Aerosol delivery of kinase-deficient Akt1 attenuates Clara cell injury induced by naphthalene in the lungs of dual luciferase mice

Conventional lung cancer therapies are associated with poor survival rates; therefore, new approaches such as gene therapy are required for treating cancer. Gene therapies for treating lung cancer patients can involve several approaches. Among these, aerosol gene delivery is a potentially more effective approach. In this study, Akt1 kinase-deficient (KD) and wild-type (WT) Akt1 were delivered to the lungs of CMV-LucR-cMyc-IRES-LucF dual reporter mice through a nose only inhalation system using glucosylated polyethylenimine and naphthalene was administrated to the mice via intraperitoneal injection. Aerosol delivery of Akt1 WT and naphthalene treatment increased protein levels of downstream substrates of Akt signaling pathway while aerosol delivery of Akt1 KD did not. Our results showed that naphthalene affected extracellular signal-regulated kinase (ERK) protein levels, ERK-related signaling, and induced Clara cell injury. However, Clara cell injury induced by naphthalene was considerably attenuated in mice exposed to Akt1 KD. Furthermore, a dual luciferase activity assay showed that aerosol delivery of Akt1 WT and naphthalene treatment enhanced cap-dependent protein translation, while reduced cap-dependent protein translation was observed after delivering Akt1 KD. These studies demonstrated that our aerosol delivery is compatible for in vivo gene delivery.     

A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate

Alaska pollack frame protein, which is normally discarded as an industrial byproduct in the processing of fish in plants, was hydrolyzed with pepsin. This was fractionated into five major types of Alaska pollack frame protein hydrolysates (APH-I, 10-30 kDa; APH-II, 5-10 kDa; APH-III, 3-5 kDa; APH-IV, 1-3 kDa; and APH-V, below 1 kDa) using an ultrafiltration membrane bioreactor system. Angiotensin I converting enzyme (ACE) inhibitory activities of the fractionated hydrolysates were investigated, and the fraction that exhibited the highest ACE inhibitory activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. Finally, we purified a novel ACE inhibitory peptide with an IC50 value of 14.7 microM, and the sequence of the peptide was Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala. In addition, the ACE inhibition pattern of the peptide was found to be noncompetitive.     

Polyozellin isolated from Polyozellus multiplex induces phase 2 enzymes in mouse hepatoma cells and differentiation in human myeloid leukaemic cell lines

Induction of cellular phase 2 detoxifying enzymes is associated with cancer preventive potential. Quinone reductase (QR) has been used as a prototype for anticarcinogenic phase 2 enzymes because of its widespread distribution in mammalian systems, large amplitude of inducer response, and ease of measurement in murine hepatoma cells. Methanol extract of Polyozellus multiplex, which shows a strong QR induction potential, was subjected to bioassay-guided fractionation, and polyozellin (PM1) appeared to be a major active component. In in vitro cultured cells (hepa1c1c7 and BPRc1 cells), polyozellin enhanced QR, glutathione S-transferase (GST) activities, and glutathione (GSH) content in a dose-dependent manner. The compound also significantly promoted differentiation of HL-60 human promyelocytic emia cells. In conclusion, polyozellin deserves further in vivo study to evaluate its potential as a cancer preventive agent.     

Possible role of plasma neurotensin in regulating the excitatory neural control of the rectum of the fowl

Effects of neurotensin (NT) applied via the blood vessel on the responses to stimulation of Remak's nerve (RNS) were investigated in the chicken isolated and perfused rectums. NT (5 ng-2 micrograms/ml) produced a concentration-dependent inhibition of the constituent contraction but not relaxation of the responses to RNS. In addition, high concentrations of NT (over 80 ng/ml) produced a contraction of the rectal muscle. Propranolol, a beta-adrenoceptor blocking agent, and guanethidine, an adrenergic neurone blocking agent, were able to reduce the inhibitory effect of NT on the response to RNS while potentiating the contractile effect of NT on the rectal muscle. NT (0.1 and 1 microgram/ml), like norepinephrine, decreased the flow rate of perfusate from the isolated rectum which was perfused at a constant pressure. Guanethidine enhanced norepinephrine-induced vasoconstriction, and phentolamine, an alpha-adrenoceptor blocking agent, plus propranolol was able to abolish it. Either of these prior applications resulted in a small but significant reduction of NT-induced vasoconstriction. These findings suggest that NT in plasma may function as a circulating hormone to exhibit an inhibitory action on the excitatory neural input to the rectum in the chicken, and that catecholamine release from adrenergic nerve terminals by NT may account for some but not all of the activity.     

Effects of partial removal of cytoplasmic lipid on survival of vitrified germinal vesicle stage pig oocytes

This study was designed to investigate whether the partial removal of cytoplasmic lipid from immature pig oocytes prior to vitrification had any positive effects on subsequent maturation, fertilization and early development. Oocytes at the germinal vesicle stage were partially freed from cumulus cells and centrifuged, and then polarized cytoplasmic lipid was removed by micromanipulation. When cultured for 44-48 h, significantly fewer of the centrifuged oocytes reached metaphase II (M-II) than did the non-centrifuged oocytes (approximately 53% vs approximately 68%, respectively); however, no further reduction in the M-II rate was observed when centrifuged oocytes were then delipated prior to culture (approximately 47%). To evaluate their sensitivity to the equilibration and vitrification solutions containing ethylene glycol, non-centrifuged, centrifuged, and delipated oocytes were cultured continuously for several minutes in those solutions, then washed and cultured further; no significant differences in the M-II rates (approximately 20-27%) were observed among the three treatment groups. When oocytes were vitrified and then warmed, significantly more delipated oocytes reached M-II in culture (approximately 15%) than did the non-delipated oocytes, whether centrifuged or not (approximately 4% in each group). When delipated, vitrified and matured oocytes were microsurgically injected with frozen-thawed spermatozoa, approximately 39% were activated and male pronucleus formation was observed in approximately 40% of activated oocytes; none developed beyond the 4-cell stage. These results show that maturation in vitro of vitrified pig oocytes can be promoted by partial removal of cytoplasmic lipid prior to vitrification and that the vitrified oocytes can be fertilized, although the embryonic development obtained in this study was limited.     

An outbreak of necrotic enteritis in the ostrich farm in Korea

An acute disease with high mortality occurred in the ostrich farm and characterized by depression, severe diarrhea and sternal recumbency. Four dead ostriches of the farm were submitted to the National Veterinary Research & Quarantine Service, and diagnosed as necrotic enteritis. In the gross and histopathological examination, extensive diffuse fibrinonecrotic enteritis was found in the small intestine, especially jejunum. Clostridium perfringens was isolated from a pure culture from the duodenum and jejunum of these birds. Based on our current knowledge, this is the first report of an outbreak of necrotic enteritis in the ostrich in Korea.     

Production of blastocysts after intergeneric nuclear transfer of goral (Naemorhedus goral) somatic cells into bovine oocytes

Interspecies cloning may be a useful method to help conserve endangered species and to study nuclear-cytoplasm interaction. The present study investigated in vitro development of goral (Naemorhedus goral) intergeneric nuclear transfer embryos produced by fusing goral fibroblasts with enucleated metaphase II (MII) bovine oocytes. After two to five passages, serum-starved or non-starved goral skin fibroblast cells were transferred into enucleated MII bovine oocytes. Couplets were electrically fused and chemically activated, and then cultured in either modified synthetic oviduct fluid (mSOF) or tissue culture medium-199 (TCM-199) supplemented with 10% FBS. Serum starvation of donor cells did not affect the fusion rate and or development to of cells to the two-cell stage, to more than 9-cells, or to morulae, regardless of culture medium. Three blastocysts from 202 fused embryos were obtained when embryos reconstructed with non- serum- starved donor cells were cultured in mSOF. However, no blastocysts were obtained when the embryos reconstructed with serum-starved donor cells were cultured in mSOF. The total cell number of goral intergeneric embryos averaged 130.3 (range 105-180). In conclusion, this study demonstrated that bovine oocytes can support blastocyst development after intergeneric SCNT with goral fibroblasts.     

Antiviral activity of Alpinia katsumadai extracts against rotaviruses

In vitro anti-rotavirus activity of Alpinia katsumadai (AK) extracts were evaluated against bovine G8P[7] and porcine G5P[7] rotaviruses in two different assay strategies, a mixed treatment assay and a post treatment assay. In the mixed treatment assay, six AK extracts [AK-1 (EtOH extract), AK-3 (H(2)O layer), AK-5 (40% methanol fraction), and AK-9-11 (H(2)O extract, polysaccharide fraction, supernatant fraction)] exhibited inhibitory activities against G5P[7] rotavirus with the EC(50) values ranging from 0.7±0.4 to 33.7±6.5 μg/mL. Extracts AK-1, AK-3, and AK-5 inhibited rotavirus infection against G8P[7] rotavirus, the with EC(50) values of 8.4±2.2 μg/mL, 6.5±0.8 μg/mL and 8.4±5.0 μg/mL, respectively. By hemagglutination inhibition (HI) assay, six AK extracts completely inhibited viral adsorption onto human RBCs in both strains of rotaviruses at less than 11 μg/mL. However, in the post treatment assay, there was no anti activity shown against both strains of rotaviruses. As a result, six AK extracts were attributed mainly to having a strong interaction with hemagglutinin protein on the outer surface of rotavirus, resulting to blockage of viral adsorption.     

Effect of supplementation of multi-microbe probiotic product on growth performance, apparent digestibility, cecal microbiota and small intestinal morphology of broilers

The present study investigated the effect of inclusion of multi-microbe probiotic product on growth performance, apparent digestibility of nutrients, cecal microbiota and small intestinal morphology in broilers. Four hundred days-old Ross chicks were randomly allotted to five treatments on the basis of body weight (BW). Each treatment had four replicates of 20 chicks in each. Experimental diets were fed in two phases, starter (day 0-21) and finisher (day 22-35). Dietary treatments were; basal diet without any antimicrobial (NC), basal diet added with 20 mg Avilamycin/kg of diet (PC), 10(7) cfu multi-microbe probiotic/kg of diet (P1), 10(8) cfu multi-microbe probiotic/kg of diet (P2), and 10(9) cfu multi-microbe probiotic/kg of diet (P3). Overall BW gain and feed conversion ratio were better (p < 0.05) for treatments PC, P2 and P3 compared with NC and P1, with P1 being better (p < 0.05) than NC. Overall feed intake in treatments PC, P1, P2 and P3 were greater (p < 0.05) than NC. Apparent digestibility of dry matter and crude protein were greater (p < 0.05) in treatments PC, P2 and P3 compared with NC, with P1 being intermediate and not different form NC, PC, P2 and P3. At d 21 and 35, treatments PC, P1, P2 and P3 showed lower (p < 0.05) cecal Clostridium and Coliforms count in relation to NC. Moreover, cecal Clostridium (d 21) and Coliforms (d 21 and 35) count were lower (p < 0.05) in treatment PC in relation to P1; with P2 and P3 being intermediate and not different from PC. However, there was no effect of dietary treatments on cecal total anaerobic bacteria and Bifidobacterium spp. count. The villus height of duodenum in treatment PC was greater (p < 0.05) than NC, with P1, P2 and P3 being intermediate. Villus height of ileum in treatment PC was greater (p < 0.05) than in treatments P1 and NC, whereas it remained comparable among treatments PC, P2 and P3. Villus height to crypt depth ratio of ileum was greater (p < 0.05) for treatment PC, P2 and P3 compared with that in P1 and NC. It is concluded that multi-microbe probiotic inclusion at 10(8) and 10(9) cfu/kg diet had beneficial effects on broilers growth performance, apparent digestibility of nutrients and intestinal morphology and can be used as replacement to antibiotics growth promoter in broiler nutrition.     

Detection and molecular characterization of porcine type 3 orthoreoviruses circulating in South Korea

Orthoreoviruses infect virtually all mammalian species, causing systemic infections including mild gastrointestinal and respiratory illnesses. However, little is known about the prevalence or genetic diversity of porcine orthoreoviruses in South Korea. We examined 237 diarrheic fecal samples collected from 78 pig farms around the country. RT-PCR utilizing primers specific for the L1 gene of mammalian orthoreoviruses showed that 45 (19.0%) samples were positive. The 10 strains isolated from orthoreovirus-positive samples formed typical perinuclear cytoplasmic inclusion bodies and had an atypical hemagglutination pattern; these are characteristics of type 3 orthoreovirus. Phylogenetic analysis of the S1 gene in these 10 Korean and other strains showed that type 3 orthoreoviruses could be divided into four lineages; the 10 Korean strains were included in porcine lineage IV, along with T3/porcine/Sichuan/2006. Sequence analysis showed that strains in lineage IV had nucleotide identities of 97.0-98.1% and deduced amino acid identities of 96.4-98.2%. Sequence analysis of the σ1 protein, a viral attachment protein, revealed that the amino acid sequences associated with neurotropism (amino acids 198-204, 249I, 350D, and 419E) were highly conserved among the Korean strains, confirming that neural tropism was present. In conclusion, our findings suggest that porcine orthoreovirus infections are endemic in pig farms in South Korea and that the 10 novel Korean porcine orthoreoviruses belong to porcine lineage IV of type 3 orthoreovirus. In addition, sequence analysis of S1 genes encoding the σ1 protein showed that the 9 of 10 Korean porcine orthoreoviruses exhibited neural tropism.