Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.     

Genetic analysis for diabetes in a new rat model of nonobese type 2 diabetes,Spontaneously Diabetic Torii rat

The Spontaneously Diabetic Torii (SDT) rat has recently been established as a new rat model of nonobese type 2 diabetes. In this study, we characterized diabetic features in SDT rats, and performed quantitative trait locus (QTL) analysis for glucose intolerance using 319 male (BNxSDT)xSDT backcrosses. Male SDT rats exhibited glucose intolerance at 20 weeks, and spontaneously developed diabetes with the incidence of 100% at 38 weeks, and glucose intolerance is well associated with the development of diabetes. The QTL analysis identified three highly significant QTLs (Gisdt1, Gisdt2, and Gisdt3) for glucose intolerance on rat chromosomes 1, 2, and X, respectively. The SDT allele for these QTLs significantly exacerbated glucose intolerance. Furthermore, synergistic interactions among these QTLs were detected. These findings indicate that diabetic features in SDT rats are inherited as polygenic traits and that SDT rats would provide insights into genetics of human type 2 diabetes.     

Flowering of Arabidopsis cop1 mutants in darkness

To elucidate the role of the COP1 gene in flowering, we analyzed flowering of cop1 mutant lines in darkness. When grown in the presence of 1% (w/v) sucrose, the cop1-6 mutant flowered in darkness, but cop1-1 and cop1-4 did not. However, cop1-1 and cop1-4 flowered in darkness when grown in the presence of 5% (w/v) sucrose. Therefore, the COP1 gene represses not only photomorphogenesis in seedlings but also flowering in darkness. Comparison of mRNAs levels of floral identity genes in cop1-6 and wild-type plants grown in darkness revealed increased mRNA levels of genes that act downstream of CO and reduced FLC mRNA level in cop1-6. Double mutants of cop1-6 and each of the late-flowering mutations cry2-1, gi-2, co-1, and ld-1 flowered in darkness. All of the double mutants except cry2-1 cop1-6 flowered later than cop1-6, demonstrating that cop1-6 is epistatic to cry2-1 for early flowering. The ld-1 cop1-6 double mutant flowered much earlier than the ld-1 mutant. The delay in flowering in the double mutants was not strongly influenced by the light conditions, whereas that of the gi-2 cop1-6 double mutant was reduced in darkness.     

Proper regulation of cyclic AMP-dependent protein kinase is required for growth, conidiation, and appressorium function in the anthracnose fungus Colletotrichum lagenarium

Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpkl-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium.     

Selective activation of human soleus and medial gastrocnemius muscles during walking in water

During walking in water (WW) the vertical component of ground reaction forces decreases, while the greater propulsive force is required to move forward against the greater resistance of water. In such reduced gravity environment, Hutchison et al. (1989) have demonstrated that the relative activation of rat medial gastrocnemius (MGAS) increased compared to that of the soleus (SOL) during swimming, suggesting different effects of peripheral information on motoneuron excitability of these muscles. It is conceivable that both buoyancy and resistance of water have different effects on the activation patterns of triceps surae muscles during WW, since the reduced weight in water might decrease the peripheral inflow relating load information while greater volitional command might be needed to propel a body forward against the water resistance. The present study was designed to assess each peripheral inflow and efferent input by adjusting the load and walking speed voluntarily during WW. The aim of this study is to investigate the dissociative activation pattern between the SOL and the MGAS during WW.     

Efficient Initiation of DNA Replication in Eukaryotes Requires Dpb11/TopBP1-GINS Interaction

Dpb11/Cut5/TopBP1 is evolutionarily conserved and is essential for the initiation of DNA replication in eukaryotes. The Dpb11 of the budding yeast Saccharomyces cerevisiae has four BRCT domains (BRCT1 to -4). The N-terminal pair (BRCT1 and -2) and the C-terminal pair (BRCT3 and -4) bind to cyclin-dependent kinase (CDK)-phosphorylated Sld3 and Sld2, respectively. These phosphorylation-dependent interactions trigger the initiation of DNA replication. BRCT1 and -2 and BRCT3 and -4 of Dpb11 are separated by a short stretch of ∼100 amino acids. It is unknown whether this inter-BRCT region functions in DNA replication. Here, we showed that the inter-BRCT region is a GINS interaction domain that is essential for cell growth and that mutations in this domain cause replication defects in budding yeast. We found the corresponding region in the vertebrate ortholog, TopBP1, and showed that the corresponding region also interacts with GINS and is required for efficient DNA replication. We propose that the inter-BRCT region of Dpb11 is a functionally conserved GINS interaction domain that is important for the initiation of DNA replication in eukaryotes.     

Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana

The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced “hedge”) genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2–1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2–2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2–2 hdg. The allele-specific effect caused by pdf2–1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domain-encoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins.     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance.

Agonist-induced activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPAR gamma was investigated by gene targeting. Homozygous PPAR gamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPAR gamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPAR gamma agonist treatment. Heterozygous PPAR gamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPAR gamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPAR gamma.