Topographic variations of soil properties and stand structure in aPinus thunbergii plantation

Soil properties and above-and belowground forest structure were studied across various topographies in a 20-year-oldPinus thunbergii Parl. plantation on Mt Tanakami, Japan. The soil properties and stand structure varied greatly with slope position fromridge top to valley floor. Soil thickness, fine soil content and soil moisture content were greater in lower slope positions. The amount of organic carbon in the forest floor was greater in upper slope positions. The organic carbon content in the mineral soil was slightly greater in lower slope positions. These changes in soil properties suggested an upslope decrease in decomposition rate and water and/or nutrient availability. The aboveground structure ofP. thunbergii was more developed at lower slope positions. The mean stem diameter, height and volume ofP. thunbergii increased downslope with decreasing tree density. However, fine root biomass increased greatly upslope. This inverse relationship between tree height and fine root biomass indicated morphological plasticity ofP. thunbergii in exploiting environmental heterogeneity. Variations in soil-plant interactions in the stand along various topographies caused spatial heterogeneity in the accumulation pattern of organic matter in plants and the soil.     

DNA helicase activity in Werner's syndrome gene product synthesized in a baculovirus system.

The gene responsible for Werner's syndrome (WRN) contains a region homologous to the Escherichia coli RecQ type DNA helicase and was thought to code for a DNA helicase belonging to this helicase family. However, no evidence has been shown before to substantiate this prediction. Here, we show data that the product of the WRN gene is indeed a DNA helicase. The gene product, a polypeptide with a relative molecular mass of 170 kDa, expressed in the insect Spodoptera frugiperda (Sf21) cell and purified by affinity column chromatography contained both the ATPase and DNA unwinding activities characteristic of DNA helicase. Expressions in Sf21, as well as in HeLa cells, showed that the WRN DNA helicase is exclusively transported to the nucleoplasm, which is consistent with its function in DNA metabolism. Our studies on strand displacement suggest that WRN helicase can unwind not only a duplex DNA, but also an RNA-DNA heteroduplex, while the latter reaction seems less efficient. Enzymological features learned from the purified WRN helicase are discussed with respect to the biological function, which remains to be clarified.     

Adjuvanticity of an Ornithine-Containing Lipid of Flavobacterium meningosepticum as a Candidate Vaccine Adjuvant

Ornithine-containing lipids (OrnL) extracted from Flavobacterium meningosepticum have been reported to have various biological activities such as B-cell mitogenicity and macrophage activation to generate interleukin-1 and prostaglandin E₂. We, using ovalbumin (OVA) as an antigen, evaluated the adjuvant activity of OrnL as an immunological adjuvant in BALB/c mice. OrnL showed the function of forming liposome-like vesicles retaining biological activities when prepared as either small unilamellar or dehydration-rehydration vesicles. Although OrnL was not shown to have enough entrapping efficacy for use as a vaccine adjuvant, phosphatidylglycerol (PG) and cholesterol (CHOL) added to stabilize the vesicle membrane increased the entrapping efficacy to the same extent as that of conventional liposomes. Furthermore, the stabilized OrnL vesicles tolerated centrifugation to remove non-entrapped antigens. Completely antigen-entrapped OrnL vesicles including PG and CHOL induced a significantly greater enhancement of IgG antibody production than did aluminum hydroxide gel in BALB/c mice from week 6. These results indicate that OrnL can be utilized as an immunological adjuvant for vaccines.     

Coral reef ecosystems as a source of atmospheric CO2: evidence from PCO2 measurements of surface waters

 Precise measurements of the partial pressure of CO₂ (PCO₂) in oceanic and lagoonal surface waters from Palau Barrier Reef and Majuro Atoll were taken to ascertain the net effect of these coral reef systems on air-sea CO₂ exchange. PCO₂ in the surface water in the lagoon of Palau Barrier Reef and Majuro Atoll was 48 and 14 μatm higher than in the surrounding oceanic water, respectively. A total carbon dioxide vs. total alkalinity diagram indicates that calcification is mainly responsible for CO₂ evasion in these coral reef systems. Accepted: 31 March 1997     

THE FRESHWATER CYANOBACTERIUM PLANKTOTHRIX SP. FP1: MOLECULAR IDENTIFICATION AND DETECTION OF PARALYTIC SHELLFISH POISONING TOXINS

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix . In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre-column and post-column derivatization high-performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage-dependent Na⁺ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na⁺ current at a toxin concentration of 200 nM), and STX was detected only by pre-column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.     

Susceptibility of various developmental stages of the maize weevil, Sitophilus zeamais Motschulsky (Col., Curculionidae) to methyl iodide in brown rice

Abstract:  The efficacy of methyl iodide (MI) as a fumigant against all developmental stages of the maize weevil, Sitophilus zeamais Motsch. was investigated. Tests were conducted with concentrations of 1.5, 1.8, 2.1, 2.4, 2.7 and 3.0 mg/l, for a 6-h exposure period. Values of LC₅₀, LC₉₅ and LC₉₉ of MI for immatures and adult stages were determined. The present laboratory tests showed that MI was toxic to various life stages of S. zeamais at relatively short exposure periods. At the LC₅₀ and LC₉₅ levels, the most susceptible stage was the egg stage followed by larvae, pupae and adults (1-day mortality). The egg was found to be most susceptible to MI, requiring 0.81 and 2.16 mg/l for 50 and 99% mortality, respectively, while the adult was most tolerant, requiring 2.30 and 3.02 mg/l for 50 and 99% mortality, respectively, based on 1-day mortality count. Pupae were less susceptible to MI than egg and larvae, requiring 1.47 and 3.19 mg/l for 50 and 99% mortality, respectively. Based on the present toxicity tests, MI has the potential for use as a fumigant to control all developmental stages of the maize weevil, S. zeamais .     

Tsukiyamaia,a new genus of the tribe Baorini (Lepidoptera,Hesperiidae, Hesperiinae)

Skippers of the tribe Baorini are evidently a monophyletic group in the subfamily Hesperiinae. In this study, a new Baorini member Tsukiyamaia albimacula gen. n. et sp. n. is described from north Myanmar, southwest China and north Vietnam. Despite its peculiar and striking wing-pattern, this new genus has some important characters of Baorini, such as a broad and bifid uncus and a well-developed gnathos. Based on an analysis of male genitalia and the molecular phylogenies inferred from both mitochondrial and nuclear genes (28 taxa, total aligned length: 2968 bp), it is proposed that the genus Tsukiyamaia is closely related to the genus Polytremis, which has high species diversity in China. This study not only describes a new skipper but also highlights that Tsukiyamaia is important in clarifying phylogenetic relationship of Polytremis and its allies.     

The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1

Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1–AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.Membrane trafficking in plants shares many fundamental features with those in yeast and animals (Bassham et al., 2008). In general, vacuolar proteins are synthesized on the rough endoplasmic reticulum and then transported to vacuoles via the Golgi apparatus (Xiang et al., 2013; Robinson and Pimpl, 2014). The vacuolar trafficking in plants has been studied by monitoring the transport of reporter proteins to lytic vacuoles in vegetative cells and tissues (Jin et al., 2001; Pimpl et al., 2003; Miao et al., 2008; Niemes et al., 2010). Recently, seed storage proteins became a model cargo for monitoring the transport of endogenous vacuolar proteins in plants (Shimada et al., 2003a; Sanmartín et al., 2007; Isono et al., 2010; Pourcher et al., 2010; Uemura et al., 2012; Shirakawa et al., 2014). During seed maturation, a large amount of storage proteins are synthesized and sorted to specialized vacuoles, the protein storage vacuoles (PSVs). To properly deliver vacuolar proteins, sorting receptors play a critical role in recognizing the vacuole-targeting signal of the proteins. VACUOLAR PROTEIN SORTING10 and Man-6-P receptor function as sorting receptors for vacuolar/lysosomal proteins in the trans-Golgi network (TGN) of yeast and mammals, respectively. The best-characterized sorting receptors in plants are VACUOLAR SORTING RECEPTOR (VSR) family proteins (De Marcos Lousa et al., 2012). VSRs have been shown to function in sorting both storage proteins to PSVs (Shimada et al., 2003a; Fuji et al., 2007) and lytic cargos to lytic vacuoles (Zouhar et al., 2010).To sort the receptors in the TGN into vacuoles/lysosomes, the adaptor protein (AP) complex binds the cytosolic domain of the receptors. The AP complexes form evolutionarily conserved machinery that mediates the post-Golgi trafficking in eukaryotic cells (Robinson, 2004). There are five types of AP complexes, AP-1 to AP-5. The functions of AP-1, AP-2, and AP-3 have been established. AP-1 appears to be involved in trafficking between the TGN and endosomes (Hirst et al., 2012), AP-2 is involved in clathrin-mediated endocytosis (McMahon and Boucrot, 2011), and AP-3 is involved in protein trafficking from the TGN/endosomes to the vacuole/lysosomes (Dell’Angelica, 2009). However, little is known about AP-4 and AP-5. Mammalian AP-4 may be involved in basolateral sorting in polarized cells and in the transport of specific cargo proteins, such as the amyloid precursor protein APP, from the TGN to endosomes (Burgos et al., 2010). The fifth AP complex, AP-5, was recently identified, and its orthologs are widely conserved in the eukaryotic genomes (Hirst et al., 2011). The AP complexes exist as heterotetrameric proteins that consist of two large subunits (β1-5 and one each of ɣ/α/δ/ε/ζ), one medium subunit (µ1-5), and one small subunit (σ1-5). The sorting mechanism is best characterized for the medium (µ) subunit, which is known to recognize the Tyr-based YXXФ motif (where Ф represents Leu, Ile, Phe, Met, or Val) that is present in the cytosolic domains of cargo proteins (Ohno et al., 1995). Mutations of the YXXФ motif abolish the interaction with µ and alter the subcellular localization of the cargo proteins.The genome of Arabidopsis (Arabidopsis thaliana) contains all five sets of putative AP genes (Bassham et al., 2008; Hirst et al., 2011). The function of AP-4 in membrane trafficking and its physiological roles in plants are largely unknown. In this study, we identified and characterized the AP-4 complex in Arabidopsis. Mutants lacking the AP-4 subunits exhibited defects in VSR1-mediated vacuolar sorting of storage proteins in seeds. Our results provide new insights into the receptor-mediated vacuolar trafficking in post-Golgi pathways.     

Natural hybridization of Yezo and Sakhalin spruce in central Hokkaido,revealed by DNA markers with contrasting modes of inheritance

Yezo spruce (Picea jezoensis var. jezoensis) and Sakhalin spruce (Picea glehnii) occur across Hokkaido and co‐occur in some forest habitats. This leads to the potential for natural hybridization between these two species, which has been shown to occur at low frequencies. The purpose of this study was to identify these hybrids and their possible mating patterns, using various Pinaceae DNA markers with different modes of inheritance. The markers used were maternally inherited mitochondrial DNA (mtDNA), paternally inherited chloroplast DNA (cpDNA) and biparentally inherited nuclear microsatellites (nSSRs). Seven putative natural hybrids, four artificially‐crossed F₁ hybrids, four parent plants from each species, and two artificially‐backcrossed hybrids of putative natural hybrids and their parents were analyzed using the diagnostic DNA markers developed in this study. We found Yezo spruce and Sakhalin spruce to be distinct (J and G types, respectively), and the modes of inheritance held true for the two species, as was previously reported to be the case in Pinaceae. Four of the seven putative natural hybrids harbored J‐type cpDNA, G‐type mtDNA and J/G‐type nSSRs, indicating that natural F₁ hybrids are likely to arise from a G (female) × J (male) crossing. One natural hybrid harbored G‐type cpDNA, J‐type mtDNA and J/G‐type nSSRs, which implies that hybrids produced by J (female) × G (male) crossings occur at low frequencies. The two remaining hybrids harbored J‐type cpDNA and mtDNA with either J/G or J/J‐type nSSRs, suggesting that they may be F₂ hybrids resulting from backcrossing between an F₁ hybrid and a Yezo spruce.