Isolation and identification of alpha-(beta-alanyl)hypusine from bovine brain

A unique dipeptide was isolated from bovine brain using five steps of ion-exchange chromatography. Its acid hydrolysate contained equimolar amounts of beta-alanine and hypusine. The structure of the peptide was elucidated as alpha-(beta-alanyl)hypusine using dansylation technique. About 1 mumol of the compound was isolated from 1090 g of bovine brain.     

Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea

We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.     

The early expression of immunoreactivity for calmodulin in the nervous system of mouse embryos

Summary Calmodulin (CaM) is a major calcium-binding protein in the brain, where its immunoreactivity is mainly localized in the neurons. In this study, ontogenical changes in the distribution of CaM in the nervous system of mouse embryos were investigated immunohistochemically using a specific antibody against CaM and an indirect immunoenzyme method. Immunoreactive staining was first observed in the marginal layer of the cranial neural tube after 9.5 days of gestation; thereafter, the amount of stained structures increased rapidly. Particularly intense staining was observed in the long neuronal processes extending from or into the brain and spinal cord primordia. Intense immunostaining was also observed in the optic nerve layer of early retinae from 12.5 days of gestation. The appearance of CaM immunoreactivity is thus an early event during neuronal differentiation, apparently concominant with the initiation of axon extension and the appearance of neurofilament proteins.     

Carbohydrate analysis by a phenol-sulfuric acid method in microplate format

Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50-450 nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10-80 nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1-150 nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15 min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions.     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

Inhibition of the Motility and Growth of B16F10 Mouse Melanoma Cells by Dominant Negative Mutants of Dok-1

Dok-1 (p62(Dok)) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed to contribute to the control of cell growth and migration through association with the Ras GTPase-activating protein and the adapter protein Nck, the role of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOH-terminal truncation mutants of this protein: one (DokPH+PTB) containing the pleckstrin homology and phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants in highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyrosine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membranes and the cytoskeleton. Overexpression of DokPH+PTB in these cells also markedly reduced both the rates of cell spreading, migration, and growth as well as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH+PTB, indicating the importance of the phosphotyrosine-binding domain. These results suggest that at least in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.     

Host-plant specificity limits the geographic distribution of thistle feeding ladybird beetles

The relationships between two phytophagous ladybird beetle species, Epilachna pustulosa K^ono and E. niponica Lewis (Coleoptera, Coccinellidae), and their main host plants, thistles (Cirsium spp., Asteraceae) were investigated in Oshima Peninsula, southern Hokkaido, northern Japan. Epilachna pustulosa was found feeding on Cirsium kamtschaticum in the northernmost part of the peninsula, whereas E. niponica was confined to the Ohno Plain and adjacent areas in the southernmost part, and occurred mainly on C. alpicola. No thistle feeding epilachnines were found in the middle part of the peninsula despite the abundance of another thistle species, C. grayanum. Both beetle species showed lower adult preference and reduced growth performance on C. grayanum compared to their respective host plants under laboratory conditions. We concluded that the distribution of thistle feeding epilachnines in Oshima Peninsula was principally determined by the availability of appropriate host plants.     

Fluorescence and electron microscopic evidence for the dual innervation of the iris sphincter muscle of the rabbit

Summary The sphincter zone of the rabbit iris sometimes contains terminals with small granular vesicles. These terminals correspond to yellowish-green fluorescent structures in the sphincter zone. The paired arrangement of a terminal containing these vesicles and one full of agranular vesicles might indicate dual innervation of sphincter muscles by sympathetic and parasympathetic fibers. The sympathetic component probably exerts an inhibitory action on the sphincter muscles.This investigation was supported by a research grant from the Ministry of Education, Japan.     

ALDH2 polymorphism for the risk of cervical carcinogenesis

To investigate the clinical significance of ALDH2 genetic polymorphisms in cervical carcinogenesis. ALDH2 polymorphisms together with human papillomavirus (HPV) types were examined in a total of 195 cervical smear in exfoliated cervical cell samples using Real-Time polymerase chain reaction (PCR) System. The frequency for the AG+AA genotype was seven in the normal group (70.0 %), 16 in the LSIL group (57.1 %), and 27 in the HSIL group (90.0 %). A significant difference was found between the LSIL and HSIL groups (P = 0.0064). Patients with HSIL lesions frequently had high-risk HPV infections and concurrently belonged to the AG+AA group. ALDH2 genotype in cervical cell samples may be associated with more severe precancerous lesions of the cervix in a Japanese population.