Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

History vs. habitat type: explaining the genetic structure of European nine‐spined stickleback (Pungitius pungitius) populations

The genetic structure of contemporary populations can be shaped by both their history and current ecological conditions. We assessed the relative importance of postglacial colonization history and habitat type in the patterns and degree of genetic diversity and differentiation in northern European nine‐spined sticklebacks (Pungitius pungitius), using mitochondrial DNA (mtDNA) sequences and 12 nuclear microsatellite and insertion/deletion loci. The mtDNA analyses identified – and microsatellite analyses supported – the existence of two historically distinct lineages (eastern and western). The analyses of nuclear loci among 51 European sites revealed clear historically influenced and to minor degree habitat dependent, patterns of genetic diversity and differentiation. While the effect of habitat type on the levels of genetic variation (coastal > freshwater) and differentiation (freshwater > coastal) was clear, the levels of genetic variability and differentiation in the freshwater sites were independent of habitat type (viz. river, lake and pond). However, levels of genetic variability, together with estimates of historical effective population sizes, decreased dramatically and linearly with increasing latitude. These geographical patterns of genetic variability and differentiation suggest that the contemporary genetic structure of freshwater nine‐spined sticklebacks has been strongly impacted by the founder events associated with postglacial colonization and less by current ecological conditions (cf. habitat type). In general, the results highlight the strong and persistent effects of postglacial colonization history on genetic structuring of northern European fauna and provide an unparalleled example of latitudinal trends in levels of genetic diversity.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Differentiation Expression in Blastomeres of Cleavage-Arrested Embryos of the Ascidian Halocynthia roretzi

The question whether two or more different genetic programs are expressed in the common cytoplasm of single blastomeres or the expression of one genetic program somehow excludes expression of the other, was analyzed by assessing the occurrence of a muscle-specific and two epidermis-specific antigens in cleavage-arrested blastomeres in early embryos of the ascidian Halocynthia roretzi . Blastomeres which had been arrested in 1- to 4-cell stages expressed only the epidermis markers. Arrested 8-cell to 32-cell embryos produced both epidermis and muscle markers, but each cell expressed only one program of differentiation, even though some possessed the potential to express both. The differentiation expressions followed their cell lineages. These results indicate that at least in this experimental system differentiation markers of the two different cell-types are expressed exclusively.
A distinct order was noticed in expression of the two epidermis markers in a single blastomeres; a marker is always superiorly expressed, whereas the other appears only when the superior marker is expressed.     

A step towards understanding plant responses to multiple environmental stresses: a genome‐wide study

In natural habitats, especially in arid areas, plants are often simultaneously exposed to multiple abiotic stresses, such as salt, osmotic and heat stresses. However, most analyses of gene expression in stress responses examine individual stresses. In this report, we compare gene expression in individual and combined stresses. We show that combined stress treatments with salt, mannitol and heat induce a unique pattern of gene expression that is not a simple merge of the individual stress responses. Under multiple stress conditions, expression of most heat and salt stress‐responsive genes increased to levels similar to or higher than those measured in single stress conditions, but osmotic stress‐responsive genes increased to lower levels. Genes up‐regulated to higher levels under multiple stress condition than single stress conditions include genes for heat shock proteins, heat shock regulators and late embryogenesis abundant proteins (LEAs), which protect other proteins from damage caused by stresses, suggesting their importance in multiple stress condition. Based on this analysis, we identify candidate genes for engineering crop plants tolerant to multiple stresses.     

DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5‐phosphatase,is required for proper development of intercalary meristem

Rice internodes are vital for supporting high‐yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell‐producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5‐phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.     

Analysis In Vitro of Capacity of Fetal Fat Pad to Support Mammary Gland Embryogenesis

Fourteen-day fetal mammary fat pad precursor tissue (FP) has the capacity to support various fetal epithelia allowing them to accomplish their characteristic development in vivo , without their own mesenchyme (1). This capacity decreases with age of fetal fat pad and is lost postnatally. To analyse the molecular mechanism of such interaction, a method for in vitro duplication of organogenesis is necessary. In the present paper, a co-culture system of fetal epithelium with prospective mammary fat pad is described. The explanted mammary epithelium started budding, then grew out forming branched mammary ducts with end buds. Ultrastructurally, the developing ductal structures exhibited the typical mammary gland morphogenesis.
³H-Thymidine incorportion assessed by autoradiography showed that the mammary gland morphogenesis in vitro was due to the proliferation of epithelial cells, not merely to a change of the shape of the epithelium. This supportive capacity of 14-day FP also decreased with aging; explanted mammary epithelium did not grow into 17-day FP. When insoluble, non-living biomatrix was used in place of living FP the epithelium grew into the matrix but the resulting structures lacked characteristic morphology of epithelium on living fetal FP. The difference of capacity between 14-day and 17-day tissues was also lost.     

Scale Formation in an Amoeba, Cochliopodium sp.

During excystment of an amoeba, Cochliopodium sp., scale formation was examined with light and electron microscopy. This amoeba was covered with scales. When the amoeba encysted, the scales remained on the external surface of the cyst wall. Soon after the induction of excystment the Golgi complex began to develop. Many vesicles were extruded from it and changed into vacuoles. Scales were observed first in the vacuole adjacent to the Golgi complex and later in inside the cyst wall. When the amoeba excysted it had been coated by the newly formed scales. It is suggested that the scale formation is dependent on the activity of the Golgi complex.     

VEGETALIZATION OF SEA URCHIN LARVAE INDUCED WITH cAMP PHOSPHODIESTERASE INHIBITORS

Treatment of sea urchin embryos with cAMP phosphodiesterase (PDE)-inhibitors such as caffeine (4×10⁻³ M), theophylline (8×10⁻³M), or nicotinamide (10⁻²M), at the morula stage for only a couple of hours, yields vegetalized larvae. Most of the embryos treated with these reagents before the morula stage develop to blastulae filled with mcsenchyme-like cells. Almost all embryos at the blastula stage develop normally even if they are treated with a PDE-inhibitor for a considerable period. The rate of ³H-valine incorporation into protein in the morulae is reduced by caffeine and theophylline, but does not decrease in the presence of nicotinamide. Actinomycin D cancels the vegetalizing effect of PDE-inhibitors on the morulae.