Using tunable diode laser spectroscopy to measure carbon isotope discrimination and mesophyll conductance to CO₂ diffusion dynamically at different CO₂ concentrations

In C₃ leaves, the mesophyll conductance to CO₂ diffusion, g_m, determines the drawdown in CO₂ concentration from intercellular airspace to the chloroplast stroma. Both g_m and stomatal conductance limit photosynthetic rate and vary in response to the environment. We investigated the response of g_m to changes in CO₂ in two Arabidopsis genotypes (including a mutant with open stomata, ost1), tobacco and wheat. We combined measurements of gas exchange with carbon isotope discrimination using tunable diode laser absorption spectroscopy with a CO₂ calibration system specially designed for a range of CO₂ and O₂ concentrations. CO₂ was initially increased from 200 to 1000 ppm and then decreased stepwise to 200 ppm and increased stepwise back to 1000 ppm, or the sequence was reversed. In 2% O₂ a step increase from 200 to 1000 ppm significantly decreased g_m by 26–40% in all three species, whereas following a step decrease from 1000 to 200 ppm, the 26–38% increase in g_m was not statistically significant. The response of g_m to CO₂ was less in 21% O₂. Comparing wild type against the ost1 revealed that mesophyll and stomatal conductance varied independently in response to CO₂. We discuss the effects of isotope fractionation factors on estimating g_m.     

A molecular mechanism for copper transportation to tyrosinase that is assisted by a metallochaperone, caddie protein

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated “caddie,” which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16–1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.     

Lineage-Specific Distribution of Insertion Sequence Excision Enhancer in Enterotoxigenic Escherichia coli Isolated from Swine

Insertion sequences (ISs) are the simplest transposable elements and are widely distributed in bacteria; however, they also play important roles in genome evolution. We recently identified a protein called IS excision enhancer (IEE) in enterohemorrhagic Escherichia coli (EHEC) O157. IEE promotes the excision of IS elements belonging to the IS3 family, such as IS629, as well as several other families. IEE-mediated IS excision generates various genomic deletions that lead to the diversification of the bacterial genome. IEE has been found in a broad range of bacterial species; however, among sequenced E. coli strains, IEE is primarily found in EHEC isolates. In this study, we investigated non-EHEC pathogenic E. coli strains isolated from domestic animals and found that IEE is distributed in specific lineages of enterotoxigenic E. coli (ETEC) strains of serotypes O139 or O149 isolated from swine. The iee gene is located within integrative elements that are similar to SpLE1 of EHEC O157. All iee-positive ETEC lineages also contained multiple copies of IS629, a preferred substrate of IEE, and their genomic locations varied significantly between strains, as observed in O157. These data suggest that IEE may have been transferred among EHEC and ETEC in swine via SpLE1 or SpLE1-like integrative elements. In addition, IS629 is actively moving in the ETEC O139 and O149 genomes and, as in EHEC O157, is promoting the diversification of these genomes in combination with IEE.     

Estimation of dieback process caused by herbivory in an endangered root-sprouting shrub species,Paliurus ramosissimus (Lour.) Poir., using a shoot-dynamics matrix model

Mortality, a critical parameter for population dynamics, is difficult to measure in long-lived trees or clonal herbaceous species because of the extremely low frequency of deaths. A model based on shoot recruitment would be helpful to estimate the population fate of species without a sufficient number of observed deaths. Existing matrix models are applicable to the dynamics of physiologically independent shoots, but not to physiologically dependent ones. We developed a shoot-dynamics model for plants with physiologically-dependent shoots, and used the model to estimate the effects of herbivory and conservation measures on the dynamics of a long-lived, shoot-sprouting shrub species, Paliurus ramosissimus (Rhamnaceae). Two populations of the endangered shrub have been severely damaged by herbivory by sika deer. The damage was mainly to new sprouting shoots. No deaths of individual plants were observed during an 8-year survey, and we could not estimate mortality. Thus, prediction of population dynamics based on births and deaths of individuals was impossible. Because P. ramosissimus is a shoot-sprouting species, we instead estimated the decline of individuals using a shoot-dynamics model. Using this model, we estimated the time to an 80 % decrease in shoot number per individual in the two populations at 37.8 and 37.2 years. These lengths suggest an immediate need for conservation measures to prevent herbivory even though no death of any individual was observed in the field survey. The estimated recovery times from the herbivory damage were 28.7 and 29.2 years if herbivory of new shoots is completely prevented by conservation measure.     

Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a “transition zone” (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium–BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.     

The antagonistic relationship between chlorophyll a concentrations and the growth areas of Trapa during summer in a shallow eutrophic lake

The relationship between concentrations of chlorophyll a in an open water area and growing areas of the macrophytes Trapa spp. and Nelumbo nucifera was investigated to clarify the role of macrophytes in phytoplankton growth in summer in a eutrophic shallow sand lake, Lake Honsagata, Japan. The chlorophyll a concentrations formed peaks in June 2007 and July 2008 in summer with cyanobacterial blooms composed of mainly Microcystis, Anabaena, Phormidium, and Oscillatoria, which decreased toward August and were maintained at different annual levels. The decline of blooms in August was caused by the rapid growth of macrophytes. The composition of phytoplankton and the level of bloom development in summertime differed characteristically from year to year. Also the total vegetation area of N. nucifera and Trapa spp. showed annually marked changes. A significant negative correlation between the concentrations of chlorophyll a and the growth areas of Trapa spp. in August was detected, indicating that the floating-leaved plants, Trapa spp., produced irregular, clear and turbid water states in this shallow eutrophicated lake. These antagonistic relations are explained based on a likely scenario of allelopathic effects on the development of cyanobacteria by Trapa spp. vegetation and the nutrient absorption competition among them. Our study demonstrated the potential of Trapa spp. to control cyanobacterial blooms producing harmful toxins.     

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met¹), Ad(Met⁻¹), Ad(Met⁻¹²), and Ad(Met⁶). The shoulder numbers represent this site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met⁻¹), were purified from the cell lysate and the ratios of A₄₁₄-to-A₂₇₆ of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450_SCC reduction catalyzed by Ad(Met⁰) was about 60% or that by Ad(Met¹), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450_SCC would need to be considered.     

The restoration of the functions of serially passaged calf hepatocytes by spheroid formation

Summary A serial cultivation system of hepatocytes was established for the first time using calf liver as a cell source and, repeating passage of more than 30 cumulative population doublings (PDs), was obtained in the presence of long-acting ascorbic acid derivative (L-ascorbic acid 2-phosphate) and epidermal growth factor. The complete purification of hepatocytes was achieved by repeating ethylenediaminetetraacetic acid (EDTA) treatment, by which hepatocytes were easily detached from the culture dish, leaving most of the nonparenchymal cells on the dish. As the population cumulatively doubled, the cell density and albumin-synthesizing ability decreased gradually, and doubling time has exceeded 120 h at about 30 cumulative PDs. In serially passaged cells, the hepatocyte-specific histochemical and biochemical markers—including glucose-6-phosphatase, ornithine carbamoyltransferase, glutamate hydrogenase, and ammonia-metabolizing activities—have been lost after 20 cumulative PDs. However, when these passaged cells were allowed to form spheroids, the morphologic and biochemical characteristics of hepatocytes have rapidly been restored to levels comparable to those in younger generations. Because no extrinsic factor was needed for this restoration, three-dimensional cell-cell interaction would be indispensable for the differentiation of the hepatocytes. The routine serial cultivation of hepatocytes and their redifferentiation by spheroid formation will be useful for studying metabolism, gene regulation, and transplantation of hepatocytes.     

Isoflavanoid constituents from Dalbergia monetaria

The structures and isolation of eight compounds from Dalbergia monetaria seeds are described. Four of them are known rotenoids. In addition to a new isoflavone and its 7-β-D-glucoside, the first 12-dihydrorotenone, 12-dihydrodalbinol, and its 8′-β-D-glucoside were identified.