Integrating elements and energy through the metabolic dependencies of gross growth efficiency and the threshold elemental ratio

Metabolic theory proposes that individual growth is governed through the mass‐ and temperature‐dependence of metabolism, and ecological stoichiometry posits that growth is maximized at consumer‐specific optima of resource elemental composition. A given consumer's optimum, the threshold elemental ratio (TER), is proportional to the ratio of its maximum elemental gross growth efficiencies (GGEs). GGE is defined by the ratio of metabolism‐dependent processes such that GGEs should be independent of body mass and temperature. Understanding the metabolic‐dependencies of GGEs and TERs may open the path towards a theoretical framework integrating the flow of energy and chemical elements through ecosystems. However, the mass and temperature scaling of GGEs and TERs have not been broadly evaluated. Here, we use data from 95 published studies to evaluate these metabolic‐dependencies for C, N and P from unicells to vertebrates. We show that maximum GGEs commonly decline as power functions of asymptotic body mass and exponential functions of temperature. The rates of change in maximum GGEs with mass and temperature are relatively slow, however, suggesting that metabolism may not causally influence maximum GGEs. We additionally derived the theoretical expectation that the TER for C:P should not vary with body mass and this was supported empirically. A strong linear relationship between carbon and nitrogen GGEs further suggests that variation in the TER for C:N should be due to variation in consumer C:N. In general we show that GGEs may scale with metabolic rate, but it is unclear if there is a causal link between metabolism and GGEs. Further integrating stoichiometry and metabolism will provide better understanding of the processes governing the flow of energy and elements from organisms to ecosystems.     

Actin Reorganization Underlies Phototropin-Dependent Positioning of Nuclei in Arabidopsis Leaf Cells

In epidermal and mesophyll cells of Arabidopsis (Arabidopsis thaliana) leaves, nuclei become relocated in response to strong blue light. We previously reported that nuclear positions both in darkness and in strong blue light are regulated by the blue light receptor phototropin2 in mesophyll cells. Here, we investigate the involvement of phototropin and the actin cytoskeleton in nuclear positioning in epidermal cells. Analysis of geometrical parameters revealed that, in darkness, nuclei were distributed near the center of the cell, adjacent to the inner periclinal wall, independent of cell shape. Dividing the anticlinal wall into concave, convex, and intermediate regions indicated that, in strong blue light, nuclei became relocated preferably to a concave region of the anticlinal wall, nearest the center of the cell. Mutant analyses verified that light-dependent nuclear positioning was regulated by phototropin2, while dark positioning of nuclei was independent of phototropin. Nuclear movement was inhibited by an actin-depolymerizing reagent, latrunculin B, but not by a microtubule-disrupting reagent, propyzamide. Imaging actin organization by immunofluorescence microscopy revealed that thick actin bundles, periclinally arranged parallel to the longest axis of the epidermal cell, were associated with the nucleus in darkness, whereas under strong blue light, the actin bundles, especially in the vicinity of the nucleus, became arranged close to the anticlinal walls. Light-dependent changes in the actin organization were clear in phot1 mutant but not in phot2 and phot1phot2 mutants. We propose that, in Arabidopsis, blue-light-dependent nuclear positioning is regulated by phototropin2-dependent reorganization of the actin cytoskeleton.Positioning organelles is essential for cellular activities. The nucleus changes its position in a programmatic way during development and the cell cycle (Britz, 1979; Nagai, 1993; Chytilova et al., 2000). For example, before asymmetrical divisions that give rise to the formation of root hair cells or guard mother cells, the nucleus migrates to the future division plane (Britz, 1979). In elongating root hair cells of Arabidopsis (Arabidopsis thaliana), the nucleus is maintained at a fixed distance from the apex (Ketelaar et al., 2002).While the nuclear migrations before mitosis and in root hairs are developmental, nuclear positioning is also regulated environmentally. In the fern, Adiantum capillus-veneris, nuclei in prothallial cells change their intracellular positions in response to light (Kagawa and Wada, 1993, 1995). The nuclei are located along the anticlinal walls in darkness and move toward the outer periclinal walls in weak light and to the anticlinal walls in strong light (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). This response is called light-dependent nuclear positioning. Since the response is induced in cells that exhibit neither cell division nor expansion, it is believed to have a physiological role, distinct from the nuclear positioning associated with development.Recently, light-dependent nuclear positioning was reported in the spermatophyte Arabidopsis (Iwabuchi et al., 2007). In epidermal and mesophyll cells of dark-treated leaves, nuclei are distributed along the inner periclinal wall. Under strong light, they become located along the anticlinal walls. In mesophyll cells, nuclear movement from inner periclinal to anticlinal walls is induced repeatedly and specifically by blue light of high-fluence rate (more than 50 μ mol m⁻² s⁻¹) and is regulated by the blue light receptor phototropin2. Interestingly, mesophyll cells of the phot2 mutant have aberrantly positioned nuclei even in darkness. By contrast, the involvement of phototropins in nuclear positioning has not yet been examined for epidermal cells.Phototropin is a blue light receptor containing two light oxygen voltage domains at the N terminus, which bind an FMN chromophore, and a Ser/Thr kinase domain at the C terminus, which undergoes blue-light-dependent autophosphorylation (Briggs et al., 2001a; Christie, 2007). Arabidopsis possesses phototropins1 and 2 (Huala et al., 1997; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Phototropins are shown microscopically and biochemically to localize to the plasma membrane region (Briggs et al., 2001b; Sakamoto and Briggs, 2002; Kong et al., 2006) and mediate several responses, including phototropism (Liscum and Briggs, 1995; Sakai et al., 2001), stomatal opening (Kinoshita et al., 2001), and chloroplast movements (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). In general, phototropin1 is more sensitive to light than its paralog and mediates low-fluence-rate light responses, whereas phototropin2 functions predominantly under higher fluence rates (Sakai et al., 2001).While the photoreceptor eliciting these nuclear movements has been revealed, the motile system responsible for moving the nuclei is still unknown. In general, organelle movements depend on the cytoskeleton, with the specific roles for actin and microtubules dependent on the organelle and species (Wada and Suetsugu, 2004). In land plants, the actin cytoskeleton plays a pivotal role in positioning organelles, including nuclei, chloroplasts, mitochondria, and peroxisomes (Wada and Suetsugu, 2004; Takagi et al., 2009).The role of the cytoskeleton in developmental nuclear movements has been investigated. In growing root hairs of Arabidopsis, the nuclear movements are driven along actin filaments (Ketelaar et al., 2002), whereas, in tobacco (Nicotiana tabacum) BY-2 cells, the cell-cycle-based nuclear migration before mitosis is found to depend on microtubules (Katsuta et al., 1990). In interphase Spirogyra crassa cells, centering of nuclei is regulated by both actin filaments and microtubules, but in distinct ways (Grolig, 1998). To the best of our knowledge, the cytoskeletal basis of environmentally induced nuclear movements in land plants has not been elucidated.The best-characterized organelle movements are the light-induced orientation movements of chloroplasts, and although exceptions have been reported, this movement depends on actin (Britz, 1979; Takagi, 2003; Wada et al., 2003). Under weak light, chloroplasts gather at the periclinal walls, perpendicular to the direction of light (accumulation response), whereas under strong light, they become positioned along the anticlinal walls, parallel to the direction of light (avoidance response). Recently, for Arabidopsis, Kadota et al. (2009) characterized the nature of the actin filaments probably involved in these movements. With the onset of either accumulation or avoidance response, short actin filaments appear at the leading edge of each chloroplast.In Arabidopsis, light-dependent nuclear positioning shows similarities to the chloroplast avoidance response, with regard to the direction of movement, relevant photoreceptor (phototropin2), and effective fluence rate (Iwabuchi and Takagi, 2008). On the other hand, nuclei are larger than chloroplasts and might require thicker, more rigid actin bundles for effective motility. Here, we investigate the involvement of the actin cytoskeleton as well as phototropin in regulatory system for nuclear positioning in epidermal cells of Arabidopsis leaves.     

Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodochrous strain S-2

A possibility has been suggested of applying the EPS produced by Rhodococcus rhodochrous strain S-2 (S-2 EPS) to the bioremediation of oil-contaminated environments, because its addition, together with minerals, to oil-contaminated seawater resulted in emulsification of the oil, increased the degradation of polyaromatic hydrocarbons (PAH) of the oil, and led to the dominance of PAH-degrading marine bacteria. To understand the underlying principles of these phenomena, we determined the chemical structure of the sugar chain of S-2 EPS. The EPS was found to be composed of D-galactose, D-mannose, D-glucose, and D-glucuronic acid, in a molar ratio of 1:1:1:1. In addition, 0.8% (w/w) of octadecanoic acid and 2.7% (w/w) of hexadecanoic acid were also contained in its structure. By 1H and 13C NMR spectroscopy, including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments, as well as chemical and enzymatic analyses, the polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (see formula in text).     

Change in substrate preference of Streptomyces aminopeptidase through modification of the environment around the substrate binding site

We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k(cat) values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively).     

Study on peptide hydrolysis by aminopeptidases from Streptomyces griseus, Streptomyces septatus and Aeromonas proteolytica

We developed a spectrophotometric assay for peptide hydrolysis by aminopeptidases (APs). The assay enables the measurement of free amino acids liberated by AP-catalyzed peptide hydrolysis using 4-aminoantipyrine, phenol, peroxidase, and l-amino acid oxidase. We investigated the specificity of bacterial APs [enzymes from Streptomyces griseus (SGAP), Streptomyces septatus (SSAP), and Aeromonas proteolytica (AAP)] toward peptide substrates using this assay method. Although these enzymes most efficiently cleave leucyl derivatives among 20 aminoacyl derivatives, in peptide hydrolysis, the catalytic efficiencies of Phe-Phe hydrolysis by SGAP and SSAP exceed that of Leu-Phe hydrolysis. Furthermore, all enzymes showed the maximum catalytic efficiencies for Phe-Phe-Phe hydrolysis. These results indicate that the hydrolytic activities of bacterial APs are affected by the nature of the penultimate residue or flanking moiety and the length of the peptide substrate.     

Deficiency in early development of the thymus-dependent cells in irradiation chimeras attributable to recipient's environment.

Allogeneic bone marrow chimeras were prepared using reciprocal combinations of AKR and C3H mice. When C3H mice were recipients, the number of thymocytes recoverable from such chimeras (C3H recipient chimeras) was small as compared with that from chimeras for which AKR mice were used as recipients (AKR recipient chimeras) regardless of donor strain. The thymocytes from C3H recipient chimeras showed a profound deficiency in generating proliferative responses to stimulation by anti-CD3 mAb (2C11) or anti-TCR (alpha, beta) mAb (H57-597), even though the expression of CD3 and TCR molecules fell within the same range as that in AKR recipient chimeras. Furthermore, after stimulation with immobilized 2C11, the proportion of IL-2R+ cells in the thymocytes from C3H recipient chimeras was much less than that in AKR recipient chimeras. However, no significant difference in proliferative responses to 2C11 plus PMA, in influx of Ca2+ after stimulation with 2C11 or IL-2 production in response to 2C11 plus PMA or PMA plus A23187 was demonstrated between C3H and AKR recipient chimeras. These findings suggest that the thymocytes from C3H recipient chimeras have a deficiency in the signal transduction system as compared with chimeras for which AKR mice are the recipients. The thymic stromal component involved in this difference in the C3H recipient chimeras is discussed.     

Accurate determination of the structural elasticity of human hair by a small-scale bending test

This paper reports on a small-scale bending method for human hair. The test sample, which is elliptical in cross-section, is fixed to a hollow steel needle using resin to form a cantilever. A loading probe is used to subject this to a lateral load, where the load is applied parallel to either the long or short axis of the elliptical cross-section. From these tests, load-displacement relationships for the hair were obtained. From the experimental data and analysis, we found that the structural elasticity determined is independent of the direction of bending, and precise measurements of the structural elasticity of human hair with scattering of less than 5% were realized using this test scheme. Finally, changes in the structural elasticity of hair due to hair treatments were detected and the changes are discussed based on a theoretical model of the multi-layered structure.     

Functional significance of the negative-feedback regulation of ATP release via pannexin-1 hemichannels under ischemic stress in astrocytes

The opening of pannexin-1 (Px1) hemichannels is regulated by the activity of P2X(7) receptors (P2X(7)Rs). At present, however, little is known about how extracellular ATP-sensitive P2X(7)Rs regulates the opening and closure of Px1 hemichannels. Several lines of evidence suggest that P2X(7)Rs are activated under pathological conditions such as ischemia, resulting in the opening of Px1 hemichannels responsible for the massive influx of Ca(2+) from the extracellular space and the release of ATP from the cytoplasm, leading to cell death. Here we show in cultured astrocytes that the suppression of the activity of P2X(7)Rs during simulated ischemia (oxygen/glucose deprivation, OGD) resulted in the opening of Px1 hemichannels, leading to the enhanced release of ATP. In addition, the suppression of the activity of P2X(7)Rs during OGD resulted in a significant increase in astrocytic damage. Both the P2X(7)Rs suppression-induced enhancement of the release of ATP and cell damage were reversed by co-treatment with blockers of Px1 hemichannels, suggesting that suppression of the activity of PX(7)Rs resulted in the opening of Px1 hemichannels. All these findings suggested the existence of a negative-feedback loop regulating the release of ATP via Px1 hemichannels; ATP-induced suppression of ATP release. The present study indicates that ATP, released through Px1 hemichannels, activates P2X(7)Rs, resulting in the closure of Px1 hemichannels during ischemia. This negative-feedback mechanism, suppressing the loss of cellular ATP and Ca(2+) influx, might contribute to the survival of astrocytes under ischemic stress.