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.     

Interaction of intracellular signalling and metabolic pathways at inhibition of mitochondrial aconitase by fluoroacetate

Mitochondrial aconitase has been shown to be inactivated under the effects of many compounds and critical states. Fluoroacetate (FA) is the best-known aconitase-inhibiting toxic agent. The biochemistry of the toxic action of FA has been rather well studied; however, no effective therapy has been developed over the past six decades. To search for new approaches to the development of possible antidotes, experiments were carried out in vitro with rat liver mitochondria, Ehrlich ascite tumor (EAT) cells, and cardiomyocytes exposed to FA or fluorocitrate (FC). FA produced its effects at much higher concentrations as compared with FC; in experiments with mitochondria these effects depended on respiratory substrates: with pyruvate, FA induced a slow oxidation and/or a leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides (PN) was prevented by the incubation of mitochondria with cyclosporin A. Studies of the PN level and dynamics of Ca²⁺ in EAT cells during activation by ATP also revealed the PN leak from mitochondria, which led to a shift in the balance of mitochondrial and cytosolic NAD(P)H under action of FA. Moreover, an increase of cytosolic Ca²⁺ was revealed in the cells exposed to FA, which could be explained by the activation of plasma membrane calcium channels. This mechanism could affect the amplitude and rate of calcium waves in cardiomyocytes under the effects of FA. We emphasize the reciprocal relationship between intracellular PN dynamics and calcium balance and discuss possible pathways of metabolic modulation in the context of development of effective therapy of poisoning with FA and other aconitase inhibitors.     

The calmodulin inhibitor R24571 induces a short-term Ca2+ entry and a pulse-like secretion of ATP in Ehrlich ascites tumor cells

The properties of the Ca2+ channel induced by a calmodulin inhibitor in Ehrlich ascites tumor cells were investigated using fluorescent indicators Indo-1 and chlortetracycline. The inhibitor of calmodulin calmidazolium (R24571) in concentrations of 1-2 microM induces a short-term Ca2+ entry and a pulse-like ATP secretion. Repeated addition of R24571 also causes a transient Ca2+ signal. Ca2+ channels induced by R24571 are permeable for Mn2+. Ca2+ entry does not depend on endoplasmic reticulum depletion by thapsigargin, ATP, or ionomycin and is suppressed by nordihydroguaretic acid (EC50 = 6.7 microM), quercetin (EC50 = 1.5 microM), dihydroquercetin (EC50 = 17 microM), arachidonic acid (AA) (EC50 = 8.6 microM), and suramin (EC50 = 0.25 +/- 0.05 MM), and weakly depends on temperature in the range of 18 - 37 degrees C. The apparent activation constant for R24571 and the Hill coefficient are 2.5 +/- 0.2 and 4 +/- 0.3 microM, respectively. The products of arachidonic acid oxidation are neither activators nor inhibitors of these channels. The inhibitory effect of nordihydroguaretic acid is indirect and is conceivably caused by the accumulation of arachidonic acid due to suppression of its lipoxygenase-catalyzed oxidation at phospholipase A2 activation. The maximal level of about 1.3 microM in the dependence of Ca2+ signal amplitude on R24571 concentration points to possible inhibition of the channel by increased Ca2+ concentration in the cytosol. The weak dependence on temperature implies that the channel is highly permeable, the chain of enzymic processes is not involved in Ca2+ entry activation, and the mutual compensation of processes with opposite contributions is possible. Using chlortetracycline fluorescence, we have shown in model experiments on calmodulin solution that Ca2+ induces cooperatively a conformational transition of calmodulin with the exposure of a hydrophobic chlortetracycline-Ca(2+)-binding site. The interaction of R24571 with the CaM-Ca2+ complex results in quenching of fluorescence to its level in water, which is interpreted as the elimination of the availability of calmodulin hydrophobic site for chlortetracycline-Ca+. Nordihydroguaretic acid, quercetin, and dihydroquercetin, but not suramin, also interact with calmodulin, but this does not result in the complete closing of its hydrophobic site. It is supposed that the activation of the Ca2+ channel occurs owing to the activation of calmodulin-dependent phospholipase A2 by R24571, which leads to the formation of a low-molecular short-lived secondary messenger, or because of the interaction of R24571 with calmodulin, which directly inhibits the channel. The termination of Ca2+ entry is probably due to the inhibition of phospholipase A2 and/or of the channel at increased concentrations of arachidonic acid and Ca2+.     

Release of mtDNA from mitochondria and activation of its replication in tissues of irradiated mice

A nessessary condition for normal functioning of mitochondria is the maintenance of certain numbers of intact mtDNA molecules. In the present study, we investigasted changes in the number of mtDNA copies in brain and spleen cells of mice subjected to irradiation. For the first time, we observed the irradiation-induced output of mtDNA fragments into brain and spleen cell cytosol. In the cytosol of these cells, examined in mice 5 h after 5 Gy irradiation, 1841 h.p. mtDNA fragments were detected able to persist for at 3 weeks. In addition, larger fragments of mtDNA (10,090 b.p.) were detected in the cytosol of brain cells of irradiated mice. The occurrence of mtDNA fragments in the cytosol of brain cells is accompanied with an increase in the number of mtDNA copies in the mitochondrial matrix. The induction of mtDNA replication in brain cells of irradiated animals may be considered as a compensatory reaction in response to mtDNA damage. A sharp decrease in the amount of mtDNA copies in the mitochondrial matrix of spleen cells on the first day after irradiation may be considered as apoptosis development. However, the compensatory reaction in brain cells was also noticed but in later terms.