Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

Number, activity and thermostability of the electrophoretic forms of acid phosphatase in Amoeba proteus, cultured at different temperatures

In free-living amoebae (Amoeba proteus, strain B), cultured at 10 and 25 degrees C, we compared the number, activity, and thermostability of separate electromorphs of Triton-soluble acid phosphatase (AcP) revealed by disc-electrophoresis in polyacrylamide gel using 2-naphthyl phosphate (pH 4.0) as a substrate. No differences in the number of AcP electromorphs and their mobility were observed at both these temperatures. The total activity of AcP electromorphas per unit of cellular protein and their total thermostability were lower in amoebae acclimated to 10 degrees C than to 25 degrees C. The above decrease may be a consequence of a simultaneous decrease in the activity and thermostability of two tartrate-sensitive electromorphs, both being of lysosomal nature. The total activity and thermostability of tartrate-resistant AcP electromorphs did not differ in amoebae acclimated to the two above temperatures. In amoebae cultured at 10 degrees C the fall of activity and thermostability of lysosomal AcP correlates with the decrease in their primary cell thermoresistance and phagocytic activity. The obtained results confirm the earlier conclusion (Vysotskaya et al., 1994) that lysosomes may be involved in acclimation of electrothermal animals to changing environmental temperatures.     

Activity and thermal stability of acid phosphatase in homogenates of Amoeba proteus, acclimated to various temperatures

Activity and thermoresistance of acid phosphatase were determined in supernatant of Amoeba proteus homogenates using 1-naphthyl phosphate (pH 4.0) and p-nitrophenyl phosphate (pH 5.5). Although tartrate-resistant and tartrate-sensitive acid phosphatases hydrolyse both substrates, the former mainly hydrolyses p-nitrophenyl phosphate and the latter 1-naphthyl phosphate. A decrease in the activity of the total and tartrate-sensitive acid phosphatases, when using 1-naphthyl phosphate, and of the total and tartrate-resistant acid phosphatases, when using p-nitrophenyl phosphate, was found in amoebae acclimated to 10 degrees C (10 degrees-amoebae) compared to those acclimated to 25 degrees C (25 degrees-amoebae). Using 1-naphthyl phosphate, the thermoresistance of the total acid phosphatase was lower in 10 degrees-amoebae than in 25 degrees-amoebae, but the thermostability of tartrate-resistant enzyme was the same in both groups of amoebae. Using p-nitrophenyl phosphate, the thermoresistance of the total and tartrate-resistant acid phosphatases was lower (the latter only slightly) in 10 degrees-amoebae than in 25 degrees-amoebae. It is suggested that at least with the use of 1-naphthyl phosphate a decrease in thermostability of the total acid phosphatase may be due to a decrease in thermoresistance of tartrate-sensitive enzyme. The results obtained confirm the author's previous data on the activity and thermostability of electrophoretic forms of acid phosphatase using 2-naphthyl phosphate in 10- and 25 degrees-amoebae (Sopina, 2001). It is the first case of discovering a correlation between changes in primary cell thermoresistance of amoebae cultured at different temperatures and changes in the activity and thermostability of acid phosphatase in their homogenates, with the number of electrophoretic forms of this enzyme and their mobility being permanent.     

Intraspecific polymorphism of glucose-6-phosphate dehydrogenase in Amoeba proteus

Seven Amoeba proteus strains of different origin were studied using a polyacrylamide gel electrophoresis micromethod in the presence of NADP+ in cathode buffer. The strains examined differ in glucose-6-phosphate dehydrogenase (G6PD) zymograms: the majority of them (A, L, F, Da and Bk) display 3 bands, whereas strains B and C show 4 and 2 bands, respectively. All but strain C have a common band of high relative mobility. Strain B shows two fast migrating bands instead of a single band typical of strains A, L, F, Da and Bk. The results obtained testify to a certain intraspecific polymorphism of electrophoretic G6PD patterns in A. proteus. These difference between strains may be used as genetic markers in the nuclear transfer experiments.     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at pH 9.0

In the free-living ameba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called “fast,” “intermediate,” and “slow” phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH values. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be either acid phosphatase, or protein tyrosine phosphatase. Based on data of inhibitor analysis, broad substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, and another localization in the ameba cell than of the fast and intermediate phosphatases, it is concluded that only the slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.