Seasonal changes in serum leptin of the feral raccoon (Procyon lotor) determined by canine-leptin-specific ELISA

Several reports have been published on blood leptin concentrations in feral animals, including members of the Carnivora, using a commercially available multi-species radioimmunoassay (RIA) kit with anti-human leptin antibody. However, we observed weak immunoreactivity between recombinant canine leptin and anti-human leptin antibody, suggesting a limitation in the applicability of the RIA kit for leptin assays in Carnivora species. We tested the applicability of RIA and sandwich enzyme-linked immunosorbent assay (ELISA) with anti-canine leptin antibody to assay blood leptin in the dog (Canis familiaris) and the raccoon (Procyon lotor). When RIA was used for recombinant canine leptin and dog sera, values were much lower than those determined by ELISA at higher concentrations (>10 ng/ml), while rather higher at lower concentrations (<2 ng/ml). A similar discrepancy between the two methods was found for serum leptin concentrations in raccoons. Clear seasonal variations were observed by ELISA, but not by RIA, with high values in autumn (3.46+/-0.45 ng/ml) and low values in spring and summer (0.71+/-0.07 ng/ml). Serum leptin concentrations in raccoons correlated positively with their body weight (r=0.753) and body mass index (r=0.755), corroborating our previous findings of a strong positive correlation between serum leptin concentrations and body fat content in dogs. Thus, the canine leptin ELISA is useful for assays of dog and raccoon leptin, and blood leptin is a good marker of nutritional condition in the species of Carnivora assayed in this study.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.     

Isolation of a cadmium-releasing bacterium and characterization of its novel protease

Microorganisms were screened for their ability to release cadmium from scallop hepatopancreas, which is the main residue after removing of the edible parts of scallop. The isolated strain, 23-0-11, identified as Arthrobacter nicotinovorans, secreted a protease which released cadmium from scallop hepatopancreas into the liquid medium. The molecular mass of the enzyme was estimated to be 27 kDa. The sequence of the 15 N-terminal amino acids of the protease showed no close similarity with any other protein. Compared with a commercial enzyme, the purified protease had greater ability to release cadmium. The enzyme activity was greatest at 50 degrees C and pH 7.0, and was enhanced in the presence of Ca(2+), Mg(2+) and Mn(2+), while being strongly inhibited by Co(2+). The inhibition profile by the serine protease inhibitor, phenylmethylsulphonyl fluoride (PMSF), confirmed that the protease belonged to the serine protease family.     

Application of an enzyme chip to the microquantification of l-phenylalanine

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.     

Incorporation of β‐amino acids into ascidiacyclamides: Effects on conformation,cytotoxicity and interaction with copper (II) ion

Seven ascidiacyclamide [cyclo(–Ile–oxazoline–d ‐Val–thiazole–)₂] (ASC) analogues incorporating the β‐amino acids βIle, βoxazoline, and/or d ‐βVal were synthesized. We then investigated the effects of the position and number of incorporated β‐amino acids on the structure, cytotoxicity, and copper binding by these seven analogues. The structural analyses revealed that both βIle and d ‐βVal favor a gauche‐type θ torsion angles, while βoxazoline favors a trans‐type θ torsion angle. Expansion of the macrocycle by incorporation of βIle or d ‐βVal readily induced molecular folding. On the other hand, the incorporation of two βoxazoline residues strongly extended the peptide conformation, and the incorporation of one was sufficient for the moderate restriction important for conformational equilibrium and cytotoxicity. Despite expansion of the macrocycles, the structure‐cytotoxicity relationships were largely maintained. In studies of complexation of the analogues with Cu (II) ion, the position and number of incorporated β‐amino acids had a large impact on the structure of the metal complex and may contribute to its stabilization.     

Inhibition of human T-lymphocyte activation by macrolide antibiotic, roxithromycin.

Effects of macrolide antibiotic, roxithromycin (RXM) on human lymphocytes in culture were studied. The drug showed a dose-dependent inhibition of 3H-thymidine and 35S-methionine uptake responding to T cell mitogens and purified protein derivative of tuberculin (PPD). Activation by PPD, as assessed by 3H-thymidine uptake, was more sensitive to inhibition than the response to T cell mitogens. The drug produced a loss of blasts when added soon after transformation commenced. Immunosuppressive effects of RXM were further characterized by using four different types of metabolized RXM, RU 28111, RU 39001, RU 44981 and RU 45179. The most potent inhibitor of lymphocyte transformation was RU 45179, followed by RU 44981, RU 39001 and RU 28111 have little activity.     

Enhanced accumulation of atropine in Atropa belladonna transformed by Rac GTPase gene isolated from Scoparia dulcis

Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.     

Novel methods for nucleotide length control in double-stranded polyinosinic-polycytidylic acid production using uneven length components

ABSTRACT

Polyinosinic-polycytidylic acid (PIC), a double-stranded RNA that induces innate immunity in mammals, is a candidate immunopotentiator for pharmaceuticals. The potency and adverse effects of PIC are strongly correlated with the nucleotide length, and the inability to precisely control the length in PIC production limits its practical use. Length extension during the annealing process is the major factor underlying the lack of control, but tuning the annealing conditions is insufficient to resolve this issue. In this study, we developed a novel method to produce accurate nucleotide length PIC at an industrial scale. The length extension was significantly suppressed by the assembly of multiple short polyinosinic acid molecules with one long polycytidylic acid molecule. A newly developed PIC, uPIC100-400, demonstrated a reproducible length and better storage stability than that of corresponding evenly structured PIC. Human dsRNA receptors exhibited equivalent responsiveness to uPIC100-400 and the evenly structured PIC with the same length.