Diversity of protein inclusion bodies and identification of mosquitocidal protein in Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis subsp. israelensis produces, during sporulation, protein inclusion bodies of wide ranging sizes, all of which are toxic to mosquitoes. Two proteins are present in the smallest protein bodies (less than 0.2 micron dia.), but the number of proteins increases with increasing size of protein bodies. The largest bodies (greater than 1.5 micron dia.) contain seven proteins. All of the proteins are synthesized at different times during sporulation and are added to developing protein bodies in a stepwise manner. The protein component responsible for mosquitocidal activity is a 65,000-dalton protein, that is present in all of the protein bodies.     

Isolation and Analysis of Protein Bodies from Cotyledons of Lablab purpureus and Phaseolus vulgaris (Leguminoseae)

Protein bodies, isolated by differential and isopycnic centrifugation, have been observed in transmission and scanning electron microscope and biochemically analysed. The powders of the axes and of the cotyledons contain numerous protein bodies, which in the scanning microscope appear to be surrounded by a more or less torn membrane. The proportion of intact, isolated protein bodies is influenced by the grinding methods, but even in the best conditions soaking disaggregates the majority of them. After isopycnic centrifugation, their debris gather in different density zones. Analyses of each zone have revealed that the caseinases are associated with particles of higher density than are peptidases and trypsin inhibitors. A minority population of small-size protein bodies resists the homogenization and fractioning modalities. A double origin of the protein bodies is considered.     

Evidence for the presence of two different types of protein bodies in wheat endosperm

Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low- and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route.     

Development of protein bodies in cotyledons of Fagus sylvatica

Electron microscopic analysis of cotyledons of beech ( Fagus sylvatica ) at different stages of seed maturation indicates that protein bodies originate by gradual subdivision of the vacuoles in which reserve proteins are deposited. The majority of protein bodies show a proteinaceous matrix and a number of globoid inclusions of different sizes. In a small number of cells, druse-like inclusions were observed. Analysis by SDS-PAGE of proteins extracted from isolated protein bodies shows that the majority correspond to beech seed storage globulins.     

两种品质类型花生品质形成与子叶细胞超微结构的关系

在大田条件下研究了两种品质类型花生(Arachis hypogaea)品质形成的动态差异及其子叶细胞超微结构的差异。结果表明, 高蛋白品种‘XB023’的蛋白质含量在籽仁发育前期较高油品种‘鲁花9号’低, 后期显著高于‘鲁花9号’, 且成熟期籽仁8种必需氨基酸组分含量均高于‘鲁花9号’, 其中谷氨酸、赖氨酸和亮氨酸含量差异极显著; ‘XB023’脂肪含量在籽仁发育期一直低于‘鲁花9号’。‘XB023’各时期的籽仁可溶性糖含量和油酸/亚油酸(O/L)值均显著低于‘鲁花9号’。两品种在果针入土10天时子叶细胞即形成淀粉粒、脂体和蛋白体, 随后脂体、蛋白体的数量不断增加, 淀粉粒先增大后逐渐缩小解体。‘XB023’的脂体达到最大的时间早于‘鲁花9号’, 而‘鲁花9号’的脂体快速积累的时间比‘XB023’长。两品种蛋白体大小都在果针入土40天时达到最大值, ‘XB023’的蛋白体在籽仁发育后期数量增加较快。高蛋白品种较高的蛋白质含量由其子叶细胞中较大蛋白体的大小和较多的蛋白体数量决定, 而高油品种较高的脂肪含量是由其较多的脂体数量决定。     

Differential subcellular localization of particular mRNAs in hippocampal neurons in culture

In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and beta-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that is selectively distributed in dendrites and cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.     

Localization by immunoelectron microscopy of antigens of Chlamydia psittaci suitable for diagnosis or vaccine development

Two different antigens of serotype 1 Chlamydia psittaci were localized using three immunoelectron microscopy techniques: non-embedding, pre-embedding and post-embedding. The antigens had previously been described as being of potential use in diagnosis (80–90 kDa protein region) and vaccine development (110 kDa protein). The results show a direct relationship between the protective capacity of the antigens and their surface localization on the elementary bodies, which are the infectious form of Chlamydia. The 80–90 kDa protein region is located on the surface of reticulate bodies but not of elementary bodies, where it was located periplasmically, while the 110 kDa protein occurs on the surface of both elementary and reticulate bodies.     

Protein accumulation in aleurone cells,sub-aleurone cells and the center starch endosperm of cereals

There are mainly three endosperm storage tissues in the cereal endosperm: aleurone cells, sub-aleurone cells and the center starch endosperm. The protein accumulation is very different in the three endosperm storage tissues. The aleurone cells accumulate protein in aleurone granules. The sub-aleurone cells and the center starch endosperm accumulate protein in endoplasmic reticulum-derived protein bodies and vacuolar protein bodies. Proteins are deposited in different patterns within different endosperm storage tissues probably because of the special storage properties of these tissues. There are several special genes and other molecular factors to mediate the protein accumulation in these tissues. Different proteins have distinct functions in the protein body formation and the protein interactions determine protein body assembly. There are both cooperation and competition relationships between protein, starch and lipid in the cereal endosperm. This paper reviews the latest investigations on protein accumulation in aleurone cells, sub-aleurone cells and the center starch endosperm. Useful information will be supplied for future investigations on the cereal endosperm development.     

Chromatographic methods for the isolation of,and refolding of proteins from,Escherichia coli inclusion bodies

New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

Comparisons of Cells, Refractile Bodies, and Spores of Bacillus popilliae

Spores of Bacillus popilliae from infected larvae and refractile bodies produced in a Trypticase-barbiturate medium were similar but distinct from vegetative cells of this organism in protein, nucleic acid, and enzyme composition. The spores and refractile bodies were found to have catalase activity, some of which was heat-resistant. This enzyme was not found in the vegetative cells. The spores contained dipicolinic acid, but the refractile bodies did not. The latter were similar to cells in having considerably higher levels of phosphate extractable with cold trichloroacetic acid and of poly-beta-hydroxybutyrate than had the spores. Electron microscopy demonstrated conclusively that the refractile bodies are distinctly different from either cells or spores of B. popilliae. The possibility that these bodies are formed as a result of an aborted sporulation process is discussed.     

棉花种子萌发过程中子叶蛋白体变化

对棉花种子萌发过程中子叶细胞内蛋白体的变化进行了详细的观察。干种子内存在仅由蛋白质基质组成无内含物的蛋白体,含有球状晶体的蛋白体和无含球状晶体和拟晶体的蛋白体。种子萌发过程中蛋白体逐渐液泡化,其降解方式可分为三种类型：（１）内部降解类型：（２）周边降解类型;（３）内部和周边同时降解类型。文中还一步进行了不同降解类型与酶的分布,蛋白体存在部位和萌发时间进程之间的关系。     

Localization and secretory pathways of a 58K-like protein in multi-vesicular bodies in callus of Arabidopsis thaliana

Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.     

Sieve-Element Characters of the Proteaceae and Elaeagnaceae: Nuclear Crystals,Phloem Proteins and Sieve-Element Plastids

The sieve-element characters of 34 species from the Proteaceae and Elaeagnaceae have been studied by transmission electron microscopy. While nondispersive protein bodies and dispersive P-protein are typical components of both families, specific forms and/or their distinctive origin accentuate some taxa. Within the Grevilloideae, subfamily of Proteaceae, a number of Australian species and genera contain protein crystals of nuclear origin arranged into rosette-like bodies, while in the other members studied from the same subfamily no nondispersive protein bodies were found. Several Australian and South African genera of the Proteoideae contain compound-spherical nondispersive protein bodies that reside in the cytoplasm from their very beginning. In the Elaeagnaceae three different P-protein bodies are present of which one is tubular and dispersing, another is nondispersive and of irregular-stellate form, and a third is globular (resembling a P-protein from Cucurbita). The great majority of the species studied from the Proteaceae contains form-Ss sieve-element plastids, Lomatia ilicifolia and Macadamia ternifolia are distinct in having form-Pcs plastids. The average diameter of stem sieve-element plastids in the family is 1.38 μm. The Elaeagnaceae (three species investigated) is a pure form-So family (average diameter: 0.8 μm). There are no specific sieve-element characters that would support any relationship between the Proteaceae and Elaeagnaceae. While affinities of the former to pre-Gondwanan parts of the Rosanae/Myrtanae are discussed, a reconsideration of the Elaeagnaceae as a possible member of the Violanae (identical features with Cucurbitaceae) is proposed.     

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.     

Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo.

Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.     

Ultrastructure, Origin, and Composition of the Protein Bodies in the Ligule of Isoetes lacustris L.

The basal cells in the ligule of Isoetes lacustris contain numerousprotein bodies, the contents of which can be digested enzymicallyby pronase and are stained red by treatment with ninhydrin Schiff'sreagent. Two types of protein bodies can be distinguished ultrastructurally:spherically-shaped bodies with granular contents and spindle-likebodies with fibrillar contents. Both are ensheathed by singlemembranes and do not show any solid inclusions within theirmatrix. The protein bodies probably arise from dilatation of the endoplasmicreticulum (ER) cisternae. This conclusion is based upon threeobservations: (a) The protein bodies occasionally show membranecontinuity with the ER; (b) ribosomes and polysomes are frequentlyattached to the protein-body membranes; (c) the contents ofthe protein bodies and of the dilated ER cisternae show similarultrastructural features. The dilatation of the ER cisternae is assumed to be a resultof protein accumulation in the intracisternal space. Based upon the results of polyacrylamide gel electrophoresis,it is likely that the spherically-shaped protein bodies storepredominately two proteins with molecular weights of 51300 and55800 D, while the spindle-like bodies store two proteins withmolecular weights of 92000 and 98000 D. The results presented do not permit a definite conclusion regardingthe function of the ligule of Isoetes lacustris but it is suggestedthat it may have a nutritive role. Isoetes lacustris L., ligule, protein bodies, endoplasmic reticulum, ultrastructure     

Evolutionary conservation of an epitope associated with striated rootlets in different epithelial ciliated cells.

In ciliated cells of metazoa, striated rootlets associated with basal bodies anchor the ciliary apparatus to the cytoskeleton. We have used here a monoclonal antibody against a 175 kDa protein associated with the striated rootlets of quail ciliated cells, to study ciliated cells of different species. In mussel gill epithelium the antibody recognized a protein of 92 kDa which shows a periodic distribution along the striated rootlets. In frog ciliated palate epithelium, two different rootlets are associated with basal bodies, both are decorated and only one protein of 48 kDa is recognized on immunoblot. The antigen is arranged in a helix around the striated rootlets. In rabbit ciliated oviduct epithelium, we detected the presence of very small and thin rootlets which are weakly labeled. We have shown that an epitope associated with the striated rootlets is preserved through evolution although the molecular weight of the peptide varies. We have also observed the appearance of this epitope on protein associated with junctional complexes in rabbit and cytoskeleton component in quail oviduct.