Lotus japonicus CASTOR and POLLUX Are Ion Channels Essential for Perinuclear Calcium Spiking in Legume Root Endosymbiosis

The mechanism underlying perinuclear calcium spiking induced during legume root endosymbioses is largely unknown. Lotus japonicus symbiosis-defective castor and pollux mutants are impaired in perinuclear calcium spiking. Homology modeling suggested that the related proteins CASTOR and POLLUX might be ion channels. Here, we show that CASTOR and POLLUX form two independent homocomplexes in planta. CASTOR reconstituted in planar lipid bilayers exhibited ion channel activity, and the channel characteristics were altered in a symbiosis-defective mutant carrying an amino acid replacement close to the selectivity filter. Permeability ratio determination and competition experiments reveled a weak preference of CASTOR for cations such as potassium over anions. POLLUX has an identical selectivity filter region and complemented a potassium transport–deficient yeast mutant, suggesting that POLLUX is also a potassium-permeable channel. Immunogold labeling localized the endogenous CASTOR protein to the nuclear envelope of Lotus root cells. Our data are consistent with a role of CASTOR and POLLUX in modulating the nuclear envelope membrane potential. They could either trigger the opening of calcium release channels or compensate the charge release during the calcium efflux as counter ion channels.     

Effects of peroxisome proliferator-activated receptor α (PPARα) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells

Effect of peroxisome proliferator-activated receptor α (PPARα) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.     

Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1

DNA methyltransferase 1 (Dnmt1) is an enzyme that recognizes and methylates hemimethylated CpG after DNA replication to maintain methylation patterns. Although the N-terminal region of Dnmt1 is known to interact with various proteins, such as methyl-CpG-binding protein 2 (MeCP2), the associations of protein kinases with this region have not been reported. In the present study, we found that a 110-kDa protein kinase in mouse brain could bind to the N-terminal domain of Dnmt1. This 110-kDa kinase was identified as cyclin-dependent kinase-like 5 (CDKL5) by LC-MS/MS analysis. CDKL5 and Dnmt1 were found to colocalize in nuclei and appeared to interact with each other. Catalytically active CDKL5, CDKL5(1-352), phosphorylated the N-terminal region of Dnmt1 in the presence of DNA. Considering that defects in the MeCP2 or CDKL5 genes cause Rett syndrome, we propose that the interaction between Dnmt1 and CDKL5 may contribute to the pathogenic processes of Rett syndrome.     

Synthesis and characterization of new piperazine-type inhibitors for mitochondrial NADH-ubiquinone oxidoreductase (complex I)

The mode of action of Deltalac-acetogenins, strong inhibitors of bovine heart mitochondrial complex I, is different from that of traditional inhibitors such as rotenone and piericidin A [Murai, M., et al. (2007) Biochemistry 46 , 6409-6416]. As further exploration of these unique inhibitors might provide new insights into the terminal electron transfer step of complex I, we drastically modified the structure of Deltalac-acetogenins and characterized their inhibitory action. In particular, on the basis of structural similarity between the bis-THF and the piperazine rings, we here synthesized a series of piperazine derivatives. Some of the derivatives exhibited very potent inhibition at nanomolar levels. The hydrophobicity of the side chains and their balance were important structural factors for the inhibition, as is the case for the original Deltalac-acetogenins. However, unlike in the case of the original Deltalac-acetogenins, (i) the presence of two hydroxy groups is not crucial for the activity, (ii) the level of superoxide production induced by the piperazines is relatively high, (iii) the inhibitory potency for the reverse electron transfer is remarkably weaker than that for the forward event, and (iv) the piperazines efficiently suppressed the specific binding of a photoaffinity probe of natural-type acetogenins ([ (125)I]TDA) to the ND1 subunit. We therefore conclude that the action mechanism of the piperazine series differs from that of the original Deltalac-acetogenins. The photoaffinity labeling study using a newly synthesized photoreactive piperazine ([ (125)I]AFP) revealed that this compound binds to the 49 kDa subunit and an unidentified subunit, not ND1, with a frequency of approximately 1:3. A variety of traditional complex I inhibitors as well as Deltalac-acetogenins suppressed the specific binding of [ (125)I]AFP to the subunits. The apparent competitive behavior of inhibitors that seem to bind to different sites may be due to structural changes at the binding site, rather than occupying the same site. The meaning of the occurrence of diverse inhibitors exhibiting different mechanisms of action is discussed in light of the functionality of the membrane arm of complex I.     

Overexpression of poly(ADP-ribose) polymerase disrupts organization of cytoskeletal F-actin and tissue polarity in Drosophila.

Poly(ADP-ribose) polymerase (PARP) may play important roles in nuclear events such as cell cycle, cell proliferation, and maintenance of chromosomal stability. However, the exact biological role played by PARP or how PARP is involved in these cellular functions is still unclear. To elucidate the biological functions of PARP in vivo, we have constructed transgenic flies that overexpress Drosophila PARP in the developing eye primordia. These flies showed mild roughening of the normally smooth ommatidial lattice and tissue polarity disruption caused by improper rotation and chirality of the ommatidia. To clarify how this phenotypical change was induced, here we analyzed transgenic flies overexpressing PARP in the developing eye, embryo, and adult in detail. PARP mRNA level and the phenotype were enhanced in flies carrying more copies of the transgene. Developing eyes from third instar larvae were analyzed by using the neural cell marker to examine the involvement of PARP in cell fate. Morphological disorder of non-neuronal accessory cells was observed in PARP transgenic flies. Interestingly, overexpression of PARP did not interfere with the cell cycle or apoptosis, but it did disrupt the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupted organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Because these phenotypes closely resembled mutants or transgenic flies of the tissue polarity genes, genetic interaction of PARP with known tissue polarity genes was examined. Transgenic flies expressing either PARP or RhoA GTPase in the eye were crossed, and co-expression of PARP suppressed the effect of RhoA GTPase. Our results indicate that PARP may play a role in cytoskeletal or cytoplasmic events in developmental processes of Drosophila.     

A new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the alderfly Sialis melania (Neuroptera: Sialidae) from Japan,with comments on its phylogeny and male wing polymorphism

A new species of Trichogramma that parasitizes Sialis melania eggs is described as Trichogramma tajimaense Yashiro, Hirose and Honda, sp. nov. from Japan. Its phylogenetic position is based on a DNA‐based analysis, and data regarding its male wing polymorphism are also presented. The view that T. tajimaense is closely related to T. semblidis, another parasitoid of Sialis eggs, is supported by the results of a phylogenetic analysis, as well as by the biological and morphological similarities between both species. Trichogramma tajimaense is also similar in male wing polymorphism to T. kurosuae, a gregarious egg parasitoid of the lepidopteran Ivela auripes, as both Trichogramma species exhibit male wing trimorphism (fully alate, brachypterous and apterous forms) in contrast to the male wing dimorphism (fully alate and apterous forms) of T. semblidis. However, no phylogenetic analysis reveals a close relationship between T. tajimaense and T. kurosuae, and a difference exists between these two species in the mean percentage of flightless (brachypterous and apterous) males that emerge from a host egg mass; 96% of T. tajimaense males are incapable of flight, whereas about 50% of T. kurosuae males are flightless. Because all or almost all males of T. semblidis parasitizing Sialis eggs are apterous, T. tajimaense is more similar to T. semblidis than to T. kurosuae in the proportion of flightless males. In addition, male wing polymorphisms of Trichogramma in relation to mating systems could also show a similarity between T. tajimaense and T. semblidis when considering both species as quasi‐gregarious parasitoids of Sialis eggs.     

The contribution of the asymmetric alpha 1beta 1 half-oxygenated intermediate to human hemoglobin cooperativity.

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O(2)] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O(2))beta(Fe-O(2))][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O(2) binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O(2) equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O(2)-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)]( 2) and [alpha(Zn)beta(Fe)]( 2) were measured by using an Imai apparatus. It was shown that the first and second O(2) equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg(-1), respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O(2) binding study.     

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION? sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.     

Allopatric speciation of Meteterakis (Heterakoidea: Heterakidae), a highly dispersible parasitic nematode,in the East Asian islands

To clarify how the species diversity of highly dispersible parasites has developed, molecular phylogenetic analyses of Meteterakis spp., multi-host endoparasitic nematodes of reptiles and amphibians from the East Asian islands, were conducted. The results demonstrated the existence of two major clades, the J- and A-groups, with exclusive geographic ranges that are discordant with the host faunal province. However, diversification within the J-group was concordant with the host biogeography and suggested co-divergence of this group with vicariance of the host fauna. In contrast, the phylogenetic pattern within the A-group was discordant with host biogeography and implied diversification by repeated colonization. In addition, the mosaic distribution pattern of a J-group and an A-group species in the Japanese Archipelago, along with comparison of population genetic parameters and the genetic distance from their closest relatives, suggested the initial occurrence of a J-group lineage followed by exclusion in the western part of this region caused by invasion of an A-group lineage. Thus, the present study suggested that the species diversity of highly dispersible parasites including Meteterakis is formed not only by co-divergence with host faunal vicariance but also by peripatric speciation and exclusive interactions between species.     

Association of lecithin-cholesterol acyltransferase activity and low-density lipoprotein heterogeneity with atherosclerotic cardiovascular disease risk: a longitudinal pilot study

Background

Lecithin-cholesterol acyltransferase (LCAT) is believed to be involved in reverse cholesterol transport, which is known to play a key role in suppression of atherosclerosis. However, recent investigations have demonstrated that higher LCAT activity, measured in terms of the serum cholesterol esterification rate by an endogenous substrate method, is associated with increased formation of triglyceride (TG)-rich lipoproteins (TRLs), leading to a decrease in the low-density lipoprotein (LDL) particle size. The purpose of this hospital-based longitudinal study was to clarify the causal relationship between changes in the LCAT activity and changes in the LDL-particle size.

Methods

The subjects were a total of 335 patients, derived from our previous study cohort, with one or more risk factors for atherosclerotic cardiovascular disease (ASCVD). For this study, we measured the LDL-particle size (relative LDL migration [LDL-Rm value]) by polyacrylamide gel electrophoresis in the subjects, along with the changes in the LCAT activity, at the end of a follow-up period of at least 1 year.

Results

The results revealed that the absolute change (Δ) in the LDL-particle size increased significantly as the quartile of Δ LCAT activity increased (p =?0.01). A multi-logistic regression adjusted-analysis revealed that Δ LCAT activity in the fourth quartile as compared to that in the first quartile was independently predictive of an increased LDL-particle size (odds ratio [95% confidence interval]: 2.03 [1.02/4.04], p =?0.04). Moreover, the ? LCAT activity was also positively correlated with ? TRL-related markers (i.e., TG, remnant particle-like cholesterol [RLP-C], apolipoprotein B, apolipoprotein C-2, and apolipoprotein C-3).

Conclusions

The results lend support to the hypothesis that increased LCAT activity may be associated with increased formation of TRLs, leading to a reduction in the LDL-particle size in patients at a high risk for ASCVD. To reduce the risk of ASCVD, it may be important to focus not only on the quantitative changes in the serum LDL-cholesterol levels, but also on the LCAT activity.

Trial registration

UMIN (https://upload.umin.ac.jp/cgi-bin/ctr/ctr_reg_list.cgi) Study ID: UMIN000033228 retrospectively registered 2 July 2018.