The decline in ascorbic acid content is associated with cadmium toxicity of rice seedlings

Cadmium (Cd) toxicity of rice (Oryza sativa L. cv. Taichung Native 1) seedlings was evaluated by the decrease in chlorophyll content and the increase in malondialdehyde (MDA) in the second leaves of rice seedlings. CdCl₂ (5 μM) treatment was accompanied by a decrease in the contents of ascorbic acid (AsA) and AsA + dehydroascorbate (DHA) and in the ratios of AsA/DHA in leaves. However, CdCl₂ treatment resulted in an increase in DHA content in leaves. Moreover, the decrease in AsA content was prior to the occurrence of chlorosis and associated with the increase in MDA content in the leaves of seedlings treated with Cd. Pretreatment with 0.5 mM AsA or l-galactono-1,4-lactone (GalL), the biosynthetic precursor of AsA, for 6 h resulted in an increase in the contents of AsA and reduced glutathione (GSH), the ratios of AsA/DHA and GSH/oxidized glutathione, and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in the leaves of rice seedlings. Quantitative RT-PCR was applied to quantify the mRNA levels for OsAPX and OsGR genes from rice leaves to examine the effect of AsA or GalL pretreatment on the expression of OsAPX and OsGR genes in rice leaves. The expression of OsAPX2, OsAPX3, OsAPX4, OsAPX5, OsAPX6, OsAPX7, and OsGR1 was increased by AsA or GalL pretreatment. Rice seedlings pretreated with AsA or GalL were observed to reduce the subsequent Cd-induced toxicity. Our results suggest that AsA content may play a role in regulating Cd toxicity of rice seedlings.     

Brome mosaic virus RNA replication proteins 1a and 2a from a complex in vitro.

Brome mosaic virus (BMV) is a positive-strand RNA virus that encodes two RNA replication proteins, the helicaselike 1a and the polymeraselike 2a. 1a and 2a share extensive sequence similarities with proteins encoded by many other members of the alphaviruslike superfamily. While further purifying enzymatically active RNA-dependent RNA polymerase from plants infected by BMV, we observed that 1a, 2a, and the polymerase activity all cofractionated through multiple independent purification steps. Moreover, using immunoprecipitation, we found that BMV 1a and 2a proteins synthesized in rabbit reticulocyte lysates or insect cells can form a specific complex in vitro. Complex formation was more efficient when 1a and 2a were cotranslated than when they were mixed after independent synthesis. In an antibody-independent assay, in vitro-translated 1a protein was also found to bind to 2a protein fixed on a nylon membrane. A three-amino-acid insertion in 1a that blocks BMV RNA replication in vivo also blocked in vitro interaction with 2a, while another two-amino-acid insertion that renders the 1a protein temperature sensitive for RNA replication interacted in vitro with 2a at 24 degrees C but not at 32 degrees C. These results and previous genetic data suggest that the 1a-2a interaction observed in vitro is required for BMV RNA replication and may have direct implications for other members of the alphaviruslike superfamily.     

Ascorbate increases the synthesis of procollagen hydroxyproline by cultured fibroblasts from chick embryo tendons without activation or prolyl hydroxylase

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 μg of enzyme protein per 10⁸ cells and 40–50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein by only 15–20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and in cultured tendon cells had the same apparent size and the same activity per μg of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme.When freshly isolated cells were incubated for 2 h in the presence of 40 μg per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 μg per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not increase the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve “activation” of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.     

Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II

The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.     

Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Culture conditions for induction of green plants from barley microspores by anther culture methods

Summary With barley a large variation in frequency of plant formation from microspores of spikes from the same plant has been observed. The highest frequency of plant formation was obtained when culturing anthers in the dark on a high Ficoll medium containing 2,4-D and kinetin to induce proembryo (or callus) formation. Subsequently the proembryos or calli were cultured in dim light on a high Ficoll-high sugar medium containing IBA and kinetin. Finally the embryos were transferred to a starch agar medium. A maximum of 13 green plants were obtained from microspores of a single anther.The ratios of green to albino microspore derived plants varied from 91 to 19 depending on culture conditions. Under anaerobic conditions, lactic acid and other organic acids may have damaged the organelles in the cells resulting in the formation of albino plants. Thus, direct embryogenesis by using a well-buffered, high Ficoll-high sugar medium and proper aeration are essential for obtaining high frequency of green plants from microspores.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA 3 indolylbutyric acid     

15-lipoxygenase metabolites of gamma-linolenic acid/eicosapentaenoic acid suppress growth and arachidonic acid metabolism in human prostatic adenocarcinoma cells: possible implications of dietary fatty acids

Although gammalinolenic acid (GLA) and eicosapentaenoic acid (EPA) have independently been reported to suppress growth of cancer cells, their relative potencies are unknown. To determine the possible attenuating efficacies of dietary GLA or EPA on prostate carcinogenesis, we hereby report the in vitro effects of GLA, EPA and their 15-lipoxygenase (15-LOX) metabolites: 15(S)-HETrE and 15(S)-HEPE, respectively, on growth and arachidonic acid (AA) metabolism in human androgen-dependent (LNCaP) and androgen-independent (PC-3) prostatic cancer cells in culture. Specifically, both cells were preincubated respectively with the above PUFAs. Growth was determined by [3H]thymidine uptake and AA metabolism by HPLC analysis of the extracted metabolites. Our data revealed increased biosynthesis of prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5(S)-HETE) by both cells. Preincubation of the cells with 15(S)-HETrE or 15(S)-HEPE more markedly inhibited cellular growth and AA metabolism when compared to precursor PUFAs. Notably, 15(S)-HETrE exerted the greatest inhibitory effects. These findings therefore imply that dietary GLA rather than EPA should better attenuate prostate carcinogenesis via its in vivo generation of 15(S)-HETrE, thus warranting exploration.