Induction of Salicylic Acid {beta}-Glucosidase in Tobacco Leaves by Exogenous Salicylic Acid

Salicylic acid (SA) has been proposed to be an endogenous signalfor systemic acquired resistance to infection by pathogens inplants. In general, most SA is found in an inactive form asSA ß-glucoside (SAG). SAG seems to be a storage formof SA from which bioactive SA can be generated. Recent reportsindicate that ß-glucosidase might be involved in regulatingthe signaling activity of phytohormones. Therefore, it seemslikely that SA ß-glucosidase, the enzyme that hydrolyzesSAG to yield free SA, might also play an important role by regulatingthe level of free SA. Since hydrolysis of SAG seems to occurin intercellular spaces, we attempted to isolate SA ß-glucosidaseactivity from the intercellular spaces of SA-treated tobaccoleaves, where we found considerable amounts of the enzymaticactivity. Furthermore, increased levels of SA and SA ß-glucosidaseactivity were found in the leaves after treatment with exogenousSA. The role of SA ß-glucosidase in plant defensesystems is discussed. (Received November 15, 1994; Accepted January 20, 1995)     

Effect of high temperature on the development of nuclear polyhedrosis virus in the silkworm,Bombyx mori

Effect of a high temperature on the development of nuclear polyhedrosis and nuclear polyhedrosis virus (NPV) was studied employing pupae and isolated pupal abdomens of the silkworm, Bombyx mori. It was shown that pupae inoculated with an NPV and incubated at 35°C survived longer than those incubated at 25°C. At lower dosages of virus, pupae at 35°C escaped death from NPV. When inoculated pupae were incubated at 35°C for varying periods and then transferred to 25°C, the longer the pupae had been kept at 35°C the longer they survived. In contrast, when inoculated pupae were transferred from 25° to 35°C, the longer the pupae had been kept at 25°C the sooner after inoculation they died. Essentially the same results were obtained in isolated abdomens which were in an arrested state of development, excluding the possibility that observed thermal inhibition of viral diseases is dependent upon the altered developmental processes at high temperatures. Virus titration experiments showed that, under experimental conditions utilized, no detectable accumulation of infectious NPV was present in abdomens inoculated with an NPV and incubated at 35°C. When inoculated abdomens were shifted up from 25° to 35°C at 3 days postinoculation, NPV accumulation was inhibited almost immediately, and when inoculated abdomens were shifted down from 35° to 25°C, infectious NPV started to accumulate as early as 1 day after the shift. It was also shown that the pattern of infectious NPV accumulation and that of nucleic acid increase in infected abdomens gave a rough correlation. These results indicate that the thermal inhibition of viral diseases is attributed, at least in part, to the restricted accumulation of infectious progeny and suggest that the virus replication mechanism itself is more sensitive to high temperatures than that related to other events necessary for viral replication to be initiated.     

Characterization of the Soybean Genome Using EST-derived Microsatellite Markers

We generated a high-density genetic linkage map of soybean usingexpressed sequence tag (EST)-derived microsatellite markers.A total of 6920 primer pairs (10.9%) were designed to amplifysimple sequence repeats (SSRs) from 63 676 publicly availablenon-redundant soybean ESTs. The polymorphism of two parent plants,the Japanese cultivar ‘Misuzudaizu’ and the Chineseline ‘Moshidou Gong 503’, were examined using 10%polyacrylamide gel electrophoresis. Primer pairs showing polymorphismwere then used for genotyping 94 recombinant inbred lines (RILs)derived from a cross between the parents. In addition to previouslyreported markers, 680 EST-derived microsatellite markers wereselected and subjected to linkage analysis. As a result, 935marker loci were mapped successfully onto 20 linkage groups,which totaled 2700.3 cM in length; 693 loci were detected usingthe 668 EST-derived microsatellite markers developed in thisstudy, the other 242 loci were detected with 105 RFLP markers,136 genome-derived microsatellite markers, and one phenotypicmarker. We examined allelic variation among 23 soybean cultivars/linesand a wild soybean line using 668 mapped EST-derived microsatellitemarkers (corresponding to 686 marker loci), in order to determinethe transferability of the markers among soybean germplasms.A limited degree of macrosynteny was observed at the segmentallevel between the genomes of soybean and the model legume Lotusjaponicus, which suggests that considerable genome shufflingoccurred after separation of the species and during establishmentof the paleopolyploid soybean genome.     

Cloning and sequencing of cDNA and genomic DNA encoding PDM phosphatase of Fusarium moniliforme

PDM phosphatase was purified approximately 500-fold through six steps from the extract of dried powder of the culture filtrate of Fusarium moniliforme. The purified preparation appeared homogeneous on SDS-PAGE although the protein band was broad. Amino acid sequence information was collected on tryptic peptides from this preparation. cDNA cloning was carried out based on the information. A full-length cDNA was obtained and sequenced. The sequence had an open reading frame of 651 amino acid residues with a molecular mass of 69,988 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA was also conducted. The deduced amino acid sequence could account for many but not all of the tryptic peptides, suggesting presence of contaminant protein(s). SDS-PAGE analysis after chemical deglycosylation showed two proteins with molecular masses of 58 and 68 kDa. This implied that the 58 kDa protein had been copurified with PDM phosphatase. Homology search showed that PDM phosphatase belongs to the purple acid phosphatase family, which is widely distributed in the biosphere. Sequence data of fungal purple acid phosphatases were collected from the database. Processing of the data revealed presence of two types, whose evolutionary relationships were discussed.     

Anoikis-resistant MDCK cells carrying susceptibilities to TNF-α and verotoxin that are suitable for influenza virus cultivation

Madin-Darby canine kidney (MDCK) cells were originally anchorage-dependent epithelial cells. Here, we have isolated a novel MDCK-derived cell population, termed 6 M-4, by means of culturing MDCK cells in suspension for nearly 6 months in the presence of Streptomyces griseus metalloendopeptidase (MEP). The isolated cells showed unique proliferation characteristics, which differed from parental MDCK cells. They proliferated adherently on a polystyrene matrix, but proliferated non-adherently both in the presence of MEP and on a non-adhesive matrix coated with poly 2-methacryloyloxyethyl phosphorylcholine (MPC). The 6 M-4 cells consisted of at least two cell types. One type, termed 6 M-4-TR7, would not grow in soft agar and showed a novel phenotype in that the cells were susceptible to both TNF-α and verotoxin 1 (VT1). In addition, the isolated adhesion-independent cells sustained epithelial traits of parental MDCK cells. We further show that these MDCK-derivative cells are suitable for influenza virus cultivation. Hemagglutination (HA) titers of influenzaviruses A and B were increased in the suspension culture of 6 M-4-TR7 cells supplemented with the MEP in comparison to adherently growing cells in the presence of trypsin.     

Mn and Cu concentrations in mixed saliva of elementary school children in relation to sex,age, and dental caries

To examine the standard Mn and Cu concentrations in mixed saliva from children and the relationship between these levels and dental caries, resting mixed saliva samples obtained from 527 children of an elementary school in Kitakyushu City were collected at 10:00–11:30 a.m. during December 2004. The Mn and Cu concentrations were determined using simultaneous multi-element atomic absorption spectrometry. The standard Mn and Cu levels were 22.0±15.2 and 3.8±4.1 ng/mL, respectively, in the sound teeth group. Mn levels were significantly higher in boys (25.4±17.4 ng/mL) than girls (19.1±12.3 ng/mL) and also higher in upper (25.5±16.4 ng/mL) than lower (19.0±13.5 ng/mL) grades. The Cu level was unaffected by sex and age in the sound teeth group. The Cu level in children with caries experience (5.7±5.3 ng/mL) was significantly higher than that of the sound teeth group. Moreover, the Cu levels in children with untreated caries were significantly higher than that of the sound teeth group, and increased with the number of untreated teeth. No significant difference was found in the Cu concentrations between the group in which all decayed teeth were treated and the sound teeth group. The Mn levels were similar with or without caries and treatment. These findings indicate that the Mn level in mixed saliva depended on sex and age, and suggest the possibility of Cu dissolving into mixed saliva by demineralization due to dental caries.     

Apoptosis of CD4+CD25high T Cells in Type 1 Diabetes May Be Partially Mediated by IL-2 Deprivation

Background

Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease targeting the insulin-producing pancreatic β cells. Naturally occurring FOXP3⁺CD4⁺CD25^high regulatory T cells (T_regs) play an important role in dominant tolerance, suppressing autoreactive CD4⁺ effector T cell activity. Previously, in both recent-onset T1D patients and β cell antibody-positive at-risk individuals, we observed increased apoptosis and decreased function of polyclonal T_regs in the periphery. Our objective here was to elucidate the genes and signaling pathways triggering apoptosis in T_regs from T1D subjects.

Principal Findings

Gene expression profiles of unstimulated T_regs from recent-onset T1D (n = 12) and healthy control subjects (n = 15) were generated. Statistical analysis was performed using a Bayesian approach that is highly efficient in determining differentially expressed genes with low number of replicate samples in each of the two phenotypic groups. Microarray analysis showed that several cytokine/chemokine receptor genes, HLA genes, GIMAP family genes and cell adhesion genes were downregulated in T_regs from T1D subjects, relative to control subjects. Several downstream target genes of the AKT and p53 pathways were also upregulated in T1D subjects, relative to controls. Further, expression signatures and increased apoptosis in T_regs from T1D subjects partially mirrored the response of healthy T_regs under conditions of IL-2 deprivation. CD4⁺ effector T-cells from T1D subjects showed a marked reduction in IL-2 secretion. This could indicate that prior to and during the onset of disease, T_regs in T1D may be caught up in a relatively deficient cytokine milieu.

Conclusions

In summary, expression signatures in T_regs from T1D subjects reflect a cellular response that leads to increased sensitivity to apoptosis, partially due to cytokine deprivation. Further characterization of these signaling cascades should enable the detection of genes that can be targeted for restoring T_reg function in subjects predisposed to T1D.     

High-density Integrated Linkage Map Based on SSR Markers in Soybean

A well-saturated molecular linkage map is a prerequisite for modern plant breeding. Several genetic maps have been developed for soybean with various types of molecular markers. Simple sequence repeats (SSRs) are single-locus markers with high allelic variation and are widely applicable to different genotypes. We have now mapped 1810 SSR or sequence-tagged site markers in one or more of three recombinant inbred populations of soybean (the US cultivar ‘Jack’ × the Japanese cultivar ‘Fukuyutaka’, the Chinese cultivar ‘Peking’ × the Japanese cultivar ‘Akita’, and the Japanese cultivar ‘Misuzudaizu’ × the Chinese breeding line ‘Moshidou Gong 503’) and have aligned these markers with the 20 consensus linkage groups (LGs). The total length of the integrated linkage map was 2442.9 cM, and the average number of molecular markers was 90.5 (range of 70–114) for the 20 LGs. We examined allelic diversity for 1238 of the SSR markers among 23 soybean cultivars or lines and a wild accession. The number of alleles per locus ranged from 2 to 7, with an average of 2.8. Our high-density linkage map should facilitate ongoing and future genomic research such as analysis of quantitative trait loci and positional cloning in addition to marker-assisted selection in soybean breeding.Key words: EST-derived SSR marker, integrated linkage map, microsatellite marker, polymorphism information content