Cell-lytic activity of tobacco BY-2 induced by a fungal elicitor from alternaria alternata attributed to the expression of a class I beta-1,3-glucanase gene

Stress-induced cell-lytic activity was found in tobacco BY-2 cells treated with various stresses. Among 14 stresses, an elicitor fraction isolated from Alternaria alternata showed the highest inducing activity. Cell-lytic activity increased for 72 h even in the control sample, treated with distilled water, and several isozymes of beta-1,3-glucanases and chitinases were found to be involved in it. In contrast, cell-lytic activity in BY-2 cells treated with a fungal elicitor reached a higher level after 60 h. The principal enzymes specifically involved in this stress-induced portion are speculated to be basic beta-1,3-glucanases. A class I beta-1,3-glucanase gene (glu1) was found to be the specific gene for the stress-induced cell-lytic activity. Its expression became observable at 24 h, and the intensity reached a maximum at about 60-72 h. The glu1 was thus assigned as a late gene. Its role in the stress response is discussed in conjunction with earlier genes such as chitinases.     

Changes of C-reactive protein levels in rainbow trout (Oncorhynchus mykiss) sera after exposure to anti-ectoparasitic chemicals used in aquaculture

Changes of serum C-reactive protein (CRP) levels in rainbow trout (Oncorhynchus mykiss) were studied after exposure to formalin, metriphonate or potassium permanganate, which are used in aquaculture as anti-ectoparasitic chemicals. The CRP level in normal trout sera is 88+/-5 microg ml(-1) according to sandwich enzyme-linked immunosorbent assay. CRP levels increased to a maximum at six or nine days after exposure to formalin for 3.5 h at 300 ppm or 9.5 h at 30 ppm, respectively; these levels are 4.3 and 18 times higher than normal. At 18 days after treatment, the CRP level had decreased to significantly below the normal level. After exposure to metriphonate (0.4 ppm for 30 min), the CRP level increased significantly to a maximum at three days after exposure (9.9 times higher than normal), then decreased to below normal. With exposure to potassium permanganate at 40 ppm for 45 min, fish showed significantly lower CRP levels than the normal level at 14 days after exposure. Fish reared at a water temperature of 16.5-19.5 degrees C showed significantly higher CRP levels than those reared at 13 degrees C. Measurement of CRP levels in trout serum can be used as a bioindicator of the health condition of the fish.     

Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs

Plant aquaporins occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We cloned cDNAs for plasma-membrane aquaporins (PAQ1, 1b, 1c, 2, 2b, and 2c) of radish (Raphanus sativus L.). The amino acid sequences of the PAQs showed on average 63% sequence identity. Their sequences were 23% identical to those of tonoplast aquaporins (γ- and δ-VM23). A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In other organs, the mRNA level of each isoform varied according to the organ. In petals, stamens, pistils and sepals of flowers, the levels of PAQ1, 1b, 1c and γ-VM23 mRNAs were high, and mRNAs of all aquaporins except for δ-VM23 were detected at high levels in pericarps. The protein levels of aquaporins on the basis of the membrane protein were determined by immunoblotting. Proteins PAQ1 and VM23 were detected in every organ except for the mature petiole. The PAQ2 protein level was especially high in green cotyledons and leaves, but was extremely low in seedling cotyledons and hypocotyls. Proteins PAQ1, PAQ2 and VM23 were highly accumulated in growing pericarps, but not in the immature seeds. These results indicate that the gene expression of the aquaporin isoforms was individually regulated in an organ- and tissue-specific manner, and that the amounts of aquaporin protein, especially PAQ2, are regulated in certain tissues at the translational level and by the rate of protein turnover. Received: 10 February 2000 / Accepted: 30 June 2000     

Systemic administration of HVJ viral coat-liposome complex containing human insulin vector decreases glucose level in diabetic mouse: A model of gene therapy

In this study, we examined the feasibility of a systemic administration of HVJ-liposome complex containing human insulin construct into the blood in mice via the tail vein. Transfection of human insulin vector resulted in a transient decrease in serum glucose in streptozotocin (SZT)-induced diabetic mice, accompanied by the detection of human insulin in the liver and spleen. In accordance with the decreased glucose, plasma immunoreactive insulin could be detected up to 14 days after a single transfection in mice transfected with insulin vector. Repeated intravenous injection of human insulin vector every week resulted in a sustained decrease in serum glucose over a 4-week period, accompanied by the detection of C-peptide fragments and a significant decrease in BUN and creatinine. Here, we demonstrated the feasibility of intravenous systemic administration of an insulin vector that results in a sustained improvement of diabetic glucose metabolism.     

Synchronization of Phosphorylation of a Chloroplast Protein with the Cell Division Cycle in Heterosigma akashiwo

Fumigation of plants of five species with NO₂ in darkness causedvisible injuries to leaves, with the most severe injuries inkidney bean plants and the least severe in spinach plants. Fumigationof these plants in the light caused virtually no visible injuries.NO₂-fumigated leaves accumulated nitrite in the darkness butnot in the light. The level of accumulated NO₂^– was decreasedby light much more rapidly in spinach leaves than in those ofkidney bean, with much less injury to spinach leaves than tothose of kidney bean. A larger amount of NO₂^– accumulatedin the trifoliate leaves of kidney bean plants cultivated withNO₃^– as a main source of nitrogen than in those of plantscultivated with NH₄⁺, and the former plants were more susceptibleto injury from NO₂ than the latter. Administration of NO₂^–to leaves of spinach and kidney bean plants induced the destructionof Chi in the light. The extent of the destruction of Chi wassmaller in spinach than in kidney bean, consistent with theirrespective responses to NO₂. The NO₂^–-induced destructionof Chi was inhibited to some extent by scavengers of free radicals.Activities of superoxide dismutase (SOD) were higher in leavesof spinach than in those of kidney bean. These results indicatethat NO₂^– is the toxic species generated by fumigationwith NO₂ and that spinach has a greater tolerance for NO₂ thankidney bean, probably as a result both of a higher capacityfor reduction of NO₂^– and a higher level of activity ofSOD. (Received October 4, 1991; Accepted January 31, 1992)     

Five isoforms of the phosphatidylinositol 3-kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations

There are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3-kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO-PDGFR cells, the associations of these regulatory subunit isoforms with the platelet-derived growth factor receptor were similar. However, when overexpressed in CHO-IR cells, p55gamma exhibited a significantly lower ability to bind with IRS-1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55alpha and p55gamma were found to be tyrosine-phosphorylated. Finally, interestingly, when overexpressed in CHO-EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85alpha, p85beta and p50alpha immunoprecipitates, but not in p55alpha and p55gamma immunoprecipitates. In addition, EGF-induced tyrosine phosphorylation was observed in p85alpha, p85beta, p55alpha and p55gamma, but not in p50alpha, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine-phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor.     

Granulogenesis in non-neuroendocrine COS-7 cells induced by EGFP-tagged chromogranin A gene transfection: identical and distinct distribution of CgA and EGFP.

We examined whether an enhanced green fluorescent protein (EGFP)-tagged chromogranin A (CgA) gene construct could serve as a marker protein to follow the synthesis of CgA and the process of granulogenesis in non-neuroendocrine (NE) cells. We transfected a CgA-EGFP expression vector into non-NE COS-7 cells and investigated the localization of a chimeric CgA-EGFP protein using confocal laser scanning microscopy (CLSM). The fluorescent signal of CgA-EGFP was distributed granularly in the cytoplasm. An immunocytochemical study using anti-CgA antibody with a quantum dot (Qd)525 shows colocalization of fluorescent signal of chimeric CgA-EGFP and CgA-Qd525 signals in granular structures, particularly at the periphery of the cytoplasm. We interpreted granules that were immunoreactive to CgA in electron micrographs as secretory. Spectral analysis of EGFP fluorescence revealed distinct EGFP signals without CgA colocalization. This is the first report to show that a granular structure can be induced by transfecting the EGFP-tagged human CgA gene into non-NE cells. The EGFP-tagged CgA gene could be a useful tool to investigate processes of the regulatory pathway. A more precise analysis of the fluorescence signal of EGFP by combination with the Qd system or by spectral analysis with CLSM can provide insight into biological phenomena.     

RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.

Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC(male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.     

Identification of the chromosomal region responsible for high-temperature stress tolerance during the grain-filling period in rice

An unusually high temperature during the grain-filling period, such as that caused by global warming, impairs the quality of rice (Oryza sativa L.) grains. This sensitivity to high-temperature stress is different among cultivars, suggesting the possibility of developing a high-temperature-tolerant cultivar. Since marker-assisted selection would reduce time and labor in breeding for such a quantitative trait, we determined the chromosomal region responsible for high-temperature tolerance during the grain-filling period. A high-temperature-sensitive japonica cultivar Tohoku 168 and a tolerant japonica cultivar Kokoromachi were selected as the parental lines of recombinant inbred lines (RILs) by high-temperature stress treatment from 5 to 10 days after anthesis, which was found to be the period most critical for grain quality. Using the RILs, whose genotypes were determined by analysis with 131 DNA markers which were selected as polymorphic markers between these two cultivars from 2,648 DNA markers tested, the quantitative trait locus (QTL) for the percentage of white-back grains was mapped on chromosome 6. The Kokoromachi allele of the QTL, which had a positive additive effect on the high-temperature tolerance, was introduced into the Tohoku 168 genome by repeated backcrossings with marker-assisted selection. Using high-temperature stress treatment of the near isogenic lines developed, the QTL on chromosome 6 was localized within a 1.9-Mb region between two DNA markers, ktIndel001 and RFT1. These DNA markers would be useful not only for breeding high-temperature-tolerant cultivars but also for map-based cloning of the QTL.     

Induction of epidermal cell fate in Arabidopsis shoots

Land plants have evolved a cuticle-bearing epidermis to protect themselves from environmental stress and pathogen attack. Despite its important role, little is known about the molecular mechanisms regulating shoot epidermal cell identity. In a recent study, we found that the Arabidopsis thaliana ATML1 gene is possibly a master regulator of shoot epidermal cell fate. We revealed that ATML1 has the ability to confer shoot epidermis-related traits to non-epidermal cells of the seedlings. These data are consistent with the previous loss-of-function mutant analyses, which implied a positive role of ATML1 in epidermal cell differentiation. Importantly, ectopic epidermal cells induced in ATML1-overexpressing lines provide a novel tool to assess the intrinsic properties of epidermal cells and to study epistatic interactions among genes involved in epidermal/mesophyll differentiation. Using this system, we obtained data revealing that ATML1 negatively influenced mesophyll cell fate. In addition, we provided a working model of how division planes in epidermal cells are determined.