Effect of nucleotides supplementation to low‐fish meal feed on long‐chain polyunsaturated fatty acid composition of juvenile rainbow trout Oncorhynchus mykiss

A feeding trial was conducted to evaluate the effect of nucleotides supplementation to low‐fish meal feed on growth and fatty acid composition of rainbow trout. Six isonitrogenous (42% crude protein) and isolipidic (18% crude lipid) diets were formulated containing fish meal and plant ingredients as main protein sources. The control diet was a basal diet without supplementation of nucleotides, and five experimental diets were prepared by supplementing one of the five different nucleotides in the form of 5′‐monophosphate (0.15%), that is inosine (IMP), adenosine (AMP), guanosine (GMP), uridine (UMP) and cytidine (CMP) onto basal diet. Two hundred forty juvenile rainbow trout with an initial average body weight 9.8 g were randomly distributed into twelve aquaria. After 15 weeks of feeding period, growth performance and feed utilization of rainbow trout were not significantly different among dietary treatments. Dietary GMP, UMP and CMP tended to accumulate crude lipid in the muscle and whole fish body. Moreover, dietary GMP, UMP and CMP significantly increased hepatic 18:3n‐3 and long‐chain homologue 18:4n‐3 and 20:4n‐3 contents. Hepatic 18:2n‐6 content showed also increase in fish fed GMP, UMP and CMP diets, but decreased in long‐chain homologue 20:3n‐6 and 20:4n‐6 contents. Decrease in 20:4n‐6, 20:5n‐3 and 22:6n‐3 contents was also found in the muscle of fish fed IMP, GMP and CMP diets. The present study clearly showed that there was no positive effect of dietary nucleotides on growth of fish, but dietary nucleotides particularly GMP, UMP and CMP altered polyunsaturated fatty acid composition of rainbow trout.     

Responses to the Standard for Exchange of Nonclinical Data (SEND) in non-US countries

The Standard for the Exchange of Nonclinical Data (SEND), adopted by the US FDA, is part of a set of regulations and guidances requiring the submission of standardized electronic study data for nonclinical and clinical data submissions. SEND is the nonclinical implementation of SDTM (Study Data Tabulation Model), the standard electronic format for clinical regulatory submissions to FDA. SEND, SDTM, and the associated Controlled Terminology have been developed by CDISC (Clinical Data Interchange Standards Consortium). In order to successfully implement SEND, interdisciplinary contributions between sponsors and CROs, need a model for task allocation. This is being undertaken by the Pharmaceutical Users Software Exchange (PhUSE). Because SEND is currently the preferred submission format of the US FDA only and will become required by it starting in December 2016, only American academic societies and companies are actively involved. An exception to this is the INHAND initiative, which leads the way in standardizing terminology for toxicological pathology. On the other hand, international globalization of other clinical and nonclinical practices is not feasible because there are substantial differences between the US and non-US countries in CRO involvement in drug development. Thus, non-US countries must consider and develop approaches to SEND that meet their needs. This paper summarizes the activities of the major organizations involved in SEND development and implementation, discusses the effective use of SEND, and details a compliance scheme (research material of the Showa University School of Medicine) illustrating how pharmaceutical companies can complete a large amount of work up to an FDA application with the effective utilization of CROs and solution providers.     

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.     

Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle

The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.     

Plectosporium blight of monkshood caused by <Emphasis Type="Italic">Plectosporium tabacinum</Emphasis>

Severe blight of potted seedlings of monkshood caused by Plectosporium tabacinum was found in glasshouses in Kagawa Prefecture in southwest Japan in May 2001. Root rot and browning of stem bases were followed by wilting and yellowing of leaves, then blighting of leaves, flower buds and stems. A fungus was isolated from diseased plants and confirmed to cause the disease. The new disease was named “Plectosporium blight of monkshood”.     

High levels of sugars and fructan in mature seed of sweet wheat lacking GBSSI and SSIIa enzymes

Sweet wheat (SW), which lacks functional granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa), accumulates high levels of free sugars in immature seeds. Here, we examined the effects of the lack of these two enzymes on mature kernel composition. Whole grain flour of SW had higher levels of sugars, particularly maltose, slightly higher ash and protein content, approximately two to three times higher lipid levels, and about twice as much total dietary fiber as parental or wild-type lines. Considerably higher levels of low-molecular-weight soluble dietary fiber (LMW-SDF), largely consisting of fructan, were also detected in SW. Although there were no differences in total amino acid levels, the free amino acid content of SW was approximately 4-fold higher than that of wild type, and the levels of certain free amino acids such as proline were particularly high. Thus, we were able to clearly demonstrate that the lack of GBSSI and SSIIa caused dramatic changes in mature seed composition in SW. These compositional changes suggest that SW flour may provide health benefits when used as a food ingredient.     

Determination of antioxidant potential of volatile extracts isolated from various herbs and spices

Antioxidant activities of volatile extracts isolated from thyme, basil, rosemary, chamomile, lavender, and cinnamon were evaluated by two independent assays: the aldehyde/carboxylic acid assay and the conjugated diene assay. The volatile extracts were prepared from dried herbs and spices using liquid-liquid continuous extraction following steam distillation under reduced pressure (55 degrees C and 95 mmHg). The antioxidant activities of the extracts decreased in the following order in both of the lipophilic assay systems: thyme > basil > rosemary > chamomile > lavender and cinnamon. Thyme and basil extracts inhibited the oxidation of hexanal for 40 days at the levels of 10 microg/mL and 50 microg/mL, respectively. The extracts of thyme and basil were effective in retarding methyl linoleate deterioration at 40 degrees C, with activity increasing with concentration in the range 10-200 microg/mL. At a concentration of 50 microg/mL, thyme extract was similar in antioxidant activity to BHT and alpha-tocopherol in the conjugated diene assay. The antioxidant potentials of the volatile extracts used in this study were accurately measured by the lipophilic systems, such as the aldehyde/carboxylic acid assay and the conjugated diene assay.     

Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by Maillard reaction

Typical heterocyclic compounds substituted with various functional groups found in Maillard reaction products were examined for antioxidant activity. Pyrroles exhibited the greatest antioxidant activity among all heterocyclic compounds tested. All pyrroles inhibited hexanal oxidation by almost 100% at a concentration of 50 microg/mL over 40 days. Addition of formyl and acetyl groups to a pyrrole ring enhanced antioxidative activity remarkably. Pyrrole-2-carboxaldehyde, 2-acetylpyrrole, 1-methyl-2-pyrrolecarboxaldehyde, and 2-acetyl-1-methylpyrrole inhibited hexanal oxidation by >80% at 10 microg/mL. Unsubstituted furan exhibited the greatest antioxidant activity among furans tested. Addition of all functional groups used in this study to furan decreased antioxidative activity. The antioxidant activity of thiophene increased with the addition of methyl and ethyl groups, but the addition of formyl or acetyl groups to thiophene decreased antioxidant activity. Thiazoles and pyrazines were ineffective antioxidants at all concentrations tested. Reaction of all heterocyclic compounds with hydrogen peroxide resulted in the formation of various oxidized products.