The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors

A system is described for the rapid generation of Bombyx mori nucleopolyhedrovirus (BmNPV)-based expression vectors. A series of novel BmNPV genomes, that include a mini-F replicon and therefore can be maintained in Escherichia coli, have been generated. These genomes lack a portion of the essential ORF1629 gene and cannot replicate independently in insect cells. However, they can be used as parental genomes for the generation of expression vectors by cotransfection with a transfer plasmid that includes an intact ORF1629. Only recombinant viruses that have acquired the ORF1629 gene from the transfer vector, and have therefore also acquired the foreign gene of interest, can replicate after cotransfection. Parental genomes with and without a polyhedrin gene are described, enabling the generation of occlusion-positive and occlusion-negative recombinant viruses. Occlusion-positive expression vectors enable the oral infection of B. mori larvae and can therefore be used for the mass production of a foreign protein in infected insects.     

Psychometric Properties of the Multidimensional Health Locus of Control Scale Form C in a Non-Western Culture

Form C of the Multidimensional Health Locus of Control Scales (MHLC-C) was designed to investigate health-related control beliefs of persons with an existing medical condition. The aim of the present study was to examine the psychometric properties of this instrument in a culture characterized by external control beliefs and learned helplessness—contrary to the societal context of original test development. Altogether, 374 Hungarian patients with cancer, irritable bowel syndrome, diabetes, and cardiovascular and musculoskeletal disorders were enrolled in the study. Besides the MHLC-C, instruments measuring general control beliefs, anxiety, depression, self-efficacy, and health behaviors were also administered to evaluate the validity of the scale. Both exploratory and confirmatory factor analytic techniques were used to investigate the factor structure of the scale. Our results showed that the Hungarian adaptation of the instrument had a slightly different structure than the one originally hypothesized: in the present sample, a three-factor structure emerged where the items of the Doctors and the Others subscales loaded onto a single common component. Internal reliability of all three subscales was adequate (alphas between .71 and .79). Data concerning the instrument''s validity were comparable with previous results from Western countries. These findings may suggest that health locus of control can be construed very similarly to Western countries even in a post-communist society—regardless of the potential differences in general control beliefs.     

The 3.5-Å CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel

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Castasterone Can be Biosynthesized from 28-homodolichosterone in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We recently demonstrated the biosynthesis of 24-ethylidene brassinosteroids in Arabidopsis thaliana. To determine the physiological role of biosynthesis of 24-ethylidene brassinosteroids, metabolism of 28-homodolichosterone as the end product of 24-ethylidene brassinosteroids biosynthesis was examined by a crude enzyme solution prepared from A. thaliana. In wild-type plants, dolichosterone and castasterone were identified as enzyme products on GC-MS analysis. In a mutant where DWARF1 was overexpressed (35S-DWF1), the conversion rate of 28-homodolichosterone to castasterone was significantly increased. These results indicate that conversion of 28-homodolichosterone to castasterone is mediated by dolichosterone in Arabidopsis. In the root growth assay, inhibitory activity was enhanced in the order of castasterone > dolichosterone > 28-homodolichosterone, demonstrating that conversion of 28-homodolichosterone to castasterone via dolichosterone is a biosynthetic reaction that increases BR activity in Arabidopsis. Compared to Arabidopsis grown under dark conditions, light-grown Arabidopsis showed up-regulated DWARF1 expression, resulting in an increased conversion rate of 28-homodolichosterone to castasterone, suggesting that light is an important regulatory factor for the biosynthetic connection of 24-ethylidene brassinosteroids and 24-methyl brassinosteroids in A. thaliana. Consequently, 24-ethylidene brassinosteroids biosynthesis to generate 28-homodolichosterone is a lightregulated alternative route for synthesis of the biologically-active BRs, castasterone and brassinolide in Arabidopsis plants.     

Functional Analysis of TiO2 Nanoparticle Toxicity in Three Plant Species

Titanium dioxide nanoparticles (nano-TiO₂) are manufactured and used worldwide in large quantities. However, phytotoxicity research on nano-TiO₂ has yielded confusing results, ranging from strong toxicity to positive effects. Therefore, in this research, the effects of nano-TiO₂ on the germination and root elongation of seed and seedlings were studied. Additionally, the uptake and physiological responses of mature plants were investigated. Physical chemistry data were analyzed to assess the availability of nano-TiO₂. Finally, a hydroponic system designed to overcome nano-TiO₂ precipitation was used to reproduce the environmental conditions of actual fields. Nano-TiO₂ did not have any effect on seed germination or on most of the plant species tested. Nano-TiO₂ had positive effects on root elongation in some species. No physiological differences in enzyme activities or chlorophyll content were detected, even though the plants absorbed nano-TiO₂. Physical chemistry data showed that nano-TiO₂ agglomerated rapidly and formed particles with much bigger hydrodynamic diameters, even in distilled water and especially in a hydroponic system. Furthermore, agglomerated nano-TiO₂ formed precipitates; this would be more severe in an actual field. Consequently, nano-TiO₂ would not be also readily available to plants and would not cause any significant effects on plants. Our results and other reports suggest that titanium itself is not phytotoxic, even though plants absorb titanium. In conclusion, nano-TiO₂ is not toxic to the three plant species, in vitro or in situ.     

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles

The initial rate of Ca²⁺ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca²⁺-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca²⁺-selective minielectrodes to determine pCa values. The initial rate of Ca²⁺ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca²⁺ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K⁺. In addition, Ca²⁺ was shown to move across the membrane vesicles in the presence of a K⁺ diffusion potential gradient. The potential-stimulated rate of Ca²⁺ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl₃, verapamil, and nifedipine had little or no effect. These results indicate that Ca²⁺ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.     

Moderate hypothermia attenuates α(1)-adrenoceptor-mediated contraction in isolated rat aorta: the role of the endothelium

Moderate hypothermia (25–31 °C) may have a significant influence on vascular tone. We investigated the cellular mechanisms by which moderate hypothermia alters α-adrenoceptor-mediated contraction in rat thoracic aortae. Cyclooxygenase inhibition by indomethacin; nitric oxide (NO) synthase inhibition by l-NAME; potassium channel and endothelium-derived hyperpolarizing factor (EDHF) inhibition by glibenclamide and TEA; G protein inhibition by pertussis toxin; α₂-adrenergic inhibition by yohimbine; and β-adrenergic inhibition by propranolol were assessed for their effect on the contractile response to the α1-adrenoceptor agonist phenylephrine (Phe) in combination with moderate hypothermia (25 °C). Moderate hypothermia produced a shift to the right for the Phe concentration–response curves in endothelium-intact (E+) and endothelium-denuded (E?) aortic rings. The maximal response to Phe in E+ rings was significantly decreased (P < 0.05) at 25 °C compared to 38 °C, whereas there was no significant difference in E? rings. Hypothermia-induced vasorelaxation in E+ rings was attenuated (P < 0.05) following combined pretreatment with l-NAME (10^?4 M) and indomethacin (10^?5 M), whereas other inhibitors had no significant effect. Importantly, the addition of TEA to rings that were pretreated with l-NAME and indomethacin exhibited no further attenuation (P > 0.05) of hypothermia-induced vasorelaxation. The concentrations of cGMP and cAMP, as measured by radioimmunoassay, were significantly increased (P < 0.05) in E+ rings at 25 °C compared to those at 38 °C, whereas there were no significant differences (P > 0.05) in E? rings. The present study demonstrated that rat aortic endothelium is stimulated during moderate hypothermia and that the NO–cGMP and prostacyclin (PGI₂)–cAMP pathways represent endothelium-dependent mechanisms of hypothermia-induced vasorelaxation. In contrast, EDHF may not be associated with hypothermia-induced vasorelaxation.