Atomic structure of mutant PPARγ LBD complexed with 15d-PGJ2: Novel modulation mechanism of PPARγ/RXRα function by covalently bound ligands

15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) activates a nuclear receptor heterodimer, peroxisome proliferators-activated receptor γ (PPARγ)/ retinoid X receptor (RXRα) through covalent binding to Cys285 in PPARγ ligand-binding domain (LBD). Here, we present the 1.9 Å crystal structure of C285S mutant LBD complexed with 15d-PGJ₂, corresponding to the non-covalently bound state. The ligand lies adjacent to a hydrogen-bond network around the helix H2 and the nearby β-sheet. Comparisons with previous structures clarified the relationships between PPARγ function and conformational alterations of LBD during the process of covalently binding ligands, such as 15d-PGJ₂, and thus suggested a mechanism, by which these ligands modulate PPARγ/RXRα function through conformational changes of the loop following helix H2′ and the β-sheet.     

Modulation of the JNK pathway in liver affects insulin resistance status

The c-Jun N-terminal kinase (JNK) pathway is known to be activated under diabetic conditions and to possibly be involved in the progression of insulin resistance. In this study, we examined the effects of modulation of the JNK pathway in liver on insulin resistance and glucose tolerance. Overexpression of dominant-negative type JNK in the liver of obese diabetic mice dramatically improved insulin resistance and markedly decreased blood glucose levels. Conversely, expression of wild type JNK in the liver of normal mice decreased insulin sensitivity. The phosphorylation state of crucial molecules for insulin signaling was altered upon modification of the JNK pathway. Furthermore, suppression of the JNK pathway resulted in a dramatic decrease in the expression levels of the key gluconeogenic enzymes, and endogenous hepatic glucose production was also greatly reduced. Similar effects were observed in high fat, high sucrose diet-induced diabetic mice. Taken together, these findings suggest that suppression of the JNK pathway in liver exerts greatly beneficial effects on insulin resistance status and glucose tolerance in both genetic and dietary models of diabetes.     

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of α-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that α-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that α-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.     

4'-Fluorinated carbocyclic nucleosides: synthesis and inhibitory activity against S-adenosyl-L-homocysteine hydrolase

4'-Fluorinated analogue of 9-[(1'R,2'S,3'R)-2',3'-dihydroxy-cyclopentan-1'-yl]adenine (DHCaA) and their related analogues were systematically synthesized under the Mitsunobu and palladium(0) coupling conditions followed by fluorination with inversion of the configuration by using diethylaminosulfur trifluoride, respectively. 4'-beta-Fluoro DHCaA and 2-amino-4'-alpha-fluoro DHCaA demonstrated slight inhibitory activity against human and Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase, respectively.     

Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.     

Physiological suppression of the larval parasitoid Glyptapanteles pallipes by the polyembryonic parasitoid Copidosoma floridanum

Precocious larvae, clonally produced together with reproductive siblings in the polyembryonic parasitoid Copidosoma floridanum, are known to physically attack competitors in multiparasitized hosts. In this study, we show that physiological suppression by C. floridanum, as well as precocious larval activity, causes death of the larval parasitoid Glyptapanteles pallipes. Approximately 70% of the hosts multiparasitized by C. floridanum and G. pallipes produced C. floridanum offspring, irrespective of the interval of multiparasitism. G. pallipes eggs or larvae died even in multiparasitized hosts that did not contain precocious larvae of C. floridanum. An injection of C. floridanum-parasitized or multiparasitized-host hemolymph into G. pallipes singly-parasitized hosts paralyzed almost all G. pallipes larvae within 70 h. In vitro analysis showed that the hemolymph factor toxic to G. pallipes eggs and larvae was present in C. floridanum-parasitized hosts through their larval stages. Heating or proteinase treatment reduced its toxicity, suggesting that the factor is a protein.     

Sugar-inducible expression of the nucleolin-1 gene of Arabidopsis thaliana and its role in ribosome synthesis, growth and development

Animal and yeast nucleolin function as global regulators of ribosome synthesis, and their expression is tightly linked to cell proliferation. Although Arabidopsis contains two genes for nucleolin, AtNuc-L1 is the predominant if not only form of the protein found in most tissues, and GFP-AtNuc-L1 fusion proteins were targeted to the nucleolus. Expression of AtNuc-L1 was strongly induced by sucrose or glucose but not by non-metabolizable mannitol or 2-deoxyglucose. Sucrose also caused enhanced expression of genes for subunits of C/D and H/ACA small nucleolar ribonucleoproteins, as well as a large number of genes for ribosomal proteins (RPs), suggesting that carbohydrate availability regulates de novo ribosome synthesis. In sugar-starved cells, induction of AtNuc-L1 occurred with 10 mM glucose, which seemed to be a prerequisite for resumption of growth. Disruption of AtNuc-L1 caused an increased steady-state level of pre-rRNA relative to mature 25S rRNA, and resulted in various phenotypes that overlap those reported for several RP gene mutants, including a reduced growth rate, prolonged lifetime, bushy growth, pointed leaf, and defective vascular patterns and pod development. These results suggest that the rate of ribosome synthesis in the meristem has a strong impact not only on the growth but also the structure of plants. The AtNuc-L1 disruptant exhibited significantly reduced sugar-induced expression of RP genes, suggesting that AtNuc-L1 is involved in the sugar-inducible expression of RP genes.     

Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.     

Growing and differentiating characterization of aortic smooth muscle cell line, p53LMAC01 obtained from p53 knock out mice

Recently we have established an aortic smooth muscle cell line, p53LMAC01 obtained from p53 knockout mice. This cell line showed some differentiated properties which were accelerated by 5-azacytidine treatment [1]. In this study, further characterization of p53LMAC01 cell line was investigated according to cell growth and differentiation, and especially focused into the changes of cell feature, actin filaments' formation, and changes of intracellular calcium concentrations to sympathetic nerve transmitter, norepinephrine. While the cell feature was changed from flattened shape to extended form during 4 days, actin filaments were developing, arranging in parallel to longitudinal direction, and gathering under the surface membrane. In 11 days many cells died and detached from substrate, while actin filaments became poor except for the surface membrane in the remained cells. Appearance of calcium response to noradrenalin needed several days after passage as well as a morphological change of the cells for the extended form and development of actin filaments. The calcium response was maintained on 11 days, which coincided with the result that the cells hold actin filaments under the surface membrane. These results suggest that p53LMAC01 cell line maintains several differentiated characters of adult smooth muscle cell and that their expression needs several days after passage.