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.     

Induction of cytogenetic damage by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenylacetic acid in human lymphocytes

The effect of modified steroids, containing alkylating agents, on SCE rates and on cell kinetics in cultured human lymphocytes was studied. The homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenylacetic acid (ASE) was found to be the most effective in causing markedly increased SCE rates and cell division delays. The androsterone ester of p-bis(2-chloroethyl)aminophenylacetic acid (AE-CAPA) was found to be next in order of effectiveness with the lactone ester (LE-CAPA), chlorambucil ester 3 beta-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam (CBC-HAAL) and chlorambucil (CBC) following. p-Bis(2-chloroethyl)aminophenylacetic acid (CAPA) had only a small effect and 3 beta-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam (HAAL) had no effect at all. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumor activity of these drugs was observed.     

Direct measurement of static chest wall compliance in animal and human neonates

The measurement of pulmonary mechanics has been developed extensively for adults, and these techniques have been applied directly to neonates and infants. However, the compliant chest wall of the infant frequently predisposes to chest wall distortion, especially when there is a low dynamic lung compliance (CL,dyn). We describe a technique of directly measuring the static chest wall compliance (Cw,st), developed initially in the newborn lamb and subsequently applied to the premature neonate with chest wall distortion. The mean CL,dyn in seven intubated newborn lambs in normoxia was 2.45 +/- 0.41 ml.cmH2O-1.kg-1, whereas Cw,st was 11.81 +/- 0.25 ml.cmH2O-1.kg-1. These values did not change significantly in seven animals breathing through a tight-fitting face mask or with hypercapnia-induced tachypnea. For the eight premature infants the mean CL,dyn was 1.35 +/- 0.36 ml.cmH2O-1.kg-1, whereas the mean Cw,st was 3.16 +/- 1.01 ml.cmH2O-1.kg-1. This study shows that, under relaxed conditions when measurements of static compliance are performed, the chest wall is more compliant than the lung. The measurement of Cw,st may thus be used to determine the contribution of the respiratory musculature in stabilizing the chest wall.     

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.     

Light-Induced Changes in the Fluorescence Yield of Chlorophyll a In Vivo: II. Chlorella pyrenoidosa

The long-term fluorescence induction in Chlorella pyrenoidosa consists of a fast rise of the fluorescence yield from the level S (of the first wave transient) to a maximum M, followed by slower decay to a terminal stationary level T. The maximum M is attained within 40 seconds from the onset of illumination while the decay to the terminal level T lasts for several minutes. The fluorescence rise (S → M) coincides with an increase in the rate of oxygen evolution, which, however, remains constant during the fluorescence decay (M → T). Poisons of photosynthesis 3, (3,4-dichlorophenyl)-1,1 dimethylurea (DCMU, o-phenathroline) inhibit the fluorescence induction, while uncouplers of photophosphorylation affect the fluorescence time course only when they function at an early stage of the coupling sequence e.g., carbonyl cyanide p-trifluoremethoxy phenylhydrazone, (FCCP, atabrin). Phosphorylation inhibitors affecting only the terminal esterification step (phlorizin) have little effect on the fluorescence kinetics. These results suggest that the fluorescence induction requires the operation of a phosphorylating electron transport and that it is possibly related to the light-induced structural changes which accompany photophosphorylation.     

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.     

A morphogen gradient model for pattern regulation. II. Time description of global morphogen formation and field compartmentalization

In a model for pattern regulation, use was made of local and global morphogens S and Sigma. Sigma is produced from the S-degradation and it is decomposed by first order kinetics while it diffuses along the field. We solve exactly the partial differential equation for the distribution of Sigma in one spatial dimension when its source S is monotonie (for simplicity, linear or generally a power function). Assuming that S and Sigma react reversibly with an allosteric protein P according to a sequential scheme, we derive the evolution in time of the field separation into compartments. At equilibrium the relative extent of each compartment is constant (for variable field size) and so pattern regulation is achieved.