Glycolysis in human erythrocytes containing elevated concentrations of 2, 3-P2-glycerate.

In studies on the mechanism of the inhibitory effect of 2, 3-diphosphoglycerate on glycolysis in human erythrocytes, the following results were obtained: 1) Glucose consumption and lactate production are reduced by 70 and 40 per cent relative to normal erythrocytes in red blood cells containing five times the normal amount of 2, 3, -P2-glycerate ("high-diphosphoglycerate" cells) at an extracellular pH of 7.4. The marked dependency of glycolysis on the extracellular pH observed in normal erythrocytes is almost completely lost in the "high-diphosphoglycerate" cells. 2) About 50 per cent of the inhibition of glycolysis in "high-diphosphoglycerate" cells can be accounted for by the 2, 3-P2-glycerate-induced decrease of the red-cell pH. This fall of the red-cell pH which occurs as a conswquence of the Donnan effect of the non-pentrating 2, 3-P2-glycerate anion leads to a reduction of the glycolytic rate due to the properties of the enzyme phosphofructokinase. 3) The remaining part of the inhibitory effect must be attributed to an inhibition by 2, 3-P2-glycerate of glycolytic enzymes. From measurements of glycolytic rates and of the concentrations of glycolytic intermediates in the absence and presence of methylene blue it is concluded that the hexokinase reaction is inhibited by an elevation of 2, 3-P2-glycerate concentration in "high-diphosphoglycerate" cells suggests that also the enzyme pyruvate kinase is inhibited by 2, 3-P2-glycerate. 4) The dependencies of net-change of 2, 3-P2-glycerate concentration on the red-cell pH are identical in normal and "high-diphosphoglycerate" cells indicating that the balance between formation and decomposition of 2, 3-P2-glycerate is the same in erythrocytes with normal and very high compositions of 2, 3-P2-glycerate.     

Visual detection of fructose-1,6-P2 aldolase after electrophoresis by its oxidative paracatalytic reaction

A simple method for detecting aldolase activity is described which is based on the reduction of the oxidation-reduction indicator porphyrindin to its leuco form by paracatalytic oxidation of an aldolase substrate intermediate.     

Resistance of human erythrocytes containing elevated levels of vitamin E to radiation-induced hemolysis

Human erythrocytes were isolated from the blood of healthy donors and then incubated in the presence of suspensions of alpha-tocopherol for 30 min at 37 degrees C. Unabsorbed tocopherol was removed by centrifugation using several washes of isotonic phosphate-buffered saline. Washed erythrocytes were resuspended to 0.05%. Hct and exposed to hemolyzing doses of 60Co gamma radiation, and hemolysis was monitored continuously by light scattering at 700 nm in a recording spectrophotometer. The extent of hemolysis with time was sigmoid and data analysis was carried out on the time taken for 50% hemolysis to occur (t50%). The vitamin E content of erythrocytes was significantly elevated by the incubation procedure and resulted in the cells exhibiting a significantly increased resistance to hemolysis as reflected by the extended t50% values. Oral supplementation of 500 IU of vitamin E per day to eight normal human subjects for a period of 16 days also resulted in their washed erythrocytes exhibiting a significant increase in resistance to radiation-induced hemolysis. When comparing vitamin E incubated cells with control cells, both the dose-reducing factor (DRF) and the time for 50% hemolysis quotient (Qt50%) were observed to increase with increasing radiation dose.     

Interaction of clonidine and clonidine analogs with human platelet α2-adrenergic receptors

Several new clonidine analogs were synthesized and their ability to inhibit [³H] phentolamine binding to human platelet α₂-adrenergic receptors was tested. The order of potency and calculated dissociation constants for clonidine and its analogs were as follows: clonidine (0.020 ± 0.005 μM) >p-aminoclonidine (0.100 ± 0.010 μM) > hydroxy-phenacetyl-aminoclonidine (0.20 ± 0.03 μM) >p-dansyl clonidine (1.00 ± 0.20 μM) >t-boc-tyrosine clonidine (1.80 ± 0.60 μM). Thus, p-amino substitution reduces α₂-adrenergic affinity in the platelet system. The effects of clonidine and its p-amino analogs on platelet adenylate cyclase were also evaluated. This enzyme is inhibited by epinephrine acting via α₂-adrenergic receptors. Both clonidine and p-aminoclonidine cause slight inhibition of basal adenylate cyclase and reverse the inhibition induced by epinephrine. These observations indicate that clonidine is a partial agonist for platelet α₂-adrenergic receptors.     

NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

Somatic variations on a yellow mutant in Nicotiana tabacum L. (a1+/a1a2+/a2) I. Non-reciprocal genetic events occurring in leaf cells

A greenish-yellow mutant was obtained after treatment of seeds of Nicotiana tabacum L. var. Xanthi n.c. with ethyl methanesulfonate (EMS). Two genetically independent mutations (a₁ and a₂) were isolated. The first mutation (a₁) antagonizes the function of its partially dominant a₁⁺ allele. The second mutation (a₂) is amorphous but strongly interacts with a₁.Among the nine possible genotypes at the two loci, five varied in somatic cells. The heterozygous state a₁⁺/a₁ strongly increased the frequency of both spontaneous and induced variations. However, two homozygotes also showed variations.Variants were isolated from induced and spontaneous non-reciprocal and reciprocal variations within paliside tissues by bud induction in vitro. They were genetically tested. In this first paper, only non-reciprocal variations are reported.Green variants from the greenish-yellow (J¹) dihybrid a₁⁺/a₁a₂⁺/a₂ clone had two genotypes: the first was due to true reversions of a₁ to a₁⁺, whereas the second was due to amorphous a₁⁰ mutations from a₁. These a₁⁰ mutations may well be deletions.The lightest yellow variants from J¹ were due to mutations either from a₁⁺ into a₁ or from a₂⁺ into a₂.Deletions at the a₁⁺?a₁ locus led to either yellow variations when a₁⁺ was lost, or to false reversions when the antagonistic allele a₁ was lost.Amorphous alleles at the a₁⁺?a₁ locus were also isolated from tissues other than J⁺. They gave zygotic lethality (s) that probably varied with the size of the deletions. Thus, true reversions and deletions at the a₁⁺?a₁ locus could be distinguished from one another by progeny tests.Other variants showed higher frequencies of spontaneous variations (instability). Somatic changes observed in these unstable systems were due to modifications at the marker loci. The genetic nature of this instability is not yet known.There is strong evidence that the genetic events involved in these non-reciprocal variations were deletions, conversions and point mutations. True reversions from a₁ into a₁⁺ and new mutations from a₁⁺ into a₁ were obtained only from a₁⁺/a₁. It was therefore supposed that the changes observed took place only in heterozygotes, and the conversion hypothesis was made. Attempts are being made to prove that conversions do exist in higher plants, and to find out if this process, as deletions, is induced by radiation.     

Nitrogen nutrition of C3 plants at elevated atmospheric CO2 concentrations

The atmospheric CO₂ concentration has risen from the preindustrial level of approximately 290 μl l⁻¹ to more than 350 μl l⁻¹ in 1993. The current rate of rise is such that concentrations of 420 μl l⁻¹ are expected in the next 20 years. For C₃ plants, higher CO₂ levels favour the photosynthetic carbon reduction cycle over the photorespiratory cycle, resulting in higher rates of carbohydrate production and plant productivity. The change in balance between the two photosynthetic cycles appears to alter nitrogen and carbon metabolism in the leaf, possibly causing decreases in nitrogen concentrations in the leaf. This may result from increases in the concentration of storage carbohydrates of high molecular weight (soluble or insoluble) and/or changes in distribution of protein or other nitrogen containing compounds. Uptake of nitrogen may also be reduced at high CO₂ due to lower transpiration rates. Decreases in foliar nitrogen levels have important implications for production of crops such as wheat, because fertilizer management is often based on leaf chemical analysis, using standards estimated when the CO₂ levels were considerably lower. These standards will need to be re-evaluated as the CO₂ concentration continues to rise. Lower levels of leaf nitrogen will also have implications for the quality of wheat grain produced, because it is likely that less nitrogen would be retranslocated during grain filling.     

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes

Summary Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.     

The effect of growth at elevated CO2 concentrations on photosynthesis in wheat

Rising levels of atmospheric CO₂ will have profound, direct effects on plant carbon metabolism. In this study we used gas exchange measurements, models describing the instantaneous response of leaf net CO₂ assimilation rate (A) to intercellular CO₂ partial pressure (C_i), in vitro enzyme activity assay, and carbohydrate assay in order to investigate the photosynthetic responses of wheat (Triticum aestivum L., cv. Wembley) to growth under elevated partial pressures of atmospheric CO₂ (C_a). At flag leaf ligule emergence, the modelled, in vivo, maximum carboxylation velocity for RuBisCO was significantly lower in plants grown at elevated C_a than in plants grown at ambient C_a (70 Pa compared with 40 Pa). By 12 d after ligule emergence, no significant difference in this parameter was detectable. At ligule emergence, plants grown at elevated C_a exhibited reduced in vitro initial activities and activation states of RuBisCO. At their respective growth C_i values, the photosynthesis of 40-Pa-grown plants was sensitive to p(O₂) and to p(CO₂) whereas that of 70-Pa-grown plants was insensitive. Both sucrose and starch accumulated more rapidly in the leaves of plants grown at 70 Pa. At flag leaf ligule emergence, modelled non-photorespiratory respiration in the light (R_d) was significantly higher in 70-Pa-grown plants than in 40-Pa-grown plants. By 12 d after ligule emergence no significant differences in R_d were detectable.     

Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes

In a number of animal species soluble NADH-cytochrome b₅ reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b₅ reductase of liver microsomes by using an antibody to purified NADH-cytochrome b₅ reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b₅ and NADH-cytochrome b₅ reductase are also present in the ghost.     

Decomposition of soybean grown under elevated concentrations of CO2 and O3

A critical global climate change issue is how increasing concentrations of atmospheric CO₂ and ground‐level O₃ will affect agricultural productivity. This includes effects on decomposition of residues left in the field and availability of mineral nutrients to subsequent crops. To address questions about decomposition processes, a 2‐year experiment was conducted to determine the chemistry and decomposition rate of aboveground residues of soybean (Glycine max (L.) Merr.) grown under reciprocal combinations of low and high concentrations of CO₂ and O₃ in open‐top field chambers. The CO₂ treatments were ambient (370 μmol mol^?1) and elevated (714 μmol mol^?1) levels (daytime 12 h averages). Ozone treatments were charcoal‐filtered air (21 nmol mol^?1) and nonfiltered air plus 1.5 times ambient O₃ (74 nmol mol^?1) 12 h day^?1. Elevated CO₂ increased aboveground postharvest residue production by 28–56% while elevated O₃ suppressed it by 15–46%. In combination, inhibitory effects of added O₃ on biomass production were largely negated by elevated CO₂. Plant residue chemistry was generally unaffected by elevated CO₂, except for an increase in leaf residue lignin concentration. Leaf residues from the elevated O₃ treatments had lower concentrations of nonstructural carbohydrates, but higher N, fiber, and lignin levels. Chemical composition of petiole, stem, and pod husk residues was only marginally affected by the elevated gas treatments. Treatment effects on plant biomass production, however, influenced the content of chemical constituents on an areal basis. Elevated CO₂ increased the mass per square meter of nonstructural carbohydrates, phenolics, N, cellulose, and lignin by 24–46%. Elevated O₃ decreased the mass per square meter of these constituents by 30–48%, while elevated CO₂ largely ameliorated the added O₃ effect. Carbon mineralization rates of component residues from the elevated gas treatments were not significantly different from the control. However, N immobilization increased in soils containing petiole and stem residues from the elevated CO₂, O₃, and combined gas treatments. Mass loss of decomposing leaf residue from the added O₃ and combined gas treatments was 48% less than the control treatment after 20 weeks, while differences in decomposition of petiole, stem, and husk residues among treatments were minor. Decreased decomposition of leaf residues was correlated with lower starch and higher lignin levels. However, leaf residues only comprised about 20% of the total residue biomass assayed so treatment effects on mass loss of total aboveground residues were relatively small. The primary influence of elevated atmospheric CO₂ and O₃ concentrations on decomposition processes is apt to arise from effects on residue mass input, which is increased by elevated CO₂ and suppressed by O₃.     

The relationship between leaf composition and morphology at elevated CO2 concentrations

The composition and morphology of leaves exposed to elevated [CO₂] usually change so that the leaf nitrogen (N) per unit dry mass decreases and the leaf dry mass per unit area increases. However, at ambient [CO₂], leaves with a high leaf dry mass per unit area usually have low leaf N per unit dry mass. Whether the changes in leaf properties induced by elevated [CO₂] follow the same overall pattern as that at ambient [CO₂] has not previously been addressed. Here we address this issue by using leaf measurements made at ambient [CO₂] to develop an empirical model of the composition and morphology of leaves. Predictions from that model are then compared with a global database of leaf measurements made at ambient [CO₂]. Those predictions are also compared with measurements showing the impact of elevated [CO₂]. In the empirical model both the leaf dry mass and liquid mass per unit area are positively correlated with leaf thickness, whereas the mass of C per unit dry mass and the mass of N per unit liquid mass are constant. Consequently, both the N:C ratio and the surface area:volume ratio of leaves are positively correlated with the liquid content. Predictions from that model were consistent with measurements of leaf properties made at ambient [CO₂] from around the world. The changes induced by elevated [CO₂] follow the same overall trajectory. It is concluded that elevated [CO₂] enhances the rate at which dry matter is accumulated but the overall trajectory of leaf development is conserved.     

Altered clearance of human α2-macroglobulin complexes following reaction with cis-dichlorodiamineplatinum(II)

Clearance studies were performed in mice using α₂-macroglobulin (α₂M), α₂M-trypsin comlex and α₂M-CH₃NH₂ complex. All three species were incubated with cis-dichlorodiamine platinum(II) (cis-DDPt) at concentrations between 9.0 μM and 1.67 mM for 4 h and then dialyzed. The clearance rate of native α₂M was unchanged following incubation with cis-DDPt. α₂M-trypsin and α₂M-CH₃NH₂ cleared rapidly from the ciruculation; however, reaction with cis-DDPt significantly decreased the plasma elimination rate of both complexes. Non-denaturing gel electrophoresis and α₂M activity assays demonstrated relative stability following incubations with cis-DDPt which markedly altered clearance. Evidence for cis-DDPt crosslinking of α₂M subunits was obtained: however, whether this crosslinking is involved in altered clearance remains undetermined. Iodoacetamide treatment of α₂M did not duplicate the effect of cis-DDPton α₂M clearance, nor did it inhibit the effect of cis-DDPt on α₂M clearance. Plasma elimination of α₂M complex was also unaltered by pretreatment of mice with intravenous free cis-DDPt.     

An inexpensive system for exposing plants in the field to elevated concentrations of CO2

An inexpensive, potentially mobile field exposure system is described which may be easily constructed by a small workshop. It may be operated as an open-top with a frustrum or covered with a polycarbonate ‘lid’. The system is cost-effective for CO₂ exposure work because the small size allows provision of CO₂-enriched atmospheres over prolonged periods at relatively low cost. A preliminary assessment of the chambers has been made and concentrations can be maintained at ±6% for a target atmosphere of 680 cm³ m^?3 CO₂ under normal operating conditions. Other chamber environmental conditions are reported.     

Carbon balance of young birch trees grown in ambient and elevated atmospheric CO2 concentrations

We constructed a carbon budget for young birch trees grown in ambient and elevated CO₂ concentrations over their fourth year of growth. The annual total of net leaf photosynthesis was 110% more in elevated CO₂ than in ambient CO₂. However, the trees in elevated CO₂ grew only 59% more biomass than the trees in ambient CO₂ over the year. Modelling studies showed that larger loss of carbon from fine-root production and growth of the root-associated mycorrhiza by the trees in elevated CO₂ probably accounted for all the remaining difference in net photosynthesis between the two treatments. Our modelling also showed that the fraction of net photosynthate consumed by respiration of nonleaf tissue was similar in the two CO₂ treatments, and was 26% and 24% for trees in ambient and elevated CO₂, respectively. Trees in elevated CO₂ had 43% more leaves, and produced 110% more net photosynthate than trees in ambient CO₂, even though the maximum rate of carboxylation per unit leaf nitrogen decreased by 21%. Sensitivity studies showed that down-regulation reduced the annual net photosynthetic production of the trees in elevated CO₂ by only 6%. Direct effects of higher CO₂ on photosynthesis and greater leaf area of the trees in elevated CO₂ increased the net photosynthesis of the trees by 68% and 60%, respectively; and together accounted for most of the difference in net photosynthesis between the two treatments.     

Binding and degradation of α2-macroglobulin by cultured fibroblasts

We studied the interactions of α₂-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human α₂-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0–4°C, binding was saturated at a concentration of 10–20 μg/ml. At 37°C, radiolabel appered in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated α₂-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated α₂-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified α₂-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less ¹²⁵I-labeled α₂-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing α₂-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade α₂-macroglobulin. Binding and endocytosis of α₂-macroglobulin by a cell may be a means of modulating proteases in the micro-environment of the cell and during endocytosis.     

Topography of human cytochrome b5/cytochrome b5 reductase interacting domain and redox alterations upon complex formation

Cytochrome b₅ is the main electron acceptor of cytochrome b₅ reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b₅ reductase flavin autofluorescence that was dependent upon the presence of cytochrome b_5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5 ± 0.1 μM and a 1:1 stoichiometry for the complex formation. In addition, a 30 mV negative shift of cytochrome b₅ reductase redox potential in presence of cytochrome b₅ was also measured. These experiments suggest that the FAD group of cytochrome b₅ reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.