Reazioni delle piante di Ricinus communis a incisioni sperimentali

Summary

The author has produced wounds in the stem of Ricinus communis, studing the regeneration and cicatrization processes.

The rapidity of the surface cicatrization is in relation with the water loss of the waunded cells.

Both in the stems woumded let free and in those in which an extra tissue has been introduced, the cicatrization processes tacke place. They only take place with different velocity. The healing process lead to the reconstruction of the cortical and the conducting tissues.

The whole reconstruction of the stele may occur, when the flow of the assimilated stuffs is slowed. Sothat it is probable that the regeneration depends more upon the tissue nutrition tham on a specifical properti of the tissues.     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes

Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This `coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO₂.     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.     

A new missense mutation (Cys297→Phe) of the low density lipoprotein receptor in Italian patients with familial hypercholesterolemia (FHTrieste)

During a survey of the mutations of the low density lipoprotein receptor (LDL-R) gene in Italian patients with familial hypercholesterolemia (FH), we identified a novel point mutation, that creates a new EcoRI site at the 5 end of exon 7, in a heterozygous FH subject (FH-100). The sequence of a cDNA fragment encompassing exon 7 showed the presence of a GT transversion in codon 297; this created a new EcoRI site and produced a missense mutation, leading to a Cys₂₉₇Phe substitution in repeat A of the epidermal growth factor (EGF) precursor homology domain of LDL-R. Since the substitution of Cys₂₉₇ disrupts the intracellular transport of the LDL-R protein, as previously demonstrated by site-directed mutagenesis, we suggest that this mutation is the cause of FH in the FH-100 proband. We screened the DNA of 303 Italian FH patients by amplification of exon 7 from genomic DNA followed by digestion with EcoRI or by Southern blotting. Two individuals (FH-64 and FH-127) were found to be carriers of the Cys₂₉₇Phe mutation. Restriction fragment length polymorphism analysis demonstrated that, in two kindreds (FH-64 and FH-100), the haplotype in linkage with the Cys₂₉₇Phe mutation was the same, suggesting the presence of a common ancestor. The Cys₂₉₇Phe mutation has been designated FH_Trieste after the name of the city in Northern Italy from which probands FH-100 and FH-127 originate.