Evidence for the presence of proteinase inhibitor I in vacuolar protein bodies of plant cells

Summary Protein bodies induced in tomato leaf cells by wounding were shown to contain proteinase Inhibitor I by using ferritin-labelled antibodies, fluorescein-labelled antibodies, and cytochrome C-labelled antibody fragments. Both pre-embedding and postembedding techniques were used. Nonspecific binding was least when p-formaldehyde was used as the initial fixative followed by treatment with cytochrome c-labelled antibody fragments.Abbreviations Fab antibody fragments - BSA bovine serum albumin - GMA glycol methacrylate - THB Tris-HCl buffer Taken in part from a doctoral (Ph.D.) dissertation submitted to Washington State University by Vivian V. Yang. This work was supported largely by NSF Grant GB-29614X (LKS) and in part by the United States Department of Agricultural Cooperative States Research Service Grant 316-15-30 (CAR), the National Science Foundation Grant GB-37972 (CAR), and the College of Agriculture Research Center, Washington State University, Pullman, WA 99163, Scientific Paper No. 4525, Project 1791.Program in Genetics and Department of Botany. To whom reprint requests should be sent.Department of Agricultural Chemistry and Program in Biochemistry and Biophysics.     

The influence of nitrogen on the development and accumulation of protein bodies in the developing endosperm of wheat caryopses

The aim of the present work was to reveal the histological changes of protein bodies (PBs) in the developing wheat endosperm under nitrogen (N) treatment. For this purpose, the development and accumulation of PBs in the dorsal and ventral regions of wheat endosperm affected by N application at booting stage were investigated using light microscopy and Image-Pro Plus 6.0 software. The endosperm without N treatment contained many smaller PBs that were scattered in endosperm cells in an unordered pattern, whereas the endosperm with N treatment contained many larger PBs or aggregations that were concentrated in a certain region of endosperm cells. The amount and relative areas of PBs in wheat varieties cvs. Xumai 30 and Yangmai 13 were significantly increased by N application. However, the cultivars differed with the degree of response to N being cv. Xumai 30 > cv. Yangmai 13. These differences also varied with position in the endosperm in the order ventral > dorsal region. The initiation of PBs occurred 3 days earlier in N-treated endosperm than the control.     

Evidence for the presence in smooth muscle of two types of Ca2+-transport ATPase.

Membrane fractions prepared from smooth muscle of the pig stomach (antral part) contain two Ca2+-dependent phosphoprotein intermediates belonging to different Ca2+-transport ATPases. These alkali-labile phosphoproteins can be separated by electrophoresis in acid medium. The 130 kDa phosphoprotein resembles a corresponding protein in the erythrocyte membrane, whereas the 100 kDa protein resembles that of the Ca2+-transport ATPase in sarcoplasmic reticulum from skeletal muscle. These resemblances are expressed in terms of Mr, reaction to La3+ and in a similar proteolytic degradation pattern. The presence of the calmodulin-stimulated ATPase in mixed membranes from smooth muscle is confirmed by its binding of calmodulin and antibodies against erythrocyte Ca2+-transport ATPase, whereas such binding does not occur with proteins present in the presumed endoplasmic reticulum from smooth muscle.     

Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum

Localization of calsequestrin in chicken ventricular muscle cells was determined by indirect immunofluorescence and immuno-Protein A-colloidal gold labeling of cryostat and ultracryotomy sections, respectively. Calsequestrin was localized in the lumen of peripheral junctional sarcoplasmic reticulum, as well as in the lumen of membrane-bound structures present in the central region of the I-band, while being absent from the lumen of the sarcoplasmic reticulum in the A-band region of the cardiac muscle cells. Since chicken ventricular muscle cells lack transverse tubules, the presence of calsequestrin in membrane bound structures in the central region of the I-band suggests that these cells contain nonjunctional regions of sarcoplasmic reticulum that are involved in Ca2+ storage and possibly Ca2+ release. It is likely that the calsequestrin containing structures present throughout the I-band region of the muscle cells correspond to specialized regions of the free sarcoplasmic reticulum in the I-band called corbular sarcoplasmic reticulum. It will be of interest to determine whether Ca2+ storage and possibly Ca2+ release from junctional and nonjunctional regions of the sarcoplasmic reticulum in chicken ventricular muscle cells are regulated by the same or different physiological signals.     

Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium

In human unbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA)_and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70°C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

Evidence for recombination between two different immunological types of foot-and-mouth disease virus.

Recombination was observed between temperature-sensitive (ts) mutants of two immunological types of foot-and-mouth disease virus which were distinguishable by two marker characteristics in addition to their antigenic type. Putative ts+ recombinants were isolated and the segregation patterns of their marker characteristics examined. The results are discussed in terms of the origin of new sub-type strains.     

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.     

A screening for the presence of four different crystal protein gene types in 25 Bacillus thuringiensis strains

25 different strains of Bacillus thuringiensis, belonging to 18 different serotypes were screened by Southern hybridization analysis for the presence of nucleotide sequences of four different gene types coding for crystal proteins showing different insecticidal spectra. Many but not all strains showed sequences homologous to any of the probes used. Homology with sequences of the gene type present in strain kurstaki HD1 occurred in all positive strains, whereas the other gene types were much less abundant. Sequences homologous to those of gene type VI, coding for a Spodoptera-specific crystal protein, appeared only in strains of serotypes aizawai and entomocidus.