Isolation of a heat-stable maize endosperm ADP-glucose pyrophosphorylase variant

Heat stress reduces maize yield and several lines of evidence suggest that the heat lability of maize endosperm ADP-glucose pyrophosphorylase (AGPase) contributes to this yield loss. AGPase catalyzes a rate-limiting step in starch synthesis. Herein, we present a novel maize endosperm AGPase small subunit variant, termed BT2-TI that harbors a single amino acid change of residue 462 from threonine to isoleucine. The mutant was isolated by random mutagenesis and heterologous expression in a bacterial system. BT2-TI exhibits enhanced heat stability compared to wildtype maize endosperm AGPase.The TI mutation was placed into another heat-stable small subunit variant, MP. MP is composed of sequences from the maize endosperm and the potato tuber small subunit. The MP-TI small subunit variant exhibited greater heat stability than did MP. Characterization of heat stability as well as kinetic and allosteric properties suggests that MP-TI may lead to increased starch yield when expressed in monocot endosperms.     

Lipids of Pinus halepensis pollen

The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.     

FINE STRUCTURAL STUDIES ON THE INTERPHASE AND DIVIDING APICAL CELLS OF SPHACELARIA TRIBULOIDES (PHAEOPHYTA)1

The apical cells of Sphacelaria tribuloides Menegh. are larger than other thallus cells, contain more organelles and appear polarized. Their tip portion, where they grow, contains a well developed Golgi apparatus, abundant endoplasmic reticulum (ER) membranes, mitochondria, chloroplasts and a large number of small vacuoles. It seems likely that a continuous flow of membranous material from the ER membranes to the dictyosomes and from the latter to the plasmalemma of the extending tip portion takes place. In contrast, the basal pole possesses fewer organelles and is occupied mainly by large-sized, sometimes central vacuoles. The apical cells undergo two distinct types of highly asymmetrical differential divisions giving rise to cells of the thallus and hair initials. During the early stages of mitosis the nuclear envelope remains intact, except for fenestrated poles. Microtubules pass through the fenestrae into the nucleoplasm. During meta-phase, a typical chromosome plate is organized. The sites of attachment of spindle microtubules to the chromosomes are structurally different from the rest of the chromosomes. At late anaphase, the nuclear envelope breaks down completely. During telophase, a new membrane encloses the chromosomes which are decondensed and the nucleoli are reorganized. Cytokinesis proceeds long after mitosis at a stage in which the nuclei have increased in size and have moved farther apart. A membranous furrow develops centripetally, without the participation of microtubules. However, microtubules traverse the thin cytoplasmic strands which, in both interphase and cytokinetic cells, meander among the vacuoles of the basal pole of the cell and the internuclear space. Dictyosomes appear to be involved in the subsequent wall deposition.     

Reversible cytoplasmic rearrangements precede wall apposition,hypersensitive cell death and defense-related gene activation in potato/Phytophthora infestans interactions

We used video microscopy techniques as a tool for live examination of the dynamic aspects of plant/fungus interactions. Early, dynamic responses of epidermal midrib cells of leaves from a potato cultivar (Solanum tuberosum L. cv. Datura) carrying resistance gene R1 to Phytophthora infestans (race 1: compatible interaction, race 4: incompatible interaction) were monitored. Similar responses were observed in both types of interaction, ranging from no visible reaction of invaded plant cells to hypersensitive cell death. The overall defense response of each individual cell exhibited a highly dynamic behavior that appeared to be tightly coordinated with the growth of the fungus. Initial localized reactions, including major rearrangements within the cytoplasm, occurred directly at the fungal penetration site, where rapid apposition of autofluorescent material and callose took place. If fungal invasion stopped at this stage, the host cell restored its normal cytoplasmic activity and survived. Hypersensitive cell death occurred only when fungal growth had proceeded to the formation of a clearly identifiable haustorium. In such cases, cytoplasm and nucleus conglomerated around the intracellular fungal structure, followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium. Only small quantitative differences between the compatible and incompatible interactions of the two fungal races were observed for these early responses of epidermal cells. In the incompatible interaction, a slightly larger number of epidermal cells responded to fungal attack. More pronounced quantitative differences between compatible and incompatible interactions occurred upon fungal invasion of the mesophyll. These differences in the number of responding cells were not reflected at the level of gene expression: the spatial and temporal activation patterns of two defense-related genes, encoding phenylalanine ammonia-lyase and pathogenesis-related protein 1, were similar in both types of interaction.Dedicated to Professor Peter Sitte, Freiburg, Germany, on the occasion of his 65th birthday     

Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

Full-Length and Truncated Alzheimer Amyloid Precursors in Chromaffin Granules: Solubilization of Membrane Amyloid Precursor Is Mediated by an Enzymatic Mechanism

Abstract: The amyloid β peptide (Aβ) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact Aβ sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37°C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca²⁺ and Zn²⁺, and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of Aβ from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing Aβ. The detection of soluble APP with intact Aβ sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions.     

Experimental studies on the function of the cortical cytoplasmic zone of the preprophase microtubule band

Summary Centrifugation of young seedlings ofTriticum durum andTriticum aestivum for 8–10 hours at 1,500–2,000 x g causes a serious disorder of the spatial organelle relationships in the interphase as well as the preprophase and mitotic subsidiary cell mother cells (SMCs). The nucleus, most organelles and cytoplasm are displaced to the centrifugal end of the cell, while the vacuoles lie at the other end. However, after centrifugation, the preprophase microtubule bands (PMBs) are nucleated and remain at the expected position close to the guard cell mother cells (GMCs). In some elongated SMCs the PMBs become completely separated from the nucleus. The mitotic spindle exhibits variable orientation and is usually formed at some distance from the PMB cortical zone.Cytokinesis in SMCs is spatially highly disturbed and the cell plate shows a variety of unpredictable dispositions, which seem to be determined by: 1. the position of the preprophase-prophase nucleus and the orientation of the mitotic spindle as well as their spatial relationships to the PMB cortical zone, and 2. the space available for cell plate growth. Many of the daughter cells exhibit a highly variable shape and size in different planes. Usually one edge of the cell plate partly or totally joins the anticlinal parent wall adjacent to the PMB cortical zone.In some SMCs ofZea mays andTriticum aestivum, the junction regions of the periclinal walls with the anticlinal ones, lined by the PMB cortical zone in normal SMCs, are detectably thickened after the arrest of mitosis and the prevention of interphase microtubule formation by a prolonged colchicine treatment. In a small number of protodermal cells of the same plants, participating in the development of stomatal complexes, irregular wall bodies or incomplete wall sheets were formed at wall regions lined by the PMB cortical zone.The presented observations are in line with the following hypotheses: 1. the PMB cortical zone interacts with the growing edges of the cell plate attracting it to fuse with the underlying parent wall when the latter approaches the former at a critical distance, and 2. in SMCs particular regions of the PMB cortical zone and/or the adjacent plasmalemma promote the local wall deposition in the absence of microtubules.     

Histochemical studies on the oil-bodies ofMarchantia paleacea bert

Summary Correlated light and electron microscope histochemistry was performed on the oil bodies (OB) ofMarchantia paleacea. It was revealed that they contain significant quantities of material positive to PAS and periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reactions. Ag deposits were localized on the matrix surrounding the globules and on the distinct one close to the inner face of the OB membrane. In addition, the lipophilic globules were peripherally impregnated with staining product. Polysaccharidic material appears to be elaborated in dictyosomes and then advanced into the OB by their vesicles.The OB show a lipophilic character and are intensely stained with Sudan black B, with an aqueous solution of osmium tetroxide and with the Nadi reaction as modified byDavid andCarde (1964).The performance of mercury bromophenol blue, aniline blue black, coomassie brilliant blue R and acid fuchsin stainings showed that the OB do not contain structurally detectable proteins. Besides, a positive reaction of the inner OB was obtained in fresh tissue with a variety of tests sensitive to phenolic or other aromatic ring compounds. On the contrary, the epidermal and scale OB containing small globules are obviously stained only with Gibbs' reagent. The colours obtained with the above reactions, were almost the same,i.e. brown with a red shade, except for the blue one of the Gibbs' reagent.The presented observations favour the conclusion that the OB matrix does not contain proteins, but substances polysaccharidic in nature. Phenolic and possibly other aromatic compounds appear to be present at least in inner OB.     

Description of a new species,Globotruncana pseudoconica and remarks on its relation to Globotruncana conicaWhite and Globotruncana falsostuartiSigal

A new, strongly spiroconvex Globotruncana species,Globotruncana pseudoconica nov. sp., is described from the Maastrichtian of Tunisia. It is distinguished from Globotruncana conicaWhite in morphology and phylogeny. Some Maastrichtian, strongly spiroconvex specimens referred to as Globotruncana conicaWhite, are shown to be identical to Globotruncana pseudoconica nov. sp. The new species reveals close affinity with Globotruncana falsostuartiSigal from which it has evolved during the middle Lower Maastrichtian.     

Guanosine-5′-diphosphate-3′-diphosphate inhibits the in, vitro synthesis of β-lactamase from pBR322 DNA

The $\text{in}\text{,}\text{vitro}$ synthesis of β-lactamase directed by pBR322 DNA is inhibited by guanosine-5′-diphosphate-3′-diphosphate.