Subcellular localization and rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids.
(25/4352)
We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed. (+info)
Fruit and vegetable consumption in later life.
(26/4352)
OBJECTIVE: to assess levels of fruit and vegetable consumption in elderly people, and to examine the socio-economic, physical and psychological factors which influence this consumption. METHODS: a three-phase survey: face to face interviews; self-completed dietary diaries with a food frequency questionnaire; and follow-up face-to-face interviews. PARTICIPANTS: 445 elderly people (aged 65+) randomly selected from general practitioner lists in urban Nottingham and rural Nottinghamshire, Lincolnshire and Leicestershire. RESULTS: the recommended target of five portions of fruit and vegetables a day was achieved by less than half the respondents: 37% of those living in the urban area and 51% of those living in the rural area. Low fruit and vegetable consumption was particularly associated with being male, smoking and having low levels of social engagement. CONCLUSIONS: most elderly people consume less than the recommended levels of fruit and vegetables. Health programmes promoting fruit and vegetable consumption may not be successfully reaching elderly people and need to target those particularly at risk of low consumption. (+info)
Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata.
(27/4352)
The Japanese pear pathotype of Alternaria alternata causes black spot of Japanese pear by producing a host-specific toxin known as AK-toxin. Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for toxin biosynthesis. Protoplasts of a wild-type strain were treated with a linearized plasmid along with the restriction enzyme used to linearize the plasmid. Of 984 REMI transformants recovered, three produced no detectable AK-toxin and lost pathogenicity on pear leaves. Genomic DNA flanking the integrated plasmid was recovered from one of the mutants. With the recovered DNA used as a probe, a cosmid clone of the wild-type strain was isolated. Structural and functional analyses of an 8.0-kb region corresponding to the tagged site indicated the presence of two genes. One, designated AKT1, encodes a member of the class of carboxyl-activating enzymes. The other, AKT2, encodes a protein of unknown function. The essential roles of these two genes in both AK-toxin production and pathogenicity were confirmed by transformation-mediated gene disruption experiments. DNA gel blot analysis detected AKT1 and AKT2 homologues not only in the Japanese pear pathotype strains but also in strains from the tangerine and strawberry pathotypes. The host-specific toxins of these two pathotypes are similar in structure to AK-toxin. Homologues were not detected in other pathotypes or in non-pathogenic strains of A. alternata, suggesting acquisition of AKT1 and AKT2 by horizontal transfer. (+info)
Cloning and expression of a hexose transporter gene expressed during the ripening of grape berry.
(28/4352)
The ripening of grape (Vitis vinifera L.) is characterized by massive sugar import into the berries. The events triggering this process and the pathways of assimilate transport are still poorly known. A genomic clone Vvht1 (Vitis vinifera hexose transporter1) and the corresponding cDNA encoding a hexose transporter whose expression is induced during berry ripening have been isolated. Vvht1 is expressed mainly in the berries, with a first peak of expression at anthesis, and a second peak about 5 weeks after veraison (a viniculture term for the inception of ripening). Vvht is strictly conserved between two grape cultivars (Pinot Noir and Ugni-Blanc). The organization of the Vvht1 genomic sequence is homologous to that of the Arabidopsis hexose transporter, but differs strongly from that of the Chlorella kessleri hexose transporter genes. The Vvht1 promoter sequence contains several potential regulating cis elements, including ethylene-, abscisic acid-, and sugar-responsive boxes. Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase, which is expressed at the same time during ripening, also allowed the identification of a 15-bp consensus sequence, which suggests a possible co-regulation of the expression of these genes. The expression of Vvht1 during ripening indicates that sucrose is at least partially cleaved before uptake into the flesh cells. (+info)
Bromelain, from pineapple stems, proteolytically blocks activation of extracellular regulated kinase-2 in T cells.
(29/4352)
Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3epsilon mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-gamma, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways. (+info)
Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host.
(30/4352)
Viroids, small single-stranded circular RNAs endowed with autonomous replication, are unique systems to conduct evolutionary studies of complete RNA genomes. The primary structure of 36 progeny variants of peach latent mosaic viroid (PLMVd), evolved from inoculations of the peach indicator GF-305 with four individual PLMVd cDNAs differing in their pathogenicity, has been determined. Most progeny variants had unique sequences, revealing that the extremely heterogeneous character of PLMVd natural isolates most probably results from the intrinsic ability of this RNA to accumulate changes, rather than from repeated inoculations of the same individual trees under field conditions. The structure of the populations derived from single PLMVd sequences differed according to the observed phenotype. Variant gds6 induced a reproducible symptomatic infection and gave rise to a more uniform progeny that preserves some parental features, whereas variant gds15, which induced a variable phenotype, showed a more complex behaviour, generating two distinct progenies in symptomatic and asymptomatic individual plants. Progenies derived from variants esc10 and Is11, which incited latent infections, followed a similar evolutionary pattern, leading to a population structure consisting of two main groups of variants, one of which was formed by variants closely related to the parental sequence. The evolution rate exhibited by PLMVd, considerably higher than that reported for potato spindle tuber viroid, may contribute to the fluctuating symptomatology of the severe PLMVd natural isolates. However, the polymorphism observed in PLMVd progenies does preserve some structural and functional elements previously proposed for this viroid, supporting the fact that they act as constraints limiting the genetic divergence of PLMVd quasispecies generated de novo. (+info)
Randomized trial testing the effect of peer education at increasing fruit and vegetable intake.
(31/4352)
BACKGROUND: The National Cancer Institute recommends that Americans eat at least five daily servings of fruits and vegetables. National strategies to increase consumption may not reach minority and lower socioeconomic populations. In a randomized trial, peer education was tested for effectiveness at increasing fruit and vegetable intake among lower socioeconomic, multicultural labor and trades employees. METHODS: Employees (n = 2091) completed a baseline survey and received an 18-month intervention program through standard communication channels (e.g., workplace mail, cafeteria promotions, and speakers). Ninety-three social networks (cliques) of employees were identified, which were pair matched on intake. At an interim survey (during months 8 and 9), 11 cliques no longer existed and 41 matched pairs of cliques containing 905 employees remained, with one clique per pair being randomly assigned to the peer education intervention. Employees who were central in the communication flow of the peer intervention cliques served as peer educators during the last 9 months of the intervention program. Fruit and vegetable intake was measured with 24-hour intake recall and with food-frequency questions in baseline, outcome (i.e., at 18 months), and 6-month follow-up surveys. All P values are two-sided. RESULTS: By use of multiple regression, statistically significant overall effects of the peer education program were seen in the intake recall (increase of 0.77 total daily servings; P<.0001) and the food-frequency (increase of 0.46 total daily servings; P =.002) questions at the outcome survey. The effect on the total number of servings persisted at the 6-month follow-up survey when measured by the intake recall (increase of 0.41 total daily servings; P =.034) but not the food-frequency (decrease of 0.04 total daily servings; P =.743) questions. CONCLUSIONS: Peer education appears to be an effective means of achieving an increase in fruit and vegetable intake among lower socioeconomic, multicultural adult employees. (+info)
Fruit and vegetable consumption and prevention of cancer: the Black Churches United for Better Health project.
(32/4352)
OBJECTIVES: This study assessed the effects of the Black Churches United for Better Health project on increasing fruit and vegetable consumption among rural African American church members in North Carolina. METHODS: Ten counties comprising 50 churches were pair matched and randomly assigned to either intervention or delayed intervention (no program until after the follow-up survey) conditions. A multicomponent intervention was conducted over approximately 20 months. A total of 2519 adults (77.3% response rate) completed both the baseline and 2-year follow-up interviews. RESULTS: The 2 study groups consumed similar amounts of fruits and vegetables at baseline. AT the 2-year follow-up, the intervention group consumed 0.85 (SE = 0.12) servings more than the delayed intervention group (P < .0001). The largest increases were observed among people 66 years or older (1 serving), those with education beyond high school (0.92 servings), those widowed or divorced (0.96 servings), and those attending church frequently (1.3 servings). The last improvement occurred among those aged 18 to 37 years and those who were single. CONCLUSIONS: The project was a successful model for achieving dietary change among rural African Americans. (+info)