PHLDA3 is a novel tumor suppressor of pancreatic neuroendocrine tumors.

The molecular mechanisms underlying the development of pancreatic neuroendocrine tumors (PanNETs) have not been well defined. We report here that the genomic region of the PHLDA3 gene undergoes loss of heterozygosity (LOH) at a remarkably high frequency in human PanNETs, and this genetic change is correlated with disease progression and poor prognosis. We also show that the PHLDA3 locus undergoes methylation in addition to LOH, suggesting that a two-hit inactivation of the PHLDA3 gene is required for PanNET development. We demonstrate that PHLDA3 represses Akt activity and Akt-regulated biological processes in pancreatic endocrine tissues, and that PHLDA3-deficient mice develop islet hyperplasia. In addition, we show that the tumor-suppressing pathway mediated by MEN1, a well-known tumor suppressor of PanNETs, is dependent on the pathway mediated by PHLDA3, and inactivation of PHLDA3 and MEN1 cooperatively contribute to PanNET development. Collectively, these results indicate the existence of a novel PHLDA3-mediated pathway of tumor suppression that is important in the development of PanNETs.

Novel p53 target gene FUCA1 encodes a fucosidase and regulates growth and survival of cancer cells.

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.

Remodeling of epithelial cells and basement membranes in a corneal deficiency model with long-term follow-up.

The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.

Prediction of bending set, wave efficacy, and hair damage using an extensional permanent waving treatment and the 20% index value.

To predict "wave efficacy" as evaluated by hairdressers, an extensional permanent waving treatment was performed on human hair fibers using various wave lotions manufactured in Japan. Glass columns devised for the purpose were equipped with a tensile tester in order to increase the measurement accuracy. Notably, the observed set agreed with the theoretical set. In addition, the data for the extensional set exhibited good correlation with the bending set and the wave efficacy assessed in a beauty parlor, and hair damage was estimated by the characteristic change in the 20% index. The following facts were experimentally determined. First, the Young's modulus of the hair fibers after extensional permanent waving treatment continually decreased with an increase in the reduction of the fibers and then abruptly decreased at 80% reduction. Second, the reduction of hair treated with the ammonium salt of thioglycolic acid followed pseudo first-order kinetics only during the initial stage of the reaction, independent of the pH level. Third, the 20% index of the individual virgin hairs remained constant in water at 30°C and also correlated with the Young's modulus of the hair after extensional permanent waving treatment.

Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-engineered cell sheets.

BACKGROUND & AIMS: The use of esophageal endoscopic submucosal dissection (ESD) to remove superficial esophageal neoplasms is gradually becoming more common in Japan. However, large-scale esophageal ESD often requires subsequent multiple balloon dilations to prevent postoperative esophageal stricture. We investigated the safety and efficacy of endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in preventing formation of strictures after ESD. METHODS: We performed an open-label, single-arm, single-institute study. We collected specimens of oral mucosal tissue from 9 patients with superficial esophageal neoplasms. Epithelial cell sheets were fabricated ex vivo by culturing isolated cells for 16 days on temperature-responsive cell culture surfaces. After a reduction in temperature, these sheets were endoscopically transplanted directly to the ulcer surfaces of patients who had just undergone ESD. All patients were monitored by endoscopy once a week until epithelialization was complete. RESULTS: Autologous cell sheets were successfully transplanted to ulcer surfaces using an endoscope. Complete re-epithelialization occurred within a median time of 3.5 weeks. No patients experienced dysphagia, stricture, or other complications following the procedure, except for one patient who had a full circumferential ulceration that expanded to the esophagogastric junction. CONCLUSIONS: Sutureless, endoscopic transplantation of carrier-free cell sheets composed of autologous oral mucosal epithelial cells safely and effectively promotes re-epithelialization of the esophagus after ESD. Patients in this study did not experience any serious complications. This procedure might be used to prevent stricture formation following ESD and improve patients' quality of life. Further study will be needed to show that stricture formation can be prevented.

Restraint of spreading-dependent activation of polymorphonuclear leukocyte NADPH oxidase in an acidified environment.

Elucidation of the mechanisms by which environmental pH affects or regulates the functions of polymorphonuclear leukocytes (PMNs) is important because severe acidification of the microenvironment often prevails at sites of inflammation where they act in host defense. In the present study, we investigated the effect of an acidic environment on spreading-dependent activation of O2- -producing NADPH oxidase in PMNs. We found that PMNs underwent spreading spontaneously over type I collagen and plastic surfaces at both neutral and acidic pH, although spreading over fibrinogen surfaces, for which cellular stimulation with H2O2 is required, was inhibited by acidic pH. At acidic pH, however, PMNs were unable to undergo spreading-dependent production of O2-. Pharmacological experiments showed that p38 mitogen-activated protein kinase (MAPK) was involved in the signaling pathways mediating the spreading-dependent activation of NADPH oxidase, and that its spreading-dependent phosphorylation of Thr-180 and Tyr-182, a hallmark of activation, was impaired at acidic pH. Furthermore, the inhibition by acidic pH of O2- production as well as p38 MAPK phosphorylation subsequent to spreading induction was reversible; environmental neutralization and acidification after induction of spreading at acidic and neutral pH, respectively, up- and down-regulated the two phenomena. Acidic pH did not affect the O2- production activity of NADPH oxidase pre-activated by phorbol 12-myristate 13-acetate (PMA). These results suggest that, in PMNs, the p38 MAPK-mediated signaling pathway functions as a pH-sensing regulator of spreading-dependent NADPH oxidase activation.

Virus-infection or 5'ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1.

In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses including production of type I and III interferon (IFN) and the subsequent expression of IFN-inducible genes. Interferon-beta promoter stimulator 1 (IPS-1, also known as MAVS, VISA and Cardif) is a downstream molecule of RLR and is expressed on the outer membrane of mitochondria. While it is known that the location of IPS-1 is essential to its function, its underlying mechanism is unknown. Our aim in this study was to delineate the function of mitochondria so as to identify more precisely its role in innate immunity. In doing so we discovered that viral infection as well as transfection with 5'ppp-RNA resulted in the redistribution of IPS-1 to form speckle-like aggregates in cells. We further found that Mitofusin 1 (MFN1), a key regulator of mitochondrial fusion and a protein associated with IPS-1 on the outer membrane of mitochondria, positively regulates RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes.

Fabrication and validation of autologous human oral mucosal epithelial cell sheets to prevent stenosis after esophageal endoscopic submucosal dissection.

OBJECTIVES: Human oral mucosal epithelial cells derived from 7 healthy volunteer donors were cultured in a clean room in a cell-processing center (CPC) according to good manufacturing practice guidelines. Cell culture and fabricated transplantable epithelial cell sheets were validated for treating ulcers after endoscopic mucosal dissection. METHODS: The clonal growth and morphology of the human oral mucosal epithelial cells seeded on temperature-responsive surfaces were observed. During the cultivation, sterilization tests were performed to validate the environment in the CPC. To validate the purity and morphology of fabricated epithelial cell sheets, cell sheets harvested from temperature-responsive surfaces by temperature reduction were examined by histology and flow cytometry. RESULTS: Human oral mucosal epithelial cells were successfully cultured and harvested as continuous cell sheets from temperature-responsive culture inserts without any animal-derived materials. During the cultivations, the sterile environment in the CPC was confirmed. The results of histological and flow cytometry analysis showed the high reproducibility of stratification and the purity of the fabricated human oral mucosal epithelial cell sheets. CONCLUSIONS: The method for fabricating epithelial cell sheets shown in this study was suitable for the validation for clinical trials and suggested usability of the fabricated cell sheets.

Localization of three types of arginine vasotocin receptors in the brain and pituitary of the newt Cynops pyrrhogaster.

The distribution of three types of arginine vasotocin (AVT) receptors in the brain and pituitary of the newt Cynops pyrrhogaster, namely, the V1a-, V2-, and V3/V1b-type receptors, was studied by means of in situ hybridization and immunohistochemistry. mRNA signals and immunoreactive cells for the V1a-type receptor were observed in the telencephalon (mitral layer of the olfactory bulb, dorsal and medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali, bed nucleus of the stria terminalis), diencephalon (anterior preoptic area, magnocellular preoptic nucleus, suprachiasmatic nucleus, ventral thalamus, dorsal and ventral hypothalamic nucleus), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (median reticular formation, nucleus motorius tegmenti). Cells expressing the V2-type receptor were found in the telencephalon (medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali), and mesencephalon (tegmentum trigemini and facialis). In the paraphysis (possibly the main site of cerebrospinal fluid production), only V2-type receptor mRNA signal and immunoreactivity were detected. V3/V1b-type receptor mRNA was expressed in the diencephalon (dorsal hypothalamic nucleus, nucleus tuberculi posterioris), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (raphe nucleus), whereas V3/V1b-type-receptor-like immunoreactivity was scarcely detectable in the entire brain. The V3/V1b-type receptor was predominantly expressed in the anterior pituitary. V3/V1b-type receptor and proopiomelanocortin mRNAs were co-localized in the distal lobe of the pituitary. This is the first report of the distribution of three types of AVT receptor in the brain and pituitary of non-mammalian vertebrates.

Fabrication of human oral mucosal epithelial cell sheets for treatment of esophageal ulceration by endoscopic submucosal dissection.

BACKGROUND: Esophageal stenosis is one of the major complications of aggressive endoscopic resection. Tissue-engineered epithelial cell grafts have demonstrated effectiveness in promoting re-epithelialization and suppressing inflammation causing esophageal scarring and stenosis after endoscopic submucosal dissection (ESD) in an animal model. OBJECTIVE: To confirm the reproducibility and efficacy of a human oral mucosal epithelial cell (hOMEC) sheet cultured on temperature-responsive surface in conformity with Good Manufacturing Practice guidelines. DESIGN: A preclinical study. SETTING: Good Manufacturing Practice grade cell-processing center, animal laboratory. SUBJECTS: Canine esophageal ulcer models, which were made by ESD. INTERVENTIONS: Oral mucosal specimens were obtained from 7 healthy volunteers. MAIN OUTCOME MEASUREMENT: Fabricated and transplanted hOMEC sheets were subjected to histological analysis. RESULTS: The reproducibility of the fabrication of hOMEC sheets was confirmed. In this method, animal-derived materials such as 3T3 feeder layer and fetal bovine serum were successfully excluded from the culture condition. Furthermore, the environment of the culture room and safety cabinet in the cell-processing center was maintained for obtaining sterility assurances during the fabrication. Transplanted hOMEC sheets after ESD were observed to graft onto canine esophageal ulcer surfaces. LIMITATIONS: Small number of subjects, animal model. CONCLUSIONS: Cultured hOMEC sheets were fabricated without animal-derived materials and demonstrated efficacy as a medical device that promotes re-epithelialization of an esophageal ulcer after ESD.

A novel harvesting method for cultured cells using iron-cross-linked alginate films as culture substrates.

The present study was conducted to assess the efficiency of a novel cell-harvesting method involving dissolution of the culture substrate composed of Fe-alginate (ferric-ion-cross-linked alginate). Cell harvesting is an essential step for recovery of cultured adherent cells, but conventional methods such as trypsinization or scraping cause considerable damage to the cells. We therefore devised an original method for harvesting cultured cells using Fe-alginate films as a culture substrate and then retrieving the cells by disintegration of the alginate gel. Fe-alginate was easily dissolved under physiological conditions by exchange of cross-linked ions using chelating agents such as citrate. The effects of this cell-harvesting method were investigated in comparison with trypsinization and scraping. Cells harvested by dissolution of the Fe-alginate substrate showed high viability and metabolic activity, high recovery rate on subculture, a low degree of membrane damage with superior expression of cell adhesion molecules (beta1 integrin and N-cadherin), high resistance to oxidative stress, and high viability, metabolic activity and recovery rate after cryopreservation, in contrast with the cells harvested by trypsinization or scraping from tissue-culture polystyrene substrates. These results suggest that our new system involving dissolution of Fe-alginate culture substrate is effective for cell harvesting and may be advantageous for biomedical applications.

Characterization of the unique regulatory mechanisms of phorbol ester-induced polymorphonuclear leukocyte spreading in an acidified environment.

In vitro studies have shown that acidic conditions impair spreading of polymorphonuclear leukocytes, which is prerequisite for activation of microbicidal functions. However, the mechanisms by which pH affects polymorphonuclear leukocytes functions remain obscure. Moreover, in vitro observations seem to contradict the fact that an acidic microenvironment often prevails at sites of inflammation where polymorphonuclear leukocytes must function for host defense. In the present study, we found three peculiar characteristics of porcine polymorphonuclear leukocyte that had been induced to spread over fibrinogen-coated surfaces by phorbol 12-myristate 13-acetate (PMA) in acidified medium. First, the PMA-induced spreading at acidic pH, but not at neutral/alkaline pH, was dependent on extracellular Ca2+. Second, the spreading at acidic pH was independent of protein kinase C (PKC), whereas that at neutral/alkaline pH was strictly PKC-dependent. Finally, the spreading at acidic pH, but not at neutral/alkaline pH, was suppressed by H2O2 produced by activated NADPH oxidase or added exogenously. As a result, polymorphonuclear leukocyte spreading at acidic pH peaked at 30 min after PMA stimulation, and declined thereafter because of negative regulation triggered by accumulated H2O2, whereas that at neutral/alkaline pH was stable for at least 90 min. The NADPH oxidase inhibitor diphenyleneiodonium or the H2O2-degradation enzyme catalase consistently stabilized the spreading at acidic pH. We conclude that PMA-stimulated polymorphonuclear leukocytes spread in an acidic environment through a mechanism different from that under neutral/alkaline conditions. This H2O2-mediated negative regulation system in an acidic environment may be crucial for avoiding tissue-damaging inflammatory actions of accumulated polymorphonuclear leukocytes in vivo.

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors.

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.

Development of a modified microtiter plate with a concave portion in the center.

We have developed a modified microtiter plate which has following advantageous features and functions to both conventional microtiter plate and protein array, such as 1) use of conventional microtiter plate reader and washer, and 2) allowance of simultaneous reaction in the same liquid for all wells. Four proteins of human serum albumin, human C-reactive protein (CRP), human plasminogen and human MIP-1alpha as sample proteins, were measured with the modified microtiter plate. Although the reaction liquid of each wells on the modified microtiter plate shares through concave portion, its antigen/antibody in each well is independent. The independence of the reaction is supported by the result that the above four proteins produced dose-response curves simultaneously. Unlike a conventional protein array, our plate does not need the drying process for antibody adhesion to the plate, preventing inactivation of the antibody. And one can detect the antigen/antibody reaction using the enzymatic amplification reaction (for example utilizing the biotin-streptavidine interaction) like a conventional plate. In addition to these features, our microtiter plate also has the merit of eliminating the so-called "edge effect".

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets.

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.

Brush biopsy of human oral mucosal epithelial cells as a quality control of the cell source for fabrication of transplantable epithelial cell sheets for regenerative medicine.

Autologous oral mucosal epithelial cell sheets have been used for treating epithelial defects such as cornea and esophagus. The cell source of patients' oral mucosal epithelial cell sheet should be examined in normality because it has individual difference. In this study, oral mucosal epithelial cells were less invasively collected by brush biopsy from the buccal, gingival, labial, and palate mucosa of four healthy volunteer donors without anesthesia, and analyzed the keratin expressions by western blotting and the obtained results were compared with those by immunohistochemistry of each of the native tissues. All of the oral mucosal epithelial cells expressed keratin 4 (K4) and K13, which were mucosal stratified squamous epithelial cell markers. K1 and K10, keratinized epithelial cell markers, were also detected in keratinized tissues such as gingival and palate mucosa. The markers of epithelial basal cells such as p63 and K15 were not detected by brush biopsy-western blotting. Although this method does not include basal layers of oral mucosa, protein expressions of upper layer of lesion area are different from normal. Therefore, brush biopsy-western blotting was extremely less invasive and would contribute to quality control of the fabrication of autologous oral mucosal epithelial cell sheets.

IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis.

The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53-dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers.

Membrane-Permeable Calpain Inhibitors Promote Rat Oral Mucosal Epithelial Cell Proliferation by Inhibiting IL-1α Signaling.

To standardise regenerative medicine using cultured cells, the use of serum-free, chemically defined media will be necessary. We have reported that IL-1α inhibits the growth of epithelial cells in culture and that recombinant IL-1 receptor antagonist (IL-1RA) significantly promotes epithelial cell growth in no feeder layer condition. In this study, we examined inhibitors of calpain, a cysteine proteinase that plays crucial roles in various cellular functions, including IL-1α maturation and secretion. The culturing of epithelial cells in serum-free media supplemented with a membrane-permeable calpain inhibitor significantly promoted growth while suppressing IL-1α maturation and secretion. By contrast, non-membrane-permeable calpain inhibitor treatment did not have these effects. Interestingly, immunoblotting analysis revealed that immature, untruncated, IL-1α expression was also downregulated by cell-permeable calpain inhibitor treatment, and the difference in IL-1α gene expression increased from day 2 to day 6. Although IL-1RA has been reported to promote epithelial cell growth, we detected no synergistic promotion of epithelial cell growth using a calpain inhibitor and IL-1RA. These findings indicate that calpain inhibitors promote epithelial cell proliferation by inhibiting IL-1α maturation at an early phase of epithelial cell culture and by suppressing the positive feedback-mediated amplification of IL-1α signalling.

In Vitro Regression of Tadpole Tail by Thyroid Hormone: (Metamorphosis/tadpole tail regression/epidermis-mesenchyme interaction).

DERBY successfully maintained the tail of tadpole (Rana pipiens) in vitro over a period of 2 weeks in a physiological salt solution (1). When we tried to apply DERBY'S methods of the tissue culture to tadpoles of bullfrog, Rana catesbeiana, it was found that the tissue regressed spontanously without stimulation of thyroid hormone. Several different media were examined in order to select a better culture medium for the bullfrog tadpole tissues. RPMI-1640 medium supplemented with insulin and transferrin was found to be satisfactory for this aim. With this improved medium, the interaction between the epidermis and the mesenchyme has been investigated during the hormone-induced tadpole tail regression and the epidermal dependence of the mesenchyme regression was demonstrated by the following three experiments. (i) Some of surgically prepared mesenchymes regressed in responce to thyroid hormone. In these cases the mesenchymes were revealed to be contaminated with the remaining epidermal cells. (ii) Complete removal of the epidermis was accomplished by the chemical treatment. The mesenchyme thus obtained ("nude tail fin") was insensible to thyroid hormone. (iii) "Skin conditioned medium" (SCM) was prepared by culturing the skin in the presence and absence of thyroid hormone. Nude tail fin regressed when cultured in the SCM containing thyroid hormone.

Elemental characterization of Daphnia resting eggs by X-ray analytical microscopy.

Resting eggs of Daphnia, a key crustacean zooplankton of freshwater food chains, can remain viable for more than a century. These eggs are able to withstand freezing and drying, and can survive the harsh environment of a predator's digestive system. Until recently little was known about the chemical composition, microanatomy, and physical properties of the resting eggs. The current study utilized a physical technique, the X-ray analytical microscope, to identify and localize component elements of the Daphnia resting egg. The analysis demonstrated that phosphorus, sulfur, potassium, and calcium were detected as elemental components of the resting egg, and detection intensities of the four elements differed according to the position of the eggs. Phosphorus and calcium were mostly detected in regions of the eggshell that surrounded the two embryos. In addition, sulfur was distributed throughout the eggshell whereas potassium was localized to the areas that corresponded to where the embryos were encased. Through the use of X-ray analytical microscopy, the current study identifies elemental characteristics in relationship to the structure of the Daphnia resting eggs.