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.     

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)     

Partial purification and characterization of taurodeoxycholate 7α-monooxygenase

Taurodeoxycholate 7α-monooxygenase was partially purified from rat liver microsomes. The enzyme was solubilized with cholate, fractionated with polyethylene glycol and chromatographed on a Sepharose 4B column with cholate as ligand. The enzyme activity was eluted from the column into the fraction eluted with 50 mM phosphate buffer containing cholate and KCl, whereas the benzphetamine demethylase activity was eluted in the non-bound fraction. Thus it was established that both enzymes are different entities. The taurodeoxycholate 7α-monooxygenase activity was reconstituted from the partially purified cytochrome P-450, highly purified NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine and NADPH.     

Commitment to Ploidy Conversion of 3y1 Cells During Metaphase Arrest By Colcemid

Abstract. Diploid rat 3Y1 fibroblasts proliferate to a saturation density, where they are arrested with a 2N DNA content. After treatment to induce ploidy conversion, the conversion rate can be estimated by determining the fraction of cells with a 4N DNA content in the confluent culture using flow cytometry. Using this method it was found that during mitotic inhibition with colcemid, 3Y1 cells were converted to tetraploids with a high efficiency (above 80%); the optimum colcemid concentration and exposure period were 40 ng/ml and 8 hr, respectively. When metaphase cells were reseeded with 40 ng/ml of colcemid, they delayed anchorage to a dish; 6 hr was required for complete adhesion (in the absence of colcemid only 1 hr was required). When reseeded metaphase cells were exposed to 40 ng/ml of colcemid for 5 hr followed by its removal, a greater fraction of the cells anchored to the substratum were converted to tetraploids, whereas most of the floating cells were not. A greater fraction of the anchored cells had formed nuclei, whereas most of the floating cells preserved condensed metaphase chromosomes. These results indicate that the cells which have formed nuclear structure without chromosome separation during mitotic inhibition are irreversibly committed to ploidy conversion, with restoration of anchorage.     

Effects of winter flooding on phosphorus dynamics in rice fields

Limnology - Controlling phosphorous (P) loads from rice fields is important for the conservation of aquatic ecosystems, in part because P is relatively concentrated at its sources. Recently, winter...     

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.     

Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Seasonal pattern of filial cannibalism by Apogon doederleini mouthbrooding males

In temperate waters of southern Japan, mouthbrooding males of the cardinal fish Apogon doederleini sometimes ate their entire brood within a day of spawning. In spite of annual variation in age structure and length of the breeding season, however, the annual cannibalism rate (broods cannibalized to total broods spawned) was nearly constant, 12–16%. Fish 1 year old frequently cannibalized early broods of the season, especially the first brood, whereas the cannibalism rate by 2 year-old fish did not vary within the season. In contrast, the cannibalism by older males increased late in the season. This tendency was attributed to their different allocation patterns to growth and reproduction. For 1 year-old fish, which can enhance future reproductive success by growth and cannibalizing on early broods instead of mouthbrooding (at relatively low temperatures), this may be a tactic for investing energy in growth. For fish 3 years, which have more breeding cycles and grow little, cannibalizing late broods may be energetic compensation for their poor somatic condition. No seasonal variation of cannibalism by 2 year-old fish can be explained by their intermediate nature of growth and reproduction. Multiple mate availability is one factor facilitating the cannibalism by 2 and 3 + year-old fish whose operational sex ratio is female-biased.     

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)