A gastric-brooding asteroid, Smilasterias multipara

The gastric-brooding asterinid sea star, Smilasterias multipara, broods from late August to early November in the shallow sublittoral zone of southeastern Australia. We observed males and females spawning in the laboratory. They shed gametes through gonopores on the sides of the arms. The eggs were orange, about 1.0 mm in diameter, and heavier than seawater. They were externally fertilized by sperm, and placed into the stomach of the female by the tube feet. Twenty-four hours after fertilization, the first cleavage occurred. Cleavage was equal, total, and radial. Development via a non-feeding lecithotrophic brachiolaria was direct, there being no planktrotrophic bipinnaria or brachiolaria larva. Embryos developed, through wrinkled blastula and gastrula stages, into brachiolariae with arms. All of the surfaces of the brachiolaria were covered by cilia. At metamorphosis, a starfish rudiment appeared on the posterior portion of the larval body, while the anterior portion of the larval body was absorbed. Two months after fertilization, metamorphosis was complete. After metamorphosis, juveniles in the stomach grew six pairs of tube feet in each arm. Juveniles, 3 mm in diameter, emerged from the mouth of the mother in early November. Developmental evidence suggests that this asteroid has evolved mechanisms for the protection of larvae and juveniles from gastric digestion.     

Headspace solid-phase microextraction and gas chromatographic determination of dinitroaniline herbicides in human blood, urine and environmental water

Solid-phase microextraction (SPME) is a unique extraction and sampling technique, and it has been used for separation of volatile organics from water or other simple matrices. In this study, we have used SPME to separate dinitroaniline herbicides from complicated matrices of human urine and blood in order to broaden its application to biomedical analysis. The SPME conditions were optimized for water, urine and blood samples, in terms of pH, salt additives, extraction temperature, and fiber exposure time. Urine or water (1.0 ml) spiked with herbicides and 0.28 g of anhydrous sodium sulfate was preheated at 70°C for 10 min, and a polydimethylsiloxane-coated fiber for SPME was exposed to the headspace at 70°C for another 30 min; while spiked blood (0.5 ml) diluted with water (0.5 ml) was treated at 90°C in the same way. The herbicides were extractable under these conditions, and could be determined by gas chromatography–electron capture detector (GC–ECD). The recoveries of the herbicides, measured at the concentrations of 0.50 and 1.0 ng/ml urine or water, or 6.0 and 20 ng/0.5 ml blood, ranged from 35 to 64% for different herbicides from water or urine, and from 3.2 to 7.2% from blood. The headspace SPME yielded clean extracts of dinitroaniline herbicides from urine, blood or water, which could be directly analyzed by GC–ECD without further purification. The peak areas of the extracted herbicides were proportional to their concentrations in the range 0.1–10 ng/ml in water or urine, or 1–60 ng/0.5 ml in blood. The lowest detectable concentration of the herbicides lay in 0.1 ng/ml water or urine, or in 0.5 ng/0.5 ml blood. The intra- and inter-day coefficients of variation were within 14% for most of the analytes. Although the recoveries of the herbicides were rather low, the linearity of calibration curve and the precision were good. The developed method is more sensitive and much simpler in sample preparation than previously reported ones. With the established SPME method, a dosed herbicide was successfully separated and determined in rats' blood.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.     

The ubiquitin ligase gene (WWP1) is responsible for the chicken muscular dystrophy

Chicken muscular dystrophy with abnormal muscle (AM) has been studied for more than 50 years, but the gene responsible for it remains unclear. Our previous studies narrowed down the AM candidate region to approximately 1Mbp of chicken chromosome 2q containing seven genes. In this study, we performed sequence comparison and gene expression analysis to elucidate the responsible gene. One missense mutation was detected in AM candidate genes, while no remarkable alteration of expression patterns was observed. The mutation was identified in WWP1, detected only in dystrophic chickens within several tetrapods. These results suggested WWP1 is responsible for chicken muscular dystrophy.     

Structural basis of aspartylglucosaminuria

To elucidate the basis of aspartylglucosaminuria (AGU) from the viewpoint of enzyme structure, we constructed structural models of mutant aspartylglucosaminidase (AGA) proteins using molecular modeling software, TINKER. We classified the amino acid substitutions responsible for AGU and divided them into three groups based on the biochemical phenotype. Then, we examined the structural changes in the AGA protein for each group by calculating the solvent-accessible surface area (ASA), the number of atoms affected, and the root-mean-square deviation (RMSD). Our results revealed that the structural changes in group 1, which exhibits folding/transport defects and a complete deficiency of AGA activity, were generally large and located in the core region of the enzyme molecule. In group 2, exhibiting the mature AGA protein but no AGA activity, the functionally important region of the enzyme molecule was seriously affected. In group 3 exhibiting residual AGA activity, the structural changes in AGA were small and localized near the surface of the enzyme molecule. Coloring of affected atoms based on the distances between the wild-type and mutant ones revealed the characteristic structural changes in the AGA protein geographically and semi-quantitatively. Structural investigation provides us with a deeper insight into the basis of AGU.     

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.     

Co-expression of matriptase and N-acetylglucosaminyltransferase V in thyroid cancer tissues--its possible role in prolonged stability in vivo by aberrant glycosylation

UDP-N-acetylglucosamine:alpha-mannoside beta-1,6-N-acetylglucosaminyltransferase (GnT-V) catalyzes the formation of beta-1-6 GlcNAc branches on asparagine-linked oligosaccharides, which is directly linked to tumorigenesis. Our recent studies indicate that the secretion of matriptase from cancer cells is increased via the action of GnT-V, as evidenced by the fact that matriptase-bearing beta-1-6 GlcNAc branching is dramatically inhibited. In this study, we report on an investigation of the expression of GnT-V and matriptase in thyroid neoplasm tissues to determine the clinical significance on the co-expression of these two proteins in thyroid cancer. Although neither GnT-V nor matriptase was expressed in normal thyroid tissue, positive staining for matriptase and GnT-V was observed in 52/68 and 66/68 cases of papillary carcinoma, 3/23 and 10/23 cases of follicular carcinoma, 5/13 and 9/13 cases of follicular adenoma, and 11/28 and 6/28 cases of anaplastic carcinoma, respectively. Immunohistochemistry, as well as western blotting, showed that the expression of matriptase paralleled the expression to GnT-V. However, the expression of matriptase mRNA was not correlated with its protein levels, suggesting that the enhancement in matriptase expression could be regulated by a posttranslational modification such as glycosylation through GnT-V-mediated glycosylation. In papillary carcinoma, the levels of expression of both GnT-V and matriptase were significantly higher in tumors 1 cm or less in size (microcarcinoma) and in those without poorly differentiated lesions, and the two proteins were significantly correlated. In contrast, the prognosis of thyroid carcinoma after surgery was neither correlated with the expression GnT-V nor matriptase, because the levels of their expression were quite low in anaplastic (undifferentiated) carcinomas. These results suggest that prolonged stabilization of matriptase is stabilized by GnT-V-mediated glycosylation in vivo, thus extending its halftime and permitting it to play role in the early phases of papillary carcinoma, but not in its later phase progression.