ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing

In epithelial cell movements, which occur during wound healing or embryonic morphogenesis, sheets of cells move together as a unit. Molecular mechanisms that regulate this sheet movement have been largely unknown, although cell locomotion or movement mechanisms for individual cells, such as for fibroblastic cells, have been extensively studied. Here, we show that, during wound healing, sheets of MDCK epithelial cells migrate coordinately as a unit, and wound-induced activation of ERK MAP kinase (ERK1/2) propagates in cell sheets in accordance with the cell sheet movement. Inhibition of ERK1/2 activation by specific MEK inhibitors or by expressing dominant-negative ERK2 results in marked inhibition of the sheet movement during wound healing, and inhibition of the cell sheet movement by disrupting actin cytoskeleton suppresses propagation of ERK1/2 activation. These results indicate that cell movement and ERK1/2 activation form a positive feedback loop, which facilitates cell sheet migration. Moreover, we find that Src family kinase inhibitors suppress both cell migration and propagation of ERK1/2 activation, suggesting that Src family kinase may participate in this feedback loop. Interestingly, neither cell sheet migration as a unit nor migration-dependent propagation of ERK1/2 activation occurs during wound healing in fibroblastic 3Y1 cells. Thus, our results identify specific requirements of ERK1/2 MAP kinase for epithelial cell sheet movement.     

120- and 160-kDa receptors for endogenous mitogenic peptide, phytosulfokine-alpha, in rice plasma membranes

Plant cells in culture secrete a sulfated peptide named phytosulfokine-alpha (PSK-alpha), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable (125)I-labeled PSK-alpha analog, [N(epsilon)-(4-azidosalicyl)Lys(5)]PSK-alpha (AS-PSK-alpha), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands was completely inhibited by unlabeled PSK-alpha and partially decreased by PSK-alpha analogs possessing moderate binding activities. In contrast, PSK-alpha analogs that have no biological activity showed no competition for (125)I-AS-PSK-alpha binding, confirming the specificity of binding proteins. Analysis of the affinity of (125)I incorporation into the protein by ligand saturation experiments gave apparent K(d) values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of (125)I-AS-PSK-alpha cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of (125)I-AS-PSK-alpha was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data suggest that the 120- and 160-kDa proteins are PSK-alpha receptors that mediate the biological activities of PSK-alpha.     

Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity

Calumin is an endoplasmic reticulum (ER)-transmembrane protein, and little is known about its physiological roles. Here we showed that calumin homozygous mutant embryos die at embryonic days (E) 10.5−11.5. At mid-gestation, calumin was expressed predominantly in the yolk sac. Apoptosis was enhanced in calumin homozygous mutant yolk sacs at E9.5, pointing to a possible link to the embryonic lethality. Calumin co-immunoprecipitated with ERAD components such as p97, BIP, derlin-1, derlin-2 and VIMP, suggesting its involvement in ERAD. Indeed, calumin knockdown in HEK 293 cells resulted in ERAD being less efficient, as demonstrated by attenuation in both degradations of a misfolded α1-antitrypsin variant and the ER-to-cytosol dislocation of cholera toxin A1 subunit. In calumin homozygous mutant yolk sac endoderm cells, ER stress-associated alterations were observed, including lipid droplet accumulation, fragmentation of the ER and dissociation of ribosomes from the ER. In this context, the ER-overload response, assumed to be cytoprotective, was also triggered in the mutant endoderm cells, but seemed to fully counteract the excessive ER stress generated due to defective ERAD. Taken together, our findings suggested that calumin serves to maintain the yolk sac integrity through participation in the ERAD activity, contributing to embryonic development.     

A hemocyte-derived Kunitz–BPTI-type chymotrypsin inhibitor,HlChI, from the ixodid tick Haemaphysalis longicornis,plays regulatory functions in tick blood-feeding processes

Inhibitors of proteases play key roles in the biological processes of vertebrate and invertebrate animals, including arthropod parasites. Here, we describe a cDNA that encodes a functionally active chymotrypsin inhibitor of the BPTI/Kunitz family of serine protease inhibitors from the hemocytes of the ixodid tick, Haemaphysalis longicornis, herein called HlChI. HlChI sequence is evolutionarily conserved and contains six cysteine residues and three disulfide bonds with a calculated molecular weight of 9.1 kDa. HlChI-specific mRNA was expressed in all developmental stages of ticks and the expression was up-regulated by host's blood-feeding processes. Endogenous HlChI was localized mainly in the hemocytes. HlChI potently inhibited bovine pancreatic α-chymotrypsin for hydrolyzing the fluorogenic substrate (IC₅₀ 8.32 nM, K_d 5.35 ± 1.01 nM) and bovine casein digestion. However, HlChI weakly inhibited bovine pancreatic trypsin and could not affect the porcine elastase activity, suggesting its narrow specificity to chymotrypsin. HlChI was stable over the pH range 2–11 and heating up to 70 °C at pH 8. HlChI was highly stable to 8 M urea and 2% SDS at pH 8.0, when treated for 24 h at 37 °C. However, 0.2 M 2-mercaptoethanol caused complete but reversible inactivation of HlChI. Knockdown of HlChI gene by RNA interference (RNAi) caused death of the feeding ticks, failure of ticks to engorge and significantly reduced body weight gain. RNAi also resulted in significantly decreased egg conversion ratio and fecundity. These results suggest that HlChI is a chymotrypsin-specific inhibitor with high stability and may play regulatory functions in host's blood-feeding processes and tick reproduction.     

Tracking long‐distance migration of marine fishes using compound‐specific stable isotope analysis of amino acids

The long‐distance migrations by marine fishes are difficult to track by field observation. Here, we propose a new method to track such migrations using stable nitrogen isotopic composition at the base of the food web (δ¹⁵N_Base), which can be estimated by using compound‐specific isotope analysis. δ¹⁵N_Base exclusively reflects the δ¹⁵N of nitrate in the ocean at a regional scale and is not affected by the trophic position of sampled organisms. In other words, δ¹⁵N_Base allows for direct comparison of isotope ratios between proxy organisms of the isoscape and the target migratory animal. We initially constructed a δ¹⁵N_Base isoscape in the northern North Pacific by bulk and compound‐specific isotope analyses of copepods (n = 360 and 24, respectively), and then we determined retrospective δ¹⁵N_Base values of spawning chum salmon (Oncorhynchus keta) from their vertebral centra (10 sections from each of two salmon). We then estimated the migration routes of chum salmon during their skeletal growth by using a state‐space model. Our isotope tracking method successfully reproduced a known chum salmon migration route between the Okhotsk and Bering seas, and our findings suggest the presence of a new migration route to the Bering Sea Shelf during a later growth stage.     

Homogeneous detection of a target nucleic acid sequence by combination of the intercalation activating fluorescence DNA probe and the isothermal sequence amplification.

We demonstrated the homogeneous real-time detection of RNA produced during isothermal TRC amplification by INAF probe.     

A serological survey of simian virus 40 in monkeys

A serological survey of simian virus 40 (SV 40) was conducted by an immune adherence hemagglutination (IAHA) test in breeding monkeys. Of a total of 356 monkeys tested, 168 (47.2%) were seropositive. All 168 seropositive monkeys were detected from 224 monkeys which were bred or kept in Japan for a long time. In contrast, none of the 132 monkeys which were newly imported from Southeast Asia was seropositive. If a comparison was made in the same breeding place, the positive rate of 80.4% (111/138) of Japanese monkeys was significantly (P less than 0.01) higher than the 59.5% (25/42) among rhesus monkeys. The positive rate and the IAHA titers were higher in older age group (greater than 5 years) but similar in male and female. These results indicated that SV 40 was highly prevalent among breeding monkeys in Japan.     

Longitudinal studies on annual changes in plasma testosterone,body weight and spermatogenesis in adult Japanese mankeys (Macaca fuscata fuscata) under laboratory conditions

The present study was undertaken to clarify the annual changes in testicular function of Japanese monkeys under laboratory conditions. Five adult males were kept in an air-conditioned room with artificial 12/12 hr lighting. Measurements of body weight and blood sampling were conducted monthly for 13 months. The concentrations of plasma testosterone were determined by radio-immunoassay. The testicular size was measured and testicular tissues taken by biopsy were examined histologically at the four seasons. The body weight and plasma testosterone levels showed coincidental annual changes with a peak in September and a nadir in March or May. The percentage of seminiferous tubules including pachytene spermatocytes and the number of pachytene spermatocytes in tubular cross-sections were significantly increased in both the autumn and winter and decreased in the spring. Electron microscopically, the seasonal change was reflected in an increased size of fat granules in Sertoli cells in the breeding season.     

Malignant lymphoma of the thyroid and Epstein-Barr virus

We have investigated the specific immune response to Epstein-Barr virus (EBV) of peripheral blood mononuclear cells (PBMC) from patients with malignant lymphoma of the thyroid. Coculture of PBMC and EBV resulted in EBV cell transformation and regression which was assayed by an EBV-induced B cell focus-regression assay technique. The EBV had been isolated from mouthwash samples. The specific immune response to EBV by outgrowth inhibition in PBMC from untreated EBV-seropositive patients with malignant lymphoma was significantly decreased when compared to PBMC from EBV-seropositive healthy subjects (p less than 0.05). This observation is at least consistent with the possibility that B-cell proliferation after continuous or recurrent EBV infection could be a causative factor or may potentiate malignant lymphoma of the thyroid.