Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus.     

Zinc Deficiency Enhances the Induction of Micronuclei and 8-Hydroxy-2′-Deoxyguanosine Via Superoxide Radical in Bone Marrow of Zinc-Deficient Rats

The aim of the present study was to examine whether zinc (Zn) deficiency augmented the frequency of micronuclei, an indicator of chromosome aberration, and the induction of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of cellular DNA damage derived from oxidative stress, in rat bone marrow cells or not. Both the frequency of micronuclei and the induction of 8-OHdG were significantly increased in rats fed with a Zn-deficient versus a standard diet for 6 weeks (p?<?0.005). The supplementation of Zn with a standard diet for 4 weeks to rats fed with a Zn-deficient diet for 6 weeks restored the enhanced induction of micronuclei and 8-OHdG to levels comparable to those seen in rats fed with a standard diet for 10 weeks, indicating that the shortage of Zn in the body is involved in the induction of micronuclei and 8-OHdG. Again, the membrane-permeable superoxide dismutase mimetic superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, treatment (100 μmol/kg, twice a day) for 10 days prior to the termination of dietary treatment reduced the induction of micronuclei and 8-OHdG in rats fed with a Zn-deficient diet for 6 weeks to levels comparable to those in rats fed with a standard diet for 6 weeks, indicating that superoxide radical participates in the induction of micronuclei and 8-OHdG. In fact, the endogenous superoxide scavenger, Cu/Zn superoxide dismutase, was significantly reduced in the bone marrow cells of rats fed with a Zn-deficient diet for 6 weeks when compared to those of rats fed with a standard diet for 6 weeks (p?<?0.005). These observations demonstrate that Zn deficiency elevates the frequency of micronuclei and the induction of 8-OHdG through an increase in the biological action of the superoxide radical. This suggests an increase in carcinogenic initiation resulting from Zn deficiency-induced oxidative stress.     

ER stress response induced by the production of human IL-7 in rice endosperm cells

Rice seed has been used as a production platform for high value recombinant proteins. When mature human interleukin 7 (hIL-7) was expressed as a secretory protein in rice endosperm by ligating the N terminal glutelin signal peptide and the C terminal KDEL endoplasmic reticulum (ER) retention signal to the hIL-7 cytokine to improve production yield, this protein accumulated at levels visible by Coomassie Brilliant Blue staining. However, the production of this protein led not only to a severe reduction of endogenous seed storage proteins but also to a deterioration in grain quality. The appearance of aberrant grain phenotypes (such as floury and shrunken) was attributed to ER stress induced by the retention of highly aggregated unfolded hIL-7 in the ER lumen, and the expression levels of chaperones such as BiPs and PDIs were enhanced in parallel with the increase in hIL-7 levels. The activation of this ER stress response was shown to be mainly mediated by the OsIRE1-OsbZIP50 signal cascade, based on the appearance of unconventional splicing of OsbZIP50 mRNA and the induction of OsBiP4&5. Interestingly, the ER stress response could be induced by lower concentrations of hIL-7 versus other types of cytokines such as IL-1b, IL-4, IL-10, and IL-18. Furthermore, several ubiquitin 26S proteasome-related genes implicated in ER-associated degradation were upregulated by hIL-7 production. These results suggest that severe detrimental effects on grain properties were caused by proteo-toxicity induced by unfolded hIL-7 aggregates in the ER, resulting in the triggering of ER stress.     

Intra‐specific pollen polymorphism in Mimosa pigra (Mimosaceae)

Five different types of pollen tetrads were detected from the short and long stamens of the male and hermaphrodite flowers of the androdioecious species, Mimosa pigra L. Light (LM) and scanning electron microscopes (SEM) were used in this study to redescribe the pollen forms and to identify the various pollen types encountered. An identification key and relative percentages of these pollen types were also presented. The study showed that the long stamens of the male flowers and those of the hermaphrodite ones have five (I, II, III, IV and V) and two (I and V) pollen types respectively, whereas the short stamens of both male and hermaphrodite flowers have only one pollen type (type I). We report here the first incidence of intra‐specific pollen polymorphism in androdioecious species.     

Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells

The anti-HIV-1 activity of GUT-70, a natural product derived from the stem bark of Chlophyllum brasiliense, was evaluated. GUT-70 inhibited HIV-1 replication in both acutely and chronically infected cells through suppression of NF-κB. Our results strengthen the idea that NF-κB pathway is one of the potential targets to control HIV-1 replication and that GUT-70 could serve as a lead compound to develop novel therapeutic agents against HIV-1 infection.     

High Concentration—Ethanol Fermentation of Raw Ground Corn

1-Pyrroline-5-carboxylate dehydrogenase was purified and crystallized from Bacillus sphaericus. The crystalline preparation gave a single band on polyacrylamide slab gel electrophoresis. The molecular weight of the enzyme was determined to be about 100,000 by gel filtration. The enzyme consists of two subunits which are identical in molecular weight (50,000), as judged on SDS slab gel electrophoresis. The enzyme shows an optimum pH of 6.5 to 7.0. Its activity was 8.1 times higher with NADP⁺ than with NAD ⁺, and the enzyme was stabilized by NADP⁺. The apparent Km values for l-l-pyrroline-5-carboxylate, NADP⁺ and NAD⁺ are 4.2 × 10^–5m (with NADP⁺), 9.5 × 10~⁶m and 2.5 × IO^-3 m, respectively. The enzyme reaction is irreversible. A simple method for the determination of l-ornithine involving ornithine ¿-aminotransferase and 1- pyrroline-5-carboxylate dehydrogenase from B. sphaericus was developed. A linear relationship was found between the absorbance at 340 nm and the amount of l-ornithine (50 ~ 400 nmol), and between the fluorescence and the amount of l-ornithine (0.2 ~ 10 nmol).