S-like ribonuclease gene expression in carnivorous plants

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.     

The importance of larval eyes in the polychaete Capitella teleta: effects of larval eye deletion on formation of the adult eye

In many marine invertebrates with biphasic life cycles, juvenile/adult traits begin to develop before metamorphosis. For structures that are present at multiple developmental stages, but have distinct larval and adult forms, it is unclear whether larval and adult structures have shared or distinct developmental origins. In this study, we examine the relationship between the larval and adult eyes in the polychaete Capitella teleta. In addition, we describe a novel marker for larval and juvenile photoreceptor cells. Infrared laser deletion of individual micromeres in early embryos suggests that the same micromeres at the eight‐cell stage that are specified to generate the larval eyes also form the adult eyes. Direct deletion of the larval eye, including the pigment cell and the corresponding photoreceptor cell, resulted in a lack of shading pigment cells in juveniles and adults, demonstrating that this structure does not regenerate. However, a sensory photoreceptor cell was present in juveniles following direct larval eye deletions, indicating that larval and adult photoreceptors are separate cells. We propose that the formation of the adult eye in juveniles of C. teleta requires the presence of the pigment cell of the larval eye, but the adult photoreceptor is either recruited from adjacent neural tissue or arises de novo after metamorphosis. These results are different from the development and spatial orientation of larval and adult eyes found in other polychaetes, in which two scenarios have been proposed: larval eyes persist and function as adult eyes; or, distinct pigmented adult eyes begin developing separately from larval eyes prior to metamorphosis.     

Suppressive effect of molybdenum on hepatotoxicity of N-nitrosodiethylamine in rats

SUMMARY

In order to elucidate the preventive mechanism of molybdenum (Mo) against carcinogenesis of N-nitroso compounds, the effects of in vivo Mo-pretreatment on N-nitrosodiethylamine (NDEA)-induced hepatotoxicity in rats were examined. Effects of in vitro Mo-pretreatment on NDEA-induced DNA strand breaks and fluctuation of the cytosolic free Ca levels in rat hepatocytes were also investigated. Male Wistar rats weighing 170–190 g were pretreated with 10 ppm Mo as Na₂MoO₄ in deionized drinking water for 21 days, and on day 22, they were exposed to NDEA (50 mg/kg body weight, once, i.p.). 3 and 5 days after NDEA exposure, serum lactic dehydrogenase (LDH) activity, and hepatic calcium (Ca) content and lipid peroxidation levels were evaluated. In vivo Mo-pretreatment prevented NDEA-induced elevations in serum LDH activity and liver Ca content but increased hepatic lipid peroxidation levels. Hepatocytes isolated from rats pretreated with sodium phenobarbital (80 mg/kg body weight, i.p., once a day for 3 days) were exposed to NDEA (0, 100, 250 and 500 μM) in vitro for 30 min at 37°C. NDEA treatment caused DNA strand breaks and a perturbation of cytosolic free Ca level. However, in vitro Mo-pretreatment (20 μM, 20 min at 37°C) suppressed the NDEA-induced DNA damage and disruption of intracellular Ca homeostasis. These results suggest Mo protected against NDEA-induced hepatotoxicity by stimulating the metabolism of the nitroso compound via a nontoxic pathway (denitration) while preventing DNA damage connected with alteration in cytosolic free Ca levels. Thus, the general protective action of Mo against N-nitroso compound-induced carcinogenesis may be explained by a common mechanism.     

Reactivity of sorbose dehydrogenase from Sinorhizobium sp. 97507 for 1,5-anhydro-d-glucitol

Purified recombinant sorbose dehydrogenase from Sinorhizobium sp. 97507 exhibited high reactivity for 1,5-anhydro-d-glucitol (1,5-AG) and l-sorbose, but little activity for the other sugars or sugar alcohols tested. Kinetic analysis revealed that its catalytic efficiency (k_cat/K_m) for l-sorbose and 1,5-AG is 1.8 × 10² and 1.5 × 10² s^?1·M^?1, respectively.     

RNA Interference towards the Potato Psyllid,Bactericera cockerelli,Is Induced in Plants Infected with Recombinant Tobacco mosaic virus (TMV)

The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.     

Slower Uncoating Is Associated with Impaired Replicative Capability of Simian-Tropic HIV-1

Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116^th position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus.     

Unintended Effects of Cardiovascular Drugs on the Pathogenesis of Alzheimer’s Disease

Alzheimer’s disease (AD) is rapidly becoming one of the leading causes of disability and mortality in the elderly. As life-expectancy increases, an increasing number of people will rely on modern medicines to treat age-associated disorders. Among these medications, some might benefit, while others might exacerbate, the pathogenesis of AD. We screened 1,600 FDA approved drugs for β-amyloid (Aβ)-modifying activity and identified drugs that can potentially influence amyloid precursor protein processing. In this study, we focused on cardiovascular drugs and demonstrated that some hypertensive medication can differentially modulate Aβ, both in vitro and in vivo. Our study suggests that some commonly prescribed drugs might exert unintended effects and modulate AD and provides the basis for continuing investigation of the role of individual drugs on a case-by-case basis. This line of investigation will lead to the identification of common medications that are potentially beneficial or detrimental to AD as a reference for physicians to consider when prescribing the most appropriate drugs for their patients, particularly for treating chronic disorders among the growing geriatric population.     

Chromosome aberrations induced by dual exposure of protons and iron ions

During space travel, astronauts will be exposed to protons and heavy charged particles. Since the proton flux is high compared to HZE particles, on average, it is assumed that a cell will be hit by a proton before it is hit by an HZE ion. Although the effects of individual ion species on human cells have been investigated extensively, little is known about the effects of exposure to mixed beam irradiation. To address this, we exposed human epithelial cells to protons followed by HZE particles and analyzed chromosomal damage using the multicolor banding in situ hybridization (mBAND) procedure. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of intra-chromosomal aberrations (inversions and deletions within a single painted chromosome) as well as inter-chromosomal aberrations (translocation to unpainted chromosomes). Our results indicated that chromosome aberration frequencies from exposures to protons followed by Fe ions did not simply decrease as the interval between the two exposures increased, but peak when the interval was 30 min.