those showing conservation in sequence as well as in secondary structure with ncRNAs in closely related species. Almost a half of the 151 selected conserved ncRNA genes were uncharacterized. Twelve ncRNA genes that did not overlap with protein-coding sequences were next chosen for biological screening that examines defects in growth or sexual differentiation, as well as sensitivities to drugs and stresses. Finally, we highlighted an ncRNA transcribed from SPNCRNA.1669, which inhibited untimely initiation of sexual differentiation. A domain that was predicted as conserved secondary structure by the computational operations was essential for the ncRNA to function. Thus, this study demonstrates that in silico selection focusing on conservation of the secondary structure over species is a powerful method to pinpoint novel functional ncRNAs.     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.     

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Ig-like receptors in human NK cells

The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.     

Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of ferryl intermediate

Hydrogen peroxide, one of the potent oxidants in muscle tissues, can induce very rapid oxidation of oxymyoglobin (MbO2) to metmyoglobin (metMb) with an apparent rate constant of 7.5 X 10(4) h-1 M-1 (i.e., 20.8 s-1 M-1) over the wide pH range of 5.5-10.2 in 0.1 M buffer at 25 degrees C. Its molecular mechanism, however, is quite different from that of the autoxidation of MbO2 to metMb. Kinetic analysis has revealed that the hydrogen peroxide oxidation proceeds through the formation of ferryl-Mb(IV) from deoxy-Mb(II), which is in equilibrium with MbO2, by a two-equivalent oxidation with H2O2. Once the ferryl species is formed, it reacts rapidly with another deoxy-Mb(II) in a bimolecular fashion so as to yield 2 mol of metMb(III). Under physiological conditions, the rate-determining step was the oxidation of the deoxy species by H2O2, its rate constant being estimated to be on the order of 3.6 X 10(3) s-1 M-1 at 25 degrees C. These findings leads us to the view that a good supply of dioxygen provides rather an important defense against the oxidation of myoglobin with hydrogen peroxide in cardiac and skeletal muscle tissues.     

Characterization of two β-decarboxylating dehydrogenases from <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg²⁺, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.