Low expression of cell-surface thromboxane A2 receptor beta-isoform through the negative regulation of its membrane traffic by proteasomes

Human thromboxane A(2) receptor (TP) consists of two alternatively spliced isoforms, TP alpha and TP beta, which differ in their cytoplasmic tails. To examine the functional difference between TP alpha and TP beta, we searched proteins bound to C termini of TP isoforms by a yeast two-hybrid system, and found that proteasome subunit alpha 7 and proteasome activator PA28 gamma interacted potently with the C terminus of TP beta. The binding of TP beta with alpha 7 and PA28 gamma was confirmed by co-immunoprecipitation and pull-down assays. MG-132 and lactacystin, proteasome inhibitors, increased cell-surface expression of TP beta, but not TP alpha. Scatchard analysis of [(3)H]SQ29548 binding revealed that the B(max) was higher in transiently TP alpha-expressing cells than TP alpha-expressing cells. In addition, TP-mediated phosphoinositide hydrolysis was clearly observed in TP alpha-, but not TP beta-expressing cells. These results suggest that TP beta binds to alpha 7 and PA28 gamma, and the cell-surface expression of TP beta is lower than that of TP alpha through the negative regulation of its membrane traffic by proteasomes.     

Comparative phylogeography of bagrid catfishes in Taiwan

A phylogeographic analysis of two bagrid catfishes in Taiwan was conducted using sequence data from a portion of the mitochondrial DNA (mtDNA) control region. For Pseudobagrus brevianalis, which is most probably endemic to Taiwan, a total of eight haplotypes were detected in 189 specimens from nine river systems covering its entire distribution range, from northern to central western areas of the island. Obvious genetic differentiation was observed among its populations (average F _ST = 0.753); in particular, the northernmost Tamsui River population was fixed for a single endemic haplotype. Nested clade phylogeographic analysis (NCPA) indicated that the dispersal center of mtDNA was the area around the Touchien River and Holong River, north to the Miaoli Plateau, in northwestern Taiwan, suggesting both northward and southward dispersal in this species. There was no evidence for the validity of P. taiwanensis, the nominal species described from Taiwan, morphologically similar to P. brevianalis. We confirmed that P. adiposalis was distributed discontinuously in three river systems; analysis of 42 specimens from the rivers indicated a total of four haplotypes and population differentiation (average F _ST = 0.876). Fixation into a largely differentiated haplotype in the northernmost Tamsui River population was also found in this species, but different processes for this phylogeographic pattern were implied for the two species. Comparison with P. ussuriensis, a widespread continental species morphologically similar to P. adiposalis, suggested the possibility that P. adiposalis is a group of local populations of P. ussuriensis in Taiwan. Two migration routes vs. random fixation scenarios for the population structure of P. adiposalis are discussed with information on other fishes and the geological history of the island.     

The accurate replacement of long genome region more than several hundreds kilobases in Bacillus subtilis

Competent cell transformation with DNA obtained by the gentle lysis of protoplasts (LP transformation) was used to replace a large genomic region in this study. Discontinuity was detected in the replacement of the donor region tested, probably due to multiple crossover events involving a single donor genome fragment. To overcome discontinuous replacement, we inverted the genomic region to be replaced in the donor used for LP transformation. The replaced region in the transformant was identified to have a continuous genomic region originating from the donor genome. Furthermore, the genome region to be replaced was inverted in the recipient, and the same region and the flanking 10 kb region of both ends was inverted in the donor genome. LP transformation was conducted with the two inversion mutants and it is possible to restrict homologous recombination to the 10 kb flanking regions. Using this method, the 99 kb yxjG-yxbA region, the 249 kb pbpG-yxbA region and the 602 kb yvfT-yxbA region were suggested to be replaced continuously and accurately.     

Structure-activity relationships of some taxoids as multidrug resistance modulator

1,7-Deoxy-4-deacetylbaccatin III (12) and its five analogues 6-9, 13, and their oxetane ring opened derivatives 14, 16, and 17, which were synthesized from taxinine, showed significant activity as MDR reversal agent by the assay of the calcein accumulation toward MDR human ovarian cancer 2780AD cells. The most effective compound 12 in this assay is actually efficient for the recovery of cytotoxic activity of paclitaxel (taxol), adriamycin (ADM), and vincristine (VCR) toward MDR 2780AD cells at the same level toward parental 2780 cells. This activity of 12 is very interesting because baccatin III (4) has no such MDR reversal activity but has cytotoxic activity. The essential functional groups inducing such a difference in biological activity between 4 and 12 are 4alpha-acetoxyl for 4 and 4alpha-hydroxyl for 12. In seven compounds possessing MDR reversal activity, compound 12 is the most desirable compound for anti-MDR cancer reversal agent, because it has the highest accumulation ability of anticancer agent in MDR cancer cells and weak cytotoxic activity. Compounds 8 and 13 showed significant cytotoxic activity toward HepG2 and VA-13, respectively, as well as MDR reversal activity. They are expected to become lead compounds for new types of anticancer agent or anti-MDR cancer agent.     

Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin

In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0microg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8mg of 99%-pure rHSA from 2g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.     

Diploid sperm produced by artificially sex-reversed clone loaches

Clone loaches reproduce unisexually in a wild population of Hokkaido Island, Japan. These clone loaches produce genetically identical unreduced eggs which develop to diploid individuals without any genetic contribution of sperm donors. In the present study, sex reversal of clone loaches was attempted and the reproductive potential of resultant clone males was examined. Clone loaches administered 0.5 ppm of 17-alpha methyltestosterone (MT) for 30 days from 1 month after hatching differentiated into physiological males. These sex-reversed clone males produced fertile spermatozoa with a diploid DNA content. Diploid spermatozoa had significantly larger heads than normal haploid sperm, but had a normal shape showing a head, mid-piece, and tail. The motility of diploid spermatozoa was low after ambient water was added. Concentration of diploid spermatozoa per unit of sperm was lower than that of control haploid spermatozoa. Microsatellite genotyping revealed that triploid progeny from the cross between a normal diploid female and a sex-reversed clone male had two alleles specific to the diploid clone male and one allele of the mother loach. These results indicated that the sex-reversed clone males produced fertile diploid spermatozoa genetically identical to the clone lineage.     

Solid Solution Domains at Phase Transition Front of LixNi0.5Mn1.5O4

Nickel‐substituted manganese spinel LiNi_0.5Mn_1.5O₄ (LNMO) is a promising 5 V class positive electrode material for lithium‐ion batteries. As micron‐sized LNMO particles show high rate capability in its two‐phase coexistence regions, the phase transition mechanism is of great interest in understanding the electrode behavior at high rates. Here, the phase transition dynamics of Li_xNi_0.5Mn_1.5O₄ is elucidated on high rate charging–discharging using operando time‐resolved X‐ray diffraction (TR‐XRD). The TR‐XRD results indicate the existence of intermediate states, in addition to the thermodynamically stable phases, and it is shown that the origin of such intermediate states is ascribed to the solid‐solution domains at the phase transition front, as supported by the analysis using transmission electron microscopy coupled with electron energy‐loss spectroscopy. The phase transition pathways dependent on the reaction rate are shown, together with possible explanation for this unique transition behavior.