Activity of a two-domain antifreeze protein is not dependent on linker sequence

The reported NMR structure of RD3, a naturally occurring two-domain antifreeze protein, suggests that the two nearly identical domains are oriented to allow simultaneous binding of their active regions to the ice surface. It is implied that the nine residues linking the two domains play a role in this alignment, but this has not been established. We have designed and expressed a modified form of RD3 that replaces the nine-residue linker with a generic sequence of one serine and eight glycine residues to test the importance of the linker amino acid sequence. The modified linker is shown to have significantly different characteristics compared to the original linker. Heteronuclear nuclear Overhauser effect experiments show that the new linker residues have more mobility than the linker residues in the native protein. Further, NMR data show that the folding of the C-terminal domain is somewhat perturbed by the altered linker. Finally, distributions of residual dipolar couplings indicate that the two domains tumble and move independently of each other. Nevertheless, the thermal hysteresis activity of the modified protein is indistinguishable from that of native RD3, proving that increased activity of the two-domain antifreeze protein is not dependent on structure of the linker.     

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.     

Phytoplankton blooms under dim and cold conditions in freshwater lakes of East Antarctica

The seasonal variations of limnological (water temperature, light availability, turbidity, and chlorophyll a concentration) parameters were recorded continuously from January 2004 to February 2005 at two freshwater lakes: Oyako-ike and Hotoke-ike, Sôya Coast, East Antarctica. Water was in a liquid phase throughout the year, with temperatures ranging from 0 to 10°C. The maximum photosynthetically active radiation in Lake Oyako-ike was 23.16 mol m^?2 day^?1 (at 3.8 m) and Hotoke-ike was 53.01 mol m^?2 day^?1 (at 2.2 m) in summer, and chlorophyll a concentration ranged from ca. 0.5 to 2.5 μg L^?1 (Oyako-ike) and from ca. 0.1 to 0.8 μg L^?1 (Hotoke-ike) during the study period. Increase in chlorophyll a fluorescence occurred under dim-light conditions when the lakes were covered with ice in spring and autumn, but the signals were minimum in ice-free summer in both the lakes. During spring and summer, as a result of decreasing snow cover, the chlorophyll a concentration similarly decreased when PAR was relatively high, following periods of heavy winds. The autumnal and spring increase occurred under different PAR levels (ca. 20-fold and 90-fold stronger, respectively, in autumn in both the lakes). Differences in the autumn and spring increases suggest that the spring algal community is more shade-adapted than the autumn algal community. Antarctic phytoplankton appears especially adapted to low-light levels and inhibited by strong light regimes.     

The chilling injury induced by high root temperature in the leaves of rice seedlings

Root temperature is found to be a very important factor forleaves to alter the response and susceptibility to chillingstress. Severe visible damage was observed in the most activeleaves of seedlings of a japonica rice (Oryza sativa cv. Akitakomachi),e.g. the third leaf at the third-leaf stage, after the treatmentwhere only leaves but not roots were chilled (L/H). On the otherhand, no visible damage was observed after the treatment whereboth leaves and roots were chilled simultaneously (L/L). Thechilling injury induced by L/H, a novel type of chilling injury,required the light either during or after the chilling in orderto develop the visible symptoms such as leaf bleaching and tissuenecrosis. Chlorophyll fluorescence parameters measured aftervarious lengths of chilling treatments showed that significantchanges were induced before the visible injury. The effectivequantum yield and photochemical quenching of PSII dropped dramaticallywithin 24 h in both the presence and absence of a 12 h lightperiod. The maximal quantum yield and non-photochemical quenchingof PSII decreased significantly only in the presence of light.On the other hand, L/H chilling did not affect the functionof PSI, but caused a significant decrease in the electron availabilityfor PSI. These results suggest that the leaf chilling with highroot temperature destroys some component between PSII and PSIwithout the aid of light, which causes the over-reduction ofPSII in the light, and thereby the visible injury is inducedonly in the light.     

Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions

The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414–12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2‐DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans.     

Cloning, expression analysis, and sequence diversity of genes encoding two different immunodominant membrane proteins in poinsettia branch-inducing phytoplasma (PoiBI)

Poinsettia branch-inducing phytoplasma (PoiBI) is a phytopathogenic bacterium that infects poinsettia, and is associated with the free-branching morphotype (characterized by many axillary shoots and flowers) of many commercially grown poinsettias. The major membrane proteins of phytoplasmas are classified into three general types, that is, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). These membrane proteins are often used as targets for the production of antibodies used in phytoplasma detection. Herein, we cloned and sequenced the imp and idpA genes of PoiBI strains from 26 commercial poinsettia cultivars. Although the amino acid sequences of the encoded IdpA proteins were invariant, those of the encoded Imp varied among the PoiBI isolates, with no synonymous nucleotide substitution. Western blotting and immunohistochemical analyses revealed that the amount of Imp expressed exceeded that of IdpA, in contrast to the case of a related phytoplasma-disease, western X-disease, for which the major membrane protein appears to be IdpA, not Imp. These results suggest that even phylogenetically close phytoplasmas express different types of major membrane proteins.