Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis , were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.     

Production of monoclonal antibodies against a novel glycoprotein synthesized and secreted by dog thyroid C-cells.

A monoclonal antibody (MAb) that reacted only with thyroid C-cells was raised against cell suspensions from dog thyroid glands, to examine a glycoprotein secreted by C-cells. After chronically-induced hypercalcemia and administration of an anti-thyroid drug, reaction products for the antibody markedly decreased in C-cells, coinciding with alterations in calcitonin immunoreactivity. The antigen recognized by the MAb appears to be a secretory protein. The MAb reacted with C-cells from a wide variety of mammalian species, including rats, mice, hamsters, cattle, cats, rabbits, and monkeys. Furthermore, tumor cells of human medullary thyroid carcinoma, which is derived from C-cells, were immunoreactive to the MAb. Exceptionally, C-cells from guinea pigs and pigs were not stained with the MAb. No crossreactivity was observed in any of the dog tissues examined. Immunoblot analysis demonstrated that the MAb recognized a single prominent band at a molecular weight of approximately 79,000. The 79 KD band reacted with various digoxigenin-labeled lectins, including GNA, DSA, SNA, and MAA; it is a glycoprotein containing mannose, N-acetylglucosamine, and sialic acid. Dog thyroid C-cells were also densely stained with these lectins. The results indicate that thyroid C-cells synthesize and secrete a specific glycoprotein in addition to peptide hormones.     

Effect of addition of polyethyleneimine on thermal stability and activity of glucose dehydrogenase

When polyethyleneimine (PEI), a water-soluble cationic polymer, was added to solutions of glucose dehydrogenase (GDH) from Bacillus megaterium IWG3 at a molar ratio of PEI to GDH greater than 10, the thermal stability of GDH markedly increased. By addition of PEI, the rate of GDH-catalysed oxidation of -d-glucose increased in a low concentration range of NAD⁺ and NADP⁺ and the Michaelis constants and inhibition constants for both NAD⁺ and NADP⁺ decreased. These results suggested that negatively charged GDH interacts with cationic water-soluble polymers to form conjugates by electrostatic attraction, and also that negatively charged coenzymes are adsorbed by the polymers, resulting in enrichment of the coenzymes in the vicinity of GDH. Addition of PEI was also found to be effective for preventing the denaturation of GDH by acrylamide.Correspondence to: M. Teramoto     

Occurrence of colloid-containing follicles and ciliated cysts in the hypophysial pars tuberalis from guinea pigs of various ages

Colloid-containing follicles and ciliated cysts in the hypophysial pars tuberalis of guinea pigs at various ages ranging from 5 days to 36 months were examined by periodic acid-Schiff (PAS) reaction, immunohistochemistry, and electron microscopy. The follicles storing PAS-positive colloid were encountered in the pars tuberalis of all guinea pigs examined, although only a few were present in young animals. The follicles gradually increased in number with age. The largest number of follicles was found in the senile male group: 141.3 +/- 11.9, about 10 times the number in the 5-day-old male group. The follicles were scattered throughout the entire length of the pars tuberalis. Follicles with enlarged luminal cavities were concentrated in the ventral caudal region surrounding the infundibular stem and merges with the pars distalis. Three different types of follicles were found by electron microscopy: 1) those surrounded by nongranulated follicular cells that may correspond to the stellate-follicular cells in the pars distalis, 2) those surrounded by specific cells that were packed with vesicular inclusions, and 3) those surrounded by granulated cells that may be gonadotropes. In the follicles lined by non-granulated follicular cells, long, prominent microvilli and cytoplasmic processes protruding into the lumen and invaginations of colloid were often observed at the apical cell region. The follicles lined by the specific cells having numerous vesicles were localized only in the ventral caudal portion. The vesicles ranged from 200 to 700 nm in diameter, and the outer surface of their limiting membrane was partly studded with ribosomes. Gonadotropes immunoreactive to the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) antisera were distributed in the guinea pig pars tuberalis. As well as the typical follicles described above, the follicles composed solely of granulated cells showed microvilli protruding into the cavities and junctional complexes at the apical lateral surface. They stored heterogeneous materials in the lumina. Some secretory granules gave the appearance of being discharged into the lumen. Ciliated cysts were frequently observed in the pars tuberalis; their incidence was 71.7%. The ciliated cysts were much larger than colloid-containing follicles. Cystic cavities were only partly filled with heterogeneous materials showing colloid-like, flocculent, and granular features.     

C-cell follicles of canine thyroid glands studied by PAS reaction and electron microscopy

Summary Small follicles composed solely of C cells were occasionally observed in large C cell groups of dog thyroid glands. The lumina of C-cell follicles were filled with, or contained peripheral depositions of PAS-positive amorphous material, which was similar in ultrastructural features to thyroglobulin-containing colloid in typical thyroid follicles. This indicates that C cells, in addition to secreting calcitonin, produce a glycoprotein that can be stored in the lumina of the follicles.     

The fine structure of ventral prostatic secretory epithelial cells in older rats

Summary The ventral prostatic secretory epithelial cells in older rats were studied by light and electron microscopy. The cells vary in height in different parts of the same organ, and ultrastructurally they show the presence of a developed secretory apparatus such as well-developed Golgi body and abundant rough endoplasmic reticulum. They also show signs of a depressed secretory activity, involving occasional emiocytosis of apical secretory vacuoles and a paucity of condensing vacuoles in the Golgi region and above it. Further, they are characterized by the frequent occurrence of supra and paranuclear pleomorphic lysosomes.     

Regulation of lettuce hypocotyl elongation by gibberellic acid. Correlation between cell elongation, stress-relaxation properties of the cell wall and wall polysaccharide content

Lettuce hypocotyl elongation caused by gibberellic acid wasstrongly inhibited by coumarin and dichlobenil, known inhibitorsof cellulose biosyndiesis. Stress-relaxation analysis of thecell wall revealed that gibberellic acid induces a decreasein both minimum relaxation time (To) and relaxation rate (b)and an increase in maximum relaxation time (Tm), when gibberellicacid stimulates hypocotyl elongation. Both coumarin and dichlobenilnullified the effect of gibberellic acid on changes in To, Tmand b values. The content of pectic, hemicellulosic and cellulosic substancesin the cell wall increased per hypocotyl but decreased per unithypocotyl length, in response to gibberellic acid treatment.Particularly, gibberellic acid caused a substantial increasein cellulose content per hypocotyl but a decrease per unit length.A good correlation existed between the decrease in To and thedecrease in hemicellulose content per unit lengdi of the cellwall. The increase in Tm was correlated with the decrease incellulose content per unit length of the cell wall. The decreasein b was correlated with the decrease in the content of bothcellulose and hemicellulose per unit length. Based on these results, we discuss the role of polysaccharidemetabolism of the cell wall in gibberellic acid-induced lettucehypocotyl elongation and the nature of gibberellic acid-inducedbiochemical modifications of the cell wall, which are representedby changes in stress-relaxation properties of the cell wall. ¹Present address: Department of Anatomy, Aichi Medical University,Nagakutecho, Aichigun, Aichi 480-11, Japan. (Received September 22, 1975; )     

Effect of photoperiod on winter and summer diapause of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae)

To understand the geographical differences between diapause systems and synchronization of adult occurrence in the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae), we examined the timing of winter diapause termination and intensity of summer diapause using univoltine and potentially bivoltine individuals in Iwate, Japan. In laboratory rearing experiments of mature larvae maintained at constant temperature (20 °C), winter diapause intensity weakened by January without photoperiodic responses. Meanwhile, summer diapause was maintained by the long day length and presumably terminated with the photoperiodic transition from long to short day length. The intensity of summer diapause was stronger for cocoons that transitioned from a 16 h light to 8 h dark (LD 16:8) to a LD 15:9 photoperiod than for those that transitioned from LD 15:9 to LD 14:10. These results suggest that populations distributed in relatively low-latitude areas, with partly or potentially bivoltine individuals, would have a weaker summer diapause or none at all. Moreover, sexual differences in the number of days to emergence were not detected when individuals experienced a photoperiodic transition from long to short day length, suggesting that the summer diapause system may function to synchronize the emergence of males and females in the population examined.     

A Novel Cell-Sheet Technology That Achieves Durable Factor VIII Delivery in a Mouse Model of Hemophilia A

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.