Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Serologic and genetic characterization of North American H3N2 swine influenza A viruses

The H3N2 subtype of influenza A viruses isolated from pigs in the United States and Canada has shown both genetic and antigenic diversity. The objective of this study was to determine the serologic and genetic characteristics of contemporary strains of these viruses. Genetic analysis of 18 reference strains and 8 selected strains demonstrated differences in 1% to 9% of the nucleotides of the hemagglutinin (HA) gene. Phylogenetic analysis of the HA gene revealed 3 genetic clusters, as well as divergence of cluster III viruses from a cluster III prototype virus (A/Swine/Illinois/21587/99). By means of 1-way cross-hemagglutination inhibition with antiserum against 5 field isolates and 3 vaccine viruses, most of 97 isolates tested could be placed in 1 of 3 serogroups. The several isolates that did not react with any antiserum were in genetic cluster III, which suggests that continuous antigenic drift in cluster III may have resulted in virus variants. The efficacy of commercial vaccines against these virus variants should be evaluated with vaccination and challenge studies.     

Compensatory growth and body composition of juvenile black rockfish <Emphasis Type="Italic">Sebastes schlegeli</Emphasis> following feed deprivation

ABSTRACT:   Compensatory growth, feeding rate, feed efficiency and chemical composition of juvenile black rockfish (mean weight 1.43 g) were investigated for 35 days after a 14-day feed deprivation treatment under four feeding conditions: one group continuously fed (control) and the other three groups fasted for 5 days (F5), 10 days (F10) and 14 days (F14). All fasted fish were re-fed from day 15. Only F5 achieved the same body weight as the control, indicating that complete compensation occurred in F5. The specific growth rate (SGR) of F5 was the highest at day 21 and then decreased thereafter, showing higher values than the control at days 21, 28 and 42. In contrast, although SGRs of F10 and F14 were higher than that of the control during the whole refeeding period except day 21, they did not catch up the control in body mass, indicating that only partial compensation occurred in F10 and F14. The feeding rate (FR) of all groups except F14 changed in a pattern similar to SGR (Spearman's rank correlation, r _s > 0.9), suggesting that SGR varied depending on FR. Similar feeding efficiencies (FEs) were found in the four groups and they did not vary significantly during the whole refeeding period, suggesting that FE was not the factor affecting SGR. At day 14, the ratios of lipid to lean body mass in F10 and F14 were lower than those in the control and F5, and there was no difference between the control and F5. At day 49, however, only F14 showed a lower value than the other three groups, and there was no difference among the three groups. These results indicate that juvenile black rockfish fasted for 5–14 days can exhibit compensatory growth after refeeding, but timing and degree vary depending on the duration of feed deprivation.     

Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H₂O₂)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H₂O₂ in a dose-dependent manner. Additionally, H₂O₂ treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H₂O₂ significantly attenuated the H₂O₂-induced decrease of cell viability. H₂O₂ exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H₂O₂-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H₂O₂-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H₂O₂-induced apoptosis by inhibiting ROS generation.     

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.     

Effects of diet and sediment type on survival, growth and moulting of juvenile tiger crab, Orithyia sinica L.

This study investigated the effects of different types of diet and sediment on the growth parameters of juvenile tiger crab Orithyia sinica under laboratory conditions, focusing on survival, growth and moulting. In the first set of experiments, juvenile tiger crabs were reared with five different types of sediment, i.e., soft sand, coarse sand, soft sand mixed with coarse sand, mud or bare bottom. Soft sand resulted in not only the highest survival rate of 78% but also the best rate of growth and feed intake. Crabs reared with a mud substrate showed the worst results in these parameters. The intermoult period, however, was not affected by sediment types. In the second set of experiments, crabs were fed different types of diet to determine their effects on the same growth parameters. Diets were composed of different combinations with Manila clam (Ruditapes philippinarum), brine shrimp (Artemia nauplii), microencapsulated feeds and marine diatom (Chaetoceros gracilis). Survival and growth in crabs fed Manila clam as a diet component were significantly higher than in those on other diets. The highest survival and growth were achieved with a diet that included all four components. These results provide valuable information on the appropriate bottom substrates and diet types necessary for artificial mass culture of the tiger crab.     

Placotylene A,an Inhibitor of the Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclast Differentiation,from a Korean Sponge Placospongia sp.

A new inhibitor, placotylene A (1), of the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation, and a regioisomer of placotylene A, placotylene B (2), were isolated from a Korean marine sponge Placospongia sp. The chemical structures of placotylenes A and B were elucidated on the basis of 1D and 2D NMR, along with MS spectral analysis and revealed as an iodinated polyacetylene class of natural products. Placotylene A (1) displayed inhibitory activity against RANKL-induced osteoclast differentiation at 10 μM while placotylene B (2) did not show any significant activity up to 100 μM, respectively.     

Antioxidant activity of glyceollins derived from soybean elicited with Aspergillus sojae

The extract of soybean exposed to biotic elicitors such as food-grade fungus is known to have antioxidant activity. Glyceollins were major bioactive compounds present in soybean elicited by fungi and shown to have antifungal and anticancer activities. The purpose of present study was to evaluate the antioxidant activities of glyceollins by measuring ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, singlet oxygen quenching, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, hydroxyl radical scavenging activity, and lipid peroxidation inhibition. In addition, the antioxidant potential of glyceollins were measured by a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFDA), and dihydroethidium (DHE) in mouse hepatoma hepa1c1c7 cells in which they were insulted with H2O2 to generate reactive oxygen species (ROS). Glyceollins showed a strong reducing power and inhibited lipid peroxidation, with significant scavenging activities of radicals including singlet oxygen, superoxide anion, ABTS, and DPPH. We also found that glyceollins significantly suppressed H2O2-induced ROS production in hepa1c1c7 cells. Therefore, glyceollins deserve further study as natural antioxidants and nutraceuticals.     

Management of pine wilt disease in Korea through preventative silvicultural control

Pine wilt disease (PWD) represents a major threat to forest ecosystems worldwide. Although PWD is now better understood, effective control measures for this disease have still not been devised. Here, we report several years of field studies on preventative silvicultural control of PWD. Silvicultural control through preventative clear-cutting and the manual removal of logs was implemented between 2005 and 2009 in 16 Korean districts that had newly PWD-infected stands. Preventative clear-cutting of neighboring asymptomatic pine trees (within a 10-50-m radius of wilt trees) and the removal of felled logs or branches suppressed spread of PWD. Occurrences of PWD wilt pines in districts (city or county) subjected to this silvicultural control method were significantly reduced compared with those in districts using conventional controls (physical or chemical treatment of wilt pine trees). Through silvicultural control, PWD was successfully suppressed in 11 of 16 districts investigated. In contrast, successful control was achieved in only 1 among 18 districts subjected to conventional control. Our results will be of considerable interest to those engaged in the very difficult battle against the global spread of PWD.