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.     

Demonstration of intermediates between deoxy and carbonmonoxy hemoglobins by anaerobic high-performance liquid chromatography

The effect of glucagon and insulin on rat liver phosphorylase phosphatase activity in vivo was investigated. The activity of phosphatase was found to decrease following the administration of glucagon and increase with insulin in a reversible manner. No change was detected in the activity of heat-stable phosphatase inhibitors in the hormone-treated samples. Liver protein kinases (regulatory subunit of cAMP-dependent protein kinase and/or Ca2+-dependent phosphorylase kinase) are suggested to regulate the activity of hepatic phosphorylase phosphatase (type 1 and 2A).     

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)     

Aggregation of hemoglobin S modified by bifunctional imidoesters

Sickle hemoglobin (Hb S) was cross-linked by two types of bifunctional imidoesters, dimethyladipimidate (DMA) and dimethyl-3,3'-dithiobispropionimidate (DTBP). These modified hemoglobins were separated into monomer, dimer and polymer fractions by gel filtration. All of these modified hemoglobins showed extremely left-shifted oxygen equilibrium curves with no cooperativity. The stabilities of these hemoglobins were also decreased. The solubilities of these modified hemoglobins in high-phosphate buffers were lower than those of native Hb S. Studies on the kinetics of the aggregation of these modified hemoglobins showed that intracross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified monomeric Hb S) still retained the capability of aggregation with a delay time, while intercross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified oligomeric Hb S) aggregated without a delay time. When the kinetics of aggregation was measured for mixtures of modified and native deoxy-Hb S, DMA-modified monomeric deoxy-Hb S shortened the delay time prior to aggregation of native deoxy-Hb S. The other modified deoxy-Hb S did not affect the delay time, suggesting that these modified oligomeric hemoglobins neither participate in the formation of nuclei nor copolymerize with native deoxy-Hb S.     

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)     

Effect of amino acid at the beta 6 position on surface hydrophobicity, stability, solubility, and the kinetics of polymerization of hemoglobin. Comparisons among Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6)

Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.     

Stimulation of in vitro motility of Chlamydomonas axonemes by inhibition of cAMP-dependent phosphorylation

When demembranated axonemes of Chlamydomonas were reactivated with Mg-ATP, the proportion of motile axonemes was significantly increased by the presence of either phosphodiesterase (PDE) or protein inhibitor of cAMP-dependent kinase (PKI). The effect of PDE was cancelled by the addition of cAMP. These findings strongly suggest that the axoneme samples have endogenous cAMP, which can reduce the proportion of motile axonemes via phosphorylation. This inhibitory effect of cAMP on Chlamydomonas axonemes is opposite to its stimulatory effect on the axonemal motility in other organisms so far reported. PKI or PDE activated the motility either in the absence of Ca2+, when the axonemes beat with an asymmetric waveform, or in 10(-5) M Ca2+, when the axonemes beat with a symmetric waveform. This cAMP-dependent regulation of motility was observed with the axonemes from which detergent-soluble material had been removed, indicating that the proteins responsible for the regulation still remained in the axonemes. Preliminary in vitro phosphorylation studies have implicated two polypeptides as candidates for the target protein of cAMP-dependent protein kinase: one with a molecular weight of 270 kD and the other with a much larger molecular weight.     

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.