Double Isotope Labeling Technique Research Articles

Phosphorus Uptake from Green Manures and Phosphate Fertilizers Applied in an Acid Tropical Soil

Quantifying the relative contribution of different phosphorus (P) sources to P uptake can lead to greater understanding of the mechanisms that increase available P in integrated P management systems. The 32P–33P double isotope labeling technique was used to determine the relative contribution of green manures (GMs) and P fertilizers to P uptake by Setaria grass (Setaria sphacelata) grown in an amended tropical acid soil (Bungor series) in a glasshouse study. The amendments were factorial combinations of GMs (Calopogonium caeruleum, Gliricidia sepium and Imperata cylindrica) and P fertilizers [phosphate rocks (PRs) from North Carolina (NCPR), China (CPR) and Algeria (APR), and triple superphosphate (TSP)]. Dry matter yield, P uptake, and P utilization from the amendments were monitored at 4, 8, and 15 weeks after establishment (WAE). The GMs alone or in combination with P fertilizers contributed less than 5% to total P uptake in this soil, but total P uptake into Setaria plants in the GM treatments was three to four times that of the P fertilizers because the GMs mobilized more soil P. Also, the GMs markedly increased fertilizer P utilization in the combined treatments, from 3% to 39% with CPR, from 6–9% to 19–48% with reactive PRs, and from 6% to 37% with TSP in this soil. Both PGM and the other decomposition products were probably involved in reducing soil P‐retention capacity. Mobilization of soil P was most likely the result of the action of the other decomposition products. These results demonstrate the high potential of integrating GMs and PRs for managing P in tropical soils and the importance of the soil P mobilization capacity of the organic components. Even the low‐quality Imperata GM enhanced the effectiveness of the reactive APR more than fourfold.

Multiple-Micronutrient Fortification Technology Development and Evaluation: From Lab to Market

At the World Summit for Children (New York, 1990), a resolution was passed to eliminate vitamin A and iodine deficiencies and significantly reduce iron-deficiency anemia by the year 2000. In responding to this urgent call, we developed a unique multiple-micronutrient fortification delivery system called "GrowthPlus/CreciPlus." Using this technology, a fortified powder fruit drink has been formulated and extensively evaluated. One serving of the product delivers the following US recommended dietary allowances: 20-30% of iron; 10-35% of vitamin A; 25-35% of iodine; 100-120% of vitamin C; 25-35% of zinc; 15-35% of folate; and 10-50% of vitamins E, B2, B6, and B12. This was accomplished through (a) identifying and selecting the right fortificants, and (b) understanding their chemical and physical properties that contribute to multiple problems (product acceptability, stability, and bioavailability). Data from a home-use test showed fortification with the "Multiple-Fortification Technology" has no effect on the appearance and taste of the eventually consumed powder fruit drink. One-year stability studies demonstrated that iodine and the vitamins have adequate stability. Bioavailability evaluation by using double-isotope labeling technique showed that the iron from the fortified powder drink has excellent bioavailability (23.4% +/- 6.7). In conclusion, a powder fruit drink has been clinically demonstrated to deliver multiple micronutrients, which include adequate levels of bioavailable iron, vitamin A, iodine, zinc, vitamin C, and B vitamins, without compromising taste, appearance, and bioavailability. The critical limiting step in the micronutrient fortification program is the production and distribution of the multiple-micronutrient-fortified product. The fortified powder drink was marketed in Venezuela under the brand name NutriStar.

Multiple-Micronutrient Fortification Technology Development and Evaluation: From Lab to Market

At the World Summit for Children (New York, 1990), a resolution was passed to eliminate vitamin A and iodine deficiencies and significantly reduce iron-deficiency anemia by the year 2000. In responding to this urgent call, we developed a unique multiple-micronutrient fortification delivery system called “GrowthPlus/CreciPlus®.” Using this technology, a fortified powder fruit drink has been formulated and extensively evaluated. One serving of the product delivers the following US recommended dietary allowances: 20–30% of iron; 10–35% of vitamin A; 25–35% of iodine; 100–120% of vitamin C; 25–35% of zinc; 15–35% of folate; and 10–50% of vitamins E, B2, B6, and B12. This was accomplished through (a) identifying and selecting the right fortificants, and (b) understanding their chemical and physical properties that contribute to multiple problems (product acceptability, stability, and bioavailability). Data from a home-use test showed fortification with the “Multiple-Fortification Technology” has no effect on the appearance and taste of the eventually consumed powder fruit drink. One-year stability studies demonstrated that iodine and the vitamins have adequate stability. Bioavailability evaluation by using double-isotope labeling technique showed that the iron from the fortified powder drink has excellent bioavailability (23.4% ± 6.7). In conclusion, a powder fruit drink has been clinically demonstrated to deliver multiple micronutrients, which include adequate levels of bioavailable iron, vitamin A, iodine, zinc, vitamin C, and B vitamins, without compromising taste, appearance, and bioavailability. The critical limiting step in the micronutrient fortification program is the production and distribution of the multiplemicronutrient-fortified product. The fortified powder drink was marketed in Venezuela under the brand name NutriStar®.

Effects of dissolved humic substances on the speciation of iron and phosphate at different pH and ionic strength

be expected to be key factors in the complexation and release of Fe and Po43-. Consequently, variation of the pH and/or the ionic composition of humic lakes may affect the speciation and cycling of Fe and P04% and hence their availability for uptake by planktonic organisms. Numerous softwater lakes with varying DHS contents are known to have undergone substantial pH shifts in recent decades due to anthropogenic acidification. The variable response of the plankton community in these lakes to acidification may be in part due to alterations in the DHS-FWPO~~interactions. We have examined the effecta of pH and ionic strength on these interactions using a double isotope labeling technique introduced in a previous paper (IO).

Effects of dissolved humic substances on the speciation of iron and phosphate at different pH and ionic strength

Download Hi-Res ImageDownload to MS-PowerPointCite This:Environ. Sci. Technol. 1993, 27, 6, 1052-1059

Photoaffinity labelling of mitochondrial nadh: Ubiquinone reductase with pethidine analogues

1.Chemically reactive derivatives of pethidine analogues—novel potent inhibitors of the mitochondrial NADH : ubiquinone reductase (complex I)—were synthesized. 2. Dose-response curves of these components revealed that the photoactivatable aryl azido derivative has retained most of the inhibitory activity displayed by the parent substance. After introduction of a radioactive iodine isotope into the molecule, it was used as a probe for the localization of the inhibitor binding polypeptides within complex I. 3. Photolysis of the radiolabelled derivative bound to isolated complex I both from Neurospora crassa and beef heart resulted in a covalent incorporation of the inhibitor into 6–7 individual subunits of the enzyme. Essentially the same labelling patterns were obtained, when whole mitchondrial membranes were incubated with the reactive derivative. 4. Applying a double isotope labelling technique, the inhibitor-binding polypeptides in N. crassa were identified as mitochondrially synthesized constituents of complex I (ND gene products). In the beef heart enzyme the ND-1 product was detected to be among the polypeptides reacting with the inhibitor. 5. Competition experiments employing either NADH or decylbenzoquinone (DB), together with the pethidine analogue, showed that both enzyme substrates interfere specifically with the inhibitor binding to complex I.

Norepinephrine Metabolism in Man Using Deuterium Labelling: Origin of 4‐Hydroxy‐3‐Methoxymandelic Acid

A double isotope labelling technique was used to simultaneously determine the in vivo turnover rates of 4-hydroxy-3-methoxyphenylglycol (HMPG) and 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) and the rate of HMPG oxidation to HMMA. Six healthy men were given intravenous injections of [2H3]HMPG and [2H6]HMMA and their plasma and urine samples analysed by gas chromatography--mass spectrometry (GC/MS) for the protium and deuterium species. HMPG and HMMA production rates were calculated by isotope dilution. The rate of HMPG oxidation to HMMA was obtained from the fraction of [2H3]HMPG recovered as [2H3]HMMA. The results showed that the entire production of HMMA, 1.11 +/- 0.21 mumol/h (mean +/- SE), could be accounted for by oxidation of HMPG, 1.49 +/- 0.31 mumol/h. In another experiment designed to avoid expansion of the HMPG body pool, a tracer dose of [14C]HMPG was given to the same subjects. The levels of [14C]HMPG and [14C]HMMA were measured in urine after extraction and separation by thin layer chromatography. Urinary excretion of endogenous HMPG and HMMA was determined by GC/MS. The results showed that the endogenous HMMA fraction of the total HMPG and HMMA urinary excretion rate, 0.57 +/- 0.04, was the same as the fraction of [14C]HMPG oxidized to [14C]HMMA, 0.62 +/- 0.01. Thus, HMPG is the main intermediate in the metabolic conversion of norepinephrine and epinephrine to HMMA in man.

Release of [3H]gamma-aminobutyric acid from glial (Muller) cells of the rat retina: effects of K+, veratridine, and ethylenediamine

In several neural systems, glial cells appear to take up and release gamma-aminobutyric acid (GABA) upon depolarization. We have studied the release of [3H]GABA from Müller (glial) cells in the rat retina by a double isotope-labeling technique in which Müller cells are preloaded with 3H-GABA while a population of neurons is prelabeled with [14C]glycine. By autoradiography, we have confirmed that [3H]GABA is taken up by the radially oriented Müller cells, whereas [3H]glycine is accumulated by a subset of amacrine cells (neurons). Using the double-labeling procedure, we have examined the effects of two depolarizing agents, high K+ and veratridine, and the GABA mimetic, ethylenediamine, on transmitter release from glial cells and neurons simultaneously. We found the following. (1) Depolarization with 56 mM K+ released both [3H]GABA and [14C]glycine. About 70 to 80% of this release was blocked in Ca2+-free medium. (2) Veratridine (10 microM) also released both of the transmitters. This release was strongly inhibited by 100 nM tetrodotoxin or 1mM procaine. Under Ca2+-free conditions, less than 20% isotope release was observed. (3) Ethylenediamine released [3H]GABA readily, whereas little [14C]glycine release was observed. Removal of Ca2+ had no significant effect on transmitter release. Furthermore, in Na+-free medium ethylenediamine failed to induce [3H] GABA or [14C]glycine release. These results suggest that high K+ and veratridine release [3H]GABA from Müller cells by a Ca2+-dependent process. Ethylenediamine, on the other hand, appears to induce [3H]GABA release by a Ca2+-independent, carrier-mediated exchange mechanism.

Changes in Wheat Leaf Polysomal Messenger RNA Populations during the Early Stages of Rust Infection

Polysomes isolated from a susceptible variety of wheat leaves (cultivar W2691) and those inoculated with the wheat stem rust fungus (f. sp. tritici, race 126-ANZ-6, 7) were incubated in a cell-free protein-synthesizing system. Under these conditions, different size classes of polypeptides, ranging in molecular weight from 10,000 to 80,000, are radiolabeled. Using double-isotope labeling technique, we show that some discrete size classes of polypeptides are synthesized in significantly greater quantitites by polysomes from inoculated leaves compared to the corresponding size classes synthesized by polysomes from healthy leaves. These results confirm our previous observation that there are significant changes in the wheat leaf polysomal messenger RNA populations at 3 days after inoculation with the rust fungus.The effects of the organelle-specific inhibitors of protein synthesis, chloramphenicol and lincomycin, on in vitro polysomal messenger RNA translation were investigated. The polypeptides synthesized by polysomes from healthy and inoculated leaves in the presence of chloramphenicol were compared. The results show that, even in the presence of this antibiotic, the polysomes from inoculated leaves synthesize greater quantities of some size classes of polypeptides. These data indicate that changes in polysomal messenger RNA populations involve, at least in part, cytoplasmic messenger RNA.

Quantitation of some amino-terminal residues in proteins using 3H-labelled dansyl chloride and 14C-labelled amino acids.

A method for quantitation of amino-terminal residues in proteins is presented. The method is a modification of a double isotope-labelling technique, using 3H-labelled dansyl chloride and 14C-labelled amino acids as internal standards. The method is demonstrated on human fibrinogen, horse myoglobin and on mouse myoloma IgA. A linear relationship between the ratio 3H/14C in the separated amino-terminal amino acid of the protein and the amount of protein added in the labelling mixture was obtained with standard deviations of +/- 7.4% +/- 3.4% and +/- 10.3%, respectively. An application of the method is demonstrated by measuring the increase in amino-terminal glycine in fibrinogen following the proteolytic action of thrombin. The method seems to be useful when 0.1 nmol or more of protein is used.

Increased synthesis of l-glycerol 3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells

Reaggregating cell cultures of neonatal mouse cerebellar cells express many of the differentiated properties of normal developing cerebellum, including the transition for the embryonic and adult isozymes of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). In order to determine the mechanism leading to increased levels of adult isozyme, aggregates in culture from 2 to 17 days were labeled with radioactive leucine and the relative rate of enzyme synthesis was measured after purification of the enzyme by affinity chromatography on Blue Sepharose 6B. During the course of in vitro differentiation, the relative rate of synthesis increased 100-fold, such that it represented 0.5% of the total protein synthesized in the cytoplasmic fraction of the cell. In vivo, BALB cBy mice have twice the level of enzyme activity in the cerebellum as do C57BL 6J mice. Reaggregating cell cultures of cerebellar cells from these strains of mice also express a difference in the activity level, but only when the cerebellar cells are taken from mice 4 days of age or less. When the relative rates of synthesis of l-glycerol 3-phosphate dehydrogenase were measured in cultures expressing the strain-dependent difference in activity, these rates were found to be approximately twofold greater in cultures of BALB cBy cells. In contrast, estimates of the relative rate of enzyme degradation by the double-isotope labeling technique indicate that neither specific enzyme degradation nor degradation of total protein is different in aggregates from the two strains of mice. The results suggest that the genetic mechanisms controlling the levels of l-glycerol 3-phosphate dehydrogenase in the cerebellum during development are intrinsic to the cells and, with the exception of serum factors, are independent of systemic influences.

Regulation of lactate dehydrogenase levels in the mouse.

BERNARDO NADAL-GINARD From the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 The number of lactate dehydrogenase molecules per cell equivalent in normal adult mouse tissues is: heart muscle 1.13 x 10s, skeletal muscle 3.6 x 1O8, liver 3.94 x lo’, and kidney 3.91 x 107. These steady state levels of lactate dehydrogenase reflect a balance between synthesis and degradation. To study the relative role of the two processes the half-lives of lactate dehydrogenase in these four mouse tissues were determined by means of a combination of the double isotope labeling technique and enzyme purification by oxamate . aminohexyl . Sepharose 4B affinity chromatog- raphy. This technique makes possible the measurement of lactate dehydrogenase turnover in single mouse organs and allows for the comparison of isotope decay between different tissues without being affected by differences in the size of the amino acid pools. All the lactate dehydrogenase isozymes in the same tissue were found to have the same half-life. The half-lives were: heart 8.2 days, skeletal muscle 43.0 days, liver 4.3 days, and kidney 6.1 days. The rates of lactate dehydrogenase synthe- sis in these tissues were calculated to be 110, 67, 74, and 52 molecules per cell per s, respectively. From these data and from the proportion of A and B subunits in each tissue, the rates of synthesis of each subunit were calculated. From the results it is concluded that the different steady state lactate dehydrogenase levels, a phenotypic expression of the lactate dehydrogenase genes, reflect mainly the differ- ing rates of degadation since the rates of synthesis are very similar; thus the regulation of the quantitative expres- sion of the lactate dehydrogenase genes in the mouse is largely post-translational.

The ‘estrogen-induced protein’: Quantitation by autoradiography of polyacrylamide gels

The increased rate of synthesis of the “estrogen-induced protein” (IP) first detected by Notides and Gorski is one of the earliest macromolecular responses to estrogen by the rat uterus. The IP detected by autoradiography of sodium dodecyl sulfate polyacrylamide gel electropherograms was shown to correspond to a stainable protein band in electropherograms of cytosol from uteri of both untreated and estrogen-treated rats. The uteri of 9–10-day-old rats showed a maximum rate of IP synthesis 1 h after estrogen-treatment, followed by a decrease to constitutive levels by 24 h. The rate of synthesis of IP was doubled by estrogen administration to 10–30-day-old rats. The improved sensitivity of the autoradiographic method permitted the detection of some stimulation of IP synthesis in uteri of 5-day-old rats, in contrast to our previous findings in Wistar rats using a double isotope labelling technique.

The State of Lysosomes and Protein Turnover in Rat Liver Effect of Excess Vitamin A

Administration of excess vitamin A to rats induces labilization of liver lysosomal membranes, as shown by the release of lysosomal cathepsins upon tissue homogenization. The effect of lysosomal labilization on liver protein turnover was investigated. The apparent turnover rate of liver proteins in the hypervitaminotic animals, as measured by a double isotope-labeling technique (Glass and Doyle (1972) J, Biol. Chem, 247, 5234-5242), was found to be the same as that in control animals. Neutral and alkaline fructose-1, 6-biphosphatase [EC 3.3.3.11] activities in the liver were also found to be unchanged in hypervitaminosis A. These data indicate that the rate of intracellular protein degradation is not determined by the level of "free cathepsins. Protein synthesis in the livers of the hypervitaminotic animals was partially imparied, as shown by the shift of polysomal profiles toward lighter aggregates.

Biosynthesis of membrane bound Ig and secretion of Ig by chicken lymphoid cells.

The metabolic turnover of membrane proteins of chicken lymphoid cells is studied, using a double isotope labeling technique (i.e., [14C]amino acid pulse and [3H]leucine chase). Compared with other membrane proteins, the metabolic turnover of membrane bound immunoglobulins (M-Ig) is very slow. There was no difference in the turnover between M-Ig and specific antigen binding receptor immunoglobulins. Immunoglobulins appear to be a stable constituent of the lumphocyte membrane. Cellular kinetic experiments show that the rate of biosynthesis of secreted immunoglobulins (S-Ig) is nearly ten times as much as that of M-Ig, suggesting that metabolic pathway leading to M-Ig are distinct from those leading to S-Ig. The difference in 3H/14C ratios between S-Ig and M-Ig reflects the rate of biosynthesis of these immunoglobulins by two types of bursa derived lymphoid cells.

Base-catalyzed decomposition of p-nitroperoxobenzoic acid

The base-catalyzed decomposition of p-nitroperoxobenzoic acid has been studied using a double isotope labeling technique. The decomposition of a sample of the peroxo acid in which 2·3% of the peroxidic oxygen was doubly labeled as − 18O 18O- led to complete retention of the double label in the product oxygen, 36O 2. This result is consistent with a decomposition mechanism involving nucleophilic attack of the conjugate base of p-nitroperoxobenzoic acid upon the carbonyl carbon of the undissociated acid.

Turnover studies on proteins of rat liver lysosomes.

The turnover of rat liver lysosomal proteins was studied by a double isotope-labeling technique. The cellular fractions investigated included soluble lysosomal proteins, lysosomal membrane proteins, highly purified lysosomal beta-glucuronidase, and for comparison, microsomal proteins and soluble cytoplasmic proteins. Both "normal" lysosomes and Triton WR-1339-filled lysosomes (tritosomes) were studied, with similar results. It was found that (a) the turnover rate of lysosomal proteins, of both the soluble and membranous compartments, was very similar to that of the proteins of the microsomal and soluble cytoplasmic fractions, and (b) the turnover rate of lysosomal proteins was asynchronous. The latter conclusion was based on two lines of evidence: (a) lysosomal beta-glucuronidase had a distinctly slower turnover rate than the average rate of the soluble lysosomal proteins, and (b) subunits of the proteins of the soluble lysosomal fraction as separated by sodium dodecyl sulfate. Sephadex G-200 gel filtration showed different rates of degradation.

Acetylation of reticulocyte ribosomal proteins at time of protein biosynthesis.

When rabbit reticulocytes were incubated in vitro with [(3)H]acetate, their ribosomal proteins were rapidly acetylated within 10 min. Polyacrylamide-urea gel electrophoresis showed that several major ribosomal protein fractions were highly acetylated. By the double-isotope labeling technique, the incorporation of [(3)H]acetate and [(14)C]aminoacid mixture into ribosomal proteins and nascent chains was found to be closely associated. Sodium fluoride abolished the acetylation of ribosomal proteins, whereas cycloheximide reduced the acetylation of ribosomal proteins to a much lower level. These findings suggest that acetylation of ribosomal proteins may be involved in the formation of the initiation complex during protein biosynthesis.

A reassessment of the collagen reutilization theory by an isotope ratio method

The theory that intact collagen molecules (or large segments thereof) may be used in restructured collagen was tested by a double isotope-labelling technique. A 3H: 14C isotope ratio was established in the mature connective tissue of ten female Wistar rats by an intraperitoneal injection of L-[ 3H]proline and [ 14C]glycine. Twentyeight days after the injection, granulation tissue was induced by subcutaneous dorsal implantation of ten hollow steel-mesh cylinders per animal. The cylinders were then removed along with skin samples at intervals over a 21-day period. During this time three animals received a diet supplemented with 15% unlabelled L-proline and three received a diet supplemented with 15% unlabelled glycine. If the granulation tissue consisted of reutilized collagen the isotope ratio would remain unchanged regardless of the diet. If the collagen came from de novo synthesis the isotope ratio would change because the 3H and 14C must pass through separate pools. Our results show that a large proportion of the radioactivity previously identified as arising from reutilized labelled collagen is actually the result of local protein catabolism and recycling of labelled amino acids for de novo synthesis. These experiments do not conclusively disprove the collagen reutilization theory but indicate the need for further investigations using a labelled compound that cannot be recycled.

DNA-synthesis time of bone marrow cells in healthy and ascites tumor-bearing mice.

DNA‐synthesis (S) times of myelocytes and nucleated erythroid cells in the bone marrow of healthy mice as well as mice bearing advanced Ehrlich ascites tumors were measured with the aid of a combined in vivo‐in vitro double isotope labeling technique. Neither the S‐period nor the rate of proliferation of these cells were influenced by the presence of the tumor in these hosts. This finding discounts the possibility that the marked retardation of DNA‐synthesis and proliferation rate observed in the tumor cells themselves with advancing tumor‐age is a nonspecific effect of the nutritional deterioration in the host.