Protein analysis during the ontogeny of normal and male sterile stamenless-2 mutant stamens of tomato (Lycopersicon esculentum Mill.)

The levels and synthesis of proteins during the ontogeny of normal and male sterile stamenless-2 (sl-2/sl-2) mutant stamens of tomato (Lycopersicon esculentum) were examined. The mutant stamens contained low levels of soluble protein which were related to reduction in protein synthesis. The mutant stamens, however, possessed many polypeptides similar to the normal and synthesized a 53-kd polypeptide at stages when there are abnormalities in tapetum development. The mutant stamens also possessed a 23-kd and some low molecular weight polypeptides that were considered as degradative proteins. Normal stamens exhibited the synthesis of many polypeptides not found in the mutant, from microspore mother cell to the preanthesis stages. In addition, at the time of pollen maturation there was a greater synthesis of several polypeptides, particularly those of 42 and 37 kd. Although the causative mechanisms of male sterility in the sl-2/sl-2 mutant are not known, the synthesis, and the lack, of specific polypeptides reported here appears to be associated with pollen degeneration.This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to V.K.S.     

Identification of surface autoantigens which appear during spermatogenesis

Autoantigens that appear during spermatogenesis in the rabbit were identified using immunoadsorbent chromatography and SDS-PAGE. To identify cell-surface proteins, samples of freshly isolated, staged cells were labeled by the lactoperoxidase or Iodo-Gen iodination procedure and run on SDS-PAGE. Autoradiograms of the stained, dried gels were prepared. By correlating the band patterns in the SDS gels of immunocolumn and surface-labeled samples with the band patterns in the autoradiograms, it was possible to show when the autoantigenic proteins appeared on the cell surface. To further support the identification of membrane autoantigens, surface-labeled, staged cell samples were lysed in Triton X-100 and immunoprecipitated with antitestis cell autoantisera. Three types of autoantigens have been identified: (1) late class antigens that are present only on late spermatids and epididymal spermatozoa, but are intracellular in early stages, (2) early class antigens which occur on the surface of pachytene spermatocytes and are present throughout subsequent stages of development, and (3) early class, transient antigens, which appear on spermatogenic cells but are not present on epididymal spermatozoa.     

Method for Lyophilizing Brain Proteolipid Preparation That Increases Subsequent Solubilization by Detergent

Abstract: A frozen mixture of solubilized brain proteolipid proteins in chloroform-methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform-methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2 mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins. In contrast to what happens when proteolipid proteins are obtained by the evaporation of the organic mixture at room temperature, the protein obtained by lyophilization was soluble in aqueous solutions of ionic and nonionic detergents. Sodium dodecyl sulfate at 0.6 to 0.7% concentration completely solubilized the proteolipid protein obtained by lyophilization. With the nonionic detergents Lubrol WX and Triton X-100, a solubilization between 50 and 65% was achieved. Sodium deoxycholate was practically ineffective. Triton X-100 showed selectivity in solubilizing certain proteins. The role of lipids in the solubilization of proteolipid proteins with detergents is discussed.     

Systematic variation in amino acid compositions of grass caryopses

Variation in amino acid patterns of 121 species (72 genera) of grass caryopses is extensively consistent with taxonomic groupings. The patterns of pooids and chloridoids are distinguishable from one another and from those of eu-panicoids and andropogonoids; the bamboos, Oryza, Stipeae, Ehrharta and Microlaena, which share certain morphological and anatomical features, also share a characteristic amino acid profile, while profiles of danthonoioids, Triodia and Aristida are clearly non-pooid. Caryopsis amino acid patterns vary independently of photosynthetic pathway. Embryos from taxonomically diverse genera all show very similar amino acid profiles, which differ strikingly from those of the endosperms, and the amino acid patterns of whole caryopses are dominated by their endosperms, which are responsible for the taxonomic variation. ‘Chemical scores’ of the caryopsis proteins, but not total protein contents, correlate to some extent with taxonomic groupings.     

Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seems to have an “inside-out” orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins lableled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic ³²P. We conclude that the phagolysosome is probably oriented in an “inside-out” configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.     

Synthesis and degradation of proteins during wheat endosperm development

Synthesis of proteins rich in lysine declines progressively with endosperm development and these proteins appear to be degraded preferentially at later stages. The proteolytic enzymes in extracts of endosperms at a late stage of development release considerably more lysine radioactivity from labelled endosperm proteins as compared with the enzymes in endosperms at an early stage.     

ISolation and identification of a sulfur-containing metabolite of spironolactone from human urine

In the urine of normal subjects Who were given an oral dose of 500 mg spironolactone (3-(3-oxo-7α-acetylthio-17β-hydroxy-4-androsten-17α-yl)-propionic acid γ-lactone; Aldactone^R) together with 100, uCi H-20, 21 spironolactone, a so far unknown major metabolite has been detected by thin layer chromatography. The metabolite then could be isolated by means of counter-current-distribution. According to masspectral and magnetic resonance data, the metabolite has been assigned the structure of 3-(3-oxo-7α-niethyl sulfonyl-6β, 17β-dihydroxy-4-androsten-17α-yl)-propionic acid γ-lactone. By oxidation of the corresponding methylsulfinyl compound — another already known metabolite of spironolactone-with m-chloroperbenzoic acid, a compound has been isolated which proved to be identical with the new metabolite according to TIC, MS and NMR.     

G4-quadruplex-binding proteins: review and insights into selectivity

There are over 700,000 putative G4-quadruplexes (G4Qs) in the human genome, found largely in promoter regions, telomeres, and other regions of high regulation. Growing evidence links their presence to functionality in various cellular processes, where cellular proteins interact with them, either stabilizing and/or anchoring upon them, or unwinding them to allow a process to proceed. Interest in understanding and manipulating the plethora of processes regulated by these G4Qs has spawned a new area of small-molecule binder development, with attempts to mimic and block the associated G4-binding protein (G4BP). Despite the growing interest and focus on these G4Qs, there is limited data (in particular, high-resolution structural information), on the nature of these G4Q-G4BP interactions and what makes a G4BP selective to certain G4Qs, if in fact they are at all. This review summarizes the current literature on G4BPs with regards to their interactions with G4Qs, providing groupings for binding mode, drawing conclusions around commonalities and highlighting information on specific interactions where available.     

Plasmodial heat shock proteins: targets for chemotherapy

Heat shock proteins act as molecular chaperones, facilitating protein folding in cells of living organisms. Their role is particularly important in parasites because environmental changes associated with their life cycles place a strain on protein homoeostasis. Not surprisingly, some heat shock proteins are essential for the survival of the most virulent malaria parasite, Plasmodium falciparum . This justifies the need for a greater understanding of the specific roles and regulation of malarial heat shock proteins. Furthermore, heat shock proteins play a major role during invasion of the host by the parasite and mediate in malaria pathogenesis. The identification and development of inhibitor compounds of heat shock proteins has recently attracted attention. This is important, given the fact that traditional antimalarial drugs are increasingly failing, as a consequence of parasite increasing drug resistance. Heat shock protein 90 (Hsp90), Hsp70/Hsp40 partnerships and small heat shock proteins are major malaria drug targets. This review examines the structural and functional features of these proteins that render them ideal drug targets and the challenges of targeting these proteins towards malaria drug design. The major antimalarial compounds that have been used to inhibit heat shock proteins include the antibiotic, geldanamycin, deoxyspergualin and pyrimidinones. The proposed mechanisms of action of these molecules and the pathways they inhibit are discussed.