Detection of transgenic and endogenous plant DNA in blood and organs of Nile tilapia, Oreochromis niloticus fed a diet formulated with genetically modified soybean meal

Anxiety regarding the fate of ingested transgenic DNA in farmed fish fed genetically modified (GM) soybean meal (SBM) has been raised with regard to human consumption. The objective of this study was to detect possibility of gene transfer of transgenic and endogenous DNA fragments in Nile tilapia (Oreochromis niloticus) blood and organs after consumption of a GM SBM diet. Nile tilapias with an average weight of 75.0 g were fed diets containing 48% GM or non-GM SBM for 21 days. During this period, a GM SBM diet was fed to fish for 12 days, and then switched to feed with non-GM SBM for 9 days for determining the residual span of the transferred cauliflower mosaic virus (CaMV) 35S promoter fragment. Blood, spleen, liver, intestine, kidney, and muscle tissues were taken (n = 10) every three days during the feeding period. Total DNA was extracted from the samples and analyzed by polymerase chain reaction (PCR) for determining the presence of a 108-bp fragment of the CaMV 35S promoter and a 144-bp fragment of the soybean chloroplast-specific DNA. Low-copy chloroplast-specific DNA fragment was detected in all organ and tissue samples and the majority of intestinal samples of fish fed GM SBM diet. Similarly, a low number and faint signals of the CaMV 35S promoter fragments were detected in all organ samples except muscle of fish fed the GM SBM diet, while none were detected 3 days after changing to a non-GM SBM diet. A very low frequency of transmittance to muscle and organs of fish was confirmed. It is recognized that the low copy number of transgenic DNA in the GM SBM diet is a challenge to their detection in tissues. These results suggested that transgenic DNA would be processed in the gastrointestinal tract in a similar manner with conventional plant DNA

CARRIER-MEDIATED UPTAKE OF H 2 -RECEPTOR ANTAGONISTS BY THE RAT CHOROID PLEXUS: INVOLVEMENT OF RAT ORGANIC ANION TRANSPORTER 3

ABSTRACT: The choroid plexus (CP) acts as a site for the elimination of xenobiotic organic compounds from the cerebrospinal fluid (CSF). The purpose of the present study is to investigate the role of rat organic anion transporter 3 (rOat3; Slc22a8) in the uptake of H 2 -receptor antagonists (cimetidine, ranitidine, and famotidine) by the isolated rat CP. Saturable uptake of cimetidine and ranitidine was observed in rOat3-LLC with K m values of 80 and 120 M, respectively, whereas famotidine was found to be a poor substrate. The steady-state concentration of the H 2 -receptor antagonists in the CSF was significantly increased by simultaneously administered probenecid, although it did not affect their brain and plasma concentrations. Saturable uptake of cimetidine and ranitidine was observed in the isolated rat CP with K m values of 93 and 170 M, respectively, whereas 50% of the uptake of famotidine remained at the highest concentration examined (1 mM). The K i value of ranitidine for the uptake of cimetidine by the isolated CP (50 M) was similar to its own K m value, suggesting that they share the same transporter for their uptake. The inhibition potency of organic anions such as benzylpenicillin, estradiol 17␤-glucuronide, p-aminohippurate, and estrone sulfate for the uptake of cimetidine by the isolated rat CP was similar to that for benzylpenicillin, the uptake of which has been hypothesized to be mediated by rOat3, whereas a minimal effect by tetraethylammonium excludes involvement of organic cation transporter(s). These results suggest that rOat3 is the most likely candidate transporter involved in regulating the CSF concentration of H 2 -receptor antagonists at the CP

Differential Effects of Methoxy Group on the Interaction of Curcuminoids with Two Major Ligand Binding Sites of Human Serum Albumin

Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA) clearly indicated that curcumin (Cur), demethylcurcumin (Dmc) and bisdemethoxycurcumin (Bdmc) bind to both Site I (sub-site Ia and Ib) and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur , Dmc , Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds

Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus

<p>Abstract</p> <p>Background</p> <p>Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (<it>Marsupenaeus japonicus</it>), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome.</p> <p>Results</p> <p>Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of <it>Daphnia </it>and <it>Drosophila </it>suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences.</p> <p>Conclusions</p> <p>Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.</p

ブラウン動力学法によるタンパク質-リガンド複合体予測構造の評価.インフルエンザノイラミニターゼと阻害剤の複合体のシュミレーション

The prediction of protein-ligand complex structures plays an important role in structures based drug design.In this study,evaluations of predicted protein-ligand structures by using Brownian dynamics simulations were carried out.The complex strycture were generated by computational docking trials,which were frequently used in actual drug design.From the obtained complex structures,"correct answer"which reproduced the experimental structure and"incorrent answers"which were different from crystal structure were selected,and simulations of them were carried out.The complex between influenza A virus neuraminidase and its inhibitor was used as test set for the test calculations. For"incorret answers",the 3D structures were broken by the simulations,and the results seem to be caused by instabilities of the predicted structures