Morphology of Gambierdiscus scabrosus sp. nov. (Gonyaulacales): a new epiphytic toxic dinoflagellate from coastal areas of Japan

A new epiphytic dinoflagellate is described, G ambierdiscus scabrosus sp. nov., from tidal pools and rocky shores along the coastal areas of Japan. Cells are 63.2 ± 5.7 μm in depth, 58.2 ± 5.7 μm in width, and 37.3 ± 3.5 μm in length. The plate formula of G . scabrosus is Po, 4′, 0a, 6′′, 6c, ?s, 5′′′, 0p, and 2′′′′. Morphologically, G . scabrosus resembles G . belizeanus as follows: anterioposteriorly compressed cell shape, narrow 2′′′′ plate, and areolated surface. Despite this similarity, the cells of G . scabrosus can be distinguishable by the presence of the asymmetric shaped 3′′ plate and the rectangular shaped 2′ plate.     

Geographic variation in the growth of white croaker,Pennahia argentata,off the coast of northwest Kyushu,Japan

We examined geographic variation in the growth of white croaker,Pennahia argentata, off the coast of northwest Kyushu, Japan, Ariake Sound, Tachibana Bay, Omura Bay and the Goto Sea by examination of otoliths. The outer margins of the otoliths showed that a opaque zone was formed once a year, with its peak in June, and could be used as an annulus. White croaker caught during this study reached a maximum age of 10years in the Goto Sea. The growth curves for both sexes in all localities were expressed by the von Bertalanffy growth equations from back-calculated total length of fish. We found significant sexual differences in growth curves in Ariake Sound, Tachibana Bay and the Goto Sea. For both sexes, white croaker in the Goto Sea reached the largest length at each estimated age of the four localities. The growth curves for both sexes showed significant differences among four localities, suggesting that several stocks may exist in the study area although the greatest distance between each locality was at most 30 km.     

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.     

Partial Sequences of Large Subunit Ribosomal DNA of a New Yeast Species,Trichosporon domesticum and Related Species

We determined the partial sequences of large subunit rDNA of a new yeast species, Trichosporon domesticum, which was isolated from the house of a summer-type hypersensitivity pneumonitis patient. Phylogenetically, T. domesticum was positioned in the taxonomic group containing T. montevideense and T. brassicae, which indicated an identical serotype. A phylogenetic relationship among all species of the genus Trichosporon which belong to the basidiomycetous yeast is clarified.     

Detection of Antibody-Coated Bacteria in Expectorated Sputum for Diagnosis of Lower Respiratory Infections

We evaluated antibody-coated bacteria (ACB) in expectorated sputum to discriminate contaminating or colonizing organisms from true pathogens. We examined 60 expectorated sputum samples from 51 patients with lower respiratory infections (chronic obstructive pulmonary disease 25, pneumonia 20, purulent tracheobronchitis 6). All samples were examined with quantitative culture and immunofluorescent demonstration of ACB. From the results of quantitative culture, we divided specimens into pathogen-isolated and pathogen-free samples. Among pathogen-isolated samples, in which we isolated accepted pathogenic organisms at ≥ 10⁷ colony-forming units per ml, 16 of 23 samples were ACB-positive (69.5%). In contrast, among pathogen-free samples, in which we isolated accepted pathogens at < 10⁷ colony forming units per ml or only upper respiratory flora, only 3 of 37 samples were ACB-positive (8.1%). The ACB-positive rate was significantly higher in pathogen-isolated than in pathogen-free samples (P < 0.001). Consequently, detecting ACB in expectorated sputum shows good potential as another criterion for distinguishing contaminating or colonizing organisms from true pathogens.     

Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells.

We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.     

Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C₁₈ column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform—isopropyl alcohol (9:1, v/v) solution. Levels of 2.5 · 10⁻⁸ M BE in urine (25 pmol/ml) could be determined.     

Dietary folic Acid promotes survival of foxp3+ regulatory T cells in the colon

Dietary compounds as well as commensal microbiota contribute to the generation of a unique gut environment. In this study, we report that dietary folic acid (FA) is required for the maintenance of Foxp3(+) regulatory T cells (Tregs) in the colon. Deficiency of FA in the diet resulted in marked reduction of Foxp3(+) Tregs selectively in the colon. Blockade of folate receptor 4 and treatment with methotrexate, which inhibits folate metabolic pathways, decreased colonic Foxp3(+) Tregs. Compared with splenic Tregs, colonic Tregs were more activated to proliferate vigorously and were highly sensitive to apoptosis. In colonic Tregs derived from mice fed with a FA-deficient diet, expression of anti-apoptotic molecules Bcl-2 and Bcl-xL was severely decreased. A general reduction of peripheral Tregs was induced by a neutralizing Ab against IL-2, but a further decrease by additional FA deficiency was observed exclusively in the colon. Mice fed with an FA-deficient diet exhibited higher susceptibility to intestinal inflammation. These findings reveal the previously unappreciated role of dietary FA in promotion of survival of Foxp3(+) Tregs that are in a highly activated state in the colon.     

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.