Adenylate regulation of photosynthetic electron transport and the coupling sites of phosphorylation in spinach chloroplasts

The regulation by adenylates of activities of various partial electron transport systems in spinach chloroplasts was studied using systems from H₂O to 2,5-dimethyl-p-benzoquinone, H₂O to 2,6-dichlorophenolindophenol, reduced 2,6-dichlorophenolindophenol to methyl viologen, and H₂O to methyl viologen or ferricyanide. Adenylates regulated all of them. The ratio of the amount of esterified Pi (P) to that of electrons transported (e) in coupling with phosphorylation manifested that there are two phosphorylation sites: one between H₂O and 2,5-dimethyl-p-benzoquinone or 2,6-dichlorophenolindophenol and another between reduced 2,6-dichlorophenolindophenol and methyl viologen, under the proposed stoichiometries,i.e., P/H⁺=0.5 and H⁺/e=1, where H⁺ is the amount of protons pumped by electron transport (= those translocated during phosphorylation), when the basal electron transport (the part not regulated by adenylates) was excluded. The effects of pH, phlorizin, and methylamine on the adenylate regulation of electron transport, and the stimulation profile of electron transport coupled with quasiarsenylation suggested no distinction between the two phosphorylation sites.     

Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori

β-DNA, a component of DNA found in the pupal fat body of the silkworm, Bombyx mori, has the same GC content but a smaller molecular weight than typical silkworm DNA (α-DNA). Its origin and time of synthesis were studied by MAK column chromatography of phenol extracts after labelling with radioactive precursors.The DNA components of the fat body changed greatly during the early pupal stage, the β-DNA showing a striking increase relative to α-DNA. Thymidine-6-³H and phosphoric acid-³²P injected into the animals 1 day before analysis caused labelling of α-DNA, but not of β-DNA of the fat body, indicating that β-DNA was not synthesized during the stage of its appearance in the fat body.On the other hand, injection of thymidine-6-³H into 2-day-old fifth instar larvae, when DNA of the silk gland was being actively synthesized, gave high incorporation of the isotope into β-DNA of the pupal fat body. The sudden appearance of highly labelled β-DNA in the fat body during the early pupal stage as well as the occurrence of β-DNA in both the silk gland and fat body suggested that DNA might move from the silk gland to the fat body.It is possible that the fat body stores DNA as a nutrient from the degenerating silk gland.     

Inhibition and uncoupling of the ADP-regulated electron transport in isolated chloroplasts

The effects of energy transfer inhibitors, electron transport inhibitors and uncouplers on the ADP-regulated and ADP-independent activity of ferricyanide reduction in isolated spinach chloroplasts were studied. Phlorizin and sulfate did not affect the ADP-independent ferricyanide reduction. In the ADP-regulated reduction, these reagents did not affect the ADP inhibition process but inhibited the activity restoration process due to phosphorylation. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and linolenic acid depressed both ADP-regulated and ADP-independent activity of ferricyanide reduction. Gramicidin S and 2-amino-1-butanol depressed ADP-regulated activity and stimulated ADP-independent activity. The decrease in the ADP-regulated ferricyanide-reducing activity (restoration) due to (incomplete) uncoupling paralleled the decrease in phosphorylation activity (P/Δe=1).     

SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

Carbon cycling and sequestration in a Japanese red pine (Pinus densiflora) forest on lava flow of Mt. Fuji

Biometric-based carbon flux measurements were conducted in a pine forest on lava flow of Mt. Fuji, Japan, in order to estimate carbon cycling and sequestration. The forest consists mainly of Japanese red pine (Pinus densiflora) in a canopy layer and Japanese holly (Ilex pedunculosa) in a subtree layer. The lava remains exposed on the ground surface, and the soil on the lava flow is still immature with no mineral soil layer. The results showed that the net primary production (NPP) of the forest was 7.3 ± 0.7 t C ha^?1 year^?1, of which 1.4 ± 0.4 t C ha^?1 year^?1 was partitioned to biomass increment, 3.2 ± 0.5 t C ha^?1 year^?1 to above-ground fine litter production, 1.9 t C ha^?1 year^?1 to fine root production, and 0.8 ± 0.2 t C ha^?1 year^?1 to coarse woody debris. The total amount of annual soil surface CO₂ efflux was estimated as 6.1 ± 2.9 t C ha^?1 year^?1, using a closed chamber method. The estimated decomposition rate of soil organic matter, which subtracted annual root respiration from soil respiration, was 4.2 ± 3.1 t C ha^?1 year^?1. Biometric-based net ecosystem production (NEP) in the pine forest was estimated at 2.9 ± 3.2 t C ha^?1 year^?1, with high uncertainty due mainly to the model estimation error of annual soil respiration and root respiration. The sequestered carbon being allocated in roughly equal amounts to living biomass (1.4 t C ha^?1 year^?1) and the non-living C pool (1.5 t C ha^?1 year^?1). Our estimate of biometric-based NEP was 25 % lower than the eddy covariance-based NEP in this pine forest, due partly to the underestimation of NPP and difficulty of estimation of soil and root respiration in the pine forest on lava flows that have large heterogeneity of soil depth. However, our results indicate that the mature pine forest acted as a significant carbon sink even when established on lava flow with low nutrient content in immature soils, and that sequestration strength, both in biomass and in soil organic matter, is large.     

Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor gamma-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates.     

Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization

To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.