Identification and characterization of proteases involved in specific proteolysis of vitellogenin and yolk proteins in salmonids.

A pepstatin A-sensitive enzyme involved in yolk formation was purified from the masu salmon (Oncorhynchus masou) ovary using in vitro generation of yolk proteins from purified vitellogenin to assay enzymatic activity. Purification of the enzyme involved precipitation of ovarian extracts by water and ammonium sulfate followed by five steps of column chromatography. After SDS-PAGE and Western blotting, the purified enzyme appeared as a single approximately 42 kDa band that was immunoreactive to anti-human cathepsin D. The course of proteolytic cleavage of the three major yolk proteins (lipovitellin, beta'-component, and phosvitin) in fertilized masu salmon and Sakhalin taimen (Hucho perryi) eggs and embryos was visualized by SDS-PAGE and Western blotting using specific antisera. Major yolk protein bands appeared in positions corresponding to 92 kDa, 68 kDa, and 22 kDa (lipovitellin-derived peptides), as well as 17 kDa (beta'-component). During embryo development, the 92 kDa and 22 kDa bands gradually decreased in intensity, becoming undetectable in alevins. The 68 kDa band and a minor 24 kDa band became more intense after the eyed stage. Two additional peptides, corresponding to 40 and 28 kDa, newly appeared in alevins. During embryonic growth, the beta'-component band (17 kDa) persisted and phosvitin appeared to be progressively dephosphorylated. In vitro analysis of lipovitellin proteolysis indicated that the enzyme involved is a Pefabloc SC-sensitive serine protease. These results demonstrate, for the first time, that a cathepsin D-like protease and serine proteases play key roles in yolk formation and degradation, respectively, in salmonid fishes.     

Purification of serum precursor proteins to vitelline envelope (choriogenins) in masu salmon,Oncorhynchus masou

Three vitelline envelope-related proteins (VERPs), very-high-molecular-weight VERP (vhVERP), high-molecular-weight VERP (hVERP) and low-molecular-weight VERP (lVERP) were purified from female masu salmon serum. The apparent molecular weights of vhVERP, hVERP and lVERP, in their native state, were 520, 88 and 54 kDa, respectively, by gel-filtration chromatography. Very-high-molecular-weight VERP comprises two subunits, corresponding to 175 and 126 kDa. On SDS-PAGE, hVERP and lVERP migrate at 53 and 47 kDa, respectively. Amino acid analysis of vhVERP and hVERP showed that they share a high content of glutamic acid and proline. By contrast, lVERP is rich in glutamic acid and asparatic acid. These features are in good agreement with the amino acid composition of the vitelline envelope. Immuno-biochemical analysis suggested that vhVERP is derived from hVERP by polymerization and/or aggregation. Antibodies against hVERP and lVERP specifically immunostained the vitelline envelope and liver of female masu salmon. In addition, both hVERP and lVERP were induced in the serum of estrogen-treated male fish. Taken together, it is suggested that hVERP and lVERP are homologous molecules with choriogenin H and choriogenin L in medaka, respectively. These results indicate that hVERP and lVERP are precursor proteins to the vitelline envelope (choriogenins) in masu salmon.     

Vitellogenin-derived yolk proteins of white perch,Morone americana: purification,characterization, and vitellogenin-receptor binding

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.     

Synthesis and structure-activity relationships of 2-substituted-8-hydroxyadenine derivatives as orally available interferon inducers without emetic side effects

Recently we reported the adenine derivatives (2-4) as new interferon (IFN) inducers. In the present study, we conducted a detailed structure and activity relationship study of 4 and its related derivatives on IFN inducing activity. From this study, we found that compound 4 exhibited the most potent IFN inducing activity in vitro with a minimum effective concentration of 0.01 microM, and 4 also showed strong IFN-inducing activity at doses of more than 0.3mg/kg by oral administration in mice. This potency was 10-fold stronger than that of Imiquimod. Moreover, 4 did not cause emesis in ferrets even at doses as high as 10mg/kg, whereas, 80% of animals were emetic when orally administered with the same dose of Imiquimod. These results indicate that compound 4 is superior to Imiquimod with respect to efficacy and safety.     

Combination therapy with PPARgamma and PPARalpha agonists increases glucose-stimulated insulin secretion in db/db mice

Although peroxisome proliferator-activated receptor (PPAR)gamma agonists ameliorate insulin resistance, they sometimes cause body weight gain, and the effect of PPAR agonists on insulin secretion is unclear. We evaluated the effects of combination therapy with a PPARgamma agonist, pioglitazone, and a PPARalpha agonist, bezafibrate, and a dual agonist, KRP-297, for 4 wk in male C57BL/6J mice and db/db mice, and we investigated glucose-stimulated insulin secretion (GSIS) by in situ pancreatic perfusion. Body weight gain in db/db mice was less with KRP-297 treatment than with pioglitazone or pioglitazone + bezafibrate treatment. Plasma glucose, insulin, triglyceride, and nonesterified fatty acid levels were elevated in untreated db/db mice compared with untreated C57BL/6J mice, and these parameters were significantly ameliorated in the PPARgamma agonist-treated groups. Also, PPARgamma agonists ameliorated the diminished GSIS and insulin content, and they preserved insulin and GLUT2 staining in db/db mice. GSIS was further increased by PPARgamma and -alpha agonists. We conclude that combination therapy with PPARgamma and PPARalpha agonists may be more useful with respect to body weight and pancreatic GSIS in type 2 diabetes with obesity.     

Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling

The ephrin/Eph system plays a central role in neuronal circuit formation; however, its downstream effectors are poorly understood. Here we show that alpha-chimerin Rac GTPase-activating protein mediates ephrinB3/EphA4 forward signaling. We discovered a spontaneous mouse mutation, miffy (mfy), which results in a rabbit-like hopping gait, impaired corticospinal axon guidance, and abnormal spinal central pattern generators. Using positional cloning, transgene rescue, and gene targeting, we demonstrated that loss of alpha-chimerin leads to mfy phenotypes similar to those of EphA4(-/-) and ephrinB3(-/-) mice. alpha-chimerin interacts with EphA4 and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, downregulation of alpha-chimerin suppresses ephrinB3-induced growth cone collapse in cultured neurons. Our findings indicate that ephrinB3/EphA4 signaling prevents growth cone extension in motor circuit formation via alpha-chimerin-induced inactivation of Rac. They also highlight the role of a Rho family GTPase-activating protein as a key mediator of ephrin/Eph signaling.     

Effective syntheses of 2′,4′-BNANC monomers bearing adenine,guanine, thymine,and 5-methylcytosine,and the properties of oligonucleotides fully modified with 2′,4′-BNANC

We efficiently synthesized 2′-O,4′-C-aminomethylene-bridged nucleic acid (2′,4′-BNA^NC) monomers bearing the four nucleobases, guanine, adenine, thymine, and 5-methylcytosine and incorporated these monomers into oligonucleotides. Initially, we carried out the transglycosylation reaction on several 2′-O-substituted 5-methyluridines to evaluate the effects of 2′-substitutions on this reaction. Under the optimized conditions, purine nucleobases were successfully introduced, and 2′,4′-BNA^NC monomers bearing adenine or guanine were obtained over several steps. In addition, the improved synthesis of the 2′,4′-BNA^NC monomers bearing thymine or 5-methylcytosine was also achieved. The obtained 2′,4′-BNA^NC monomers were subsequently incorporated into oligonucleotides and the duplex-forming abilities of the modified oligonucleotides were investigated. Duplexes containing 2′,4′-BNA^NC monomers in both or either strands were found to possess excellent thermal stabilities.     

Factors Affecting Cerebral Oxygenation in Hemodialysis Patients: Cerebral Oxygenation Associates with pH,Hemodialysis Duration,Serum Albumin Concentration,and Diabetes Mellitus

BackgroundPatients undergoing hemodialysis (HD) often develop cerebral disease complications. Furthermore, cerebral regional saturation of oxygen (rSO₂) was previously reported to be significantly lower in HD patients than in healthy subjects. We aimed to identify the factors affecting the cerebral rSO₂ in HD patients.MethodsFifty-four HD patients (38 men and 16 women; mean age, 67.7 ± 1.2 years, HD duration, 6.5 ± 1.9 years) were recruited. Cerebral rSO₂ was monitored at the forehead before HD using an INVOS 5100C (Covidien Japan, Tokyo, Japan).ResultsThe rSO₂ levels were significantly lower in HD patients compared with healthy controls (49.5 ± 1.7% vs. 68.9 ± 1.6%, p <0.001). Multiple regression analysis showed that cerebral rSO₂ independently associated with pH (standardized coefficient: -0.35), HD duration (standardized coefficient: -0.33), and serum albumin concentration (standardized coefficient: 0.28). Furthermore, the rSO₂ was significantly lower in HD patients with diabetes mellitus (DM), compared with patients without DM (46.8 ± 1.7% vs. 52.1 ± 1.8%, p <0.05).ConclusionsIn HD patients, cerebral rSO₂ was affected by multiple factors, including pH, HD duration, and serum albumin concentration. Furthermore, this is the first report describing significantly lower levels of rSO₂ in HD patients with DM than in those without DM.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.