Uptake and efflux of copper-64 in Menkes''-disease and normal continuous lymphoid cell lines.

The accumulation of copper over 2 h by normal lymphoid cells and those from Menkes'-disease patients (Menkes' cells) was found to be biphasic, with an initial phase of rapid uptake, an approach to steady state at around 40-60 min, followed by a further accumulation phase. The accumulation of copper was not diminished by the addition of a variety of metabolic inhibitors, suggesting that copper uptake is not an active process. The presence of carbonyl cyanide m-chlorophenylhydrazone in the culture medium stimulated the uptake and accumulation of copper in both normal and Menkes' cells to the same absolute level. This effect appeared to be specific for copper, since the accumulation of Zn and Cd was unaffected. Menkes' cells did not differ from normal in their initial rate of copper uptake. Analysis of the uptake curve suggested that the membrane transport of copper involves both passive and facilitated diffusion. Initial rate of efflux from the cells was approximated by two methods. Menkes' cells did not appear to be affected in this function. It seems likely that the basic defect in Menkes' disease involves a step in intracellular copper transport rather than the membrane transport of copper.     

The Interaction of pH and Aeration in CI Uptake by Barley Roots

The uptake of Cl by excised roots of barley (Hordeum vulgare L.) from KC1 solution maintained at high pH was markedly reduced by high rates of aeration, whereas K uptake was scarcely affected. Aeration rate had relatively minor effects at low pH. The effect of high aeration rate at pH 9 could be overcome by the use of buffered solutions. In unbuffered solutions the H resulting from the excess cation uptake together with that produced from respiratory CO₂ was sufficient to materially reduce the pH of the solution. The reduction in pH favored the uptake of Cl which is adversely affected by high pH. The effect of aeration rate could be explained in terms of root induced pH changes and film diffusion involving the solution film adjacent to the root surface.     

Uptake of 67Cu by isolated human trophoblast cells.

The isolated human trophoblast was used as a system to analyze the effects of different physiological ligands on cellular uptake of copper. The results show that the uptake of copper by these cells follows a similar pattern for the ligands tested (histidine, albumin and ceruloplasmin) as that for copper chloride. The process follows a typical hyperbolic curve at 37 degrees C. The initial phase of uptake follows a linear pattern during 30 min at 37 degrees C and at least 60 min at 4 degrees C from which the uptake rate is calculated. However, a significant decrease in the uptake rate is observed for albumin. The effect of histidine on stimulating copper transport is observed in the presence of serum, a phenomenon which is considered to be due to the release of copper that is bound to albumin. These results support the role of ceruloplasmin as a copper transport protein which releases copper at the cell surface, and a subsequent transport of the released copper in a manner similar to that of copper chloride or copper-histidine complexes.     

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii.

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.     

Trace element uptake in liver cells. 2. Effect of different proteins in the medium on the uptake of copper and zinc by hepatoma cells

Cultured rat hepatoma cells (HTC-cells) were used to study the uptake of copper and zinc from a minimal salt-glucose medium, supplemented with albumin from different species or with ovalbumin. Competitive equilibrium dialysis showed that at low molar ratios of metal/protein (less than 1) the affinity for copper of human and bovine albumin was about equal, but that of dog albumin or ovalbumin was much lower. Only a small difference in affinity for zinc could be detected between human albumin and ovalbumin. Supplementing the medium with the different proteins the rate of copper uptake in the cell at a given molar Cu/protein ratio increased as follows: human albumin congruent to bovine albumin less than dog albumin less than ovalbumin. When the molar Cu/protein ratio was increased, a discontinuity was seen with all three albumin species at a ratio of about 1. In contrast, the zinc uptake mimics that of Cu/ovalbumin, and no discontinuity was observed using different molar Zn/protein ratios. These results indicate that the rate of copper and zinc uptake depends strongly on its affinity for the protein: a low affinity leads to a high uptake. The results suggest further that at physiologic concentrations zinc is taken up by a mechanism different from that for copper.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa.

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

缺Cu~（2＋）和Zn~（2＋）对水稻OsNRAMP家族基因表达与主要金属离子吸收的影响

对野生型水稻进行缺Cu2＋和Zn2＋处理,利用定量RT-PCR技术分析OsNARAMP家族不同基因的表达情况,并采用ICP-MS技术检测在水稻根、茎叶中Fe、Zn、Cu和Mn等元素的积累情况。结果表明水稻中OsNRAMP家族有9个基因,可分为3个亚类;OsNRAMP5可能参与了Cu2＋的吸收和转运,而OsNRAMP2和OsNRAMP3可能参与Zn2＋吸收和转运。同时缺Cu2＋和Zn2＋能刺激Fe2＋离子在水稻根和茎叶中的积累,减缓Mn2＋离子在根中的吸收;而缺Cu2＋减缓水稻中Zn2＋离子的吸收。这些金属离子吸收的情况和OsNRAMP家族可能存在密切关系。     

Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability.

A saturable and temperature-dependent copper uptake pathway has been identified in Chlamydomonas reinhardtii. The uptake system has a high affinity for copper ions (Km approximately 0.2 microM) and is more active in cells that are adapted to copper deficiency than to cells grown in a medium containing physiological (submicromolar to micromolar) copper ion concentrations. The maximum velocity of copper uptake by copper-deficient cells (169 pmol h-1 10(6) cells-1 or 62 ng min-1 mg-1 chlorophyll) is up to 20-fold greater than that of fully copper-supplemented cells, and the Km (approximately 2 x 10(2) nM) is unaffected. Thus, the same uptake system appears to operate in both copper-replete and copper-deficient cells, but its expression or activity must be induced under copper-deficient conditions. A cupric reductase activity is also increased in copper-deficient compared with copper-sufficient cells. The physiological characteristics of the regulation of this cupric reductase are compatible with its involvement in the uptake pathway. Despite the operation of the uptake pathway under both copper-replete and copper-deficient conditions, C. reinhardtii cells maintained in fully copper-supplemented cells do not accumulate copper in excess of their metabolic need. These results provide evidence for a homeostatic mechanism for copper metabolism in C. reinhardtii.     

Albumin has no role in the uptake of copper by human fibroblasts

The mechanism of copper uptake by cells has been the subject of controversy for some time. This paper examines the possibility of a role for albumin in the uptake of copper by fibroblasts. Although the cells could accumulate copper from a copper-albumin complex, there was no evidence for either copper-albumin or albumin receptors on the cell surface. The possibility of a surface exchange mechanism for copper was examined. While copper uptake showed saturation with increasing concentrations of labelled copper-albumin, adding unlabelled copper to the incubation medium did not inhibit uptake. Adding albumin or histidine to the copper-albumin complex resulted in an inhibition of copper uptake. The results can only be explained by the cell taking up free copper from the incubation medium, with the albumin then releasing its copper to maintain the equilibrium between free and bound metal. Since, in vivo there is essentially no free copper in serum, it is concluded that albumin is most unlikely to play a role in the uptake of copper by fibroblasts.     

Copper uptake by two sympatric species of Killifish Fundulus heteroclitus (L.) and F. majalis (Walbaum)

Differential copper uptake within brain, visceral, gill, somatic and residual tissue was examined in the two sympatric species of killifishes Fundulus heteroclitus and F. majalis . Various salinities and copper concentrations were tested on both juvenile and adult stages to examine their effects on copper uptake. Salinity played a significant role in copper uptake in both species of killifishes. However, the effect of salinity was specific for each species and life history stage. Copper content of various tissues was directly related to the uptake pattern exhibited by whole adult fishes. Fundulus majalis brain tissue and F. heteroclitus muscle tissue were exceptions. Severe body lesions and subcutaneous haemorrhaging were observed after 2–3 day exposure to both 2 and 8 ppm copper.     

Potential subsoil utilization by roots

Summary By training root systems through narrow plastic tubes down to below predetermined levels the potential capabilities for uptake from deeper soil layers was investigated.In the experiment, carried out in asbestos pipes of 1 meter height, a soil column with an increase in moisture content downwards coupled with an inverse aeration gradient was established.Uptake per unit of roots in contact with soil was higher in the deeper layers. The increase in activity must partly be attributed to the method of calculation, but is in line with other observations in the literature. Roots growing in soil layers, where aeration is limited, still perform well, demonstrating that growth and uptake have about the same oxygen requirements.Any root in the subsoil can thus be considered as a potential absorbing unit for nutrient uptake.     

Trace metal uptake in liver cells: 1. Influence of albumin in the medium on the uptake of copper by hepatoma cells

Cultured rat hepatoma cells (HTC-cells) were used to study the transfer of copper from a well-defined medium to and across the cell membrane and particularly the role of albumin in this process. HTC-cells, maintained in a minimal salt-glucose medium, accumulated far more copper than when maintained in the same medium, but supplemented with albumin. In the latter case, the Cu uptake strongly depended on the molar Cu/albumin ratio. The results suggest a role of albumin in the uptake of trace metals. The results indicate the presence of two types of binding sites for copper on the cell membrane. The sites of the first type bind copper very strongly and are probably responsible for the uptake of copper under physiological conditions. Their number was estimated to be about 10⁶ per cell. Those of the second type only bind copper when the molar Cu/albumin ratio exceeds a value of about 1, i.e., under extreme, unphysiological conditions. Furthermore, the results suggest a direct interaction of the Cu-albumin complex with these strong binding sites as a first step in Cu uptake processes.     

Regional differences in the uptake of exogenous copper into rat brain after acute treatment with sodium diethyldithiocarbamate. A histochemical and atomic absorption spectrophotometric study

Since sodium diethyldithiocarbamate (SDEDTC) is known to increase the tissue uptake of copper, we have examined its effect on copper accumulation in the rat cerebellum, hypothalamus, parietal cortex and hippocampus by means of atomic absorption spectrophotometry. Acute SDEDTC (1,000 mg/kg i.p.) administration alone did not alter the regional concentration of copper in the cerebellum, hypothalamus and parietal cortex, but significantly increased it in the hippocampus, 5 h after treatment. Copper acetate (5 mg/kg) given i.p. has a stimulatory effect on copper uptake only in the hypothalamus and hippocampus. When copper acetate was administered to rats which were pretreated with SDEDTC, an especially high significant increase in the hippocampal copper level could be observed (approximately 70%), while the enhancement in cerebellar copper concentration was much more lower (approximately 20%), but yet significant. These data suggest that SDEDTC enhances the uptake of exogenous copper in all brain regions examined since the lipophilic SDEDTC-copper complexes easily penetrate the cell membranes. Furthermore, our histochemical findings indicate that--under normal conditions--copper is stored predominantly in glial cells, while following an excessive uptake this metal is also accumulated in neurons (e.g. pyramidal cells of the hippocampus and cortex).     

Respiratory inhibition by copper in Tetrahymena pyriformis GL

An excess of copper incorporated into Tetrahymena cells was mainly distributed in mitochondria, and inhibited oxygen uptake of Tetrahymena cells. The inhibition of oxygen uptake was clearly to copper uptake in mitochondria. Succinate was most favorable as a substrate stimulating oxygen uptake in mitochondria, and oxygen uptake was most strongly inhibited by copper (0.1 mM) in the presence of succinate among various substrates. The copper incorporated into mitochondria was in the fraction with the inner membranes. Succinate dehydrogenase (SDH) was inhibited at the lowest copper concentration (0.1 mM) among respiratory related enzymes. The redox potential of respiratory components was raised by copper. These results suggest that respiratory inhibition of Tetrahymena cells by copper may be mainly cause by inhibition of SDH as a FAD-protein and oxidation of electron carriers. At higher copper concentrations, MDH, cytochrome c reductase, and ATP synthesis were also inhibited. Growth inhibition may be due to these effects of copper in mitochondria. Mercury affected both oxygen uptake and SDH more strongly than copper. Zinc (0.1 mM) also affected oxygen uptake in mitochondria and a little in whole cells, however, it did not inhibit SDH. Cobalt, manganese, and nickel affected both oxygen uptake and SDH only a little at the same concentration (0.1 mM) as copper.     

Iron metabolism is modified by the copper status of a human erythroleukemic (K562) cell line.

Copper deficiency is known to result in a microcytic, hypochromic anemia. Red cells of copper-deficient animals have less hemoglobin than their copper-adequate counterparts. The objective of this work was to determine what role copper plays in maintaining hemoglobin levels. It was hypothesized that the primary defect lies in intracellular iron metabolism. The influence of copper supplementation on iron uptake and storage was examined in a cell line capable of hemoglobin synthesis. The results demonstrated that copper supplementation of human K562 cells was associated with higher cytosolic iron levels and ferritin levels. Copper supplementation of the cell culture altered the initial rate of iron uptake from transferrin and enhanced iron uptake in noninduced cells; however, in hemin-induced K562 cells, which express fewer transferrin receptors on the cell surface, copper appeared to reduce iron uptake. Subsequent studies showed that the cells were able to take up the same amount of iron from transferrin when incubated over a longer period of time (24 hr). In the noninduced (non-hemoglobin synthesizing) cells, proportionally more iron was associated with the ferritin. We concluded from these studies that copper affects both uptake and storage of iron and that copper supplementation reduces cellular iron turnover.     

Oxygen transfer kinetics of riboflavin fermentation by Ashbya gossypii in agitated fermentors

Effects of agitation and aeration rates on volumetric oxygen transfer coefficient and oxygen uptake rate of a riboflavin broth containing Ashbya gossypii were investigated in three batch, sparged, and agitated fermentors having the working volumes of 0.42, 0.85, and 2.5 l. The change of oxygen uptake rate with time at 250 rev min⁻¹ stirring and vvm aeration rates was shown. The volumetric oxygen transfer coefficients and maximum oxygen uptake rates obtained have been correlated to mechanical power inputs per unit volume of the fermentation broth and the superficial air velocities.     

Mechanism of copper transport from plasma to hepatocytes

The effects of plasma components on the kinetics of copper transport by rat hepatocytes were examined in an attempt to determine how copper is mobilized from plasma for uptake by the liver. Specific protein-facilitated transport was indicated by saturation kinetics, competition by related substrates, and similar kinetic parameters for uptake and efflux. For copper uptake, Km = 11 +/- 0.6 microM and Vmax = 2.7 +/- 0.6 nmol Cu/(min X mg protein). Zinc is a competitive inhibitor of copper uptake, and copper competes for zinc uptake. Copper efflux from preloaded cells is biphasic. The kinetic parameters for the initial rapid phase are similar to the parameters for uptake. Copper transport by hepatocytes is strictly passive. A variety of metabolic inhibitors have no effect on uptake and initial rates are solely dependent on extracellular-intracellular concentration gradients. Albumin markedly inhibits copper uptake by a substrate removal mechanism, and histidine facilitates albumin-inhibited copper uptake. The active species that delivers copper to hepatocytes under conditions of excess albumin and excess histidine is the His2Cu complex. Experiments with [3H]His2 64Cu showed that the transported species is free ionic copper. The kinetic parameters of copper transport by hepatocytes isolated from the brindled mouse model of Menkes' disease are normal. However, these cells show a decreased capacity to accumulate copper on prolonged incubation. An intracellular metabolic defect seems to be involved.     

Effect of root zone aeration on the growth and bioactivity of cucumber plants cultured in perlite substrate

This study was aimed at investigating the growth and nutrient uptake of cucumber plants affected by forced aeration of supplying oxygen and stimulating gas exchange rate in root zone in a substrate. Five aeration levels during the growth (0, 0.5, 1.0, 1.5 and 2.0 L/min) were applied. Maximum leaf area and leaf fresh and dry weights were obtained at an aeration level of 0.5 L/min. Excessive aeration in root zone inhibited leaf area expansion, relative leaf growth rate and crop growth rate. An optimum leaf area index of 3.0 to 3.5 was estimated in range of 0 and 0.5 L/min. The highest fruit yield was measured of 1.13 kg/plant at 0.5 L/min, whereas at 2.0 L/min it was 0.62 kg/plant. Potassium concentration in petiole sap was lower at 63 days after transplanting than that at 32 days after transplanting. Ethylene concentrations increased with higher aeration values, however, CO₂ concentration reduced with increased aeration. All bioactive compounds (polyphenols, flavonoids, flavanols, tannins and ascorbic acid) and the levels of antioxidant activities by ferric-reducing/antioxidant power and cupric reducing antioxidant capacity in ethanol extracts of cucumbers differed significantly in the investigated samples and were the highest at aeration level of 0.5 L/min in comparison with other samples (P <0.05). In conclusion, antioxidant status (bioactive compounds and antioxidant activities) improved with the appropriate aeration, which is effective for higher fruit yield and bioactivity. Excessive aeration inhibited root respiration, nutrients, bioactivity, and water uptake, and it resulted in the reduction of plant growth and fruit yield.     

A transport kinetic concept for ion uptake by plants

Summary A previously presented transport kinetic model for ion uptake by plants was tested by using data for copper uptake by barley grown in soil or water culture and corresponding data for copper concentration in the soil solution or culture solution. As no significant differences were obtained between experimentally determined values for copper uptake and those calculated by using the model, it was concluded that the experimental evidence was consistent with the proposed transport kinetic model. The model allows the Michaelis-Menten Constant of ion uptake and the Mean Maximal Rate of ion uptake to be calculated for various time intervals of the growth period of plants. The Michaelis-Menten Constant for copper uptake by barley grown in soil was the same as that for plants grown in water culture, provided that the soil solution and the water culture solution had the same chemical composition. The concentration of copper in the soil solution, at which the rate of copper uptake was zero, was 2–4 times larger than corresponding values obtained in water culture solution. re]19751028     

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.