Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross‐talk between excess zinc and iron deficiency

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.     

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.     

Variation of hepatic methotrexate 7-hydroxylase activity in animals and humans

This study deals with individual and species variations in the converting activity of methotrexate (MTX) to 7-hydroxymethotrexate in animals and humans. When MTX 7-hydroxylase was assayed in six human liver cytosols, a 48-fold range of intersubject variation of the activity was observed. The variations were correlated to the concentrations of aldehyde oxidase activity in human subjects assayed with benzaldehyde as a substrate. Species differences of liver MTX 7-hydroxylase activity were also observed. The activity was highest in rabbits, followed by rats, hamsters, and monkeys but was undetectable in dogs. Strain differences of MTX 7-hydroxylase activity based on aldehyde oxidase activity were also observed in rats and mice. The results suggest that aldehyde oxidase functions as MTX 7-hydroxylase in livers of animals and humans, and the observed differences of MTX 7-hydroxylase activity are due to variations in the amount of aldehyde oxidase present.     

Heterostyly: speciation within a species

Almost all organisms in nature show nonrandom mating to different degrees. Two extreme results of nonrandom mating are speciation and sexual differentiation. Heterostyly is a form of sexual differentiation considered to have evolved to resolve conflicts between male and female functions of hermaphrodite flowers. Our study examines necessary and sufficient conditions for establishment of heterostyly using a configuration individual-based model. Previous models assume invasion of a mutant phenotype into a population with monomorphic wild phenotype. In contrast, our model demonstrates that heterostyly can be established from a population with continuous phenotypic variation, a proposition that requires simpler assumptions than the previous hypotheses. Results of our simulation show that genetic linkage between stigma and anther heights is essential for establishment of heterostyly. Dominance effects on the genes for stamen or stigma heights are not necessary, but they promote evolution of heterostyly. Probability of evolution of heterostyly also depends on the functional relationship between stigma–anther distance and strength of sexual interference, and the distance and probability of pollen deposition success. Parallelity and difference between speciation and sexual differentiation are also discussed.     

Molecular isolation and characterization of novel four subisoforms of ECE-2

Although the four polypeptides of blasticidin S (BS) deaminase (BSD) are packed rather tightly coordinated to the "structural and catalytic" zinc atom of each subunit, the C-terminal region of the enzyme was suggested to be somewhat molten and flexible [M. Kimura, S. Sekido, Y. Isogai, and I. Yamaguchi (2000) J. Biochem. 127, 955-963]. To understand roles of this flexible region, we constructed five C-terminal deletion variants of BSD (each successively deleted from the C-terminal end up to five residues) and analyzed their biochemical properties focusing on the structure and activity of the enzyme. BSD and all of the deletion mutants showed the unique rigid conformation (e.g., characterized by their stabilities in SDS solution) and high levels of resistance against protease digestions. Furthermore, both the wild-type and deletion apoenzymes exhibited similar physical properties in thermodynamic refolding into the stable tetramer conformation. However, these small C-terminal deletions exerted deleterious effects on the catalytic efficiency of the enzyme as indicated by their strongly reduced k(cat)/K(m) value. Judging from the altered kinetic parameters and unaltered structural properties of the deletion variants, these C-terminal residues appear to be directly involved in enzyme-substrate interaction. In this short flexible region, Tyr-126, Trp-128, and Gly-130 were the key residues. Most notably, removal of Gly-130 markedly increased K(m) for BS without affecting its k(cat) value. These results indicate that the flexible C-terminal region is important for catalytic function and that a single Gly residue at the C-terminal end of BSD contributes significantly in facilitating access of a substrate to the active site.     

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.     

Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote

Mitochondrial ribosomal RNAs (mtrRNAs) have been reported to translocate extra-mitochondrially and localize to the germ cell determinant of oocytes and zygotes in some metazoa except mammals. To address whether the mtrRNAs also localize in the mammals, expression and distribution of mitochondrion-encoded RNAs in the mouse oocytes and zygotes was examined by whole-mount in situ hybridization (ISH). Both 12S and 16S rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte. This distribution pattern was rearranged toward the second polar body in zygotes after fertilization. The amount of mtrRNAs decreased around first cleavage, remained low during second cleavage and increased after third cleavage. Staining intensity of the 12S rRNA was weaker than that of the 16S rRNA throughout the examined stages. Similar distribution dynamics of the 16S rRNA was observed in strontium-activated haploid parthenotes, suggesting the distribution rearrangement does not require a component from sperm. The distribution of 16S rRNAs did not coincide with that of mitochondrion-specific heat shock protein 70, suggesting that the mtrRNA is translocated from mitochondria. The ISH-scanning electron microscopy confirms the extra-mitochondrial mtrRNA in the mouse oocyte. Chloramphenicol (CP) treatment of late pronuclear stage zygotes perturbed first cleavage as judged by the greater than normal disparity in size of blastomeres of 2-cell conceptuses. Two-third of the CP-treated zygotes arrested at either 2-cell or 3-cell stage even after the CP was washed out. These findings indicate that the extra-mitochondrial mtrRNAs are localized in the mouse oocyte and implicated in correct cytoplasmic segregation into blastomeres through cleavages of the zygote.     

A simple method of intestinal anastomosis (ileocolostomy) in rats.

A simple method of ileocolostomy was performed in rats. The colon near the cecum was ligated, including its arteries and veins. Main artery and vein of the cecum were ligated. Then, the cecum was cut out. A longitudinal incision was made on the anti-mesenteric side of the proximal end of the colon, approximately 7-8 mm long. A 21-G needle was inserted toward the incision 2 cm away from the proximal end of the anti-mesenteric side of the colon. A nylon suture was knotted once to the distal end of the ileum and was introduced into the tip of the needle which had previously been passed through the colon. Then, the needle was removed. The suture was pulled to introduce the distal end of the ileum into the colonic lumen. Then, the suture was knotted once on the colon again to fix the ileum to the colon. The incision in the proximal end of the colon was not closed. At the 2nd week after the operation, X-ray examinations demonstrated that the ileocolonic passages with no leakage at the anastomotic site were quite satisfactory. At the 4th week after the operation, there were no macroscopic or microscopic complications at the anastomotic site. The mucosal and serosal epithelia of the ileum and colon continued smoothly. This simple method may be very effective in preparing anastomosis in the gastrointestinal tract, especially in small laboratory animals for nutritional and surgical experiments.     

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.