Effect of γ-cyclodextrin as a lyoprotectant for freeze-dried actinidin.

Actinidin (ATD) is a cysteine protease found in kiwifruit. It is used to tenderize meat and to enhance the digestion of proteins in the small intestine. However, ATD is unstable during freeze-drying, which alters its bioactivity. It is well known that sugars have the ability to protect proteins from the stress of freeze-drying. In this study, we investigated the protective effect of various saccharides on the stability of ATD during freeze-drying. The ATD activities of the samples containing γ-cyclodextrin (CyD) showed only a small decrease, and compared with trehalose and sucrose, γ-CyD was a more effective stabilizer for ATD. Secondary structural changes in freeze-dried ATD were observed by circular dichroism spectroscopy and compared with the changes in stabilized samples. There was a close relationship between the α-helix content and the stabilization. The sugars stabilized the protein by suppressing the changes in the α-helix. Fourier transform infrared spectroscopy measurement showed that the amide I band of ATD with γ-CyD was shifted to a lower wavenumber compared with other sugars. Therefore, stronger hydrogen bonds may be formed between ATD and γ-CyD than between ATD and other sugars. The suppression of changes in the protein secondary structure accompanying the formation of hydrogen bonding between the protein and the sugar also contributed to the protective effect of the sugars.

Characterization of ion-irradiated poly-L-lactic acid using nano-cutting.

Effects on the mechanical strength of poly-L-lactic acid (PLLA) upon irradiation with 150 keV He(+) ion were studied. Changes in the irradiated surface were investigated using a surface texture and contour measuring instrument and an atomic force microscope. Observations made with the atomic force microscope revealed that the irradiated surface subsided significantly as the fluence increased. In order to investigate the dependence on fluence of the depth of the Bragg peak for the ion implantation, the cutting strength, Σ, was analysed [F. Saito, I. Nishiyama and T. Hyodo, Mater. Lett., 2012, 66, 144-146]; this value is an indicator of the strength of a material against cutting, and is obtained from the cutting resistance. The averaged ion projected range increased from about 1.1 µm for a fluence of 1 × 10(15) He(+)/cm(2) to about 4 µm for a fluence of 1 × 10(16) He(+)/cm(2). The density of the region following irradiation was estimated using a combination of cutting resistance measurements and positron annihilation γ ray Doppler broadening measurements made with an energy-variable positron beam. The density decreased from the value of 1.27 g cm(-3) to about 0.6 g cm(-3) after irradiation with a fluence of 3 × 10(15) He(+)/cm(2). By considering the decrease in the density and the subsidence of the surface, it is concluded that only 30% of the original weight remained in the irradiated region after exposure to the He(+) ions. Anisotropic change in the cutting resistance suggests that mechanical strength in the direction normal to the surface increased while that in the lateral direction decreased.

An electrically-stabilized liquid-crystalline phase: origin and application.

An azobenzene liquid-crystalline compound possessing two chiral centres at both peripheral ends of the molecular structure exhibits electric-field-induced birefringence in the isotropic liquid phase, which was found to be attributable to the stabilization of the liquid-crystalline organization by the emergence of the polar ferroelectric molecular ordering. The optically isotropic texture changes into the homogeneous birefringent texture by the application of the in-plane electric field, which is a new type of switching mode applicable for the liquid crystal displays. The resulting birefringence can be erased by the irradiation of UV light, due to the photoinduced isomerization of the azobenzene compound, thus a dual controlled birefringent structure, by the irradiation of light and/or by the application of the electric field, is reported for the first time.

A protein kinase inhibitor H-7 induces process extrusion in fetal rat thyroid C-cells in vitro.

Calcitonin-producing cells (C-cells) are endocrine cells derived from the neural crest. We examined the effects of three types of protein kinase inhibitors on the induction of neuronal phenotypes in the rat thyroid C-cells in vitro. In a primary culture of 16-day-old fetal rat thyroid glands, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 25-75 microM) induced both process extrusion and expression of highly polysialylated neural cell adhesion molecule (NCAM) in the C-cells. These effects of H-7 were completely prevented by okadaic acid, a potent protein phosphatase inhibitor. In contrast to H-7, selective inhibitors for cyclic nucleotide-dependent protein kinases such as N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004, 25-200 microM) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89, 0.25-20 microM) failed to induce process extrusion or the expression of highly polysialylated NCAM in fetal rat C-cells. In cultured C-cells of adult origin, H-7 failed to induce marked process elongation or the expression of highly polysialylated NCAM. These results suggest that the morphological plasticity of the fetal C-cells depends upon the degree of phosphorylation of some proteins, and that the plasticity of adult C-cells are more restricted than that of fetal origin.

Developmental change in expression of highly polysialylated neural cell adhesion molecule in C-cells in rat thyroid gland.

The expression of the neural cell adhesion molecule (NCAM), highly polysialylated NCAM, and E-cadherin was immunohistochemically studied in the calcitonin-producing cells (C-cells) of developing and adult rat thyroid glands of varying ages. In fetal and neonatal rat thyroids, almost all the C-cells displayed immunoreactivity for highly polysialylated NCAM, whereas most of the follicular cells were negative. The highly polysialylated NCAM-positive C-cells markedly decreased in number between 5 and 14 days after birth. From day 14 onward, immunoreactivity for highly polysialylated NCAM was almost negative in thyroid glands. On the other hand, the expression of immunoreactivity for NCAM peptide persisted in thyroidal C-cells throughout the life span. These results suggest that conversion of the highly polysialylated NCAM into a less sialylated form occurs in the thyroid C-cells between postnatal days 5 and 14. Intense immunoreactivity for E-cadherin was observed in the entire cell surfaces of all the C-cells and follicular cells in the rats of all ages tested. In the course of thyroid organogenesis, C-cells transiently form a cell mass, an ultimobranchial body, which is fated to disappear as the C-cells migrate diffusely into the thyroid. The duration of the polysialic acid expression in the C-cell surfaces appears to coincide with the period of C-cell migration. It is possible that the expression of highly polysialylated NCAM allows the C-cells to migrate into the thyroid by reducing the cell-to-cell adhesion of C-cells with adjacent C-cells and/or with the surrounding follicular cells.

Localization of neutral and acidic glycosphingolipids in rat lens.

Rat lens was found to contain several neutral and acidic glycosphingolipids in lens epithelia, cortex and nucleus, and showed developmental changes in their content and localization. TLC-immunostaining of gangliosides revealed the enrichment of some ganglio-series gangliosides (GM3, GM1, GD3 and GD1b) in lens epithelia and the presence of GM3 and GD3 in the lens nucleus. Immunohistochemical studies confirmed the distribution of GM3 and GM1 in anterior lens epithelial cells and the cortex, with expression decreasing toward the lens nucleus. Immunoreaction to GD3 was more intense in the lens nucleus than in epithelial cells. In contrast, the expression of neolacto-series glycosphingolipids was restricted to the lens nucleus. In order to investigate the pathological changes of glycosphingolipids in cataract, galactose-induced cataractous lenses were examined. However, no significant changes were observed in the content and composition of glycosphingolipids. In addition, Lewisx epitopes found in human cataractous lenses were not detected in the cataractous lenses of galactosaemic rats and hereditary cataractous Emory mice.

Comparative study of glycosphingolipid composition in mammalian lenses.

The carbohydrate epitope Gal alpha 1-3Gal-R (alpha-galactosyl epitope), which is detectable by its binding with Bandeiraea simplicifolia-IB4 lectin, was found in glycosphingolipids (GSLs), both neutral and acidic (gangliosides), from lens tissues of non-primate mammals, but not in those of human senile cataracts and Old World monkeys. Instead, human cataractous and Old World monkey non-cataractous lenses expressed Lewisx (Le(x)) epitopes (Gal beta 1-4(Fuc alpha 1-3)GlcNAc-R) in neutral GSLs. Sialylated Le(x) epitopes were found in rat and pig lenses as well as in human and Old World monkey lenses. Ganglio-series gangliosides, consisting mainly of GM3, GM1, GD1a and GD3, were detected in a species-specific fashion. On the other hand, alpha-galactosyl epitopes were expressed in lens tissues only in water-insoluble proteins of non-primate mammals, but Le(x) and sialylated Le(x) epitopes were not detectable in lens proteins. Among the several mammalian lenses examined, humans and Old World monkeys showed similar GSL compositions, in particular the presence of Le(x) and sialylated Le(x) epitopes and the absence of alpha-galactosyl epitopes, in lens tissue.

Expression of highly polysialylated neural cell adhesion molecule in calcitonin-producing cells.

Calcitonin-producing cells are endocrine derivatives of the neural crest and have several neuron-like properties. Expression of the neural cell adhesion molecule in calcitonin-producing cells was examined using two types of antibodies to neural cell adhesion molecule: monoclonal antibody 12E3 recognizes the polysialic acid portion of highly polysialylated neural cell adhesion molecule, and monoclonal antibody AF11 and polyclonal antiserum react with the polypeptide portion common to three major isoforms of neural cell adhesion molecule. An immunohistochemical study revealed that highly polysialylated neural cell adhesion molecule was expressed both in fetal rat thyroidal calcitonin-producing cells and in a calcitonin-producing cell line, rMTC 6-23, established from explantable neoplasm of rat calcitonin-producing cells. The neural cell adhesion molecule in the rMTC 6-23 cells was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Two anti-neural cell adhesion molecule monoclonal antibodies, 12E3 and AF11, revealed a broad positive band around 200,000-250,000 mol. wt in solubilized proteins. When the polysialic acids were eliminated by neuraminidase treatment, the immunoreactivity to monoclonal antibody 12E3 was completely abolished, and core polypeptide corresponding to neural cell adhesion molecule with a molecular weight of 120,000 was detected by monoclonal antibody AF11. These results suggest that cells of the calcitonin-producing cell line express on their surfaces highly polysialylated 120,000 mol. wt form of neural cell adhesion molecule polypeptide.

Egg jelly components responsible for histone degradation and acrosome reaction in the starfish, Asterina pectinifera.

In the starfish, Asterina pectinifera, egg jelly induces the degradation of sperm histones as well as the acrosome reaction. We have isolated histone degradation-inducing components from the egg jelly. The histone degradation and the acrosome reaction are induced by a co-operative action of ARIS, which is an extremely large, sulfated glycoprotein with diffusible substance(s) in the jelly. Co-ARIS I, a steroidal saponin of the jelly, is effective to induce both reactions in the presence of ARIS.

Laminin-induced process outgrowth from isolated fetal rat C-cells.

A method for isolation of C-cells from rat fetuses was developed, and the morphological plasticity of the cells in primary culture systems was tested. Thyroid-parathyroid-ultimobranchial body (UB) complexes from 16-day rat fetuses were treated with 0.1% collagenase and 1000 PU/ml Dispase at 37 degrees C for 1 h. After dissociation by pipetting, UBs were obtained as remaining cell aggregates with diameters of 150-200 microns. The isolated UBs were cultured on untreated, fibronectin-coated, or laminin-coated substratum in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) supplemented with 5% fetal calf serum. In some experiments, the medium was changed to serum-free medium after 24 h of incubation, until the UBs had formed cell sheets. At Day 4 in vitro, the cultures were subjected to immunostaining using anti-calcitonin antiserum. On untreated or fibronectin-coated substratum, most of the C-cells exhibited polygonal or ovoid shapes, and 5-8% of them were found to project processes. On laminin-coated substratum, the ratio of process-bearing C-cells to total C-cells was 23% in serum-supplemented medium and 51% in serum-free medium. The longest processes reached 150 microns in length. The processes were intensely reactive with anti-alpha-tubulin antibody and were completely disintegrated by colcemid, suggesting that the microtubule cytoskeleton participated in the maintenance of the processes. Thus it was demonstrated that fetal rat C-cells are still responsive to environmental signals, such as laminin, and extend neuritic processes.

Developmental change in calcitonin secretory capacity of fetal rat thyroid C-cells.

Developmental changes in calcitonin (CT) secretory capacity of C-cells were studied by using primary cultures prepared from the thyroid glands of rat fetuses at 16th, 18th and 20th day of gestation and the thyroid glands of 28-day-old rats. Both CT content of C-cells and high Ca2(+)-stimulated CT secretion increased with the age of the rats. The ratio of secreted CT to the CT content of C-cells also increased according to the age. These results demonstrated the functional development of C-cells during the fetal period.

Physiological inducers of the acrosome reaction.

This article reviews recent studies on physiological inducers of the acrosome reaction in starfish. Upon encountering the jelly coat of eggs, starfish sperm undergo the acrosome reaction in response to a cooperation of three jelly components: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of steroidal saponins named Co-ARIS, and an oligopeptide presumably having an activity to increase the intracellular pH of sperm. ARIS induces the acrosome reaction in high Ca2+ or high pH sea water. In normal sea water, both ARIS and Co-ARIS are required for the induction. In addition to ARIS and Co-ARIS, a third jelly component, the oligopeptide, is necessary to mimic the full capacity of the jelly coat to induce the acrosome reaction. ARIS and Co-ARIS cooperatively increase the intracellular Ca2+ by stimulating Ca2+ channels, while the oligopeptide increases the intracellular pH by stimulating Na+/H+ exchange systems. When sperm meet the eggs, both changes are simultaneously achieved in them and thus they undergo the acrosome reaction.