Carbon budget and methane and nitrous oxide emissions over the growing season in a Miscanthus sinensis grassland in Tomakomai,Hokkaido, Japan

Species in the Miscanthus genus have been proposed as biofuel crops that have potential to mitigate elevated atmospheric carbon dioxide (CO₂) levels and nitrous oxide (N₂O) and methane (CH₄) emissions. Miscanthus sinensis is widespread throughout Japan and has been used for biomass production for centuries. We assessed the carbon (C) budget and N₂O and CH₄ emissions over the growing season for 2 years in a M. sinensis‐dominated grassland that was naturally established around 1972 in Tomakomai, Hokkaido, Japan, which is near the northern limit for M. sinensis grassland establishment on Andisols. Average C budget was ?0.31 Mg C ha^?1, which indicates C was released from the grassland ecosystem to the atmosphere. Dominant components in the C budget appeared to be aboveground net primary production of plants (1.94–2.80 Mg C ha^?1) and heterotrophic respiration (2.27–3.11 Mg C ha^?1). The measurement of belowground net primary production (BNPP) of plants in the M. sinensis grassland was extremely variable, thus only an approximate value could be calculated. Mean C budget calculated with the approximated BNPP value was 1.47 and ?0.23 Mg C ha^?1 for 2008 and 2009, respectively. Given belowground biomass (9.46–9.86 Mg C ha^?1) was 3.1–6.5 times higher than that of aboveground biomass may provide additional evidence suggesting this grassland represents a C sink. Average CH₄ emissions across years of ?1.34 kg C ha^?1 would indicate this grassland acts as an atmospheric CH₄ sink. Furthermore, average N₂O emissions across years were 0.22 kg N ha^?1. While the site may contribute N₂O to the atmosphere, this value is lower compared with other grassland types. Global warming potential calculated with the approximated BNPP value was ?5.40 and 0.95 Mg CO₂ Eq ha^?1 for 2008 and 2009, respectively, and indicates this grassland could contribute to mitigation of global warming.     

p21-activated kinase improves cardiac contractility during ischemia-reperfusion concomitant with changes in troponin-T and myosin light chain 2 phosphorylation

p21-activated kinase 1 (Pak1) is a serine/threonine kinase that activates protein phosphatase 2a, resulting in the dephosphorylation of cardiac proteins and increased myofilament Ca(2+) sensitivity. Emerging evidence indirectly indicates a role for Pak1 in ischemia-reperfusion (I/R), but direct evidence is lacking. We hypothesize that activation of the Pak1 signaling pathway is a cardioprotective mechanism that prevents or reverses the detrimental effects of ischemic injury by inducing posttranslational modifications in myofilament proteins that ultimately improve cardiac contractility following ischemic insult. In the present study, we subjected ex vivo hearts from wild-type (WT) and Pak1-knockout (KO) mice to 20 min of global cardiac ischemia followed by 30 min of reperfusion. In the absence of Pak1, there was an exacerbation of the increased end-diastolic pressure and reduced left ventricular developed pressure occurring after I/R injury. ProQ analysis revealed an increase in troponin-T phosphorylation at baseline in Pak1-KO hearts compared with WT. Significantly decreased myosin light chain 2 (MLC2) phosphorylation in Pak1-KO hearts compared with WT after I/R injury was confirmed by Western immunoblotting. These data indicate that Pak1-KO hearts have reduced recovery of myocardial performance after global I/R injury concomitant with changes in troponin-T and MLC2 phosphorylation. Finally, a protein-protein association between Pak1 and MLC2, and Pak1 and troponin-T, was determined by coimmunoprecipitation. Thus, results of our study provide a basis for targeting a novel pathway, including Pak1, in the therapies for patients with ischemic events.     

Activation mechanism of rod outer segment cyclic GMP phosphodiesterase. Release of inhibitor by the GTP/GTP-binding protein

The physiological regulation of light-activated cyclic GMP phosphodiesterase (EC 3.1.4.17) in rod outer segments has been shown to depend upon a heat-stable inhibitor and upon the reversal of its effect by a specific GTP/GTP-binding protein complex (Hurley, J. B. (1980) Biochem. Biophys. Res. Commun. 92, 505-510; Yamazaki, A., Bartucca, F., Ting, A., and Bitensky, M. W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3702-3706). Washing of illuminated disc membranes with an isotonic buffer released 86% of the peripheral proteins without any release of inhibitor. Subsequent washing with the same isotonic buffer containing GTP released 80% of the inhibitor. When inhibitor was eluted with guanosine-5'-(beta, gamma-imino)triphosphate, it had an apparent molecular weight of 60,000 on Sephadex G-100. The release of inhibitor by guanosine-5'-(beta, gamma-imino)triphosphate was also demonstrated with sucrose density gradient centrifugation. Inhibitor release from the disc membrane by GTP or its analogue was accompanied by the release of the GTP-binding protein and an increased phosphodiesterase activity in the membrane. However, following GTP hydrolysis, both inhibitor and GTP-binding protein returned to the membrane and phosphodiesterase activity in the membrane decreased proportionally. In contrast, incubation of disc membranes with guanosine-5'-(beta, gamma-imino)-triphosphate produced an increase of inhibitor activity in the supernatant and an increase of phosphodiesterase activity in the pellet which remained constant after the initial increase. These data clearly show that the activation of phosphodiesterase by the GTP/GTP-binding protein complex resulted from the release of inhibitor. Hydrolysis of GTP resulted in the reassociation of inhibitor with and concomitant inhibition of disc membrane phosphodiesterase.     

Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.     

Amyloidogenic domains, prions and structural inheritance: rudiments of early life or recent acquisition?

Amyloids are self-assembled fibre-like beta-rich protein aggregates. Amyloidogenic prion proteins propagate amyloid state in vivo and transmit it via infection or in cell divisions. While amyloid aggregation may occur in the absence of any other proteins, in vivo propagation of the amyloid state requires chaperone helpers. Yeast prion proteins contain prion domains which include distinct aggregation and propagation elements, responsible for these functions. Known aggregation and propagation elements are short in length and composed of relatively simple sequences, indicating possible ancient origin. Prion-like self-assembled structures could be involved in the initial steps of biological compartmentalization in early life.     

Evidence for a protein mutator in yeast: role of the Hsp70-related chaperone ssb in formation, stability, and toxicity of the [PSI] prion

Propagation of the yeast protein-based non-Mendelian element [PSI], a prion-like form of the release factor Sup35, was shown to be regulated by the interplay between chaperone proteins Hsp104 and Hsp70. While overproduction of Hsp104 protein cures cells of [PSI], overproduction of the Ssa1 protein of the Hsp70 family protects [PSI] from the curing effect of Hsp104. Here we demonstrate that another protein of the Hsp70 family, Ssb, previously implicated in nascent polypeptide folding and protein turnover, exhibits effects on [PSI] which are opposite those of Ssa. Ssb overproduction increases, while Ssb depletion decreases, [PSI] curing by the overproduced Hsp104. Both spontaneous [PSI] formation and [PSI] induction by overproduction of the homologous or heterologous Sup35 protein are increased significantly in the strain lacking Ssb. This is the first example when inactivation of an unrelated cellular protein facilitates prion formation. Ssb is therefore playing a role in protein-based inheritance, which is analogous to the role played by the products of mutator genes in nucleic acid-based inheritance. Ssb depletion also decreases toxicity of the overproduced Sup35 and causes extreme sensitivity to the [PSI]-curing chemical agent guanidine hydrochloride. Our data demonstrate that various members of the yeast Hsp70 family have diverged from each other in regard to their roles in prion propagation and suggest that Ssb could serve as a proofreading component of the enzymatic system, which prevents formation of prion aggregates.     

Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae.

Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process.