Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.     

New approach for the establishment of an hepatocyte cell line derived from rat early embryonic stem cells

A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and 1 ng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a bio-hybrid typed liver apparatus.     

Characterization of histone H2A.X expression in testis and specific labeling of germ cells at the commitment stage of meiosis with histone H2A.X promoter-enhanced green fluorescent protein transgene

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the -600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.     

Loss of NECROTIC SPOTTED LESIONS 1 associates with cell death and defense responses in Arabidopsis thaliana

We isolated a lesion mimic mutant, n ecrotic s potted l esions 1 (nsl1), from Ds-tagged Arabidopsis thaliana accession No-0. The nsl1 mutant exhibits a growth retardation phenotype and develops spotted necrotic lesions on its rosette and cauline leaves. These phenotypes occur in the absence of pathogens indicating that nsl1 mutants may constitutively express defense responses. Consistent with this idea, nsl1 accumulates high levels of callose and autofluorescent phenolic compounds localized to the necrotic lesions. Furthermore RNA gel blot analysis revealed that genes associated with disease resistance activation are upregulated in the nsl1 mutants and these plants contain elevated levels of salicylic acid (SA). Crossing nsl1 with an SA deficient mutant, eds16-1, revealed that the nsl1 lesions and growth retardation are dependent upon SA. The nsl1 phenotypes are not suppressed under either the rar1-10 or sgt1b-1 genetic background. NSL1 encodes a novel 612aa protein which contains a membrane-attack complex/perforin (MACPF) domain, which is conserved in bacteria, fungi, mammals and plants. The possible modes of action of NSL1 protein in negative regulation of cell death programs and defense responses are discussed.     

Isoproterenol directs hair follicle-associated pluripotent (HAP) stem cells to differentiate in vitro to cardiac muscle cells which can be induced to form beating heart-muscle tissue sheets

Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of the follicle. Previous studies have shown that HAP stem cells can differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. HAP stem cells effected nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. The differentiation potential to cardiac muscle cells was greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol. In the present study, we observed that isoproterenol directs HAP stem cells to differentiate to cardiac muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. These results demonstrate that HAP stem cells have great potential to form beating cardiac muscle cells in tissue sheets.     

Rare sugar d-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats

A rare sugar, d-psicose has progressively been evaluated as a unique metabolic regulator of glucose and lipid metabolism, and thus represents a promising compound for the treatment of type 2 diabetes mellitus (T2DM). The present study was undertaken to examine the underlying effector organs of d-psicose in lowering blood glucose and abdominal fat by exploiting a T2DM rat model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats were fed 5% d-psicose or 5% d-glucose supplemented in drinking water, and only water in the control for 13 weeks and the protective effects were compared. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. After 13 weeks feeding, d-psicose treatment significantly reduced the increase in body weight and abdominal fat mass. Oral glucose tolerance test (OGTT) showed the reduced blood glucose and insulin levels suggesting the improvement of insulin resistance in OLETF rats. Oil-red-O staining elucidated that d-psicose significantly reduced lipid accumulation in the liver. Immunohistochemical analysis showed d-psicose induced glucokinase translocation from nucleus to cytoplasm of the liver which enhances glucokinase activity and subsequent synthesis of glycogen in the liver. d-psicose also protected the pathological change of the β-cells of pancreatic islets. These data demonstrate that d-psicose controls blood glucose levels by reducing lipotoxicity in liver and by preserving pancreatic β-cell function.     

Dimeric Coiled-coil Structure of Saccharomyces cerevisiae Atg16 and Its Functional Significance in Autophagy

Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5·Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 Å resolution and its CCD at 2.5 Å resolution. The CCD and full-length Atg16 each exhibit an extended α-helix, 90 and 130 Å, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy.     

A physiological and morphological study on the injury caused by exposure to the air pollutant,peroxyacetyl nitrate (PAN), based on the quantitative assessment of the injury

A method for the numerical assessment of the foliar injury caused by the photochemical oxidant, peroxyacetyl nitrate (PAN), was devised, using three injury indices: fresh weight (FW) loss, decreased photosynthetic pigment content, and increased ion leakage, which can be measured using the same leaves. The injury indices clearly indicated a larger number of PAN-sensitive leaves and a more severe level of injury in the PAN-sensitive variety of Petunia hybrida, White Champion (WHITE), compared to the PAN-tolerant variety, Blue Champion (BLUE). FW and photosynthetic pigment content decreased correlatively in both varieties, but ion leakage increased only in WHITE. Morphological observations revealed that ion leakage started concurrently with the start of plasmolysis-like symptoms at the mesophyll cells of injured WHITE leaves, whereas FW loss corresponded to the shrinkage of cells without loss of their round shape in BLUE leaves. PAN injuries measured by the injury indices were markedly increased in the presence of light, and the morphological changes following PAN exposure were similar to those caused by the superoxide-generating chemical, paraquat. The results suggested that PAN injuries indicated by the three injury indices are all light-dependent, but are caused through several independent mechanisms.