Novel receptor-like kinase ALE2 controls shoot development by specifying epidermis in Arabidopsis

The epidermis plays crucial roles in the development of various organs and in water retention in both animals and plants. In Arabidopsis thaliana, the subtilase ABNORMAL LEAF SHAPE 1 (ALE1) and the Arabidopsis homolog of the Crinkly4 (ACR4) receptor-like protein kinase (RLK) have been implicated in the intercellular communication that is required for surface functions of the epidermis. We have identified a novel mutant gene in Arabidopsis, ale2, which is associated with various epidermal defects, including disorganization of epidermis-related tissues, defects in the leaf cuticle and the fusion of organs. ALE2 encodes a previously uncharacterized RLK with a cluster of basic amino acid residues followed by a cysteine-containing sequence in the putative extracellular domain. Our genetic investigations suggest that ALE2 and ACR4 function in the same process, whereas ALE1 has a different mode of action, and that these three genes play partially overlapping roles in positively regulating protoderm-specific gene expression and for the formation of leafy organs. We propose that at least two modes of intercellular communication facilitate the specification of epidermis, thereby promoting shoot organogenesis in Arabidopsis.     

Replication of IMR‐32‐adapted JC virus clones in human embryonic kidney cells

It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT‐PCR assay revealed that large T antigen expression in cells transfected with IMR‐32‐adapted JCVs was significantly greater than in those transfected with Mad‐1 or CY. DNA replication assay and viral load verified that the IMR‐32‐adapted JCVs were replication‐competent in 293 cells, but not Mad‐1 or CY JCVs. These results suggest that a 293 culture system with IMR‐32‐adapted JCVs may be a useful tool for assessing replication of JCV in vitro.     

CPT11 prevents virus replication in JCI cells persistently infected with JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of the demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML), which occurs in immunocompromised patients. Moreover, patients treated with natalizumab for multiple sclerosis or Crohn disease can develop PML, which is then termed natalizumab‐related PML. Because few drugs are currently available for treating PML, many antiviral agents are being investigated. It has been demonstrated that the topoisomerase I inhibitors topotecan and β‐lapachone have inhibitory effects on JCPyV replication in IMR‐32 cells. However, both of these drugs have marginal inhibitory effects on virus propagation in JC1 cells according to RT‐PCR analysis. In the present study, the inhibitory effect of another topoisomerase I inhibitor, 7‐ethy‐10‐[4‐(1‐piperidino)‐1‐piperidino] carbonyloxy camptothecin (CPT11), was assessed by investigating viral replication, propagation, and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using real‐time PCR combined with Dpn I treatment in IMR‐32 cells transfected with JCPyV DNA. It was found that JCPyV replicates less in IMR‐32 cells treated with CPT11 than in untreated cells. Moreover, CPT11 treatment of JCI cells persistently infected with JCPyV led to a dose‐dependent reduction in JCPyV DNA and VP1 production. Additionally, the inhibitory effect of CPT11 was found to be stronger than those of topotecan and β‐lapachone. These findings suggest that CPT11 may be a potential anti‐JCPyV agent that could be used to treat PML.

     

Associations between cardiorespiratory fitness and lifestyle-related factors with DNA methylation-based ageing clocks in older men: WASEDA'S Health Study

DNA methylation-based age estimators (DNAm ageing clocks) are currently one of the most promising biomarkers for predicting biological age. However, the relationships between cardiorespiratory fitness (CRF), measured directly by expiratory gas analysis, and DNAm ageing clocks are largely unknown. We investigated the relationships between CRF and the age-adjusted value from the residuals of the regression of DNAm ageing clock to chronological age (DNAmAgeAcceleration: DNAmAgeAccel) and attempted to determine the relative contribution of CRF to DNAmAgeAccel in the presence of other lifestyle factors. DNA samples from 144 Japanese men aged 65–72 years were used to appraise first- (i.e., DNAmHorvath and DNAmHannum) and second- (i.e., DNAmPhenoAge, DNAmGrimAge, and DNAmFitAge) generation DNAm ageing clocks. Various surveys and measurements were conducted, including physical fitness, body composition, blood biochemical parameters, nutrient intake, smoking, alcohol consumption, disease status, sleep status, and chronotype. Both oxygen uptake at ventilatory threshold (VO₂/kg at VT) and peak oxygen uptake (VO₂/kg at Peak) showed a significant negative correlation with GrimAgeAccel, even after adjustments for chronological age and smoking and drinking status. Notably, VO₂/kg at VT and VO₂/kg at Peak above the reference value were also associated with delayed GrimAgeAccel. Multiple regression analysis showed that calf circumference, serum triglyceride, carbohydrate intake, and smoking status, rather than CRF, contributed more to GrimAgeAccel and FitAgeAccel. In conclusion, although the contribution of CRF to GrimAgeAccel and FitAgeAccel is relatively low compared to lifestyle-related factors such as smoking, the results suggest that the maintenance of CRF is associated with delayed biological ageing in older men.     

Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution

We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.     

Mechanisms of crown gall formation: T-DNA transfer fromAgrobacterium tumefaciens to plant cells

Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. WhenAgrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circulation of T-DNA and expression ofvir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds excreted from the protoplasts. The circularization and formation of crown gall both require the expression of thevirD locus, one of the induciblevir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection byAgrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression ofvir genes, while they contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place whenAgrobacterium invades seedlings and seeds of monocotyledonous plants. Recipient of the Botanical Society Award for Young Scientists, 1987.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Two Types of Clathrin-Coated Vesicles Isolated from Rat Brain: Analysis of Biochemical Properties and Cellular Origin

Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.