UV-B and heat shock-induced changes in the wild type and UV-B heat shock-tolerant (UV-HSt) strain of the unicellular cyanobacterium Anacystis nidulans

Ultraviolet-B and heat shock (HS)¹) induced changes in growth kinetics, NO^?₃ reductase (NR), glutamine synthetase (GS), NO^?₃ uptake and NO^?₂ efflux activities, have been studied in the wild type Anacystis nidulans and its UV-HS¹ strain. The application of UV-B and HS stresses either used separately or in combination shows a drastic changes in growth rates of wild type, and insignificant effect on the growth of the UV-HS^t strain. The wild type cells, in contrast to its UV-HS^t strain exhibits insignificant effect on NR and GS activities upon UV-B radiation followed by heat shock treatment. Similar treatments to the wild type cells resulted in maximum reduction of NR and GS activities. The NO^?₃ uptake and NO^?₂ efflux activities are found to be lower in the UV-HS^t strain than in the wild type counterpart and both the systems consisted of an initial rapid phase followed by a slower one. NH⁺₄ grown cells when transferred to NO^?₃ medium in presence of streptomycin showed significant inhibition in the development of both the NO^?₃ uptake and NO^?₂ efflux systems indicating that de novo protein synthesis is required for the development of NO^?₃ uptake and NO^?₂ efflux systems. Whereas the same cells in the presence of L-methionine-DL-sulfoximine (MSX) showed marginally higher NO^?₃ uptake but, exhibits only 42% NO^?₂ efflux to that of MSX-devoid both the cells. It is suggested that NH⁺₄ assimilation via GS is necessarily required for NO^?₂ efflux system to be functional.     

Phage-induced development of a site-specific endonuclease in Anacystis nidulans, a cyanobacterium

Anacystis nidulans infected by cyanophage AS-1 produces a site-specific endonuclease cleaving double-stranded DNA, as judged from the gel electrophoretic analysis of the reaction products and the determination of the terminal nucleotide at the 5'-end of the fragments. The enzyme was purified to near electrophoretic homogeneity. It has a molecular weight of 40,000 +/- 4,000. Only Mg2+ ions are required for enzyme activity. Limit digestion was not obtained even after extensive digestion of substrate DNA with large amounts of the purified enzyme. This suggests that the endonuclease splits the substrate at more than one nucleotide sequence with a different efficiency for each sequence. The following observations suggest that the endonuclease takes part in the breakdown of host DNA in the infected cell: purified host DNA is degraded by, while cyanophage AS-1 DNA is protected against, the enzyme, and the appearance of the endonuclease during the lytic cycle coincides with the onset of intensive degradation of host DNA.     

Mutagenic action of methyl methane sulphonate on the bluegreen alga Anacystis nidulans

Growth of the blue-green alga Anacystis nidulans treated with methyl methane sulphonate (MMS) at pH 5 or 6 was better than that at pH 7 or 8, both in regard to the duration of the lag-phase and the growth rate. Toxicity of MMS was directly proportional to the temperature at which the cells had been treated. Growth of cells incubated at 5000 lux during MMS treatment was better than those that had been treated in the dark or at 15000 lux. Ten successive treatments with MMS were ineffective in significantly shortening the lagphase of treated cells. The frequency of ultraviolet-resistant mutants but not of streptomycin-resistant mutants was substantially increased following single treatment with MMS of 40-60 minute duration; treatments of longer duration resulted in a steep fall in the number of mutants suggesting thereby that treatments of up to 60 min induce genetic changes which are largely unaccompanied by physiological damage.     

Degradation of Anacystis nidulans cells by the action of cyanophage AS-1

The sequence of degradation of the photosynthetic apparatus of Anacystia nidulans after virus infection was studied using different methods. In the first stages of pathogenesis changes in the structure and functional activity of the photosynthetic apparatus were shown to be insignificant. At the stages of marked degradation the system of oxygen release and light collecting complex are destroyed gradually. The reaction center of photosystem I is the most resistant to degeneration.     

Light-induced oscillations of the glutamate pool size and of the respiratory oxygen uptake in the blue-green alga Anacystis nidulans

The glutamate and glutamine pool contains about 75% of the free amino acids of Anacystis nidulans harvested from a growing culture. Light pulses induced oscillations of the rate of oxygen uptake and of the size of the glutamate pool under certain physiological conditions. The frequencies of these oscillations were similar. During the oscillation the size of the glutamate pool was transiently reduced to about 50% of the dark level. The pool size of 2-oxoglutarate did not show similar oscillations. A phase plane plot of the glutamate against the respiration oscillation supported a connection of both oscillations.     

Ca2+ transport in digitonin-permeabilized trypanosomatids

The use of digitonin to permeabilize Leishmania mexicana mexicana, Leishmania agamae, and Crithidia fasciculata plasma membranes enabled us to study Ca2+ transport in situ. The present results show that the mitochondria of these trypanosomatids are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by inorganic phosphate. Large amounts of Ca2+ were retained by C. fasciculata mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butylhydroperoxide and diamide. In contrast, Ca2+ was not retained in the matrix of Leishmania sp. mitochondria for long periods of time. In addition to the mitochondrial Ca2+ uptake, a vanadate-sensitive Ca2(+)-transporting system was also detectable in these trypanosomatids.     

Formation,growth and regeneration of incomplete cell wall in spheroplasts of the blue-green alga Anacystis nidulans in liquid media

Growing cells of blue-green alga, Anacystis nidulans, were treated with lysozyme in osmotically stabilized nutrient medium. Their development and regeneration ability was studied in liquid media. During 5–8 hrs about 95% of cells were converted to osmotically sensitive spheroplasts having a residual wall layer on their surface. The highest viability of spheroplast population was achieved using egg-white lysozyme for spheroplast preparation and gradual dilution by nutrient medium instead of centrifugation for lysozyme removal. Most of such prepared spheroplasts survived for 3 days of cultivation in nutrient medium, and started to die gradually from 3 to 10 days. Spheroplasts were growing spherically, about 30% of them were growing for 8 days up to 40-fold increase in volume. Multiplication of thylakoid cisternae in growing spheroplasts was proved by freezeetching. Electron microscopy further revealed newly synthesized amorphous walls of isodiametrical appearance by growing spheroplasts resembling the original cell walls of cells. In liquid media blue-green alga spheroplasts of Anacystis nidulans can grow for a number of days, however, only regeneration of incomplete wall occur unable to promote reversion of spheroplasts to cells.     

Structural variations of DNA + Mn2+ complexes during helix-coil transition

The helix-coil transition in Phage T2 DNA in the presence of 6,4 · 10^?3 mol/l Mn²⁺ is studied using light scattering and UV spectroscopy. The transition range is about 0,5°C. Near the temperature of the end of melting T_f the molecular weight M_w and the radius of gyration R_z of the complex are observed to decrease to about one half. At a temperature 0,1–0,25°C higher than T_f, M_w and R_z pass through a minimum, which implies that aggregation is preceded by unwinding of DNA strands. Thus, rise in temperature rather than Mn²⁺ -induced aggregation causes DNA + Mn²⁺ melting.     

Increased Cd2+ biosorption capability of Aspergillus nidulans elicited by crpA deletion

The P-type ATPase CrpA is an important Cu²⁺/Cd²⁺ pump in the Aspergilli, significantly contributing to the heavy metal stress tolerance of these ascomycetous fungi. As expected, the deletion of crpA resulted in Cu²⁺/Cd²⁺-sensitive phenotypes in Aspergillus nidulans on stress agar plates inoculated with conidia. Nevertheless, paradoxical growth stimulations were observed with the ΔcrpA strain in both standard Cu²⁺ stress agar plate experiments and cellophane colony harvest (CCH) cultures, when exposed to Cd²⁺. These observations reflect efficient compensatory mechanisms for the loss of CrpA operating under these experimental conditions. It is remarkable that the ΔcrpA strain showed a 2.7 times higher Cd biosorption capacity in CCH cultures, which may facilitate the development of new, fungal biomass-based bioremediation technologies to extract harmful Cd²⁺ ions from the environment. The nullification of crpA also significantly changed the spatial distribution of Cu and Cd in CCH cultures, as demonstrated by the combined particle-induced X-ray emission and scanning transmission ion microscopy technique. Most important, the centers of gravity for Cu and Cd accumulations of the ΔcrpA colonies shifted toward the older regions as compared with wild-type surface cultures.     

Peroxynitrite affects Ca2+ transport in Trypanosoma cruzi

Macrophages play an important role against Trypanosoma cruzi infection, via superoxide, nitric oxide, and peroxynitrite production. Peroxynitrite has been shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. Calcium is involved in many vital functions of the parasites, being its intracellular concentration governed by several transport systems, involving mitochondrial and non-mitochondrial compartments. In this paper, we report the effect of peroxynitrite on the calcium uptake systems, as studied by digitonin-permeabilized trypanosomes in the presence of arsenazo III. Peroxynitrite, at biologically relevant concentrations produced within phagosomes (250-750 microM), inhibited calcium uptake in a dose-dependent manner. Peroxynitrite decreased the mitochondrial membrane potential obtained in the presence of tetramethyl-p-phenylenediamine (TMPD)/ascorbate. In addition, a decrease of the non-mitochondrial Ca(2+)-uptake, concomitant with the inactivation of a Ca(2+)-dependent ATPase activity, was observed. HPLC analyses of the cellular adenine nucleotide pool showed a time-dependent decrease of ATP content and energy charge of the parasite; however this drop in ATP levels was significantly delayed with respect to decrease of the ATP-dependent Ca(2+)-transport. We conclude that the disruption of calcium homeostasis by peroxynitrite may contribute to the observed cytotoxic effects of macrophages against T. cruzi.     

Characterization of temperature-induced changes in the photosynthetic properties of Anacystis nidulans grown at elevated temperature: A differential response to heat shock

Photosynthetic properties of high temperature (38 °C) grown (HTG)¹ and low temperature (25 °C) grown (LTG) cells of Anacystis were examined. The results revealed several characteristic changes in the HTG cells which included light absorption property of the pigments, DCMU binding affinity of the photosynthetic apparatus and photosynthetic yield. There was a time course dependent heat shock (47 ± 1 °C) induced inhibition of the O₂ evolution in both the types of cells. However, the response of the HTG cells was sluggish as compared to the LTG cells. Unlike the LTG cells, the heat shock treatment (47 ± 1 °C, 5 min) of the HTG cells did not result into a change in the pigment absorption spectra. The rise kinetics of fluorescence induction curve of chlorophyll a in the presence of DCMU and DCMU + NH₂OH suggested for heat-induced damage to the oxidizing side of photosystem II in both the HTG and LTG cells. Further, results on the Hill activity in the heat shocked HTG cells showed that the exogenous electron donors DPC and NH₂OH not only retrieved the heat shock induced inactivation of the Hill activity, but stimulated the rate of activity. On the other hand, the rate of Hill activity in the heat shocked LTG cells was restored by only NH₂OH an electron donor to PS II reaction centre, while DPC-an electron donor to water oxidation complex was ineffective against the heat shock induced inactivation of the electron transport. These results, together, suggested for the presence of less number of heat susceptible sites in the HTG cells than that in the LTG cells, which might be accounting for the high degree of thermostability to the former type of cells.     

Dnases of cell nuclei: Mn2+-dependent endonuclease

Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 10, pp 370–372, October, 1990.     

兔膈肌肌浆网Ca2+-ATPase活性及钙离子的转运

本文分别用定磷法测定兔膈肌SR Ca 2 -APTase 活性、Fura-2 荧光法测定SR Ca 2 释放、摄取动力学和[ 3 H] -Ryanodine 与RyR 结合实验测定SR RyR 的量,分析其功能特性。 结果显示兔隔肌、心肌和骨骼肌SR Ca 2 -APTase 活性分别为70.13 ±8.25、 41.25 ±6.25 和120.17± 17.03 m mol/L pi/mg 蛋白/h1 。膈肌的SRCa 2 -APTase 活性显著高于心肌P<0.01 。但明显低于骨骼肌P<0.01; 膈肌SR Ca 2 释放量和摄取速度显著快于心肌（P<0.01）,但明显低于骨骼肌（P<0.01）;膈肌SR RyR 同[ 3 H] Ryanodine 的最大结合值（Bmax）是0.78 ±0.05pmol/mg 蛋白,其解离常数（KD）是6.93 1.13nmol/L,分别位于心肌和骨骼肌范围内。本文认为膈肌Ca 2 释放单位、SR Ca 2 -APTase 和SR Ca 2 释放摄取动力学分别具有心肌和骨骼肌的一些特征,其Ca 2 释放可能具有变构偶联和CICR 偶联两种形式,心肌型DHPR 亚型,RyR 3 和SERCA 2 a 的存在可能是膈肌ECC 依赖于细胞外Ca 2 的主要原因。     

A novel Mn2+-oxidizing enzyme system in a freshwater bacterium

Manganese oxidation by cell suspensions and cell extracts of a freshwater bacterium, designated strain FMn 1, was investigated. Manganese appeared to be oxidized in the periplasmic space. A conventional, membrane-bound electron transport system was not utilized. An enzyme or enzyme complex and a cofactor, each of different molecular size, were located in different parts of the cell envelope. Results suggest that the cofactor reacts with manganese in the periplasmic space and that in the presence of oxygen it is reoxidized by the reoxidized by the enzyme. The enzyme is probably loosely bound to the membrane. A combination of enzyme and cofactor in a crude preparation exhibited a pH optimum at around 7.0. The enzyme exhibited a temperature optimum at around 30 °C. No temperature optimum was found for the cofactor. The enzyme was heat-labile and inhibited by mercuric chloride and para-chloromercuribenzoate. The cofactor was heat-stable and could oxidize manganese under anaerobic conditions. The enzyme system appears to be different from others so far described.     

Depression of monosynaptic excitatory postsynaptic potentials by Mn2+ and Co2+ in cat spinal cord.

In cats anesthetized with allobarbitone-urethane, Mn²⁺ and Co²⁺ (and occasionally La³⁺) were released extracellularly from micropipettes while recordings were made of monosynaptic excitatory postsynaptic potentials evoked in lumbosacral motoneurones by Ia afferent stimulation. These excitatory postsynaptic potentials consistently showed a marked depression in their rate of rise (by an average of 44%) and an increase in half-amplitude duration (by an average of 50%). There was a less pronounced reduction in peak amplitude and increase in time-to-peak. After applications of Mn²⁺ or Co²⁺, presynaptic potentials recorded in motoneurones showed no sign of any depression but the synaptic delay was clearly increased.It is concluded that the monosynaptic excitatory postsynaptic potential evoked by Ia afferents in cat motoneurones is probably mediated by chemical transmission.     

Mitochondrial Ca2+ transport in frog early distal tubule

A global and transient rise of intracellular Ca2+ (Ca2+i) is central to the operation of pump-leak coupling in the frog early distal tubule (EDT). The endoplasmic reticulum (ER) is the site of this Ca2+ release and reuptake; however, it is likely that other intracellular pools, such as mitochondria, also contribute to cellular Ca2+ homeostasis. The present study was performed to seek evidence of mitochondrial Ca2+ transport in the frog EDT. Experiments were performed on isolated and permeabilized EDT segments from the frog kidney loaded with the low-affinity, Ca2+-sensitive fluorescent indicator, mag-fura-2. Ca2+ uptake in the absence of SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) activity (inhibition by 2,5-di-t-butyl hydroquinone, TBQ) was evident at a bath [Ca2+] of 1 microm, but not at 200 nm, in the presence of ATP. This uptake was sensitive to the protonophore FCCP and the ATP-synthase inhibitor oligomycin. Ca2+ uptake was also stimulated by respiratory substrates; this uptake was enhanced by oligomycin and reversed by the application of FCCP. These findings provide the first evidence of mitochondrial Ca2+ transport in renal tubules, which appears to occur via a low-affinity pathway and which will act as a physiological Ca2+ buffer, protecting the cell from large increases in Ca2+i.     

Ca2+ transport in diluting segment of frog kidney

In the isolated-perfused frog (Rana pipiens) kidney the question of whether transepithelial transport of Ca²⁺ is a passive voltage driven process or involves active mechanisms was investigated. With conventional and ion-sensitive microelectrodes transepithelial electrical and electrochemical potential differences were measured. Luminal activities and transepithelial net fluxes of Ca²⁺ and Cl^– were evaluated. Different transepithelial electrical voltages in a wide range (+20 to–4 mV) were generated by chemical voltage clamping and the dependence of Ca²⁺ net fluxes on these voltages investigated. The hormonal control of both Cl^– and Ca²⁺ transport was studied by evaluating the effect of the cell-permeable cAMP analogue, db-cAMP and of the adenylate cyclase stimulator, forskolin. The experiments reveal that: (a) Ca²⁺ is reabsorbed along the diluting segment of frog kidney. (b) Ca²⁺ reabsorption is inhibited by furosemide because of the elimination of the transepithelial voltage. (c) There is a direct relationship between transepithelial voltage and Ca²⁺ reabsorption. (d) Neither Cl^– nor Ca²⁺ reabsorption are affected by db-cAMP or forskolin.We conclude that Ca²⁺ reabsorption is passive, driven by the lumen-positive transepithelial voltage. It most likely occurs via the paracellular shunt pathway.