Picosecond transient absorption spectroscopy in the blue spectral region of photosystem I

Picosecond transient absorption difference spectroscopy in the blue wavelength region (380-500 nm) was used to study the early electron acceptors in photosystem I. Samples were photosystem I core particles with about 100 chlorophylls per reaction center isolated from the cyanobacterium Synechocystis sp. PCC 6803. After excitation at 590 nm at room temperature, decay-associated spectra (DAS) were determined from global analysis in the blue region, yielding two transient components and one nondecaying component. A 3 ps decay phase is interpreted as primarily due to antenna excited-state redistribution. A 28 ps decay phase is interpreted as due to overall excited-state decay by electron transfer. The nondecaying component is ascribed to the difference spectrum of P(700) and the quinone or A(1) electron acceptor (P(700)(+)A(1)(-) - P(700)A(1)). Decay curves on the millisecond time scale at different wavelengths were measured with an autoxidizable artificial electron acceptor, benzyl viologen, and the (P(700)(+) - P(700)) difference spectrum was constructed. The (A(1)(-) - A(1)) difference spectrum was obtained by taking the difference between the above two difference spectra. A parallel picosecond experiment under strongly reducing conditions was also done as a control experiment. These conditions stabilize the electron on an earlier acceptor, A(0). The nondecaying component of the DAS at low potential was assigned to (P(700)(+)A(0)(-) - P(700)A(0)) since the electron-transfer pathway from A(0) to A(1) was blocked. The [(P(700)(+)A(0)(-) - P(700)A(0)) - (P(700)(+) - P(700))] subtraction gives a spectrum, interpreted as the (A(0)(-) - A(0)) difference spectrum of a chlorophyll a molecule, consistent with previous studies. The (A(1)(-) - A(1)) spectrum resolved on the picosecond time scale shows significant differences with similar spectra measured on longer time scales. These differences may be due to electrochromic effects and spectral evolution.     

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.     

Spatial genetic structure in populations of the terrestrial orchid Cephalanthera longibracteata (Orchidaceae)

Orchid seeds are unusual for being the smallest among flowering plants. These dust-like seeds are wind-borne and, thus, would seem to have the potential for long-distance dispersal (a common perception); this perception has led to a prediction of near-random spatial genetic structure within orchid populations. Mathematical models (e.g., simple ballistic model) for wind-dispersed seeds and wind-tunnel experiments, in contrast, indicate that most seeds of orchids should fall close to the maternal plant (<6 m), supporting a prediction of significant fine-scale genetic structure within populations. In reality we do not know much about seed dispersion in orchids. To determine which of these two predictions is more appropriate, Wright's F statistics and spatial autocorrelation analysis were used to examine the genetic structure within two adult populations of the terrestrial orchid Cephalanthera longibracteata (Orchidaceae) in southern Korea. In results comparable to those of other self-compatible, mixed-mating plant species, C. longibracteata populations exhibited low levels of genetic diversity (mean H(e) = 0.036) and a significant excess of homozygosity (mean F(IS) = 0.330), consistent with substantial inbreeding via selfing and/or mating among close relatives in a spatially structured population. Spatial autocorrelation analysis revealed significant positive genetic correlations among plants located <10 m, with relatedness at <3 m comparable to that expected for half sibs and first cousins. This genetic structure supports the prediction that the majority of seed dispersal occurs over distances of less than 10 m and is responsible for generating substantial overlap in seed shadows within C. longibracteata populations.     

Telomeric DNA damage by topoisomerase I. A possible mechanism for cell killing by camptothecin

Topoisomerase I adjusts torsional stress in the genome by breaking and resealing one strand of the helix through a transient covalent coupling between enzyme and DNA. Camptothecin, a specific topoisomerase I poison, traps this covalent intermediate, thereby damaging the genome. Here we examined the activity of topoisomerase I at telomeric repeats to determine whether telomere structures are targets for DNA damage. We show that topoisomerase I is catalytically active in cleaving the G-rich telomeric strand in vitro in the presence of camptothecin but not in cleaving the C-rich strand. The topoisomerase I cleavage site is 5'-TT (downward arrow) AGGG-3' (cleavage site marked by the downward arrow). We also show that endogenous topoisomerase I can access telomeric DNA in vivo and form camptothecin-dependent covalent complexes. Therefore, each telomeric repeat represents a potential topoisomerase I cleavage site in vivo. Because telomere structures are comprised of a large number of repeats, telomeres in fact represent a high concentration of nested topoisomerase I sites. Therefore, more telomeric DNA damage by camptothecin could occur in cells with longer telomeres when cells possess equivalent levels of topoisomerase I. The evidence presented here suggests that DNA damage at telomeric repeats by topoisomerase I is a prominent feature of cell killing by camptothecin and triggers camptothecin-induced apoptosis.     

Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect

The prophenoloxidase (proPO) cascade is a major innate immune response in invertebrates, which is triggered into its active form by elicitors, such as lipopolysaccharide, peptidoglycan, and 1,3-beta-D-glucan. A key question of the proPO system is how pattern recognition proteins recognize pathogenic microbes and subsequently activate the system. To investigate the biological function of 1,3-beta-D-glucan pattern recognition protein in the proPO cascade system, we isolated eight different 1,3-beta-D-glucan-binding proteins from the hemolymph of large beetle (Holotrichia diomphalia) larvae by using 1,3-beta-D-glucan immobilized column. Among them, a 20- and 17-kDa protein (referred to as Hd-PGRP-1 and Hd-PGRP-2) show high sequence identity with the short forms of peptidoglycan recognition proteins (PGRPs-S) from human and Drosophila melanogaster. To be able to characterize the biochemical properties of these two proteins, we expressed them in Drosophila S2 cells. Hd-PGRP-1 and Hd-PGRP-2 were found to specifically bind both 1,3-beta-D-glucan and peptidoglycan. By BIAcore analysis, the minimal 1,3-beta-D-glucan structure required for binding to Hd-PGRP-1 was found to be laminaritetraose. Hd-PGRP-1 increased serine protease activity upon binding to 1,3-beta-D-glucan and subsequently induced the phenoloxidase activity in the presence of both 1,3-beta-D-glucan and Ca(2+), but no phenoloxidase activity was elicited under the same conditions in the presence of peptidoglycan and Ca(2+). These results demonstrate that Hd-PGRP-1 can serve as a receptor for 1,3-beta-D-glucan in the insect proPO activation system.     

Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate

High-resolution crystallographic analysis of a complex of the serine-carboxyl proteinase sedolisin with pseudo-iodotyrostatin revealed two molecules of this inhibitor bound in the active site of the enzyme, marking subsites from S3 to S3('). The mode of binding represents two products of the proteolytic reaction. Substrate specificity of sedolisin was investigated using peptide libraries and a new peptide substrate for sedolisin, MCA-Lys-Pro-Pro-Leu-Glu#Tyr-Arg-Leu-Gly-Lys(DNP)-Gly, was synthesized based on the results of the enzymatic and crystallographic studies and was shown to be efficiently cleaved by the enzyme. The kinetic parameters for the substrate, measured by the increase in fluorescence upon relief of quenching, were: k(cat)=73+/-5 s(-1), K(m)=0.12+/-0.011 microM, and k(cat)/K(m)=608+/-85 s(-1)microM(-1).     

Reproduction and development of the conspicuously dimorphic brittle star Ophiodaphne formata (Ophiuroidea)

Ophiodaphne formata is a conspicuously dimorphic ophiuroid; the disk diameters are approximately 1 mm for males and 5 mm for females. The dwarf male clings to the larger female, with the oral surfaces and bursae of the paired ophiuroids closely appressed. Moreover, the female of each pair adheres aborally to the oral surface of a host sand dollar, Astriclypeus manni. Spawning and external fertilization occur in August, at Tsuruga Bay, Sea of Japan. Development of the dimorphic brittle star O. formata is described for the first time, from spawning through metamorphosis, with special attention to the formation of the skeletal system and the external morphology of early juveniles. Fertilized eggs are about 90 microm in diameter, pale pink, and negatively buoyant. The embryos undergo equal, total, and radial cleavage, and the larval skeleton first forms as a pair of tetraradiate spicules. Larval development proceeds to an 8-armed planktotrophic ophiopluteus, with skeletal elements that consist of a body rod and two recurrent rods. Three weeks after fertilization, all the pluteal arms, except for the postero-lateral arms, are absorbed, and the metamorphosing larvae sink to the bottom. Metamorphosis is completed 21.5 days after fertilization, and the resulting juvenile is pentagonal and approximately 270 microm in diameter. The smallest specimen (480 microm in disk diameter) collected by field sampling exhibited male features on the skeletal plates of the jaw and disk. Sexual dimorphism, the peculiar pairing behavior, and the close relationship with the host sand dollar may have evolved as distinct reproductive characteristics in this ophiuroid with its typical ophiopluteus larvae.     

Helix stability confers salt resistance upon helical antimicrobial peptides

Salt sensitivity of antimicrobial peptides poses a major obstacle in their development as novel antibiotics. Here we report the use of helix-capping motifs to confer salt resistance upon helical antimicrobial peptides. The helical content of the template peptide [RLLR](5) was almost completely destroyed at salt concentrations over 200 mm NaCl, leading to a 8-32-fold decrease in antimicrobial activity. However, the introduction of helix-capping motifs at the helix termini resulted in a structurally stable peptide, which retained membrane-permeabilizing and antimicrobial activities upon exposure to salt. Furthermore, the peptide with helix-capping motifs directly inhibited the in vivo growth of Streptococcus pyogenes, which causes localized fasciitis in mice, and prevented the necrosis of the epidermis, dermis, and subcutaneous muscle layers. Results indicate that the adoption of helix-capping motifs into salt-sensitive antimicrobial peptides provides the necessary structural stability for the peptides to permeabilize cell membranes and cause cell death at physiological salt concentrations.