Tributyltin-binding protein type 1 (fish acid glycoprotein) is a potential gatekeeper of ethinylestradiol action in fish.

Tributyltin (TBT)-binding protein type 1 in Japanese medaka (Oryzias latipes) (O.latTBT-bp1) is a fish lipocalin implicated in TBT binding and detoxification. We purified recombinant O.latTBT-bp1 (rO.latTBT-bp1; ca. 30 kDa) by using a baculovirus expression system and His- and Strep-tag chromatography process. Then, we examined O.latTBT-bp1 binding to several endo/exogenous steroid hormones by means of competitive binding assay. The dissociation constants for the binding of rO.latTBT-bp1 to DAUDA and ANS, two fluorescent ligands of lipocalin, were 7.06 and 13.6 µM, respectively. Multiple model validations indicated that a single-binding-site model was the most appropriate for evaluating rO.latTBT-bp1 binding. In the competitive binding assay, testosterone, 11-ketotestosterone, and 17ß-estradiol were each bound by rO.latTBT-bp1; rO.latTBT-bp1 showed the strongest affinity for testosterone (inhibition constant, Ki = 3.47 µM). Endocrine-disrupting chemical (synthetic steroid) also bound to rO.latTBT-bp1; the affinity for ethinylestradiol (Ki = 9.29 µM) was stronger than that for 17ß-estradiol (Ki = 30.0 µM). To determine the function of O.latTBT-bp1, we produced TBT-bp1 knockout medaka (TBT-bp1 KO), which we exposed to ethinylestradiol for 28 days. After exposure, the number of papillary processes in TBT-bp1 KO genotypic male medaka was significantly fewer (3.5), compared to that in wild-type male medaka (22). Thus, TBT-bp1 KO medaka were more sensitive to the anti-androgenic effects of ethinylestradiol than wild-type medaka. These results indicate that O.latTBT-bp1 may bind to steroids and act as a gatekeeper of ethinylestradiol action by regulating the androgen-estrogen balance.

The Effects of Brown Algae-Derived Monosaccharide L-Fucose on Lipid Metabolism in C57BL/6J Obese Mice.

Obesity is a global public health problem and a risk factor for several metabolic disorders as well as cancer. In this study, we investigated the effects of L-fucose on lipid metabolism through chronic and acute in vivo experiments in mice. In the chronic test, mice were fed a high-calorie diet (HCD) containing 0.0001%, 0.001%, 0.01%, and 0.1% L-fucose for one month. The L-fucose supplementation inhibited body weight and visceral fat mass gain in HCD-fed mice. The results of the acute test showed that L-fucose increased the ratio of serum high molecular weight adiponectin and enhanced glucose and lipid catabolism. Furthermore, L-fucose also decreased the expression of adipogenic genes (peroxisome proliferator-activated receptor γ and cluster of differentiation 36). In conclusion, this study provides a new approach to combat obesity and the related diseases.

Tetrodotoxin- and tributyltin-binding abilities of recombinant pufferfish saxitoxin and tetrodotoxin binding proteins of Takifugu rubripes.

We investigated the ability of recombinant pufferfish saxitoxin and tetrodotoxin binding protein types 1 and 2 of Takifugu rubripes (rTrub.PSTBP1 and rTrub.PSTBP2) to bind to tetrodotoxin (TTX) and tributyltin. Both rTrub.PSTBPs bound to tributyltin in an ultrafiltration binding assay but lost this ability on heat denaturation. In contrast, only rTrub.PSTBP2 bound to TTX even heat denaturation. This result suggests that the amino acid sequence of PSTBP2 may be contributed for its affinity for TTX.

Transcriptional responses of a bicarbonate-tolerant monocot, Puccinellia tenuiflora, and a related bicarbonate-sensitive species, Poa annua, to NaHCO3 stress.

Puccinellia tenuiflora is an alkaline salt-tolerant monocot found in saline-alkali soil in China. To identify the genes which are determining the higher tolerance of P. tenuiflora compared to bicarbonate sensitive species, we examined the responses of P. tenuiflora and a related bicarbonate-sensitive Poeae plant, Poa annua, to two days of 20 mM NaHCO3 stress by RNA-seq analysis. We obtained 28 and 38 million reads for P. tenuiflora and P. annua, respectively. For each species, the reads of both unstressed and stressed samples were combined for de novo assembly of contigs. We obtained 77,329 contigs for P. tenuiflora and 115,335 contigs for P. annua. NaHCO3 stress resulted in greater than two-fold absolute expression value changes in 157 of the P. tenuiflora contigs and 1090 of P. annua contigs. Homologs of the genes involved in Fe acquisition, which are important for the survival of plants under alkaline stress, were up-regulated in P. tenuiflora and down-regulated in P. annua. The smaller number of the genes differentially regulated in P. tenuiflora suggests that the genes regulating bicarbonate tolerance are constitutively expressed in P. tenuiflora.

Expression and functional characterization of recombinant tributyltin-binding protein type 2.

Tributyltin-binding proteins (TBT-bps) are members of the fish lipocalins that were isolated from the blood of Japanese flounder (Paralichthys olivaceus) and function in the binding and detoxification of TBT. In this study, we constructed a baculovirus-silkworm expression system and obtained recombinant TBT-bp2 (rTBT-bp2; 31.5 kDa) from the hemolymph of silkworm larvae injected with a recombinant baculovirus containing the TBT-bp2 gene. The binding potential of rTBT-bp2 was investigated and compared to that of the previously available recombinant TBT-bp1 (rTBT-bp1). Both rTBT-bp2 and rTBT-bp1 bound to DAUDA, a typical fluorescent ligand of lipocalins, with dissociation constants of 0.97 and 1.75 µM, respectively. The Hill coefficient value indicated that rTBT-bp2 may have multiple binding sites and strong negative cooperativity. These results suggest that the typical central cavity of lipocalins composed of eight specific ß-sheets is conserved in rTBT-bp2, as it is in rTBT-bp1, although rTBT-bp2 has different effects than rTBT-bp1 in TBT binding. In a competition assay, rTBT-bp2 displayed exponential binding affinity to TBT with an inhibition constant of 0.29 µM, demonstrating that TBT binds to the central ligand pocket of rTBT-bp2. However, three fatty acids did not show any affinity to rTBT-bp2. Further studies are required to elucidate the endogenous function of TBT-bps as fish lipocalins and their function in responding to xenobiotics.

Tributyltin in blood of marine fish collected from a coastal area of northern Kyushu, Japan.

We investigated levels of the pollutant tributyltin (TBT) in blood of pufferfishes (six species), Japanese sea perch, red sea bream, Japanese common goby, Japanese flounder, rockfish, conger eel, and sea mullet collected off the coast of northern Kyushu, Japan. We found considerable levels of TBT (1.4-190 ng/mL) accumulated in the blood of these fish. Blood TBT concentrations were 1.3-22.5 times liver concentrations and 4.9-78 times muscle concentrations, except in conger eel and mullet. We detected TBT (16-111 ng/mL-blood) in the plasma of the fine-patterned puffer (Takifugupoecilonotus) year-round, without any apparent seasonal trend. These results suggest that fish inhabiting coastal areas of Kyushu, Japan, continue to be contaminated with TBT.

Molecular cloning, sequencing, and gene expression analysis of tributyltin-binding protein type 1 in Japanese medaka fish, Oryzias latipes.

The full-length cDNA sequence of tributyltin-binding protein type 1 in Japanese medaka (Oryzias latipes) (Olat.TBT-bp1) was determined by means of rapid amplification of cDNA ends (RACE) of liver tissue. Analysis of the structure of the gene encoding Olat.TBT-bp1 revealed that the exonintron organization of this gene corresponds to that of the genes encoding lipocalin superfamily proteins, suggesting that Olat.TBT-bp1 can be categorized as a member of the lipocalin superfamily, which may play an important role in transportation, detoxification, and excretion of xenobiotic compounds. Reverse transcription - PCR revealed that Olat.TBT-bp1 was expressed mainly in the liver, and upregulation of its expression was detected 1, 2, and 4 weeks post hatching. Relative expression of the Olat.TBT-bp1 gene was significantly downregulated, compared with that in the solvent control, by exposure to tributyltin at 0.01 mg/l or triclosan at 1.7 mg/l. Further studies on Olat.TBT-bp1 expression in conjunction with other biochemical and physiological toxicities in response to chemical exposures are needed to increase our understanding and information of TBT-bps mechanisms and as molecular biomarkers of chemical exposures. The role of Olat.TBT-bp1 in xenobiotic detoxification and/or excretion needs more investigations.

Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales.

Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT.

Induction of tributyltin-binding protein type 2 in Japanese flounder, Paralichthys olivaceus, by exposure to tributyltin-d27.

In this study, individual Japanese flounder were intraperitoneally injected with 2 µg tributyltin-d27 (TBT-d27) fish⁻¹. Blood samples were collected on day 7 after injection. TBT-binding protein types 1 and 2 (TBT-bp1, -bp2) in the blood serum were quantified by western blotting analysis. As a result, the concentration of TBT-bp2 in TBT-d27 treated group increased to 220% of that in the solvent control, whereas the TBT-bp1 concentration decreased to 65% of that in the solvent control. Additionally, a positive relationship between the concentrations of TBT-bp2 and TBT was observed in blood sera of wild and cultured flounder. We suggest that TBT-bp2 is produced in response to TBT exposure and may play an important role in fish physiology.

Purification and characterization of tributyltin-binding protein of tiger puffer, Takifugu rubripes.

We successfully purified Trub.TBT-bpα, a tributyltin (TBT) binding protein (bp) of the tiger puffer, Takifugu rubripes. Tiger puffer was injected intraperitoneally with TBT (1.0mg/kg body weight) and Trub.TBT-bpα was purified from serum by ammonium sulfate fractionation, gel filtration chromatography and polyacrylamide gel electrophoresis. Gel electrophoresis revealed that the Trub.TBT-bpα has a molecular mass of approximately 48.5kDa and contains at least 40% N-glycan. The deduced 212 amino acid sequence of the protein showed the highest identity (41%, 212 amino acid overlap and E-value: 9e-42) with TBT-binding protein type 1 (TBT-bp1) of Paralichthys olivaceus (Japanese flounder). Analysis of the gene structure of Trub.TBT-bpα suggests that this protein belongs to the lipocalin superfamily, which may be important in the accumulation and elimination of TBT. Phylogenetic analysis suggests that functionalization of TBT-bps has occurred during evolution, and that the functions of this group of proteins might be important for fish survival.

Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder, Paralichthys olivaceus.

Tributyltin-binding protein type 1 (TBT-bp1) is a newly discovered protein that binds with TBT in the blood of the Japanese flounder, Paralichthys olivaceus. We determined the genomic sequence of TBT-bp1 and found that this protein has a conserved exon-intron structure that is common to the lipocalin protein family. The secondary and tertiary structures of TBT-bp1, predicted from amino acid sequence, included at least two alpha-helices and eight beta-sheets that are conserved in all lipocalins and form a barrel structure that may bind with ligands. Analysis of the gene structure, secondary structure, and tertiary structure demonstrated that TBT-bp1 could be classified as a lipocalin. A homology search revealed the presence of TBT-bp1-like proteins in eight species of teleost. When flounder were injected intraperitoneally with TBT-d27 at 11.6mug/fish, TBT-d27 was detected in the blood and in the skin mucus. The concentration of TBT-d27 in mucus was approximately 1/100 of that in the serum. Western blotting analysis revealed that TBT-bp1 was present in the skin mucus. These results suggest that TBT-bp1 in Japanese flounder binds with TBT and is excreted from the body via the mucus.

Tributyltin causes abnormal development in embryos of medaka, Oryzias latipes.

We examined the effects of tributyltin (TBT) on embryonic development, hatching success and sexual differentiation in Japanese medaka (Oryzias latipes). Embryos (within 8h after fertilization) were exposed to TBT in ovo via nanoinjection at concentrations of 0 (control), 0.16, 0.80, 3.96, 19.2 and 82.1 ng/egg. Embryonic survival, development and hatching were observed. Hatched fry were reared until 60 days when they sexually matured, and sexual differentiation was also examined by accordance of genetic and phenotypic sex, based on existence of DMY (a male determining gene in medaka) and secondary sex characteristics. As results, TBT caused a concentration-dependent mortality and impaired the embryonic development. However, no masculinization was detected at 60 dph medaka adults. Lowest observed effective concentration for inducing abnormal embryonic development was estimated to 0.16 ng/egg (ca. 160 ng/g egg).

Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus.

We used gel filtration chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis to purify tributyltin-binding protein type 2 (TBT-bp 2) from plasma of Japanese flounder (Paralichthys olivaceus) injected intraperitoneally with TBT (5.0 mg/kg body weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular mass of TBT-bp 2 was approximately 48 kDa, and isoelectric focusing-polyacrylamide gel electrophoresis indicated that the isoelectric point was approximately 3.0. TBT-bp 2 contained 40% N-glycan. The complete cDNA nucleotide sequence and the genome sequence of TBT-bp 2 were determined by means of rapid amplification of cDNA ends of liver tissue of Japanese flounder and a genome-walking technique, respectively. The 216 amino acid sequence of TBT-bp 2 showed 47% identity to the sequences of puffer fish (Takifugu pardalis) saxitoxin- and tetrodotoxin-binding protein but only 27% similarity to the sequence of TBT-bp 1. Analysis of the motif sequence of the amino acid sequence and the structure of the gene encoding TBT-bp 2 suggested that this protein belongs to the lipocalin superfamily.