Tsukushi is involved in the wound healing by regulating the expression of cytokines and growth factors

During the wound-healing process, macrophages, fibroblasts, and myofibroblasts play a leading role in shifting from the inflammation phase to the proliferation phase, although little is known about the cell differentiation and molecular control mechanisms underlying these processes. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions as a key extracellular coordinator of multiple signalling networks. In this study, we investigated the contribution of TSK to wound healing. Analysis of wound tissue in heterozygous TSK-lacZ knock-in mice revealed a pattern of sequential TSK expression from macrophages to myofibroblasts. Quantitative PCR and in vitro cell induction experiments showed that TSK controls macrophage function and myofibroblast differentiation by inhibiting TGF-β1 secreted from macrophages. Our results suggest TSK facilitates wound healing by maintaining inflammatory cell quiescence.     

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.     

Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells

Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα_i/o inhibitor, but not by NF449, a Gα_s inhibitor, or YM-254890, a Gα_q inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα_ο1 and Gα_i3 by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey^TM assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα_i/o activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα_i/o upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.     

Changes of mRNA expression of vascular endothelial growth factor, angiopoietins and their receptors during the periovulatory period in eCG/hCG-treated immature female rats

Angiogenic factors can induce the perifollicular capillary network in the theca interna that shows marked changes in and around the preovulatory luteinizing hormone (LH) surge. To get more information on their functional crosstalk, the aim of the present study was to investigate the manner of mRNA expression of vascular endothelial growth factors (VEGFs) 120, 164, angiopoietin (Ang)-1, Ang-2 and their specific receptors during the periovulatory phase. We used an established equine and human chorionic gonadotropins (eCG/hCG)-derived experimental model capable of stimulating naturally occurring follicular maturation, ovulation and corpus luteum (CL) formation. On day 28 postpartum, immature female rats were administrated s.c. with 10 IU of eCG to promote follicular development, followed 48 hr later by i.p. administration of 20 IU of hCG. Ovaries were dissected at 0, 6, 12, 18 and 24 hr after hCG treatment, and were obtained on day 30 in the untreated control. After induction of follicular growth by the eCG treatment, each mRNA expression of VEGF 120, VEGF 164, Neuropilin-1 and Flt-1 significantly increased. The peaks in mRNA expressions of VEGF120 and VEGF164 were both found at 18 hr after hCG treatment. Flk-1 mRNA expression maintained up to 6 hr after hCG treatment, and then decreased at 12, 18 and 24 hr after hCG treatment. Ang-2 mRNA expression increased in the ovaries at 6 and 12 hr after hCG treatment. Tie-2 mRNA expression decreased at 24 hr after the treatment of gonadotropins. Our findings suggest that ovarian vascular formation during the periovulatory period including preovulatory follicles, ovulation and CL formation may develop via crosstalk of the VEGF-Flt-1 and Ang-Tie2 systems.     

Rapid establishment of highly migratory cells from cancer cells for investigating cellular functions

Abstract

Cell migration is closely involved in cancer cell invasion into surrounding tissue and metastasis to the distant organs. It is crucial for understanding the molecular mechanisms that regulate cell migration in cancer cells. The aim of this study is to establish a rapid induction method of highly migratory cells from cancer cells. Osteosarcoma MG-63 and colon cancer DLD1 cells were seeded at 1?×?10⁵ cells in 6-well plates. After 10?min, unattached cells were washed off three times with PBS. The cells which remained attached on the bottom of plates were cultured in DMEM containing 10% FBS. When the cells reached approximately 80% confluence, cells were harvested using trypsin/EDTA. The harvested cells were seeded in other 6-well plates and incubated for 10?min. The unattached cells were washed off and attached cells were further cultured. By repeating this procedure 11–12 times for 2?months, highly migratory MG63-A12 and DLD-A11 cells were obtained from MG-63 and DLD1 cells, respectively. In cell motility assay, the cell motile activities of MG63-A12 and DLD-A11 cells was 10.3 and 13.7 times higher than those of the parental cells, respectively. This procedure is useful to generate highly migratory cells for investigating cellular functions during tumor progression in cancer cells.     

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.     

Dispersible chitosan particles showing bacteriostatic effect against Streptococcus mutans and their dental polishing effect

ABSTRACT

Nontoxic and biodegradable chitosan is potentially useful in various applications. We prepared submicron chitosan particles with high dispersibility in aqueous solution utilizing the electrostatic interaction phase separation method described in a previous report, but using citric acid as the polyvalent anionic compound instead of sodium sulfate. The submicron chitosan particles showed significant antibacterial activity and anti-adhesive action against Streptococcus mutans, even at around neutral pH. However, chitosan granules showed no antibacterial activity under the same conditions. The addition of the chitosan particles to dental polishing paste provided stainless steel discs (the same hardness as dental enamel) with a smoother surface than polishing paste without additives. In view of their submicron size and antibacterial activity, chitosan particles could potentially be multifunctional components of oral and dental cleaning materials.     

National surveillance of <Emphasis Type="Italic">Salmonella enterica</Emphasis> in food-producing animals in Japan

A total of 518 fecal samples collected from 183 apparently healthy cattle, 180 pigs and 155 broilers throughout Japan in 1999 were examined to determine the prevalence and antimicrobial susceptibility of Salmonella. The isolation rates were 36.1% in broilers, 2.8% in pigs and 0.5% in cattle. S. enterica Infantis was the most frequent isolate, found in 22.6% of broiler fecal samples. Higher resistance rates were observed against oxytetracycline (82.0%), dihydrostreptomycin (77.9%), kanamycin (41.0%) and trimethoprim (35.2%). Resistance rates to ampicillin, ceftiofur, bicozamycin, chloramphenicol and nalidixic acid were <10%. CTX-M-2 β-lactamase producing S. enterica Senftenberg was found in the isolates obtained from one broiler fecal sample. This is the first report of cephalosporin-resistant Salmonella directly isolated from food animal in Japan.