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.     

Inhibition of diacylglycerol acyltransferase by alkamides isolated from the fruits of Piper longum and Piper nigrum

Pharmacological inhibition of acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) has emerged as a potential therapy for the treatment of obesity and type 2 diabetes. Bioassay-guided isolation of CHCl3 extracts of the fruits of Piper longum and Piper nigum (Piperaceae), using an in vitro DGAT inhibitory assay, lead to isolation of a new alkamide named (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (2), together with four known alkamides: retrofractamide C (1), pipernonaline (3), piperrolein B (4), and dehydropipernonaline (5). Compounds 2-5 inhibited DGAT with IC50 values of 29.8 (2), 37.2 (3), 20.1 (4), and 21.2 (5) microM, respectively, but the IC50 value for 1 was more than 900 microM. This finding indicates that compounds possessing piperidine groups (2-5) can be potential DGAT inhibitors.     

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.     

Photosynthetic characteristics of highbush blueberry (Vaccinium corymbosum cv. Bluecrop) leaves in response to water stress and subsequent re-irrigation

SUMMARY

Gas exchange and photosystem II (PSII) activities in the leaves of 2-year-old ‘Bluecrop’ highbush blueberry (Vaccinium corymbosum) were monitored during water stress and subsequent re-irrigation to investigate the effects of the intensity of water stress on changes in photosynthetic characteristics. The blueberry shrubs were not irrigated for 3 to 5 weeks, then re-irrigated daily up to 8 weeks. The decrease in soil water potential during water stress caused a progressive decrease in leaf water potential. Soil water potentials decreased to -0.26 MPa and -0.34 MPa at 3 and 5 weeks, respectively, following water stress, but recovered following subsequent re-irrigation, while the soil water potential in daily-irrigated shrubs was maintained at over -0.13 MPa throughout the experiment. Chlorophyll concentrations decreased with an increasing duration of water stress. Chlorophyll concentrations in leaves on shrubs subjected to water stress for 5 weeks did not recover following re-irrigation, unlike those subjected to water stress for 3 weeks. The leaves on shrubs subjected to water stress for 5 weeks maintained lower levels of chlorophyll during reirrigation. The net rate of CO₂ assimilation (A_n) decreased significantly with an increasing duration of water stress. Reirrigation reversed the decrease in A_n in leaves on shrubs subjected to water stress for 3 weeks. Stomatal conductance (g_s) exhibited a similar pattern to A_n. The actual quantum yield of photosystem II (Φ_PSII) and the electron transport rate (ETR) also decreased significantly with an increasing duration of water stress, although the F_v/F_m ratio was not affected. Φ_PPSII and ETR values in the leaves on shrubs subjected to water stress for 5 weeks did not recover after reirrigation, unlike those subjected to water stress for 3 weeks. Non-photochemical quenching increased with an increasing duration of water stress, but subsequent re-irrigation did not reverse the increase. These results indicate that the timing of re-irrigation of water-stressed ‘Bluecrop’ highbush blueberry is critical in order to maintain their photosynthetic capacity. Among the photosynthetic characteristics measured, Φ_PSII and ETR could be used as sensitive indicators to assess the physiological status of leaves of ‘Bluecrop’ highbush blueberry growing under water stress conditions.     

Effects of activation methods on DNA synthesis and development of parthenogenetic porcine embryos

This study investigated the timing of DNA synthesis and patterns of pronuclear (PN) formation during the first cell cycle, and its influence on developmental competence, velocity and proliferation index of porcine parthenote blastocysts produced by different activation treatments. Oocytes were activated as follows: electrical stimulation (EST), EST combined with 7.5 μg/ml cytochalasin B (EST + CCB), 10 μg/ml cycloheximide (EST + CHX) and 1.9 mm 6-dimethylaminopurine (EST + 6-DMAP) for 3 h. DNA synthesis and PN formation were evaluated using 1 mm 5'bromo-2'deoxy-uridne (BrdU) at 2 h intervals from 1 to 13 h or 5 to 13 h of post-activation (hpa), respectively. In EST, DNA synthesis started at 3 hpa, reached the peak at 11 hpa and decreased at 13 hpa. Treatment with 6-DMAP resulted in an early increase of DNA synthesis at 3 hpa, whereas CCB delayed DNA synthesis for 2 h. In EST and EST + 6-DMAP, most of the eggs showed 1PN, whereas, incidence of 2PN in EST + CCB was higher than 1PN. EST + CHX was observed with 1PN, 2PN and multiple PN. Blastocyst rate in EST + CCB and EST + 6-DMAP were significantly (p<0.05) higher than EST + CHX. But, the developmental velocity was not different among groups. Proliferation index of blastocysts, as indicated the number of blastomere at S-phase of the cell cycle was low in all groups. In conclusion, CCB, CHX and 6-DMAP used for producing porcine parthenogenetic embryos induced different onset of DNA synthesis and PN, but they did not affect the subsequent embryo development.     

Influenza virus neuraminidase inhibitory activity of phlorotannins from the edible brown alga Ecklonia cava

Influenza A virus infections continue to pose a major threat to humans and several animal species. Neuraminidase (NA) is one of the most promising targets for the development of drugs against influenza viruses because of its critical role in the viral life cycle. During the course of a search for NA inhibitors from edible natural sources, we found that the ethyl acetate layer of ethanol extracts of Ecklonia cava showed extremely high NA-inhibitory activity (72.1% inhibition at 30 μg/mL). Bioactivity-guided fractionation of the ethyl acetate layer yielded five phlorotannins, identified as phloroglucinol (1), eckol (2), 7-phloroeckol (3), phlorofucofuroeckol (4), and dieckol (5). The inhibitory activities of these compounds (1-5) against NAs from group-1 (A/Bervig_Mission/1/18 [H1N1], A/PR/8/34 [H1N1]) and group-2 (A/Hong Kong/8/68 [H3N2], A/Chicken/Korea/MS96/96 [H9N2]) influenza A were evaluated to determine potencies and kinetic behavior. Analyses using various in vitro influenza A virus NA assays showed that all five phlorotannin derivatives were selective NA inhibitors. Of the phlorotannins, phlorofucofuroeckol (4) exhibited the most potent inhibitory activities toward group-1 NAs (IC?? values, 4.5 and 14.7 μM), whereas dieckol (5) potently inhibited group-2 NAs. Kinetic analyses indicated that compounds 1-5 were all noncompetitive. Notably, these noncompetitive inhibitors synergized with oseltamivir to enhance the NA-inhibitory effects of oseltamivir.     

One year's study of dynamic and evolution of types I and II PRRSV in a swine farm

This study was to investigate dynamic and evolution of PRRSV in a seed-stock farm by monitoring PRRSV status from 11 June 2009 to 4 August 2010. For laboratory test, around 18-24 umbilical cords from farrowed sows and 5-95 sera from nursery and grow/finish pigs were submitted around every 2 weeks interval during the study. The submitted samples were tested for PRRSV using IDEXX PRRS 2XR ELISA kit, RT-nested PCR. The PRRSV-positive samples were further sequences based on ORF5 and analyzed using MEGA 3.1 program and Beast 1.5.4 package. The surveyed farm was first infected with type II PRRSV but it was infected newly with type I PRRSV of unknown origin, showing rapid substitution to type I PRRSV as a dominant strain in 2 weeks. The type I PRRSV was first detected from umbilical cord of a farrowed sow in 12 January 2010, and secondly from nursery pigs in 26 January 2010. Although sudden increase of mean S/P ratio was found in grow/finish pigs around 2 months earlier than first type I PRRSV detection, no type I PRRSV viremia was found. Thirty three ORF5 full sequences from 14 type II to 19 type I PRRSVs were obtained chronologically in this farm and the genetic characteristics and evolution rates of those sequences were analyzed. The substitution rates (/site/day) of two types were 4.03×10(-5) (type I), 3.09×10(-5) (type II), respectively, which was more frequent than previous reports. The calculated divergence time of type I PRRSV was consistent with the time when the sudden elevation of serum IgG in grow/finish barn was first observed. This study provided fundamental data for type I PRRSV dynamic in a previously type II PRRSV-infected farm and suggested grow/finisher barn could be a primary site for PRRSV introduction.     

Differential regulation of senescence and in vitro differentiation by 17β-estradiol between mesenchymal stem cells derived from male and female mini-pigs

The characterization and potential of mesenchymal stem cells (MSCs) are gender dependent and estrogen influences these properties. This study demonstrated that supplementation with 17β-estradiol (E2) increases the proliferation of bone marrow-MSCs derived from male and female mini-pigs (Mp- and Fp-BMSCs) in a concentration-dependent manner, with 10^-12 M E2 suggested as the optimal dose of E2 that led to the greatest improvement in BMSCs proliferation. Supplementation of 10^-12 M E2 resulted in down-regulation of β-galactosidase activity and pro-apoptotic activity in both BMSCs, while anti-apoptotic activity was up-regulated in only Fp-BMSCs. Further, E2 increased the osteogenic ability of Fp-BMSCs. Based on these findings, optimal utilization of E2 can improve cellular senescence and apoptosis, as well as in vitro osteogenesis of BMSCs, and could therefore be useful in stem cell therapy, particularly in bone regeneration for adult females.     

Modulation of cyclizing activity and thermostability of cyclodextrin glucanotransferase and its application as an antistaling enzyme

Cyclodextrin glucanotransferase from Bacillus stearothermophilus ET1 (CGTase ET1) is a potential antistaling enzyme with cyclodextrin (CD)-forming activity. To reduce cyclization activity of CGTase ET1, phenylalanine residues at 191 and 255 were replaced with a glycine (F191G-CGTase ET1) and an isoleucine (F255I-CGTase ET1), respectively. Temperature optima of both mutant enzymes were lower than that of the wild-type. Cyclization activities of both mutants decreased dramatically, but F255I-CGTase ET1 showed a 2-fold higher hydrolytic activity than the wild-type enzyme. CD content of bread loaf treated with F191G-CGTase ET1 was 28.6% of that treated with wild-type, whereas no CD was detected in the loaf treated with F255I-CGTase ET1. Loaves treated with CGTase ET1 or either of the two mutants contained more of the larger maltooligosaccharides such as maltopentaose and maltohexaose than the control and the commercial antistaling enzyme-treated loaves. Retrogradation rates decreased significantly in the loaves treated with either mutant, which indicates the applicability of CGTase ET1 in the bread industry by modulating the cyclizing and hydrolyzing activities of the enzyme.