Chlorate-resistant rose cells: influx, efflux and reduction of [36Cl]-chlorate

Clones of Rosa damascena Mill. cv. Gloire de Guillan, selected for growth in solid medium containing 56 m M NaClO₃, were studied to determine the reason for their resistance to this toxic salt. The cells grew on medium containing nitrate as the only nitrogen source, and they synthesized nitrate reductase (EC 1.6.6.2) in the presence of nitrate. The cells were resistant in the presence of nitrate. However, their resistance was greatly increased by the presence of glutamate in the medium. The cells took up [³⁶Cl]-ClO₃- and reduced it to ClO₂⁻, but the fraction of ClO₃⁻ that they reduced under our experimental conditions was less than that reduced by wild type. The slower production of ClO₂⁻ apparently accounted for the resistance of the cells to ClO₃⁻. We suggest several possible reasons for the low rate of reduction of ClO₃⁻.     

Decreased endo-lysosomal acidification capacity in methylene diphosphonate-resistant mutants of Dictyostelium discoideum

Abstract The in vivo capacity for endo-lysosomal acidification has been monitored in Dictyostelium discoideum amoebae with acridine orange, a fluorescent weak base dye commonly used to probe transmembrane pH gradients. In the presence of aerobic amoebae, the initial rate of fluorescence quenching was found to be proportional to cell density between 5 × 10⁵ and 2.5 × 10⁶ cells ml⁻¹ and independent of acridine orange concentration in the 1.5 to 7.5 μM range. The dye response was sensitive to agents that perturb endo-lysosomal acidification such as NaN₃, nigericin or imidazole. Several mutant cell lines whose growth was resistant to methylene diphosphonate were found to be partially deficient in the acridine orange quenching test, suggesting that endo-lysosomal acidification was altered in these mutants.     

Effects of catalase on the accumulation of H2O2 in rice cells inoculated with rice blast fungus, Magnaporthe oryzae

Roles of H₂O₂ in the infection process of Magnaporthe oryzae on rice were investigated. In a leaf sheath assay for up to 48 h post-inoculation, the absence or presence of catalase in the conidia suspension was correlated with the level of accumulated H₂O₂ in infected leaf cells, as observed by staining with 3',3-diaminobenzidine tetrahydrochloride. In the incompatible interaction, the appearance of autofluorescence or frequency of cell death characterized by granulation (symptoms characteristic of hypersensitive responses) was not significantly affected by the presence of catalase in the conidia suspension. In the leaf blade assay, inoculation of compatible conidia in the presence of catalase produced more severe symptoms than that of conidia in the absence of catalase at 6 days post-inoculation. These results suggest that, in this host–parasite interaction, the primary role of host-produced H₂O₂ is in limiting hyphal growth after penetration through toxic action. Furthermore, in incompatible interactions, H₂O₂ is implied not to be a major mediator of hypersensitive cell death.     

Tributyltin-resistant Methanothermobacter thermautotrophicus mutant with mutational substitutions in the A1A0-ATP synthase operon

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to tributyltin chloride (TBT) was isolated. TBT, the inhibitor of the A₀ domain of A₁A₀-ATP synthase, inhibits methanogenesis in the wild-type cells; however, the TBT-resistant mutant exhibited methanogenesis even in the presence of 800 μM TBT. ATP synthesis driven by methanogenic electron transport was markedly diminished in the mutant strain. While TBT profoundly inhibited ATP synthesis driven by methanogenic electron transport in the wild type, only a slight inhibition was observed in the mutant strain. These results suggested a modification in the ATP-synthesizing system of the mutant strain. The sequence of the complete A₁A₀-ATP synthase operon ( Mth952 – Mth961 ) in the wild-type and mutant strains was determined and compared. Three mutations leading to amino acid substitutions in two A₁A₀-ATP synthase subunits were identified – Val₃₃₈Ala in subunit A and Leu₂₅₂Ile and Ser₂₉₃Ala in subunit B. Moreover, this study revealed the differential expression of several proteins that may contribute to TBT resistance. The results imply that change of TBT sensitivities of TBT-resistant mutant is due to mutational substitutions in the A₁A₀-ATP synthase operon.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

The F1FO ATP synthase genes in Methanosarcina acetivorans are dispensable for growth and ATP synthesis

There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A₁A_O together with the bacterial F₁F_O ATP synthase. To address a potential role of the bacterial F₁F_O ATP synthase, we have exchanged the F₁F_O ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F₁F_O ATP synthase is dispensable for the growth of M. acetivorans .     

Effects of Extracellular ATP on Fe2+-Induced Cytotoxicity in PC-12 Cells

Abstract: Extracellular ATP is known to cause a variety of changes, including the alteration of ion fluxes, cell growth, and other physiological activities. Recently, it has been suggested that ATP acts as an excitatory synaptic transmitter, which may produce a Ca²⁺ influx via the activation of a P_2y purinoceptor. Rat pheochromocytoma (PC-12) cells are known to resemble rat sensory neurons and to possess a P_2y purinoceptor. In this study, we demonstrated that extracellular ATP dose-dependently increased PC-12 cell death in the presence of ferrous ions. Voltage-sensitive calcium channel blockers and calpain and xanthine oxidase inhibitors were found to be effective at protecting PC-12 cells from Fe²⁺/ATP-induced lipid peroxidation and cell death. These results suggest that xanthine oxidase activation induced by calpains and subsequent free radical formation may be responsible for Fe²⁺/ATP-induced neuronal cell death.     

Genetic study of resistance to inhibitory effects of UV radiation in rice (Oryza sativa)

Genetic analysis of resistance to the inhibitory effects of UV radiation on growth of rice ( Oryza sativa L.) cultivars was carried out. Some experimental plants were grown in visible radiation supplemented with UV radiation containing a large amount of UV-B and a small amount of UV-C in a phytotron, while others were grown without UV radiation. The degree of resistance to UV radiation was estimated in terms of the degree of reduction caused by supplemental UV radiation in the fresh weight of the aboveground plant parts and the chlorophyll content per unit fresh weight. Fresh weight and chlorophyll content in F₂ plants generated by reciprocally crossing cv. Sasanishiki, a cultivar more resistant to UV radiation, and Norin 1, a cultivar less resistant to such radiation exhibited a normal frequency distribution. The heritabilities of these two properties in F₂ plants were low under conditions of non-supplemental UV radiation. Under elevated UV radiation, the F₂ population shifted to the lower range of fresh weight and chlorophyll content, and the means were close to those of Norin 1. The heritabilities of these two properties were the same in the reciprocal crosses, indicating that maternal inheritance was not involved. Inheritance of chlorophyll content per unit fresh weight was further determined in F₃ lines generated by self-fertilizing F₂ plants of Sasanishiki and Norin 1. The results showed that the F₃ population was segregated into three genotypes, namely, resistant homozygotes, segregated heterozygotes and sensitive homozygotes, with a ratio of 1:65:16.
It was thus evident that the resistance to the inhibitory effect of elevated UV radiation in these rice plants was controlled by recessive polygenes.     

Catalytic properties of Na+-translocating NADH:quinone oxidoreductases from Vibrio harveyi, Klebsiella pneumoniae, and Azotobacter vinelandii

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na⁺-NQRs) from the marine bacterium Vibrio harveyi , the enterobacterium Klebsiella pneumoniae , and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na⁺-NQR from A. vinelandii is not sensitive to low 2- n -heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na⁺-NQR from K. pneumoniae is fully resistant to either Ag⁺ or N-ethylmaleimide. All the Na⁺-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme.     

Mechanism of Cd2+ resistance in Euglena gracilis

The characteristics of Cd²⁺ accumulation by Euglena gracilis L. strain Z have been studied using sensitive and resistant cells. In both strains Cd²⁺ is mainly absorbed by a temperature- and light-dependent process. Resistance to Cd²⁺ is associated with a lower accumulation of Cd²⁺ and with a decreased affinity for Cd²⁺. Gel filtration on Sephadex G75 of the soluble fraction shows that resistance is not linked to an induction of metallothioneins.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

The Content of Alternative Oxidase of Euglena Mitochondria is Increased by Growth in the Presence of Cyanide and is not Cytochrome o.

ABSTRACT A study of the effect of respiratory inhibitors on O₂ uptake of Euglena gracilis mitochondria, isolated from cells grown in the presence of cyanide or with ethanol as carbon source, was undertaken. The contents of cytochrome c oxidase and alternative oxidase were also determined. Inhibition of respiration by antimycin and cyanide was only partial and it was dependent on the oxidizable substrate used. Succinate oxidation was the most sensitive to cyanide whereas lactate oxidation was the most resistant. Cell growth in the presence of cyanide or with ethanol as carbon source brought about an enhanced content of alternative oxidase without a concomitant increase in cytochrome aa₃ content. However, a correlation between cyanide-resistant respiration and alternative oxidase content was not found. Analysis of heme types in mitochondrial membranes revealed the absence of heme O. The data suggest the presence of an inducible alternative oxidase in Euglena mitochondria which has high resistance to cyanide and contains heme B. A close relationship between Euglena alternative oxidase and bacterial quinol oxidases containing B-type heme is proposed.     

Spiral growth and cell wall properties of the gibberellin-treated first internodes in the seedlings of a wheat cultivar tolerant to deep-sowing conditions

The Hong Mang Mai wheat cultivar is tolerant to deep-sowing conditions because it has an elongated first internode that is sensitive to gibberellin (GA₃). The cells in the GA-treated first internodes were approximately 4.2 mm long, twice as long as the untreated Hong Mang Mai first internode cells. The elongation of the first internode of Hong Mang Mai, particularly when treated with GA₃, was accompanied by remarkable spiral growth. In contrast, the first internodes of the GA-insensitive cultivar Norin 10 did not exhibit GA₃-induced elongation or spiral growth. The walls of the first internode cells of GA₃-treated Hong Mang Mai seedlings showed increased extensibility and higher (1→3), (1→4)- β - d -glucanase activity, autolysis and glucan contents than the cell walls of untreated Hong Mang Mai first internodes. The changes in the cell wall extensibility due to GA₃ treatment correlated strongly with the GA₃-induced changes in cell wall glucan content, autolysis, and glucanase activity. GA₃-treated Hong Mang Mai seedlings showed elevated expression of Glucanase EI gene in the first internode compared to GA₃-treated Norin 10. Thus, GA aids first internode elongation in Hong Mang Mai by enhancing glucan turnover and thus increasing cell wall loosening. The spiral growth of the first internode also helps the plant elongate against soil resistance, thereby promoting the deep-sowing tolerance of this cultivar.     

Heat injury and recovery of Streptococcus faecium associated with the souring of chub-packed luncheon meat

The presence of NaCl in the heating medium provided some protection from lethal heat damage for cells of a Streptococcus faecium strain isolated from luncheon meat whereas the presence of NaNO₂ either alone or in addition to NaCl, had no significant effect on cell survival. Subsequent recovery and growth of heat-damaged cells was retarded by the presence of NaCl. When NaNO₂ was present in addition to NaCl the inhibitory effect of the latter was reduced. These principal components of the luncheon-meat-cure are apparently opposed in their activities on post-heating recovery and growth of Strep. faecium . Product stability, i.e. duration of the lag before growth occurs, is directly related to the severity of the heat treatment and to the concentration of NaCl in the product. Therefore the resistance of pasteurized chub-packed luncheon meat to streptococcal spoilage during storage at temperatures conducive to microbial growth results from a prolonged heat-induced salt-maintained pre-growth adjustment phase rather than to any inherent inhibitory property of the luncheon meat to the growth of non-heat-damaged Strep. faecium cells.     

A novel growth stimulating activity from BRL-3A cell conditioned medium

The R⁻ cell line is a 3T3-like cell line originating from mouse embryos with a homozygous disruption of the type 1 insulin-like growth factor receptor (IGF-IR) genes. Although R⁻ cells cannot grow at all in serum-free medium (SFM) supplemented by several known growth factors, either singly or in combination, they are able to grow in 10% serum, albeit at a reduced rate. These findings suggested that serum contains an unknown, or unidentified, growth factor that can promote cell growth even in cells devoid of IGF-IRs. In an effort to identify such growth factor, we searched, using R⁻ cells, for a growth and DNA synthesis stimulating activity in SFM conditioned by different cell lines. We found that the BRL-3A cell line secreted an activity capable of stimulating DNA synthesis and cell proliferation in R⁻ cells. This activity (which is concentration-dependent) can be collected and concentrated by ultrafiltration, it is heat-labile, proteinase K-sensitive and has a size larger than 10 kDa. Because of the resistance of R⁻ cells to stimulation by known growth factors, we believe that this activity is due to a novel polypeptide secreted by BRL-3A cells. Further characterization of the active component(s) is in progress.     

Normal G1/S transition and prolonged S phase within one cell cycle after seeding cells in the presence of an ornithine decarboxylase inhibitor

Abstract. We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G₁/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine-flow cytometric method to more specifically study the G₁/S transition, the S phase length, and the progression of cells from S phase through G₂+ M and into G₁, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G₁/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G₁ phase occurred later than the effect on S phase progression. These results imply that the G₂+ M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.     

Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells

Abstract: We have expressed and biochemically characterized the human D2_long (D2_L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2_L receptors expressed in mammalian cell lines. Dopamine binding to D2_L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2_L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2_L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2_L receptor prelabeled with ³²P_i or [³H]-palmitate. These results constitute the first direct evidence for D2_L receptor phosphorylation and palmitoylation.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.