The Effect of Toxic Doses of Copper upon Respiration, Photosynthesis and Growth of Chlorella vulgaris

When cells of Chlorella vulgaris absorb copper under anaerobic conditions, subsequent respiration, photosynthesis and growth of the cells are all severely inhibited. This does not occur when the metal is absorbed under aerobic conditions. When, after aerobic absorption of copper, the cells are exposed to a period of anaerobiosis, respiratory inhibition is as profound as when the uptake is anaerobic. In this case, however, respiration must eventually recover, for growth is not affected so severely as it is when copper is taken up under anaerobic conditions. It is concluded that the extra copper absorbed under anaerobic conditions is directly or indirectly responsible for the greatly increased toxicity to growth, and that this copper is bound to sites not normally available under aerobic conditions. Some aspects of the apparently unique toxic effect of copper suggest that these extra sites are sulphydryl groups.     

Inhibition of respiration by sodium fluoride and monoiodoacetic acid in wheat leaves at different oxygen tensions and in the presence of methylene blue and L-cysteine

Six-day leaves of wheat (Triticum vulgare) were used to test the effect of oxygen, nitrogen and different mixtures of these gases, methylene blue and L-cysteine on the inhibition of respiration by sodium fluoride and monoiodoacetic acid using the Warburg manometric method. In an atmosphere of 100% oxygen total respiration was increased by an average of 45%, 10% oxygen led to a decrease in respiration by 20%. However, no simultaneous changes were found in the degree of inhibition by these enzyme poisons. On decreasing the oxygen content to 5% inhibition of respiration was almost doubled while at the same time respiration fell by an average of 23%. In nitrogen, the respiration rate varied around 10% and inhibition was almost 100%. The results showed that the action of enzyme poisons is only influenced by different oxygen content at low oxygen tension and is evidently associated with the dominance of a given energy system (glycolysis). On using methylene blue and L-cystein in concentrations of 10-3M a decrease in the relative inhibition of respiration by enzyme poisons was also found. The total respiration rate, however, was only raised by 6–8%. Since the change in the degree of inhibition on using these substances is caused by an increase in the participation of respiratory processes resistant to glycolytic inhibitors it can, in this case be assumed that there is an increase in the activity of the pentose cycle in such tissue.     

Terminal oxidation and the effects of zinc in prostate versus liver mitochondria

Although the total zinc content of cells generally approximates 0.2 mM, the cytosolic free zinc ion concentration is negligible (subnanomolar concentrations). However, all reported studies of effects of zinc on cellular respiration and terminal oxidation involved microM-mM levels of free zinc ions. Prostate cells and their mitochondria accumulate 3-10 fold more zinc than other mammalian cells. We considered that a cytosolic pool of mobile reactive low molecular weight zinc ligands could inhibit respiration and terminal oxidation. The effects of ZnLigands, especially ZnCitrate, versus free Zn++ ions on respiration and terminal oxidation were studied with prostate and liver mitochondria. ZnLigands were equally as effective as free Zn++ ions in the inhibition of respiration and terminal oxidation of both prostate and liver mitochondria, which supports our concept that zinc can be transferred from cytosolic donor ZnLigands directly to zinc-binding sites of terminal oxidation components. Also, the respiration and specific activities of terminal oxidation components of prostate mitochondria are 20-50% of liver mitochondria. Zinc inhibition and inherently low levels of electron transport components are likely major factors responsible for the low respiration that characterizes prostate cells.     

A LATENT PERIOD IN THE ACTION OF COPPER ON RESPIRATION

1. When copper chloride is allowed to act on Aspergillus niger there is at first a period during which there is no change in the rate of the production of carbon dioxide, following which the rate of respiration falls. The interval of no change is called the latent period. 2. When the copper is removed from the external solution before the end of the latent period this interval is prolonged. The rate of respiration then falls to a new level below the normal level. 3. Experiments on Nitella and on Valonia indicate that the copper penetrates the cell almost immediately. 4. The length of the latent period varies inversely as a constant power of the concentration of the copper. 5. These results are explained by assuming that the copper is made active in the respiration system by means of a reversible reaction. By using appropriate velocity constants the experimental curves can be duplicated by calculated curves.     

Blocking of human T lymphocyte activation by channel antagonists

It has been established that early events in lymphocyte activation involve a rise in intracellular Ca++ as well as changes in the flux of other ions. Although a Ca++ channel has been postulated to participate in the early Ca++ rise, its presence in lymphocytes remains controversial. Also although yet undetected, electrophysiological data suggest the presence of a Ca++ activated K+ channel on human peripheral blood lymphocytes (HPBL). Here we report on the effect of specific channel blockers as an approach to the identification of these channels on HPBL. At 40 nM nifedipine, an inhibitor of voltage-gated Ca++ channels, fully inhibits the PHA-promoted activation of HPBL. This effect is concentration dependent with a half maximum effect at approximately 10 nM and is demonstrable whether the drug is added at the same time as or up to 18 h after the addition of the mitogen. This inhibition of activation is not seen if the lymphocytes are activated using IL-2 instead of PHA. Charybdotoxin a toxin which blocks a Ca++ activated K+ channel of muscle cells also blocks to almost 100 per cent the PHA-induced activation of HPBL. This inhibition can be demonstrated regardless of whether the blocker is added together with or up to 4 h after PHA. As opposed to nifedipine charybdotoxin shows no effect if added 18 h after the initiation of the activation process. When nifedipine and charybdotoxin were tested on mice splenocytes we found that nifedipine fully inhibits the LPS-promoted activation of these cells while charybdotoxin has no effect on their activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and electron transfer specificity of copper proteins from the denitrifier "Achromobacter cycloclastes"

A blue copper protein (Mr 12,000) was purified from cells of "Achromobacter cycloclastes" grown as a denitrifier. When reduced, the blue copper protein transferred electrons to the copper protein nitrite reductase purified from the same cells, whereas a variety of cytochromes from denitrifiers failed to do so. Inclusion of a protease inhibitor, phenylmethylsulfonyl fluoride, in the buffers employed during preparation yielded purified blue copper protein with 18 more amino acid residues and two times more specific enzyme activity than other researchers have found.     

Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.     

Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii.

In Azotobacter vinelandii cells, the short-term inhibition of nitrogenase activity by NH4Cl was found to depend on several factors. The first factor is the dissolved oxygen concentration during the assay of nitrogenase. When cells are incubated with low concentrations of oxygen, nitrogenase activity is low and ammonia inhibits strongly. With more oxygen, nitrogenase activity increases. Cells incubated with an optimum amount of oxygen have maximum nitrogenase activity, and the extent of inhibition by ammonia is small. With higher amounts of oxygen, the nitrogenase activity of the cells is decreased and strongly inhibited by ammonia. The second factor found to be important for the inhibition of nitrogenase activity by NH4Cl was the pH of the medium. At a low pH, NH4+ inhibits more strongly than at a higher pH. The third factor that influenced the extent of ammonia inhibition was the respiration rate of the cells. When cells are grown with excess oxygen, the respiration rate of the cells is high and inhibition of nitrogenase activity by ammonia is small. Cells grown under oxygen-limited conditions have a low respiration rate and NH4Cl inhibition of nitrogenase activity is strong. Our results explain the contradictory reports described in the literature for the NH4Cl inhibition of nitrogenase in A. vinelandii.     

Effects of Fluoride on Mitochondrial Activity in Higher Plants

The effects of fluoride on respiration of plant tissue and mitochondria were investigated. Fumigation of young soybean plants (Glycine max Merr. cv. Hawkeye) with 9–12 μg × m^?3 HF caused a stimulation of respiration at about 2 days of treatment followed by inhibition 2 days later. Mitochondria isolated from the stimulated tissue had higher respiration rates, greater ATPase activity, and lower P/O ratios, while in mitochondria from inhibited tissue, all three were reduced. Treatment of etiolated soybean hypocotyl sections in Hoagland's solution containing KF for 3 to 10 h only resulted in inhibition of respiration. Mitochondria isolated from this tissue elicited increased respiration rates with malate as substrate and inhibited respiration with succinate. With both substrates respiratory control and ADP/O ratios were decreased. Direct treatment of mitochondria from the etiolated soybean hypocotyl tissue with fluoride resulted in inhibition of state 3 respiration and lower ADP/O ratios with the substrates succinate, malate, and NADH. Fluoride was also found to increase the amount of osmotically induced swelling and cause a more rapid leakage of protein with mitochondria isolated from etiolated corn shoots (Zea mays L. cv. Golden Cross Bantam). The results are discussed with respect to possible effects of fluoride on mitochondrial membranes.     

铜离子胁迫下玉米根边缘细胞数量及存活率

采用悬空培养及离体培养的方式,观察玉米的根边缘细胞在铜离子胁迫下发生的形态、数量及存活率变化情况,结果表明,在玉米根长为32mm的时候,边缘细胞的数量达到最大,为4125个,但其存活率此时最低。玉米的根边缘细胞随着铜离子浓度增加,边缘细胞的数量发生变化。当处理浓度为50umol·L-1时,边缘细胞的死细胞数量达到758个（存活率为24．80％）,随后铜离子处理浓度增加到100umol·L-1时,存活率降低了17．54％。玉米根边缘细胞的数量随着铜离子浓度的增加呈现出逐渐减少的趋势,而存活率逐渐降低,说明在重金属铜离子胁迫下,根边缘细胞的释放起到对根际区域的保护作用。     

Stabilization of ascorbate solution by chelating agents that block redox cycling of metal ions

Among the seven chelating agents tested, ethylenediamine di(o-hydroxyphenylacetic acid) and diethylenetriamine pentaacetic acid were found to almost completely inhibit ascorbate oxidation catalyzed by iron ions. The inhibition with the former chelator is due to the prevention of the reduction of Fe3+ by ascorbate, while the inhibition with the latter is caused by the strong inhibition of both this reductive reaction and the oxidation of Fe2+ by O2. These chelators almost completely inhibit ascorbate oxidation catalyzed by copper ions as well. These results indicate that the blocking of redox cycling of metal ions is important to prevent the oxidation of ascorbate.     

Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices

1. Simultaneous measurement of tissue NAD(P)H fluorescence and respiration was used to elucidate some of the early chemical changes after depolarization of the membranes of cerebral-cortical cells. Depolarization was effected by the application of a train of short-duration voltage pulses across a slice of guinea-pig cerebral cortex. The pulses cause a biphasic change in fluorescence corresponding to an early (within 1s) oxidation and later (about 10s) reduction of nicotinamide nucleotide. The major portions of both these redox changes are unrelated to the accompanying increase in slice respiration of about 75%. 2. The early oxidation requires Ca(2+) and phosphate in the bathing medium and appears to be largely due to an early mitochondrial uptake of these ions. 3. The ensuing reduction occurs in the cytosol, as judged by its almost complete elimination in the presence of exogenous pyruvate. It becomes markedly attenuated with increasing time of incubation. This and the ability of exogenous 6-N-2'-O-dibutyryl cyclic AMP to cause a reduction in the absence of electrical pulses suggest that it results from a cyclic AMP-mediated activation of glycogenolysis. 4. Further results, obtained in the presence of exogenous pyruvate, indicate that in addition to the above effects a net transfer of NADH from the mitochondrial to cytosolic space (presumably via a shuttle mechanism) is activated by the electrical pulses. 5. The lack of any sizeable (more than 2% of basal fluorescence) fluorescence change associated with the respiratory increase caused by the pulses, and the fact that any change which may be associated with it corresponds to a small reduction of mitochondrial nicotinamide nucleotide, show that a simple state 4 --> state 3 mitochondrial transition cannot account for the increased respiration. The results suggest, rather, a co-ordinated control of respiration, at more than one site.     

Inhibition of cholinesterases by fluoride in vitro

1. Series of colorimetric dynamic assays allowed the study of the inhibition of cholinesterases by F(-) ions in vitro, by using, as sources of enzyme, whole human blood, human serum, homogenized rat brain and two preparations of red blood cells (human and bovine) whose enzymic purity was ascertained. 2. The first evidence of inhibition of human serum pseudocholinesterase by fluoride was noticed at 15-25mum-fluoride. Ten times as much fluoride was needed to start inhibition of acetylcholinesterase of the red blood cells. 3. The action of fluoride on the enzymic reaction was immediate. The reversibility of the inhibition was shown by dialysis and dilution. 4. Kinetic measurements showed that the inhibition under study was not dependent on the substrate concentration and was of the uncompetitive type, similar to that observed in the presence of a heavy metal (cadmium). 5. The activity of serum cholinesterase did not change in the absence of Mg(2+) and Ca(2+) ions. Fluoride was shown to inhibit the enzyme in the absence of these ions as well as of phosphate. 6. Fluoride could inhibit cholinesterases in the presence of three different substrates and had no action on the non-enzymic hydrolysis. 7. It is thought that the halide is bound reversibly to the enzyme molecule, with the probable exclusion of the active site, but no firm conclusion could be reached on this point.     

Effect of nitrate on respiration ofMentha arvensis

The supply of nitrate nitrogen caused a marked increase in the rate of respiration of Japanese mint. Sodium azide strongly inhibited the rate of respiration at all the nitrate levels, but the inhibition was more marked at higher levels. Besides this, inhibition caused by azide treatment was less marked in older and nitrogen deficient tissues than in younger ones at higher nitrogen levels. The addition of sodium diethyl dithio-carbamate (DIECA), an inhibitor of copper containing enzymes resulted in an increase in the respiration of mint leaves which increased further with an increase in the nitrate supply. The same concentration of DIECA which stimulate the respiration of leaves caused an inhibition in the respiration of roots. The inhibition was greater at lower levels and decreased consistently as the supply of nitrate increased. The sensitivity of root respiration to DIECA observed with varying levels of nitrate indicated that unlike the leaf, the roots contain copper-containing enzymes which get decreased as the nitrate supply is increased. An increased supply of nitrogen up to 16 me NO₃ ^- -N was associated with an increase in respiratory quotient.     

Pyrrolidine dithiocarbamate induces cyclooxygenase-2 expression in NIH 3T3 fibroblast cells

Pyrrolidine dithiocarbamate (PDTC) is a metal chelating compound that can exert either pro-oxidant or antioxidant effects in different situations. Several studies demonstrate that it can inhibit cyclooxygenase-2 (COX-2) expression, which may be due to its antioxidant activity. Here, we found that PDTC rather increased COX-2 expression in NIH 3T3. The increase of COX-2 expression was inhibited by adding bathocuproline disulfonic acid, a non-permeable specific copper chelator, in the incubation medium. This result suggests that PDTC exerts its effect by transporting redox-active copper ions into the cells. In support of this observation, PDTC did not induce COX-2 expression in a serum-free environment. When PDTC was added with copper in the serum-free medium, it acted as the inducer of COX-2 expression. In addition, pretreatment of N-acetyl-L-cystein or dithiothreitol, other antioxidants, inhibited the PDTC-induced COX-2 expression. Our data indicate that PDTC induces COX-2 expression in NIH 3T3 cells, which may be due to its activities as a copper chelator and a pro-oxidant.     

THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.     

Fluoride Inhibition of Respiration and Fermentation in Cultured Cells of Acer pseudoplatanus

Low concentrations of sodium fluoride severely inhibit anaerobic CO₂ evolution in Acer pseadoplatanus L. cells but have relatively little effect on aerobic respiration. The insensitivity of respiratory O₂ uptake to fluoride is due in part to the fact that fluoride reduces the intracellular pyruvate concentration to only a relatively minor extent under aerobic conditions, although it prevents the several fold increase in endogenous private which is normally brought about by anoxia. The respiratory insensitivity is also ascribable to the existence of a respiratory component which is unaffected by the decrease in endogenous private resulting from fluoride treatment. The extremely severe respiratory inhibition brought about by fluoride plus dinitrophenol is not appreciably relieved by exogenous private, indicating that this inhibition is the result of interference with the aerobic oxidation process per se and is not solely a consequence of glycolytic inhibition.     

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reaction mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both .O2- and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H2O2 and .O2- are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (less than 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal chelating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (less than 5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (less than 10%) inhibition, but inhibited by 30% in the presence of catalase + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalase + superoxide dismutase + DETAPAC.     

The influence of metal ions on malic enzyme activity and lipid synthesis in Aspergillus niger

In the presence of copper significant induction of citric acid overflow was observed, while concomitantly lower levels of total lipids were detected in the cells. Its effect was more obvious in a medium with magnesium as sole divalent metal ions, while in a medium with magnesium and manganese the addition of copper had a less pronounced effect. Since the malic enzyme was recognised as a supplier of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate for lipid biosynthesis, its kinetic parameters with regard to different concentrations of metal ions were investigated. Some inhibition was found with Fe(2+) and Zn(2+), while Cu(2+) ions in a concentration of 0.1 mM completely abolished malic enzyme activity. The same metal ions proportionally reduced the levels of total lipids in Aspergillus niger cells. A strong competitive inhibition of the enzyme by Cu(2+) was observed. It seemed that copper competes with Mg(2+) and Mn(2+) for the same binding site on the protein.