Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Exocytotic release of secretory granules from endocrine cells in the midgut of insects

Summary Exocytotic release of the secretory granules of the endocrine cells in the midgut of a cockroach, Periplaneta americana, was studied by means of fixation with tannic acid in combination with glutaraldehyde and osmium tetroxide. A sequence of images indicative of exocytosis suggests the following steps in this process: (1) A delicate connection appears between the granule-limiting membrane and the plasma membrane. (2) The plasma membrane approaches the granule, forming a concave indentation. (3) The granule-limiting membrane fuses with the plasma membrane and opens to give rise to an omega profile. (4) The granule content is voided into extracellular space. Exocytosis occurs not only at the base of the cell but occasionally at its side facing adjacent cells. (5) The exocytotic invagination after release becomes smaller and narrower; sometimes a coated pit with bristles appears. Multiple exocytosis, and exocytosis in the endocrine cells of the nidus, i.e., the regenerative cell mass, are also described.     

Changes in spatial distribution pattern during the larval stage of the fall webworm,Hyphantria cunea drury (Lepidoptera: Arctiidae)

Summary The changes in spatial distribution pattern during larval stage of the fall webworm,Hyphantria cunea were quantitatively investigated in the field experimental populations. The female adult deposits eggs as a cluster and the hatchlings make a compact colonial-web. In this period, the all-or-none type mortality which is characteristic in gregarious insect species was occasionary recognized before spinning a compact colonial-web. Once making a compact colonial-web, the larvae feed the leaves in the colonial-web up to about 5th instar. In this period, the movement of larvae occurred due to the local food shortage in a colonial-web and the expansion of colonial-web. As the larvae developed, the colonial-web was separated into several small groups. These larvae began to disperse about 5th instar. In this period, the local food shortage seems to be an important trigger for the larval dispersal. The mean concentration of larvae on leaves abruptly decreased, and finally the larvae became solitary at the 6th or 7th instars. The dispersal process in later larval stage is not necessarily due to the complete food shortage. The dispersal prior to the occurrence of food shortage may be a safety mechanism to protect the larvae from the food shortage.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

Time-resolved resonance Raman study on ultrafast structural relaxation and vibrational cooling of photodissociated carbonmonoxy myoglobin

A localized small structural change is converted to a higher order conformational change of protein and extends to a mesoscopic scale to induce a physiological function. To understand such features of protein, ultrafast dynamics of myoglobin (Mb) following photolysis of carbon monoxide were investigated. Recent results are summarized here with a stress on structural and vibrational energy relaxation. The core expansion of heme takes place within 2 ps but the out of plane displacement of the heme iron and the accompanying protein conformational change occur in 10 and 100 s of the picosecond regimes, respectively. Unexpectedly, it was found from UV resonance Raman spectra that Trp7 in the N-terminal region and Tyr151 in the C-terminal region undergo appreciable structural changes upon ligand binding-dissociation while Tyr104, Tyr146, and Trp14 do not. Because of the communication between the movements of these surface residues and the heme iron, the rate of spectral change of the iron-histidine (Fe- His) stretching band after CO photodissociation is influenced by the viscosity of solvent. Temporal changes of the anti-Stokes Raman intensity demonstrated immediate generation of vibrationally excited heme upon photodissociation and its decay with a time constant of 1-2 ps.     

Molecular phytogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes

We investigated the molecular phylogeny of conifers using restriction endonuclease fragment length polymorphism of six polymerase chain reaction-amplified chloroplast genes — frxC, rbcL, psbA, psbD, trnK, and 16S. We detected 227 total site changes among species, representing 23, 26, 38, 48, 67, and 25 site changes in frxC, psbA, psbD, rbcL, trnK and 16S, respectively. The mean nucleotide substitution was 10.75% (SD 0.573) among species in five families. Forty maximally parsimonious trees were obtained using the Wagner parsimony method, and a 50% majority-rule consensus tree was obtained from them. Data analysis produced similar basic patterns when both the Wagner parsimony and the neighbor-joining methods were applied, and the main lineages were clearly separated. Taxaceae and Cephalotaxaceae species were used as the out-groups when applying Wagner parsimony methods. With the Wagner method, the consistency index was 0.510, the retention index was 0.879, and tree length was 435 steps. Our results indicated that Cupressaceae and Taxodiaceae are closely related families and that Sciadopitys verticillata is the basal lineage of Cupressaceae and Taxodiaceae. The neighbor-joining tree is similar to the 50% majority-rule consensus of the 40 Wagner parsimony trees except for the position of Keteleeria daversifolia, the Picea and Cedrus group, and the divergence within Cupressaceae.     

Mass mortality of the Japanese pearl oyster Pinctada fucata martensii in relation to water temperature, chlorophyll a and phytoplankton composition

Mass mortalities of the Japanese pearl oyster Pinctada fucata martensii have widely occurred in western Japan since 1994. The causes of these mass mortalities are at present not thoroughly understood. In this study, we investigated oyster survival in relation to some environmental factors such as water temperature, concentration of chlorophyll a and density or composition of phytoplankton. The examined mass mortality occurred from September to December 1998, and the color on the adductor muscle of the oysters was red-brown, suggesting an infectious disease. Oysters that became moribund during the experiment lost weight, while the weight of unaffected oysters increased. The cell density of Nitzschia spp., an inedible algae for the oyster, in Uchiumi Bay increased before and during the mass mortality event. From the results of our study, we hypothesize that P. fucata martensii was weakened by starvation because of the dominance of inedible food and then contracted an infectious disease that resulted in mortality.     

Development and characterization of EST‐SSR markers for the genus Rhododendron section Brachycalyx (Ericaceae)

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Rhododendron section Brachycalyx in order to elucidate its evolutionary processes and reproductive ecology. Nineteen polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense. Polymorphisms for these markers were assessed using four species of section Brachycalyx. The number of alleles ranged from 1 to 14, and the observed and expected heterozygosity ranged from 0.000 to 0.931 and 0.000 to 0.904, respectively. The EST‐SSR markers developed in this study will be useful for elucidating population genetic structure and breeding systems in section Brachycalyx.     

Intraspecies host specificity of a single-stranded RNA virus infecting a marine photosynthetic protist is determined at the early steps of infection

Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.     

Settlement of Planulae of the Moon Jellyfish Aurelia aurita onto Hydrophilic Polycarbonate Plates Modified by Atmospheric Plasma Treatment

It has been reported that planula larvae of some jellyfish prefer artificial substrates for settlement. This research focused on the relationship between the settlement of planulae and the wettability of artificial substrate surfaces. We used atmospheric plasmas to change the wettability of the surfaces of polycarbonate (PC) plates because plasma treatment has no chemical side effects. The treatment made the surfaces hydrophilic, as evidenced by the decrease of contact angle from 85° to 35°. X-ray photoelectron spectroscopy revealed that the change of wettability of the PC plates could be attributed to N₂, which was probably ionized in the air above the plates. Scanning electron microscopy revealed no difference in the surface morphology of the plates before and after plasma treatment. Results of bioassays using treated PC plates showed that planulae tended to preferentially settle on hydrophobic surfaces.