Phylogeny and biogeography of the genus Ainsliaea (Asteraceae) in the Sino-Japanese region based on nuclear rDNA and plastid DNA sequence data

BACKGROUND AND AIMS: The flora of the Sino-Japanese plant region of eastern Asia is distinctively rich compared with other floristic regions in the world. However, knowledge of its floristic evolution is fairly limited. The genus Ainsliaea is endemic to and distributed throughout the Sino-Japanese region. Its interspecific phylogenetic relationships have not been resolved. The aim is to provide insight into floristic evolution in eastern Asia on the basis of a molecular phylogenetic analysis of Ainsliaea species. METHODS: Cladistic analyses of the sequences of two nuclear (ITS, ETS) and one plastid (ndhF) regions were carried out individually and using the combined data from the three markers. KEY RESULTS: Phylogenetic analyses of three DNA regions confirmed that Ainsliaea is composed of three major clades that correspond to species distributions. Evolution of the three lineages was estimated to have occurred around 1.1 MYA during the early Pleistocene. CONCLUSIONS: The results suggest that Ainsliaea species evolved allopatrically and that the descendants were isolated in the eastern (between SE China and Japan, through Taiwan and the Ryukyu Islands) and western (Yunnan Province and its surrounding areas, including the Himalayas, the temperate region of Southeast Asia, and Sichuan Province) sides of the Sino-Japanese region. The results suggest that two distinct lineages of Ainsliaea have independently evolved in environmentally heterogeneous regions within the Sino-Japanese region. These regions have maintained rich and original floras due to their diverse climates and topographies.     

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments

By affinity chromatography utilizing alpha-cobrotoxin from digitonin-solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin-dystrophin-associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802-807), alpha-actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X-100 solubilization released dystrophin-DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5-4 microM) significantly activated Mg(2+)-ATPase activity of digitonin-solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg(2+)-ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system.     

Lipopolysaccharide enhances the production of nicotine-induced prostaglandin E2 by an increase in cyclooxygenase-2 expression in osteoblasts

Previous studies have indicated that lipopolysaccharide(LPS)from Gram-negative bacteria inplaque induces the release of prostaglandin E_2(PGE_2),which promotes alveolar bone resorption in periodontitis,and that tobacco smoking might be an important risk factor for the development and severity of periodontitis.We determined the effect of nicotine and LPS on alkaline phosphatase(ALPase)activity,PGE_2 production,and the expression of cyclooxygenase(COX-1,COX-2),PGE_2 receptors Ep1-4,and macrophage colonystimulating factor(M-CSF)in human osteoblastic Saos-2 cells.The cells were cultured with 10~(-3)M nicotinein the presence of 0,1,or 10μg/ml LPS,or with LPS alone.ALPase activity decreased in cells cultured withnicotine or LPS alone,and decreased further in those cultured with both nicotine and LPS,whereas PGE_2production significantly increased in the former and increased further in the latter.By itself,nicotine did notaffect expression of COX-1,COX-2,any of the PGE_2 receptors,or M-CSF,but when both nicotine and LPSwere present,expression of COX-2,Ep3,Ep4,and M-CSF increased significantly.Simultaneous addition of10~(-4)M indomethacin eliminated the effects of nicotine and LPS on ALPase activity,PGE_2 production,and M-CSF expression.Phosphorylation of protein kinase A was high in cells cultured with nicotine and LPS.Theseresults suggest that LPS enhances the production of nicotine-induced PGE_2 by an increase in COX-2 expres-sion in osteoblasts,that nicotine-LPS-induced PGE_2 interacts with the osteoblast Ep4 receptor primarily inautocrine or paracrine mode,and that the nicotine-LPS-induced PGE_2 then decreases ALPase activity andincreases M-CSF expression.     

Alternative and cyclic appearance of H2 and O2 photoproduction activities under non-growing conditions in an aerobic nitrogen-fixing unicellular cyanobacterium Synechococcus sp.

With synchronously grown cells of an aerobic unicellular marine cyanobacterium Synechococcus sp. Miami BG 043511, changes in the activities of anoxygenic, nitrogenase-dependent hydrogen photoproduction and oxygen photoevolution were measured under non-growing hydrogen production conditions. Interestingly, synchronously grown cells, incubated in light under an argon atmosphere, exhibited cyclic changes in the activity of nitrogenase-catalyzed hydrogen production for approximately 20- to 25-h intervals. Cyclic photosynthetic oxygen production activity also appeared in approximately the same intervals. However, changes in hydrogen production and oxygen production activities were inversely correlated and temporally separated. Stepwise accumulation of hydrogen in closed vessels was also observed in approximately 24-h cycles in these non-growing cells. These observations in non-growing cells suggest that this unicellular aerobic, nitrogen-fixing cyanobacterium may have an endogenous system to control the exhibition of cyclic rhythms, in addition to the previously studied cell cycle-oriented system. Expression and switching between these two systems may be related to the sufficiency or insufficiency of nitrogen growth nutrients. The possibility of the existence of a common control factor of the two systems involving glycogen is also discussed.     

Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells

The characteristics of glycosaminoglycan (GAG) synthesis in normal and transformed human endothelial cells were analyzed by the incorporation of [3H]glucosamine and by the activities of GAG synthetases. The GAG synthesized by normal endothelial cells consisted of mainly heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate but little hyaluronic acid (HA) (less than 1%). The characteristics of GAG synthesis by normal cells reflected the synthetic enzyme activities for each individual GAG: the activity of HA synthetase was very low. In spite of this, the activity of HA synthetase inhibitor, induced in growth-retarded fibroblasts with low HA synthetase activity (Matuoka et al. (1987 J. Cell Biol., 104, 1105-1115), was very low in endothelial cells. In contrast to normal cells, transformed endothelial (ECV304) cells synthesized mainly HA (62% of total GAGs). These findings suggest that the regulatory system of GAG metabolism is cell type specific, and that transformation is accompanied by high levels of HA synthesis in endothelial cells.     

A cytokinin-binding protein complex from tobacco leaves

A cytokinin binding protein complex (CBP130) has been purified from tobacco leaves (Nicotiana sylvestris). It contains two protein species of 57 and 36 kDa (CBP57 and CBP36). The cDNAs encoding CBP57 have been isolated from a tobacco cDNA library. Their predicted amino acid sequences showed significant homology between CBP57 and S-adenosyl-L-homocysteine (SAH) hydrolase, which catalyzes the reversible hydrolysis of SAH, a methyltransferase inhibitor. A combination of gel filtration an western blot analysis revealed that both CBP57 and benzyladenine (BA)-binding activity were eluted at a peak of 130 kDa. A purified CBP130 fraction contains SAH hydrolase activity. We discuss possible CBP57 as a cytokinin receptor subunit and its possible role as a regulator of methylation.     

Extremely Low D/H Ratios of Photoproduced Hydrogen by Cyanobacteria

Cyanobacteria, having primary photosynthetic reactions similarto higher plants, are capable of producing large quantitiesof molecular hydrogen by nitrogenase and/or hydrogenase deliveringelectrons to hydrogen ions via ferredoxin or oxidation of NADPH.We measured the deuterium/hydrogen (D/H) ratios of the hydrogengas photoproduced by Synechococcus sp. Miami BG 043511 and Anabaenasp. TU 37-1, and demonstrate that D values of their hydrogengas are extremely low (about – 600%) when compared withthat of available water (–7%).This depletion gives a meanfractionation factor (a) of 0.43, which is similar to that calculatedfor hydrogen ions at equilibrium with water (0.35) and hydrogenproduced by electrolysis of water (0.24) but significantly differentfrom those of carbon bound hydrogens (>0.83). Thus hydrogenions available for protonation of NADP⁺ may be extremely deuteriumdepleted. Our results may explain why D/H ratios of nitratedcellulose or lipids from most plants are always depleted relativeto water available for photosynthesis. ³ On leave from School of Marine Science and Technology, TokaiUniversity, 3-20-1 Orido, Shimizu, 424 Japan (Received April 1, 1991; Accepted June 21, 1991)     

New crystal form of recombinant murine interferon-beta

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.     

Crystal structure at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN'. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region"). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).