Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Specific Features of the Formation of a Halo during Pathogenesis as a Response of Cereal Epidermal Cells to Penetration of Powdery Mildew Causative Agents

A cytophysiological study was carried out of the functional status of a halo as a response of the host plant to contact with a powdery mildew pathogen. Interactions of the powdery mildew causative agents with barley, wheat, wheat–wheat-grass hybrids, wheat-aegilops lines, and aegilops with different genotypic resistance lead to the expression of haloes during pathogens, which are induced by infection pegs of the primary growth tubes, appressoria, and hyphal lobes. Haloes are visualized using cytochemical reactions to proteins and scanning electron microscopy. The observed differences in the size of haloes and intensity of their staining (uniform or zonal) are related, to a great extent, to individual reactions of the plant cell at the penetration site and, to a lesser extent, to the level of genotypic resistance. An analysis of electron microscopy and cytochemistry studies suggests that the halo as a physiologically active zone is localized at the level of the plant cell plasmalemma. Active taxis of the cell organelles to the site of infection during the formation of a halo suggests that some kind of informational signals to changes in the cell metabolism are spread from the halo zone, which lead to compatible or incompatible interactions.     

Changes in Targeting Efficiencies of Proteins to Plant Microbodies Caused by Amino Acid Substitutions in the Carboxy-terminal Tripeptide

It has been demonstrated that the carboxyl terminus of microbodyenzymes functions as a targeting signal to microbodies in higherplants. We have examined an ability of 24 carboxy-terminal aminoacid sequences to facilitate the transport of a cytosolic passengerprotein, ß-glucuroni-dase, into microbodies in greencotyledonary cells of trans-genic Arabidopsis. Immunoelectronmicroscopic analysis revealed that carboxy-terminal tripeptidesequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as amicrobody-targeting signal, although tripeptides with prolineat the first amino acid position and isoleucine at the carboxylterminus show weak targeting efficiencies. All known micro-bodyenzymes that are synthesized in a form similar in size to themature molecule, except catalase, contain one of these tripeptidesequences at their carboxyl terminus. (Received April 14, 1997; Accepted April 8, 1997)     

Economical Speed and Energetically Optimal Transition Speed Evaluated by Gross and Net Oxygen Cost of Transport at Different Gradients

The oxygen cost of transport per unit distance (CoT; mL·kg^-1·km^-1) shows a U-shaped curve as a function of walking speed (v), which includes a particular walking speed minimizing the CoT, so called economical speed (ES). The CoT-v relationship in running is approximately linear. These distinctive walking and running CoT-v relationships give an intersection between U-shaped and linear CoT relationships, termed the energetically optimal transition speed (EOTS). This study investigated the effects of subtracting the standing oxygen cost for calculating the CoT and its relevant effects on the ES and EOTS at the level and gradient slopes (±5%) in eleven male trained athletes. The percent effects of subtracting the standing oxygen cost (4.8 ± 0.4 mL·kg^-1·min^-1) on the CoT were significantly greater as the walking speed was slower, but it was not significant at faster running speeds over 9.4 km·h^-1. The percent effect was significantly dependent on the gradient (downhill > level > uphill, P < 0.001). The net ES (level 4.09 ± 0.31, uphill 4.22 ± 0.37, and downhill 4.16 ± 0.44 km·h^-1) was approximately 20% slower than the gross ES (level 5.15 ± 0.18, uphill 5.27 ± 0.20, and downhill 5.37 ± 0.22 km·h^-1, P < 0.001). Both net and gross ES were not significantly dependent on the gradient. In contrast, the gross EOTS was slower than the net EOTS at the level (7.49 ± 0.32 vs. 7.63 ± 0.36 km·h^-1, P = 0.003) and downhill gradients (7.78 ± 0.33 vs. 8.01 ± 0.41 km·h^-1, P < 0.001), but not at the uphill gradient (7.55 ± 0.37 vs. 7.63 ± 0.51 km·h^-1, P = 0.080). Note that those percent differences were less than 2.9%. Given these results, a subtraction of the standing oxygen cost should be carefully considered depending on the purpose of each study.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Measurement of Serine Acetyltransferase Activity in Crude Plant Extracts by a Coupled Assay System Using Cysteine Synthase

Serine acetyltransferase (SATase) (EC 2.3.1.30 [EC] ) catalyzes theformation of Oacetyl-L-serine (OAS) from L-serine in the presenceof acetyl-CoA. A novel assay method was developed for measuringthis enzyme activity in extracts from plant tissues. The assayconsists of a coupled system in which the OAS formed is convertedto cysteine by the addition of cysteine synthase (CSase) (EC4.2.99.8 [EC] ). Cysteine thus formed is determined colorimetricallyand serves as a measure for SATase activity. This method israpid, simple and sensitive, and can be readily adapted formeasurement of SATase activity in crude tissue extracts or homogenates. (Received January 14, 1987; Accepted April 27, 1987)     

Factors controlling induction of commitment of murine erythroleukemia (TSA8) cells to CFU-E (colony forming unit-erythroid)

On addition of DMSO, the MEL cell line TSA8 becomes committed into erythroid progenitor cells (CFU-E) which can form differentiated colonies in the presence of erythropoietin. To understand the mechanism of cellular commitment, the number and the affinity of the receptors for erythropoietin were estimated. The affinity of the receptors did not change before or after induction. The number of receptors changed depending on the growth phase, but was not dependent on the addition of the inducer. Thus, the presence of the receptors for erythropoietin may be required, but are not essential for responsiveness to erythropoietin. Further examination of the optimum conditions for commitment suggests that the concomitant actions of induced factor(s) with the receptors may control commitment of TSA8 cells to CFU-E.     

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.     

The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Summary The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.     

Three species of Trebius Krøyer, 1838 (Copepoda: Siphonostomatoida) parasitic on Pacific elasmobranchs

The paratypes of Trebius akajeii Shiino, 1954, originally collected from Dasyatis akajei (Müller & Henle) at Owase, Japan, and the paratypes and newly collected specimens of T. latifurcatus Wilson, 1921, previously collected from Urolophus and Myliobatis at Venice, California, are redescribed. New host records for T. latifurcatus are: Torpedo californica Ayres, Squatina californica Ayres, Rhinobatos productus (Ayres), Platyrhinoides triseriata (Jordan & Gilbert), Raja inornata (Jordan & Gilbert) and Gymnura marmorata (Cooper). A new species, Trebius heterodonti, is described from the horn shark Heterodontus francisci (Girard) collected from southern California waters. These three species of Trebius are apparently closely related to each other. Diagnostic characters of the species are provided along with a discussion on the taxonomic value of selected features.