GHRP-6 mimics ghrelin-induced stimulation of food intake and suppression of locomotor activity in goldfish

Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue (GHS) receptor (GHS-R). Ghrelin also acts as an orexigenic factor and regulates energy balance in rodents. In goldfish, native ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide with n-octanoic acid modification (n-octanoyl ghrelin17), and intraperitoneal (IP) administration of n-octanoyl ghrelin17 induces central actions such as stimulation of food intake and suppression of locomotor activity through capsaicin-sensitive afferents. Four types of GHS-Rs (1a-1, 1a-2, 2a-1 and 2a-2) have been identified in goldfish, and one GHS, GHRP-6, can activate only GHS-R2a-1 in vitro. However, there is no information about the effect of GHRP-6 on food intake and locomotor activity in goldfish in vivo. Therefore, in the present study, we examined whether IP-administered GHRP-6 would mimic the orexigenic action of n-octanoyl ghrelin17 and its suppression of locomotor activity. IP administration of GHRP-6 at 1pmol/g body weight (BW) stimulated food intake, and was equipotent to the orexigenic action of n-octanoyl ghrelin17 at 10 pmol/g BW. IP-injected GHRP-6 at 1 pmol/g BW also induced a significant decrease of locomotor activity, as was the case for IP-injected n-octanoyl ghrelin17 at 10 pmol/g BW. The action of GHRP-6 was blocked by IP-preinjected capsaicin at 160 nmol/g BW. These results suggest that the central action of GHRP-6 might be mediated via the GHS-R2a-1-signaling pathway, and subsequently through capsaicin-sensitive afferents in goldfish.     

A novel and efficient assay for identification and quantification of Acidithiobacillus ferrooxidans in bioleaching samples

Acidithiobacillus ferrooxidans is a Gram-negative, acidophilic, and chemolithotrophic bacterium that is active in bioleaching. The leaching efficacy is directly influenced by the biomass changes of this specie in bioleaching microbial community. In order to perform a simple and sensitive assay on A. ferrooxidans from mixed strains in this process, a novel assay was developed based on sandwich hybridization assay with the aid of S1 nuclease treatment and fluorescent labeling. In the work, a designed DNA probe complementary to the conservative region of its 16S rRNA was synthesized, which showed high accuracy for distinguishing homologous species with the exclusion of even-only two base pairs difference. The specificity of this assay was verified in different systems with mixed strains, and the quantitative result was proved by comparison of microscopic cell counting. The detection sensitivity was about 8 × 10(2) cells/ml and the inter-assay coefficient of variation of three independent assays was from 3.8 to 7.7 %, respectively. In addition, the cycle of assay was about 3-4 h when the cost estimated was less than $0.5 per sample. This assay method might be applied for identifying and monitoring any kind of bacterial strain from a mixed microbial flora in bioleaching or other areas.     

Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate

The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k(cat)/K(M) up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity.     

Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

Large‐scale gene expression reveals different adaptations of Hyalopterus persikonus to winter and summer host plants

Host alternation, an obligatory seasonal shifting between host plants of distant genetic relationship, has had significant consequences for the diversification and success of the superfamily of aphids. However, the underlying molecular mechanism remains unclear. In this study, the molecular mechanism of host alternation was explored through a large‐scale gene expression analysis of the mealy aphid Hyalopterus persikonus on winter and summer host plants. More than four times as many unigenes of the mealy aphid were significantly upregulated on summer host Phragmites australis than on winter host Rosaceae plants. In order to identify gene candidates related to host alternation, the differentially expressed unigenes of H. persikonus were compared to salivary gland expressed genes and secretome of Acyrthosiphon pisum. Genes involved in ribosome and oxidative phosphorylation and with molecular functions of heme–copper terminal oxidase activity, hydrolase activity and ribosome binding were potentially upregulated in salivary glands of H. persikonus on the summer host. Putative secretory proteins, such as detoxification enzymes (carboxylesterases and cytochrome P450s), antioxidant enzymes (peroxidase and superoxide dismutase), glutathione peroxidase, glucose dehydrogenase, angiotensin‐converting enzyme, cadherin, and calreticulin, were highly expressed in H. persikonus on the summer host, while a SCP GAPR‐1‐like family protein and a salivary sheath protein were highly expressed in the aphids on winter hosts. These results shed light on phenotypic plasticity in host utilization and seasonal adaptation of aphids.     

Evaluating the potential of the extract of Perilla sp. as a natural insecticide for Bemisia tabaci (Hemiptera: Aleyrodidae) on sweet peppers

The sweetpotato whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), is a major pest on greenhouse crops including sweet pepper (Capsicum annuum L.), which is one of the leading greenhouse crops in South Korea. Synthetic insecticides, especially the neonicotinoids, have been used to conventionally control this pest. There have been continuous efforts to develop plant‐derived compounds as insecticides, deterrents, and repellents to reduce spraying synthetic insecticides. To develop new plant‐extract insecticides, we investigated the insecticidal effects of Perilla sp. (Perilla frutescens var. crispa) extract on B. tabaci in laboratory conditions. The Perilla sp. extract induced 90 % mortality within one hour, but phytotoxicity symptoms on sweet pepper leaves were also observed. We monitored the population change and spatial distribution of adult B. tabaci in an experimental sweet pepper greenhouse using yellow sticky traps, and analyzed distribution patterns by spatial analysis with distance indices (SADIE). Based on monitoring data and SADIE analysis, we concluded that B. tabaci aggregated near the greenhouse entrances, and it showed aggregation and association pattern as time passed. Therefore, we recommend spraying Perilla sp. extract near the entrances or wild host before the pest population penetrates. It will be one of the alternative pest management strategies to reduce B. tabaci population with fewer negative effects from chemical insecticide. Further study is required to reduce the phytotoxicity symptoms from Perilla sp. extract spray and insecticidal effect should be evaluated under field conditions.     

Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel

The hERG (human ether‐a‐go‐go related gene) potassium channel is a voltage‐gated potassium channel containing an N‐terminal domain, a voltage‐sensor domain, a pore domain and a C‐terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2–S3 linker, S3, S4 and the S4–S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso‐myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4–S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3–S4 and S4–S4–S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Heat stress-induced modulation of host defense against Toxoplasma gondii infection in mice

This study examined the effects of burn injury on murine immune response against Toxoplasma gondii infection. Male C57BL/6 mice were divided into 3 groups: T. gondii infection (group T), burn injury (group B), and burn injury followed by T. gondii infection (group BT). The survival of group BT was significantly lower than those of group B and group T. Parasite abundance in the tissues was determined by quantitative competitive-polymerase chain reaction. Group BT exhibited significantly higher numbers of T. gondii than group T. Antibody production against T.g.HSP30 in group BT was significantly lower than that in group T, whereas no significant difference was observed in SAG1-specific antibody production. Delayed-type hypersensitivity (DTH) specific for 2,4-dinitrofluorobenzene (DNFB) of both group B and group BT was significantly lower than that of group T. One week after infection, serum interferon-gamma (IFN-gamma) and interleukin (IL)-10 levels in group BT were significantly lower, whereas serum IL-6 levels were significantly higher than in group T Serum TNF-alpha levels in both group T and group BT were elevated at 1 wk after infection, although there was no significant difference between them. Serum IFN-gamma, IL-10, and TNF-alpha levels in group B were not elevated during the experimental term. In conclusion, the impaired antigen-specific antibody production and DTH response, together with the modulated patterns of cytokine responses, seemed to be strongly involved in the development of burn-induced immunosuppression and the consequent increased susceptibility to T. gondii infection in mice.     

Functional response of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) to Aphis gossypii Glover (Homoptera: Aphididae) in the Laboratory

Stage specific functional response of Harmonia axyridis (Pallas) to varying densities of Aphis gossypii Glover was examined in a simplified cucumber leaf arena under laboratory conditions. All stages of H. axyridis were isolated individually for 24 h with different prey densities at 25 °C and a photoperiod of 16:8 (L:D) h. The number of prey consumed by the predator was checked at 3, 6, 12, and 24 h. All stages of H. axyridis showed a Type II functional response. Based on the random predator equation, estimated attack rates of H. axyridis at 24 h were 0.0037, 0.0442, 0.3590, 0.3228, and 0.1456, and estimated handling times were 4.1001, 2.4575, 0.7500, 0.2132, and 0.1708 h for the first, second, third, and fourth instars, and female adult, respectively.