Application of SSR markers and real-time PCR for variety identification in azuki-bean [Vigna angularis (Willd.) Ohwi and Ohashi]

The azuki bean in Korea consists of seven domestic varieties which have been developed and registered for the public during last 25 years. Here, we present a simple but reliable method to screen and identify Korean azuki bean varieties. A method based on simple sequence repeat (SSR) markers is widely used for prominent gene identification and variety discrimination. In molecular biology, real-time polymerase chain reaction (PCR) is a laboratory technique based on the polymerase chain reaction that is used to amplify and simultaneously quantify a targeted DNA molecule. It enables easy detection of a specific sequence in a DNA sample without performing electrophoresis and further processes. For separation of seven Korean azuki bean varieties, 110 unique azuki bean SSR markers from an (AG)n-enriched library were selected, synthesized and used for polymerase chain reaction (PCR). Data were taken through acrylamide gel electrophoresis and automated multi-capillary electrophoresis system for selection of specific markers and then changed into proper formats for data mining analysis. Ten primer pairs that showed high polymorphism were chosen for the indepth study. These ten primers were re-amplified with real-time PCR and checked the cycle threshold (Ct) and temperature (Tm) for comparison of amplification sequence in seven varieties. Consequently, a total of 20 alleles and 6 SSR primers were detected from the standard PCR amplification. Within these 6 primers, 7 alleles of 3 SSR primers were isolated for variety identification. From real-time PCR results, 3 SSR primers were selected as efficient markers for discrimination of seven Korean azuki bean varieties. The approach described here could be applied in monitoring our varieties and can be adapted in the azuki bean breeding program.     

Sprayed intraperitoneal bupivacaine reduces early postoperative pain behavior and biochemical stress response after laparoscopic ovariohysterectomy in dogs

This study investigated the use of sprayed intraperitoneal bupivacaine to relieve postoperative pain behavior and biochemical stress response after laparoscopic ovariohysterectomy (LOVH) in dogs. Sixteen sexually intact female dogs were randomly assigned to two groups. The sprayed intraperitoneal bupivacaine (SIB) group received 4.4 mg/kg of sprayed intraperitoneal bupivacaine diluted to 0.25% with an equivalent volume of saline after pneumoperitoneum. The control group received 1.76 mL/kg of saline in a similar fashion. Both groups received preoperative periportal 5% bupivacaine (1 mL) before incision. Postoperative pain was measured using the short form of the Glasgow composite measures pain scale (CMPS-SF, 0-24). Serum cortisol and glucose concentrations were measured preoperatively and 0.5, 1, 2, 4, 6, 12, and 24h postoperatively. The SIB group had significantly lower CMPS-SF compared to the control group 1, 2, 4, 6, and 12h after the operation. Cortisol concentrations were significantly increased from preoperative concentrations in the control group at 0.5, 1, 2, and 4h post operation and at 0.5 and 1h post operation in the SIB group. No significant differences were seen in serum glucose within each group. This report suggests that the use of sprayed intraperitoneal bupivacaine can be used as part of a multimodal approach for pain management after LOVH in dogs.     

Use of canine small intestinal submucosa allograft for treating perineal hernias in two dogs

Here, we describe two dogs in which canine small intestinal submucosa (SIS) was implanted as a biomaterial scaffold during perineal herniorrhaphy. Both dogs had developed severe muscle weakness, unilaterally herniated rectal protrusions, and heart problems with potential anesthetic risks. Areas affected by the perineal hernia (PH) located between the internal obturator and external anal sphincter muscles were reconstructed with naïve canine SIS sheets. In 12 months, post-operative complications such as wound infections, sciatic paralysis, rectal prolapse, or recurrence of the hernia were not observed. Symptoms of defecatory tenesmus also improved. Neither case showed any signs of rejection or specific immune responses as determined by complete and differential cell counts. Our findings demonstrate that canine SIS can be used as a biomaterial scaffold for PH repair in dogs.     

Cloning of avian G(0)/G(1) switch gene 2 genes and developmental and nutritional regulation of G(0)/G(1) switch gene 2 in chicken adipose tissue

Adipose triglyceride lipase (ATGL), a newly identified lipase, is a rate-limiting enzyme for triglyceride hydrolysis in adipocytes. The regulatory proteins involved in ATGL-mediated lipolysis in fat tissue are not fully identified and understood. The G(0)/G(1) switch gene 2 (G0S2) is an inhibitor of ATGL activity by interacting with ATGL through the hydrophobic domain of G0S2. Here, for the first time, we have cloned the coding sequence of G0S2 cDNA for the chicken, turkey, and quail. Sequence comparisons with mammals revealed that the avian G0S2 also have a conserved hydrophobic domain. Avian G0S2 is predominantly expressed in adipose tissues relative to other tested tissues. Within the adipose tissue, G0S2 is expressed 20-fold greater in the adipocyte than in the stromal-vascular (SV) fraction (P < 0.001). Expression of G0S2 mRNA gradually increased during differentiation of chicken adipocytes in culture (P < 0.05). However, there is G0S2 expression in embryonic adipose tissue, SV fraction, and primary preadipocytes before confluence that generally have an increased capacity of cell proliferation, which indicates it has an important role in adipocyte differentiation rather than proliferation. For a better understanding of how G0S2 responds to environmental stimuli, chickens were fasted for 24 h and then refed. Expression of G0S2 in adipose tissue was dramatically decreased (P < 0.05) in the chickens and quail after a 24-h fasting period, and increased to the control level after refeeding. In contrast to G0S2 expression, ATGL expression was induced (P < 0.05) after the 24-h fasting period and rapidly returned to the control level during the refeeding period. These data indicate that changes in lipolytic activities of adipose tissue in vivo can be regulated by G0S2 expression, as an inhibitor of ATGL.     

Cloning of comparative gene identification-58 gene in avian species and investigation of its developmental and nutritional regulation in chicken adipose tissue

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis in chicken adipose tissue. Its regulation is not fully understood. Recent studies suggest ATGL may be regulated by physical protein-protein interactions. Comparative gene identification 58 (CGI-58) has been identified as an activator of ATGL in mice. The purpose of the current study was to clone and sequence the CGI-58 gene in avian species and to investigate its regulation during development, fasting, and refeeding. Here, we report the cloning and sequencing of the complete coding sequence of CGI-58 and the deduced AA sequences for the domestic chicken, turkey, and Coturnix quail. The CGI-58 protein is a 343-AA protein in the chicken and quail, and a 344-AA protein in the turkey. Sequence comparisons with the human and mouse show that the CGI-58 gene is highly conserved among avian and mammalian species, with complete identities at the predicted lipid-binding site. Cell fractionation of chicken fat cells and stromal-vascular cells revealed that CGI-58 is expressed primarily in mature adipocytes (P < 0.01). When compared in multiple organs and tissues, avian CGI-58 is expressed predominantly in the adipose tissue (P < 0.001), similar to ATGL. To understand CGI-58 expression during adipose tissue development, its mRNA expression was measured along with ATGL and stearoyl CoA desaturase (SCD-1) mRNA, an adipogenic marker, in embryos and adults. Messenger RNA expression of CGI-58 increased (P < 0.05) immediately after hatching, concurrent with peak ATGL expression. It is interesting that CGI-58 remained somewhat increased at posthatch d 11 and 33 as SCD-1 mRNA expression increased (P < 0.05). To evaluate the response of CGI-58 to nutritional status, chickens and quail were fasted for 24 h and subsequently refed. After the fasting period, CGI-58 mRNA was induced (P < 0.05) for both chickens and quail and was returned to control levels upon refeeding. The ATGL mRNA responded similarly, increasing dramatically after fasting and quickly decreasing with refeeding. The direct relationship between CGI-58 and ATGL mRNA expression indicates a role for CGI-58 in activating ATGL-mediated lipolysis in avian species.     

Socio-economic factors affecting smallholder tree planting and management intentions in Leyte Province,Philippines

Survey data have been collected from four rural communities in Leyte Province, the Philippines, on household tree planting and management intentions, as well as the socio-economic characteristics, attitudes to tree planting and management, farming practices and the number of trees planted. In relation to intended tree planting and management activities, respondents were asked a series of structured questions as to what tree species they intend to plant, how many individual trees of each of those species they plan to plant, and for what purpose they propose to plant each of the tree species. This paper reports the results of analyses of the stated tree management intentions of households in the four communities and identifies the socioeconomic factors that influence householders’ tree management intentions. Respondents were generally enthusiastic about the possibility for further tree planting on their land, 75% indicating they would undertake planting. About 60% indicated an interest in commercial tree farming, with no significant differences in this level of interest between communities. It was found that a number of socio-economic variables indicating higher levels of land ownership and previous experience in the forestry industry are related to the intention to plant trees to produce timber for sale. These include the use of materials from public lands, participation in community organisations and community forestry programs, and the present management of trees to produce timber for sale.     

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus,Containing Lactose-Binding Molecules,on Human Leukemic K562 Cells

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.     

Analysis of phenolic compounds by high-performance liquid chromatography and liquid chromatography/mass spectrometry in potato plant flowers, leaves, stems, and tubers and in home-processed potatoes

Potato plants synthesize phenolic compounds as protection against bruising and injury from bacteria, fungi, viruses, and insects. Because antioxidative phenolic compounds are also reported to participate in enzymatic browning reactions and to exhibit health-promoting effects in humans, a need exists for accurate methods to measure their content in fresh and processed potatoes. To contribute to our knowledge about the levels of phenolic compounds in potatoes, we validated and used high-performance liquid chromatography and liquid chromatography/mass spectrometry to measure levels of chlorogenic acid, a chlorogenic isomer, and caffeic acid in flowers, leaves, stems, and tubers of the potato plant and in home-processed potatoes. The total phenolic acid content of flowers (626 mg/100 g fresh wt) was 21 and 59 times greater than that of leaves and stems, respectively. For all samples, chlorogenic acid and its isomer contributed 96-98% to the total. Total phenolic acid levels (in g/100 g fresh wt) of peels of five potato varieties grown in Korea ranged from 6.5 to 42.1 and of the flesh (pulp) from 0.5 to 16.5, with peel/pulp ratios ranging from 2.6 to 21.1. The total phenolic acid content for 25 American potatoes ranged from 1.0 to 172. The highest amounts were present in red and purple potatoes. Home processing of pulp with various forms of heat induced reductions in the phenolic content. The described methodology should facilitate future studies on the role of potato phenolic compounds in the plant and the diet.     

Flowering and changes in respiration in Asiatic hybrid lilies as influenced by bulb vernalization

Asiatic hybrid lilies, Lilium × elegans Thunb., ‘Red Carpet’ and ‘Sunray’ were used to investigate the effect of bulb vernalization at 2.5 °C on plant growth, flowering, and CO₂ production (respiration), and to use the CO₂ production pattern to monitor the time of flower bud initiation and development. Lily shoot emergence and flowering were accelerated when bulbs received 2.5 °C bulb vernalization; however, flowering was delayed when bulbs were stored at 20 °C before treatment at 2.5 °C; this indicated that bulbs were de-vernalized. The maximum CO₂ level, and the minimum level, reached in 78 h in non-vernalized bulbs and in 110 h in 6 weeks of 2.5 °C (6 weeks/2.5 °C) treated bulbs, was increased as the 2.5 °C duration was increased; this indicated that CO₂ level can be an useful parameter to measure the cold stimulus (i) accumulated in bulbs following bulb vernalization. The respiration rate higher than the predicted values of the best-fit curves derived from the quadratic equations was designated as Blip A and this was correlated to the time of flower bud initiation and development. Shoot elongation may follow the rise in carbon dioxide levels after reaching the minimum level. It is proposed that increased carbon dioxide levels higher than the predicted levels (Blip A), was correlated to the time of flower bud initiation and development. Measurement of carbon dioxide production upon receipt of bulbs may be a useful technique to provide important information for optimum vernalization treatments for bulbs that have accumulated different levels of low temperature stimulus after bulb vernalization.