Clk2 and B56β mediate insulin-regulated assembly of the PP2A phosphatase holoenzyme complex on Akt

Akt mediates important cellular decisions involved in growth, survival, and metabolism. The mechanisms by which Akt is phosphorylated and activated in response to growth factors or insulin have been extensively studied, but the molecular regulatory components and dynamics of Akt attenuation are poorly understood. Here we show that a downstream target of insulin-induced Akt activation, Clk2, triggers Akt dephosphorylation through the PP2A phosphatase complex. Clk2 phosphorylates the PP2A regulatory subunit B56β (PPP2R5B, B'β), which is a critical regulatory step in the assembly of the PP2A holoenzyme complex on Akt leading to dephosphorylation of both S473 and T308 Akt sites. Since Akt plays a pivotal role in cellular signaling, these results have important implications for our understanding of Akt regulation in many biological processes.     

Effects of low atmospheric CO2 and elevated temperature during growth on the gas exchange responses of C3, C3–C4 intermediate, and C4 species from three evolutionary lineages of C4 photosynthesis

This study evaluates acclimation of photosynthesis and stomatal conductance in three evolutionary lineages of C(3), C(3)-C(4) intermediate, and C(4) species grown in the low CO(2) and hot conditions proposed to favo r the evolution of C(4) photosynthesis. Closely related C(3), C(3)-C(4), and C(4) species in the genera Flaveria, Heliotropium, and Alternanthera were grown near 380 and 180 μmol CO(2) mol(-1) air and day/night temperatures of 37/29°C. Growth CO(2) had no effect on photosynthetic capacity or nitrogen allocation to Rubisco and electron transport in any of the species. There was also no effect of growth CO(2) on photosynthetic and stomatal responses to intercellular CO(2) concentration. These results demonstrate little ability to acclimate to low CO(2) growth conditions in closely related C(3) and C(3)-C(4) species, indicating that, during past episodes of low CO(2), individual C(3) plants had little ability to adjust their photosynthetic physiology to compensate for carbon starvation. This deficiency could have favored selection for more efficient modes of carbon assimilation, such as C(3)-C(4) intermediacy. The C(3)-C(4) species had approximately 50% greater rates of net CO(2) assimilation than the C(3) species when measured at the growth conditions of 180 μmol mol(-1) and 37°C, demonstrating the superiority of the C(3)-C(4) pathway in low atmospheric CO(2) and hot climates of recent geological time.     

A two-site immunometric assay for the detection of the expression level of α2(C2)-adrenergic receptor produced in Escherichia coli

Human ₂(C2)-adrenergic receptor was expressed in Escherichia coli as a fusion protein with Bacillus circulans var. alcalophilus cyclomaltodextrin glucanotransferase. For the determination of the expression level (0.6 mg of solubilized fusion protein l^–1 of E. coli culture), a two-site immunometric assay based on two monoclonal antibodies with different epitopes was developed.     

Association of HL-A antigens andβ 2-microglobulin at the cellular and molecular level

Surfaces of cultured human lymphoid cells RPMI 1788, RPMI 4098, RPMI 8866, Raji, and WI-L2 were found to contain bothβ ₂-microglobulin (β ₂-μ) and HL-A determinants when tested by direct complement-dependent cytotoxicity andquantitative absorption with different cytotoxic antiβ ₂-μ antisera and specific HL-A alloantisera. The same antigenic specificities were found in 3M KCl extracts of these cultured cells with a sensitiveβ ₂-μ radioimmunoassay and an HL-A antigen blocking assay. Daudi cells provided a contrast, since noβ ₂-μ or HL-A determinants were found on their surfaces or in 3 M KCl extracts prepared from them. Results from specific antibody blocking tests suggest a close association betweenβ ₂-μ and HL-A determinants on plasma membranes of cultured human lymphoid cells. A solid state immunoadsorbent containing antiβ ₂-μ antibodies effectively removed all detectable HL-A antigenic activity from some 3M KCl extracts of cultured human lymphoid cells as well as from some sera. Adsorption of HL-A antigens to these immunoadsorbents was specific since it was blocked only by prior addition ofβ ₂-μ. Once on the antiβ ₂-μ immunoadsorbents, HL-A antigens still reacted specifically with HL-A alloantibodies in quantitative absorption experiments. HL-A antigens andβ ₂-μ could be eluted from antiβ ₂-μ immunoadsorbents with a variety of chaotropic reagents and detergents, but thus far potassium bromide and sodium dodecyl sulfate (SDS) appear to be the most effective. SDS-PAGE of these eluates indicated that HL-A antigens were considerably purified by adsorption to antiβ ₂-μ immunoadsorbents and that two major molecular size fragments were distinguishable, i.e., ∼33,000 for HL-A and ∼ 12,000 forβ ₂-μ.     

The single-process biochemical reaction of Rubisco: a unified theory and model with the effects of irradiance, CO₂ and rate-limiting step on the kinetics of C₃ and C₄ photosynthesis from gas exchange

Photosynthesis is the origin of oxygenic life on the planet, and its models are the core of all models of plant biology, agriculture, environmental quality and global climate change. A theory is presented here, based on single process biochemical reactions of Rubisco, recognizing that: In the light, Rubisco activase helps separate Rubisco from the stored ribulose-1,5-bisphosphate (RuBP), activates Rubisco with carbamylation and addition of Mg²⁺, and then produces two products, in two steps: (Step 1) Reaction of Rubisco with RuBP produces a Rubisco-enediol complex, which is the carboxylase-oxygenase enzyme (Enco) and (Step 2) Enco captures CO₂ and/or O₂ and produces intermediate products leading to production and release of 3-phosphoglycerate (PGA) and Rubisco. PGA interactively controls (1) the carboxylation-oxygenation, (2) electron transport, and (3) triosephosphate pathway of the Calvin-Benson cycle that leads to the release of glucose and regeneration of RuBP. Initially, the total enzyme participates in the two steps of the reaction transitionally and its rate follows Michaelis-Menten kinetics. But, for a continuous steady state, Rubisco must be divided into two concurrently active segments for the two steps. This causes a deviation of the steady state from the transitional rate. Kinetic models are developed that integrate the transitional and the steady state reactions. They are tested and successfully validated with verifiable experimental data. The single-process theory is compared to the widely used two-process theory of Farquhar et al. (1980. Planta 149, 78-90), which assumes that the carboxylation rate is either Rubisco-limited at low CO₂ levels such as CO₂ compensation point, or RuBP regeneration-limited at high CO₂. Since the photosynthesis rate cannot increase beyond the two-process theory's Rubisco limit at the CO₂ compensation point, net photosynthesis cannot increase above zero in daylight, and since there is always respiration at night, it leads to progressively negative daily CO₂ fixation with no possibility of oxygenic life on the planet. The Rubisco-limited theory at low CO₂ also contradicts all experimental evidence for low substrate reactions, and for all known enzymes, Rubisco included.     

A new β-globin gene from the zebrafish, β E1 , and its pattern of transcription during embryogenesis

In a search for novel, developmentally regulated genes we screened randomly picked cDNA clones, obtained from zebrafish mRNA, by in situ hybridization with digoxigenin-labelled riboprobes. Out of 150 clones tested, 1 codes for a new beta-globin gene and is expressed during embryogenesis. Here we describe its pattern of expression and its use as a marker for early zebrafish erythropoiesis.     

Implications for Collagen Binding from the Crystallographic Structure of Fibronectin 6FnI1�C2FnII7FnI

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, ⁶FnI^1–2FnII^7–9FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains ^8–9FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains ⁶FnI^1–2FnII⁷FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains ²FnII and ⁷FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the ^8–9FnI·collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.     

Mandshunosides C–E from the roots and rhizomes of Clematis mandshurica

Three new triterpene saponins, mandshunosides C–E (1–3) together with four known triterpene saponins (4–7) were isolated from the roots and rhizomes of Clematis mandshurica. Their structures were elucidated on the basis of spectroscopic evidence and hydrolysis products. Those compounds showed inhibitory activities against two human colorectal cancer cell lines HCT-116 and HT-29.     

A dual fluorinated and iodinated radiotracer for PET and SPECT imaging of β-amyloid plaques in the brain

We report a fluorinated and iodinated radiotracer as a probe for PET/SPECT to detect of β-amyloid (Aβ) plaques in the brain of patients with Alzheimer's disease (AD). We successfully designed and synthesized the fluorinated and iodinated aurone derivative (3) and its radiolabels ([(125)I]3 and [(18)F]3). In binding experiments in vitro, 3 showed high affinity for Aβ aggregates (K(i)=6.81nM). In brain sections of AD model mice, 3 intensely stained Aβ plaques. Furthermore, a specific plaque labeling signal was observed on the autoradiography of postmortem AD brain sections using [(125)I]3. In biodistribution experiments using normal mice, [(125)I]3 and [(18)F]3 displayed good uptake into and a rapid washout from the brain, properties highly desirable for Aβ imaging agents. These results suggest that 3 may function as a PET/SPECT dual imaging agent for detecting Aβ plaques in AD brains.