Acetyl- and butyrylcholinesterase inhibitory activities of sterols and phlorotannins from Ecklonia stolonifera

ABSTRACT:   As part of this study on the isolation of cholinesterase inhibitors from natural marine products, the bioactivity of the ethanolic extracts from 27 Korean seaweeds were screened using acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory assays. Ecklonia stolonifera exhibited promising inhibitory properties against both AChE and BChE. Bioassay-guided fractionation of the active n -hexane and ethyl acetate (EtOAc) soluble fractions, obtained from the ethanolic extract of E. stolonifera , resulted in the isolation of the sterols; fucosterol ( 1 ) and 24-hydroperoxy 24-vinylcholesterol ( 2 ), from the n -hexane fraction and the phlorotannins; phloroglucinol ( 3 ), eckstolonol ( 4 ), eckol ( 5 ), phlorofucofuroeckol-A ( 6 ), dieckol ( 7 ), triphlorethol-A ( 8 ), 2-phloroeckol ( 9 ) and 7-phloroeckol ( 10 ), from the EtOAc fraction. Of these, compounds 2 , 9 and 10 were isolated from E. stolonifera for the first time. Compounds 4 – 7 , 9 and 10 exhibited inhibitory potential against AChE, with 50% inhibition concentration (IC₅₀) values of 42.66 ± 8.48, 20.56 ± 5.61, 4.89 ± 2.28, 17.11 ± 3.24, 38.13 ± 4.95 and 21.11 ± 4.16 μM, respectively; whereas, compounds 1 , 2 , 4 and 6 were found to be active against BChE, with IC₅₀ values of 421.72 ± 1.43, 176.46 ± 2.51, 230.27 ± 3.52 and 136.71 ± 3.33 μM, respectively. It has been suggested that the inhibition of these enzymes by the sterols and phlorotannins derived from marine brown algae could be a useful approach for the treatment of Alzheimer's disease.     

Association of macrophage and lymphocyte infiltration with outcome in canine osteosarcoma

Immunotherapeutic strategies have shown promise for the treatment of canine osteosarcoma (cOSA). Very little is known about the immune microenvironment within cOSA, however, limiting our ability to identify potential immune targets and biomarkers of therapeutic response. We therefore prospectively assessed the disease‐free interval (DFI) and overall survival time (ST) of 30 dogs with cOSA treated with amputation and six doses of adjuvant carboplatin. We then quantified lymphocytic (CD3+, FOXP3+) and macrophage (CD204+) infiltrates within the primary tumours of this cohort using immunohistochemistry, and evaluated their association with outcome. Overall, the median DFI and ST were 392 and 455 days, respectively. The median number of CD3+ and FOXP3+ infiltrates were 45.8 cells/mm² (4.6‐607.6 cells/mm²) and 8.5 mm² (0‐163.1 cells/mm²), respectively. The median area of CD204+ macrophages was 4.7% (1.3%‐23.3%), and dogs with tumours containing greater than 4.7% CD204+ macrophages experienced a significantly longer DFI (P = 0.016). Interestingly, a significantly lower percentage of CD204+ macrophages was detected in cOSA arising from the proximal humerus compared to other appendicular bone locations (P = 0.016). Lymphocytic infiltrates did not appear to correlate with outcome in cOSA. Overall, our findings suggest that macrophages may play a role in inhibiting cOSA progression, as has been suggested in human osteosarcoma.     

Molecular identification of the vaccine strain from the inactivated oil emulsion H9N2 low pathogenic avian influenza vaccine

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.     

Molecular cloning and expression of caspase-3 in the protandrous cinnamon clownfish, Amphiprion melanopus, during sex change

The caspase-3 appears to be a key protease in the apoptotic pathway. We identified caspase-3 complementary DNAs from the ovaries of the protandrous cinnamon clownfish (Amphiprion melanopus), and investigated its mRNA and proteins, and activity levels during the sex change (I, mature male; II, male at 90 days after removing of the female; and III, mature female). The nucleotide sequence of the caspase-3 cDNA was 969 base pairs in length with open reading frames encoding peptides of 282 amino acids. The caspase-3 mRNA and protein, and activity levels in stages of the mature gonad are higher than those of the development gonad stage. To understand the effect of gonadotropin-releasing hormone (GnRH) on gonad apoptosis, we examined expression of genes caspase-3 mRNA and activity level in immature cinnamon clownfish gonads after GnRH analogue (GnRHa). The findings support the hypothesis that caspase-3 expression is associated with both testicular and ovarian development, and suggests that it may play a role in the control of ovarian development in cinnamon clownfish. Also, we demonstrate that GnRH agonists stimulate caspase-3 production which can in turn stimulate apoptosis. The present study provides a framework for better understanding of the role of caspase-3 during sex change processes in fish.     

Effects of Culture Area,Stocking Density,and Shrimp and Fish Polyculture on the Cost Efficiency of Hard Clam,Meretrix meretrix,Culture: A Case Study of Hard Clam Farms in Yunlin,Taiwan

Using the hard clam farms in Yunlin, Taiwan as examples, this study analyzes the effects of culture area, stocking density, and shrimp and fish polyculture on the cost efficiency (CE) of hard clam farming. A data envelopment analysis (DEA) is used to estimate the farms' technical efficiency (TE), allocative efficiency (AE), and CE values. The findings show that the mean TE, AE, and CE values of hard clam farms are 0.74, 0.57, and 0.43, respectively. The TE value is higher than the AE value, which indicates that cost inefficiency is mainly attributable to poor AE. Tobit regression results suggest that there are positive relationships between CE and both culture area and operator experience. Hard clam stocking density and age of the operators have negative effects on CE. Hard clam culture areas larger than 2 ha and a stocking density of 1.1–1.2 million hard clams per hectare have higher CEs than smaller culture areas and smaller or larger stocking densities. The use of sand shrimp, white shrimp, kuruma shrimp, and grass shrimp for polyculture and the use of milkfish and silver sea bream for polyculture are both associated with high CEs.     

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.     

Comparative stomach contents and growth of the juvenile black seabream, Acanthopagrus schlegeli, reared in illuminated and unilluminated cages

The present study was designed to determine the positive effects of artificial illumination on the juvenile black seabream, Acanthopagrus schlegeli , by comparing stomach contents and growth between juveniles exposed to light and those maintained in the absence of light. The major prey items for juvenile black seabream reared in illuminated cages were amphipods (IRI%=50.5), copepods (IRI%=44.7) and polychaetes (IRI%=3.0), whereas those for the juveniles maintained in unilluminated cages were copepods (IRI%=96.0), amphipods (IRI%=3.4) and polychaetes (IRI%=0.6). The specific growth rates (SGR) of the juveniles reared in illuminated cages (0.99%) were significantly higher than those of the juveniles maintained in unilluminated cages (0.78%).     

Comparative evaluation of MCP gene in worldwide strains of Megalocytivirus (Iridoviridae family) for early diagnostic marker

Members of the Iridoviridae family have been considered as aetiological agents of iridovirus diseases, causing fish mortalities and economic losses all over the world. Virus identification based on candidate gene sequencing is faster, more accurate and more reliable than other traditional phenotype methodologies. Iridoviridae viruses are covered by a protein shell (capsid) encoded by the important candidate gene, major capsid protein (MCP). In this study, we investigated the potential of the MCP gene for use in the diagnosis and identification of infections caused Megalocytivirus of the Iridoviridae family. We selected data of 66 Iridoviridae family isolates (53 strains of Megalocytivirus, eight strains of iridoviruses and five strains of Ranavirus) infecting various species of fish distributed all over the world. A total of 53 strains of Megalocytivirus were used for designing the complete primer sets for identifying the most hypervariable region of the MCP gene. Further, our in silico analysis of 102 sequences of related and unrelated viruses reconfirms that primer sets could identify strains more specifically and offers a useful and fast alternative for routine clinical laboratory testing. Our findings suggest that phenotype observation along with diagnosis using universal primer sets can help detect infection or carriers at an early stage.     

Replacement of fish meal with two fermented soybean meals in diets for rainbow trout (Oncorhynchus mykiss)

The present study investigated the effect of fish meal (FM) replacement with fermented soybean meal (FSM) on growth and feed utilization of rainbow trout. Two FSM products, FSM1 (more fermentation with more small peptide and acid than FSM2) and FSM2 were used to replace 20%, 40% and 60% of FM in control diet (250 g/kg FM), respectively (FSM1‐20, FSM1‐40, FSM1‐60, FSM2‐20, FSM2‐40 and FSM2‐60). Then the seven diets were fed to rainbow trout (18.1 g) for 8 weeks. Weight gain (WG), feed conversion ratio (FCR) and digestibility of crude protein and dry matter showed no significant difference among the groups of FSM1‐20, FSM1‐40, FSM2‐20, FSM2‐40 and the control, but WG significantly decreased and FCR increased when 60% FM was replaced by both FSMs (p < .05). The replacement of 40%, 60% FM resulted in lower villus height than the control (p < .05), and intestinal protease activity was lower in FSM2‐40, FSM2‐60 and FSM1‐60 groups than the control group (p < .05). In addition, the activity of alkaline phosphatase and superoxide dismutase increased with increasing levels of FSM (p < .05). In conclusion, dietary fish meal could be replaced by 40% with both FSMs without adverse effects on growth and feed utilization of rainbow trout based on an eight weeks feeding trial.     

Therapeutic trial of granulocyte-colony stimulating factor for dilated cardiomyopathy in three dogs

Three dogs were presented to us for evaluation of cardiac problems. Electrocardiographic recordings revealed severe tachyarrhythmia and atrial fibrillation with ventricular tachycardia in 2 of the 3 dogs. The echocardiographic findings of the 3 dogs revealed markedly decreased fractional shortening and a marked increase in E-point septal separation. Based on the results of electrocardiographic and echocardiographic evaluation, the 3 dogs were diagnosed as dilated cardiomyopathy (DCM). The dogs were treated with conventional cardiac medication, but cardiac function did not improve and the clinical signs remained. We subsequently attempted treatment with granulocyte-colony stimulating factor (G-CSF; 10 microg/kg, subcutaneously). The specific purpose of G-CSF therapy for DCM was to improve cardiac function and a significant improvement in cardiac function was confirmed. The three dogs had no treatment side effects. This case report suggests that G-CSF might have therapeutic effects for medically refractory DCM in dogs.