Intra‐trophic isotopic discrimination of 15N/14N for amino acids in autotrophs: Implications for nitrogen dynamics in ecological studies

The differential discrimination of nitrogen isotopes (¹⁵N/¹⁴N) within amino acids in consumers and their diets has been routinely used to estimate organismal tropic position (TP). Analogous isotopic discrimination can occur within plants, particularly in organs lacking chloroplasts. Such discrimination likely arises from the catabolic deamination of amino acids, resulting in a numerical elevation of estimated TP, within newly synthesized biomass. To investigate this phenomenon, we examined the ¹⁵N/¹⁴N of amino acids (δ¹⁵N_AA) in spring leaves and flowers from eight deciduous and two annual plants. These plants were classified on the basis of their time of bloom, plants that bloomed when their leaves were absent (Type I) versus plants that bloomed while leaves were already present (Type II). Based on the δ¹⁵N_AA values from leaves, both plant types occupied comparable and ecologically realistic mean TPs (=1.0 ± 0.1, mean ± 1σ). However, the estimated TPs of flowers varied significantly (Type I: 2.2 ± 0.2; Type II: 1.0 ± 0.1). We hypothesize that these results can be interpreted by the following sequence of events: (1) Type I floral biomass is synthesized in absence of active photosynthesis; (2) the catabolic deamination of amino acids in particular, leaves behind ¹⁵N in the residual pool of amino acids; and (3) the incorporation of these ¹⁵N‐enriched amino acids within the biomass of Type I flowers results in the numerical elevation of the TPs. In contrast, the actively photosynthesizing Type II leaves energetically sustain the synthesis of Type II flower biomass, precluding any reliance on catabolic deamination of amino acids. Amino acids within Type II flowers are therefore isotopically comparable to the Type II leaves. These findings demonstrate the idiosyncratic nature of the δ¹⁵N_AA values within autotrophic organs and have implications for interpreting trophic hierarchies using primary producers and their consumers.     

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.     

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.     

Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells

Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin β1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion.     

Mass mortality of Japanese macaques in a western coastal forest of Yakushima

The mass mortality of wild Japanese macaques (Macaca fuscata Blyth) was observed in a warm temperate forest of Yakushima, southern Japan. Demographic changes of eight troops between August 1998 and August 1999 were studied and 56% of macaques disappeared from the five intensively studied troops. Mortality varied among troops: two troops became extinct, while another troop did not decrease in size. The rate of mortality of the other troops was between 33 and 80%. The variation in mortality among the troops was either the outcome of local concentrations of mortality or of intertroop competition. The rate of mortality decreased with increasing distance from the two extinct troops and with increasing troop size; these two factors could not be separated statistically. The direct cause of death was diagnosed as pneumonia for four out of five fresh carcasses. The fleshy fruit production in autumn 1998 was the lowest in 14years, and macaques had relied on leaves earlier than in usual years. It was exceptionally hot and dry in the summer of 1998. The exceptionally poor fruit production and hot summer of this year, with the resulting shortage of high-quality foods, was consistent with the scenario that mass mortality was due to the poor nutritional conditions. However, the possibility that epidemics caused the mass mortality cannot be ruled out. Our findings proved that primates in a seemingly stable habitat experience fluctuations in demographic parameters under natural conditions.     

Synthesis and in vitro characterization of antigen-conjugated polysaccharide as a CpG DNA carrier

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) are known as an immune adjuvant. CpG DNAs coupled with a particular antigen enabling both CpG DNA and antigen delivery to the same antigen-presenting cell have been shown to be more effective. Based on our previous finding that beta-(1-->3)-D-glucan schizophyllan (SPG) can be used as a CpG DNA carrier, here we present the synthesis of an antigen-conjugated SPG and the characterization of the conjugate. Ovalbumin (OVA, 43 kDa) was used as a model antigen, and two OVA were conjugated to one SPG molecule (M(w) = 150,000), denoted by OVA-SPG. Circular dichroism and gel electrophoresis showed that OVA-SPG could form a complex with a (dA)(40)-tailed CpG DNA at the 3' end (1,668-(dA)(40)). When OVA-SPG was added to macrophages (J774.A1), the amount of the ingested OVA-SPG was increased compared with that of OVA itself, suggesting that Dectin-1 (proinflammatory nonopsonic receptor for beta-glucans) is involved to ingest OVA-SPG. Furthermore, the complex of the conjugate and DNA was co-localized in the same vesicles, implying that OVA (antigen) and CpG DNA (adjuvant) were ingested into the cell at the same time. This paper shows that OVA-SPG can be used as a CpG DNA carrier to induce antigen-specific immune responses.     

Isolation and Chemical Characterization of a Mating Pheromone Produced by Saccharomyces kluyveri

The pullulanase gene (pul) of Klebsiella aerogenes was transferred in vivo to Escherichia coli by using RP4:: Mu cts. The pul gene was expressed in E. coli, although the level of pullulanase activity in E. coli was lower than that in K. aerogenes, and the Pul⁺ transconjugants were relatively unstable in an unselective medium. Production of pullulanase, which is used to make maltose from starch, was induced in E. coli by pullulan, waxy maize amylopectin, soluble starch and maltose. When the transconjugant cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Retransfer of the pul_k gene from E. coli to K. aerogenes by conjugation resulted in an increase of the production of extracellular pullulanase.     

Nucleic Acid Metabolism Involved in Auxin-induced Elongation of Yeast Cells

The following results were obtained using a variant yeast strain, N55, which can respond to the cell-elongating action of auxin. Base analogs of nucleic acids (2-thiouracil, 8-azaguanine, and 5-fluorouracil) inhibited the auxin-induced elongation of yeast cells only when they were added to the preculture prior to auxin treatment. The inhibitory effect of 2-thiouracil and 5-fluorouracil was reversed by uracil and that of 8-azaguanine by guanine. Actino-mycin D inhibited the auxin-induced elongation when given to the culture containing auxin, but not when given to the preculture. The similarity in these respects between yeast and tissues of higher plants is discussed.