High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system

Bioactive recombinant bovine interleukin-18 (rboIL-18) was expressed using a baculovirus system. Normally, IL-18 is translated as a precursor form of a 24kDa polypeptide and processed by IL-1beta converting enzyme (ICE) to a mature bioactive form of 18kDa protein. Hence, to express active form IL-18, we constructed two recombinant baculoviruses containing boIL-18 and human ICE (hICE) genes, respectively, and superinfected these viruses into insect cells. Superinfection of both recombinant viruses into the cells resulted in the expression of a 24kDa precursor form and an 18kDa mature form detectable in the supernatant by immunoblotting using anti-porcine IL-18 antibody. Culture supernatant from the superinfected cells showed a synergistic effect with recombinant boIL-12 for production of interferon-gamma (IFN-gamma) in bovine peripheral mononuclear cells. By addition of histidine hexamer at the C-terminal of boIL-18, the mature IL-18 was purified. Bioactivity remained after purification.     

Production and in vivo testing of a recombinant bovine IL-12 as an adjuvant for Salmonella typhimurium vaccination in calves

A recombinant bovine interleukin-12 (boIL-12) that contains a histidine hexamer, rboIL-12His, was produced, purified and administered to calves. We first tried the purification of heterodimer IL-12 from a mixture of p40 homodimer, p40 monomer, and p40-p35 heterodimer with a p35 subunit tagged with a histidine hexamar at its C-terminal (p35His). A recombinant baculovirus expressing p35His was generated and used for superinfection with a recombinant baculovirus expressing p40 subunit. The expressed subunits, p40 and p35His, were assembled into a 70kDa heterodimer in insect cells, released into culture medium, and then purified using a nickel chelate column. The purified rboIL-12His was bioactive for induction of IFN-gamma in bovine peripheral blood mononuclear cells (PBMCs) in vitro.The purified rboIL-12His was then administered to calves with inactivated Salmonella Typhimurium (ST). When sera were assayed by ELISA, specific anti-ST IgG1 antibodies were detected in all ST immunized calves, but, specific anti-ST IgG2 antibodies were detected only in calves administered ST along with rboIL-12His, indicating a possible switch to a Th1 response. Administration of commercially available Salmonella vaccine did not elicit IgG2 antibodies in calves. These results suggest that co-administration of IL-12 with inactivated ST cells could induce a Th1-type response in calves.     

Bacterial Leaf Spot of Ivy Caused by Xanthomonas campestris pv. hederae

A bacterial leaf spot disease was observed on Hedera helix (English ivy) and H. canariensis (Algerian ivy) in Japan. The causal agent was identified as Xanthomonas campestris pv. hederae (Arnaud 1920) Dye 1978. Received 13 May 2002/ Accepted in revised form 3 July 2002     

The antiproliferative effect of bovine lactoferrin on canine mammary gland tumor cells

Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.     

Effect of bovine lactoferrin on functions of activated feline peripheral blood mononuclear cells during chronic feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum gamma-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIV-infected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConA-induced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection.     

Dehydroeffusol Rescues Amyloid β25–35-Induced Spatial Working Memory Deficit

Plant Foods for Human Nutrition - Amyloid β (Aβ) peptides produced from the amyloid precursor protein, a transmembrane protein, are neurotoxic and blocking the neurotoxicity may lead to...     

Comparisons of Ophiostomatoid fungi associated withTomicus piniperda andT. minor in Japanese red pine

Five species ofOphiostoma, twoLeptographium species and aGraphium species were isolated from two morphologically and ecologically similar bark beetle species,Tomicus piniperda andT. minor, and their infested Japanese red pine (Pinus densiflora) in Yamanashi Prefecture, central Honshu, Japan. An underscribedOphiostoma species andO. minus were isolated mainly fromT. piniperda and its galleries.Ophiostoma canum which was found for the first time in Japan was mainly fromT. minor and its galleries. Specific relationships between the beetles and fungal species are suggested. Contribution No.140, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba. Part of this study was presented at 108th Annual meeting of Japanese Forestry Society, April 2, 1997, Fukuoka, Japan.     

Development of a system for measuring calcitonin in the stingray Dasyatis akajei (a cartilaginous fish): the possible involvement of stingray calcitonin in gonadal development

To elucidate the physiological role of calcitonin (CT) in stingrays (cartilaginous fish), an enzyme-linked immunosorbent assay (ELISA) system using a specific antibody against stingray CT has been developed. Synthetic stingray CT was subcutaneously injected into mice four times—once every 2 weeks—together with an adjuvant. We purified the IgG antibody fraction using the protein A affinity chromatography from collected antiserum. Evaluating the antibody titer, we found the antibody’s optimum dilution ratio to be 600 times. Competitive ELISA has been developed using the antibody diluted 600 times. Our antibody did not cross-react with teleost CTs and muscle extraction, but cross-reacted with stingray plasma and the extract of the ultimobranchial gland, the secretary organ of stingray CT. Using this ELISA, we measured the plasma CT level in stingrays and examined its correlation with several mineral concentrations. Plasma CT did not show significant correlation to calcium, magnesium, inorganic phosphorus, sodium, chlorine, or urea, although there was a correlation among the factors involved in osmoregulation, such as sodium, chlorine, and urea. On the other hand, plasma CT was significantly correlated to body weight and length. Furthermore, there was a significant correlation between plasma CT and gonad weight. Since plasma CT was correlated with the weight of liver, which is involved in the synthesis of egg yolk protein, we examined the influence of 17β-estradiol (E2) on CT secretion. After E2 injection, the plasma CT level increased significantly. This is the first study to demonstrate that E2 induced plasma CT secretion in cartilaginous fish.     

Assessment of the measurement of canine and feline serum fibroblast growth factor-23 concentrations by automated chemiluminescence immunoassay

This study compared canine and feline fibroblast growth factor (FGF)-23 concentration measurements between automated chemiluminescence assay (CLEIA) and enzyme-linked immunosorbent assay (ELISA). Seventy serum samples each from dogs and cats were evaluated. FGF-23 measurements by CLEIA significantly correlated with those of ELISA in both dogs and cats. The Bland–Altman test showed that FGF-23 between CLEIA and ELISA had fixed and proportional biases, respectively, in both dogs and cats. Measurements by CLEIA were lower than those of ELISA, especially in higher serum FGF-23 concentrations. This study showed that FGF-23 concentrations in dogs and cats can be evaluated by automated CLEIA. However, FGF-23 cannot be directly compared between CLEIA and ELISA.     

Temperature and metal ions-dependent activity of the family I inorganic pyrophosphatase from the swine roundworm Ascaris suum

Temperature dependence, heat stability and metal ions-dependent activity were examined on the Family I inorganic pyrophosphatase (PPase) recently identified from Ascaris suum. Recombinant A. suum PPase (rAsPPase) showed an optimal activity at 55 degrees C. The rAsPPase was heat stable at 40 degrees C in the absence of added Mg(2+) and at 50 degrees C in its presence. The enzyme required divalent metal ions for its activity. The preferences for the metal ions (5 mM concentration) were in the order: Mg(2+)> Co(2+)> Cu(2+)> Fe(2+)> Zn(2+)> Mn(2+). On the contrary, enzyme activity was inhibited by Ca(2+). These findings suggest that catalytic features of AsPPase are consistent with the Family I PPases reported from a wide range of organisms.