Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.

The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same composition. This level of similarity fails to support the suggested gene fusion.     

The alteration of membrane proteins in human erythrocyte membranes induced by quinolinic acid, an endogenous neurotoxin. Correlation of effect with structure

Mixtures containing subfractions of human plasma high-density lipoproteins (HDL) and human lipoprotein-free plasma were incubated in vitro at 37 degrees C. Esterification of cholesterol was observed both in incubations containing HDL-subfraction 3 (HDL3) and in those containing HDL-subfraction 2 (HDL2). The implication that the lecithin: cholesterol acyltransferase in lipoprotein-free plasma may therefore interact with lipoproteins in both HDL subfractions was developed further by proposing a simple model in which the two HDL subfractions may compete for interactions with the enzyme. This model was described mathematically and tested in experiments in which a constant amount of the enzyme was incubated with a wide range of concentrations of HDL2 and HDL3 present either alone or in combination. The model was able to predict experimentally observed rates of cholesterol esterification with great accuracy. The best fit was obtained with a Vmax for HDL3 that was 2.4-4-times greater than that for HDL2 and values of the apparent Km for HDL3 free cholesterol and HDL2 free cholesterol of 43-60 nmol/ml and 167-391 nmol/ml, respectively. The model thus predicts that, at physiological concentrations of lipoproteins, HDL2 will function as a competitive inhibitor of the cholesterol esterification reaction by displacing lecithin: cholesterol acyltransferase from a more effective substrate, HDL3, to a less effective substrate, HDL2.     

Studies in cryoprotection II: Propylene glycol and glycerol

Propylene glycol (PG), glycerol (G), or a combination of both were introduced into and eluted from 116 rabbit kidneys. The function of the kidneys was then studied on an ex vivo shunt by determining bloodflow and creatinine clearance. The cryoprotective agents (CPAs) were introduced and eluted at the rate of about 30 mM/min in an albumin-Ringer's lactate perfusate. In this system, 1.75 to 2.25 M cryoprotectant could be introduced and removed with no demonstrable loss of function. Significant injury was incurred at and above 2.5 M concentrations. When a combination of PG and G (equal proportions) was utilized, both 2.5 and 2.75 M total concentrations were tolerated by the kidney, with no loss of function. These studies demonstrate that in this very rigorous test system, cryoprotectant-induced injury can be significantly diminished by combining two agents of low toxicity. This suggests that osmotic injury may be enhanced by specific toxicities of cryoprotective agents, which can be minimized by using combinations, such as was done in these studies. Also, it has been shown that PG is at least as low in toxicity in the rabbit kidney as is G.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Characterization of the amino acids of bovine fibrinogen involved in the fibrinogen-thrombin interaction of the blood clotting process. Comparison with the milk clotting process

Summary Bovine fibrinogen and the A and B chains of bovine fibrinogen have been subjected to chemical modification by a number of reagents and the effects of these procedures on the susceptibility of the proteins to thrombin hydrolysis is described. The reagents used were rose bengal (for photo-oxidation), 2-hydroxy-5-nitrobenzyl bromide, N-acetylimidazole, iodoacetic acid and diethyl pyrocarbonate. Evidence is presented which indicates that the tryptophan and tyrosine residues of fibrinogen are not involved to any great extent in the interaction of this protein with thrombin. Modification with iodoacetic acid suggests that methionine residues play a major role in such interactions, but the fibrinogen chains on which the important residues reside remain uncertain. The use of diethyl pyrocarbonate indicates the participation also of histidine in fibrinogen-thrombin interactions and that, whereas the histidine residues of the B chain are involved to a great extent, it appears that those of the Aa chain are not. The similarities which exist between the fibrinogen-thrombin and the -casein-chymosin systems are discussed.Abbreviations used DEP diethyl pyrocarbonate (ethoxyformic anhydride) - HNBB 2-hydroxy-5-nitrobenzyl bromide - N-Acl N-acetylimidazole - PTC phenylthiocarbamyl - PTH 3-phenyl-2-thiohydantoin.     

The influence of plant growth regulators on the growth of the embryonic axes of red- and far-red-treated lettuce seeds

The influence of several plant growth regulators on the growth of the embryonic axes from red- and far-red-(R- and FR-)treated lettuce (Lactuca sativa L., cv. Grand Rapids) seeds was examined; as shown previously, the water potential of the axes from R-treated seeds has been lowered by 3.5–5.6 bars compared to that in axes from FR-treated ones. Kinetin and abscisic acid (ABA), when included in the incubation medium, reduced the elongation of the axes whereas fusicoccin stimulated it; however, these effects were the same in axes of both R- and FR-treated seeds. In contrast, elongation of axes from FR-treated seeds was stimulated by gibberellic acid (GA₃, but elongation of axes from R-treated ones was not affected by this hormone. This latter result indicates that gibberellins may be involved in the phytochrome-mediated growth responses in lettuce axes.When the root caps of the embryos were removed prior to light treatment, R was still able to induce a water-potential decrease in the embryonic axes, indicating that at least a portion of the active Pfr resides in the axis and not the root cap.Abbreviations ABA abscisic acid - FR far red light - GA₃ gibberellic acid - PEG polyethylene glycol - Pfr far-red-absorbing form of phytochrome - R red light     

Radiation activation of respiration inBacillus cereus T spores

The effects of gamma-irradiation on glucose oxidase activity were studied inBacillus cereus T spores. Radiation was found to activate oxidase in native spores. This activation was greatly enhanced when irradiated spores were subsequently heat-activated. Although activation by heat alone was 1.6 times that by radiation alone, the combined treatment with both radiation and heating resulted in a 12-fold enhancement of oxidase activity over that obtainable by optimum heat activation. Activation by combined treatment of gamma-rays and heat depended on the presence of exchangeable calcium during heating but not during irradiation.