The rosettazyme: A synthetic cellulosome

Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds.Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg²⁺ assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin–rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.     

Polyhydroxylated fullerene C60(OH)44 suppresses intracellular lipid accumulation together with repression of intracellular superoxide anion radicals and subsequent PPARγ2 expression during spontaneous differentiation of OP9 preadipocytes into adipocytes

Reactive oxygen species has been suggested to be one of the key factors associated with the development of obesity. During spontaneous differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular superoxide anion radicals (O (2) (-.) ) level markedly increases and is accompanied by a significant elevation of intracellular lipid accumulation. This differentiation-dependent increase in intracellular O (2) (-.) level positively correlated with the intracellular augmentation of the lipid level. Super-highly hydroxylated fullerene (SHH-F; C(60)(OH)(44)), a novel polyhydroxylated fullerene derivative, quenched intracellular O (2) (-.) , and lipid accumulation to 38.7 and 42.7 % of that in the control, respectively. By thin-layer chromatographic analysis of extracted cellular lipid components, SHH-F clearly decreased the triglycerides ratio in the whole lipid droplet fraction, but scarcely influenced other lipids components. PPARγ2 expression, which plays a key role in regulating adipogenic differentiation, was significantly suppressed by SHH-F at the late stage of differentiation, with unaltered PPARγ1 expression. The intracellular superoxide anion radical augmentation preceded expression of PPARγ2, strongly suggesting that the primary O (2) (-.) generation was closely associated with lipid accumulation and subsequent PPARγ2 induction. These results indicate that SHH-F suppresses intracellular lipid accumulation, particularly in lipid droplets, and decreases O (2) (-.) level and subsequent PPARγ2 upregulation during spontaneous differentiation of OP9 preadipocytes into adipocytes.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

The coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks to the reaction of human renin and sheep angiotensinogen

The pH dependence and kinetics parameters of renin-angiotensinogen reactions were determined using wild-type and S84G mutant human renins and wild-type and H13Y mutant sheep angiotensinogens. It is explained in this report that (i) renin catalyzes acidic and basic reactions of which the optimum pHs are 5.5 and 7.5-8.2 respectively, both of which produce angiotensin I; (ii) Ser84 specific to human renin accelerates the acidic reaction by 75-110% through elevation of V(max), and shifts the optimum pH of the basic reaction from 7.5 to 8.0-8.2; and (iii) His13 specific to sheep angiotensinogen accelerates the acidic and basic reactions by 25-42% through reduction of K(m). It is concluded from these results that the coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks at pHs 5.5 and 8.2 to the reaction of human renin and sheep angiotensinogen.     

Modeling daily flowering probabilities: expected impact of climate change on Japanese cherry phenology

Understanding the drivers of phenological events is vital for forecasting species’ responses to climate change. We developed flexible Bayesian survival regression models to assess a 29‐year, individual‐level time series of flowering phenology from four taxa of Japanese cherry trees (Prunus spachiana, Prunus × yedoensis, Prunus jamasakura, and Prunus lannesiana), from the Tama Forest Cherry Preservation Garden in Hachioji, Japan. Our modeling framework used time‐varying (chill and heat units) and time‐invariant (slope, aspect, and elevation) factors. We found limited differences among taxa in sensitivity to chill, but earlier flowering taxa, such as P. spachiana, were more sensitive to heat than later flowering taxa, such as P. lannesiana. Using an ensemble of three downscaled regional climate models under the A1B emissions scenario, we projected shifts in flowering timing by 2100. Projections suggest that each taxa will flower about 30 days earlier on average by 2100 with 2–6 days greater uncertainty around the species mean flowering date. Dramatic shifts in the flowering times of cherry trees may have implications for economically important cultural festivals in Japan and East Asia. The survival models used here provide a mechanistic modeling approach and are broadly applicable to any time‐to‐event phenological data, such as plant leafing, bird arrival time, and insect emergence. The ability to explicitly quantify uncertainty, examine phenological responses on a fine time scale, and incorporate conditions leading up to an event may provide future insight into phenologically driven changes in carbon balance and ecological mismatches of plants and pollinators in natural populations and horticultural crops.     

Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambdamax, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.