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).     

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.     

RNA-Induced Interference with Animal Virus Infection

Some RNAs, including both single- and double-stranded RNAs, when incubated with chick embryo cell culture induce cellular resistance against viruses. Evidence was now obtained indicating that the induction of cellular resistance by RNA depends on the cellular metabolic activity, especially on the synthesis of cellular RNA and protein. Thus, inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, were found to inhibit the development of an antiviral state when added before, or during the relatively early period of, incubation of the cells with RNA. In the course of induction of cellular resistance, three stages may be distinguished, the priming stage, the developing stage, and the established resistant stage.     

Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot (Daucus carota L.)

Carbohydrate metabolism in Al-phosphate utilizing cells of carrot[designated as IPG, Koyama et al. (1992) Plant Cell Physiol.33: 171], which grow normally in Al-phosphate medium accompaniedby citrate excretion, was investigated. The excretion of citratewas strongly related to the availability of sucrose in medium,indicating that citrate excretion was severely limited by sucrosein medium. The ratio of the amount of carbon in the excretedcitrate to the consumed sucrose, was significantly higher inIPG cells than in wild-type cells. When 50% of the sucrose inthe medium was consumed, the ratio was 0.6% for the IPG cellsand 0.2% the wild-type cells. Under these conditions, IPG cellsshowed altered citrate synthesis metabolism, which resultedin increased citrate production. Specific activity of mitochondrialcitrate synthase was higher in IPG cells than in wild-type cells,whereas the activity of cytosolic NADP-specific isocitrate dehydrogenasewas lower in IPG cells than in wild-type cells. (Received August 27, 1998; Accepted February 21, 1999)     

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.     

Distribution and evolution of C4 syndrome inRhynchospora (Rhynchosporeae-Cyperaceae)

In order to elucidate the evolution of C₄ syndrome, the taxonomic relationships, leaf anatomy, and ecological and global distribution of C₃ and C₄ species in the genusRhynchospora were investigated. The anatomical observation for 181 species revealed that 26 C₄ species occurred within theCapitatae group of the subgenusHaplostyleae, a natural group showing highly advanced morphological characteristics, together with several C₃ species. In spite of there being rather few C₄ species, they possessed two kinds of Kranz anatomical structure differing from each other in the location of Kranz cells. Some C₃ species ofCapitatae showed radial arrangement in mesophyll cells surrounding vascular bundles, which is distinguished from typical non-Kranz anatomy. The C₄ species extended their ecological ranges from wet habitats to dry savanna grasslands, while the C₃ species showed the best development in wet habitats. The C₃ species were widespread from tropical to temperate regions with partial range extension into subarctic regions of both hemispheres, showing conspicuously high concentration of species in the New World, but being absent from arid climatic regions. The C₄ species were distributed mostly in tropics and subtropics, showing two separate distributional centers in South and Central America and in Tropical Asia and Australia. The range of C₄ species was nearly completely included in the C₃ range. In conclusion, it seems that inRhynchospora the C₄ syndrome evolved relatively recently, and arose in at least two separate phylogenetic trends in the tropics and the subtropics, more probably in the Neotropics.     

Aminergic and peptidergic amplification of intracellular cyclic AMP levels in a molluscan neural network

1. Serotonin (5-hydroxytryptamine; 5-HT), dopamine (DA), and small cardioactive peptide B (SCPB) can activate adenylate cyclase and increase the intracellular cyclic AMP (cAMP) levels in the Limax procerebrum (PC), with differing time courses and to differing extents. 5-HT and SCPB are potent stimulators of adenylate cyclase, and when both were applied simultaneously, an additive effect was observed. 2. In contrast, DA shows a great variability in the time course of cAMP synthesis and is a weak stimulator. Ergonovine, a DA antagonist, failed to inhibit cyclase activation, indicating that ergonovine-sensitive receptors are absent or ergonovine-sensitive DA receptors are not coupled to adenylate cyclase. 3. 5-HT and SCPB cause a rapid synthesis of cAMP, reaching the maximum 20- to 30-fold increase within a minute. DA's effect is slow in onset and very prolonged, reaching a maximum of only a two- to three-fold increase in the cAMP level. Reasons for variability in DA action are discussed.