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M C Linder

Publications and source records attributed to M C Linder.

At least 19 recordsLinked to original sources
PubMed1994

Copper transport in the Nagase analbuminemic rat.

The copper binding components of serum and potential importance of albumin to copper transport were investigated in adult Nagase analbuminemic and Sprague-Dawley rats of both sexes. There was a sex difference in total plasma copper concentrations, which were 60 and 130% higher than in the parent strain, in male and female Nagase rats, respectively. The higher levels of plasma copper were accounted for by two- and threefold increases in ceruloplasmin, as measured by p-phenylenediamine oxidase activity and copper by atomic absorption after gel chromatography. Other nonalbumin plasma proteins were also present in higher concentrations. Albumin concentrations were one-four-thousandth that of Sprague-Dawley rats, at 15 micrograms/ml (determined by rocket immunoelectrophoresis and comparative Western blotting). The tissue distribution and rate of uptake of intravenously injected 67 Cu(II) were unaffected by the lack of circulating albumin. 67Cu entered the liver of Nagase rats at least as rapidly as in the parent strain and reemerged in the blood on ceruloplasmin at an accelerated rate. The initial binding of 67Cu(II) to plasma components was primarily to transcuprein compared with albumin in the case of Sprague-Dawley rats. In the Nagase rats, the rest of the 67Cu bound primarily to nonalbumin proteins with about the same size as albumin; in Sprague-Dawley rats, it bound primarily to transcuprein. Limited analyses of tissue copper confirmed previous reports showing no striking differences from the parent strain. We conclude that albumin is not critical to the normal distribution and metabolism of copper and that it may serve more as a reservoir for excess plasma copper than as a specific conduit for the delivery of this element to hepatocytes or other cells.

Animals↗
PubMed1992

Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver.

We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.

Animals↗
PubMed1989

Heart tissue contains small and large aggregates of ferritin subunits.

Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits.

Animals↗
PubMed1989

Ferritin mRNA is found on bound as well as on free polyribosomes in rat heart.

Free and endoplasmic reticulum-bound polyribosomes from rat heart were examined for their ferritin mRNA content. A procedure for separation and purification of the two ribosome populations that produced good yields of homogeneous mono- and polyribosomes with no contaminating ultrastructures and gave distinctive sedimentation profiles in 15-50% sucrose gradients was developed. 14C-labeled free and bound polyribosomes added to heart preparations indicated that only 3% of free and 5.5% of bound polyribosomes cross-contaminated the bound and free fractions, respectively. RNA from both polyribosome populations hybridized with [32P]cDNA for rat ferritin. The extent of hybridization with mRNA from endoplasmic reticulum (ER)-derived polyribosomes was much greater than what could be accounted for by cross-contamination with free polyribosomes. This indicates that heart ferritin is synthesized not only on free polyribosomes for internal use in iron storage but also on ER-bound polyribosomes, where it may be destined for secretion into the plasma.

Animals↗
PubMed1989

Translational regulation of ferritin synthesis in rat spleen: effects of iron and inflammation.

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.

Animals↗
PubMed1989

Dissociation of ferritins.

Apoferritins prepared from horse spleen and heart and rat heart and liver were dissociated by treatment with acetic acid (pH 1.3-3.0). Sedimentation velocity studies showed that apoferritins of spleen and liver (16-17 S) and heart (18-19 S) dissociated into material sedimenting near 3.2 S. Sedimentation equilibrium measurements determined that most of the material had a molecular weight of 38,000-43,000, corresponding to subunit dimers. Failure to dissociate into subunit monomers was confirmed by gel chromatography on Sephadex G-75 and G-150. With the exception of boiling in sodium dodecyl sulfate, further treatments with 0.1-0.4 M KCl, NaCl, 4-9 M urea, 0.01-0.5 M KSCN, 0.1-0.5% Triton X-100, 5-52% dimethylsulfoxide, 10% ethylene glycol, or 0.1% trifluoroacetic acid all failed to cause dissociation into individual subunits, as did exposure to 6 M guanidine-HCl or formic acid, or prior succinylation and/or nitration of the protein. Reassociation occurred between pH 4 and 7 but was not aided by the addition of Fe(II) or reducing agents. It is concluded that ferritins readily dissociate to subunit dimer units and that further dissociation does not occur without full denaturation of the protein.

Animals↗
PubMed1986

Binding and uptake of copper from ceruloplasmin.

Specific binding of [67Cu]ceruloplasmin to plasma membrane containing preparations from rat tissues was shown in the presence of an excess of nonradioactive Cu(II) or ceruloplasmin. With Cu(II) there was positive cooperativity and an apparent KD of 10(-7) M. The effects of both "cold" ligands was partly additive. No "specific" binding was shown with Zn(II), unrelated proteins and after boiling the membranes. Total and specific binding of [67Cu]ceruloplasmin were 2-7 fold greater for heart and brain than for liver preparations, per g tissue or per mg protein, +/- correction for yield of 5'-nucleotidase. Cu(II) also inhibited uptake of [67Cu] from ceruloplasmin by CHO cells, but monensin did not, suggesting uptake of ceruloplasmin Cu occurs at the cell surface.

Animals↗
PubMed1986

Non-ferritin, non-heme iron pools in rat tissues.

Concentrations of intracellular, low molecular weight (LMW) and desferrioxamine B (DF) chelatable Fe, in tissues of normal, Fe-deficient and Fe-loaded female rats, were determined. Ice cold, high speed supernatants were rapidly fractionated on Ultrogel AcA202 or by filter centrifugation. After correction for residual blood and DF effects on Fe proteins, liver, kidney, heart and spleen contained 3-8 micrograms/g LMW Fe, brain 20 micrograms/g, with DF; two-thirds of this was detected without DF. There was little variation with Fe status. MW standardization and fractionation on Sephadex G-25 indicated components of apparent MW 13,000, 1400 and 350; the latter two were rapidly labeled with in vivo 59Fe.

Animals↗
PubMed1986

Turnover and excretion of copper in rats as measured with 67Cu.

We have examined the whole-body turnover and excretion of copper in rats, using 67Cu. Female Fischer rats, 3-4 mo of age, were injected intraperitoneally with tracer doses of 67CuCl2, and whole-body radioactivity was monitored over the next 3-6 days. The rate of loss was exponential and biphasic, with a half-life of 67 h for the first 70 h, followed by a much slower rate over the following days. A small percentage (2-7%) of the early loss was in the urine, the rest in the feces. In sexually immature animals, turnover was slower in the first and faster in the second phases. Pretreatment of rats with a large dose of copper had no significant effect on the initial turnover rate. Ligation of the common bile duct cut the rate in half, but did not block turnover. It is concluded that there are at least two pools of copper in the rat that turn over at different rates and that excretion of copper occurs via the bile and also by other intestinal routes.

Animals↗
PubMed1985

Copper transport in rats involving a new plasma protein.

The time course of distribution of high-specific activity 67CuCl2 to tissues and plasma components was followed in adult, female rats. Immediately after intubation or injection, tracer 67Cu associated with two components of the blood plasma separable on columns of Sephadex G-150: albumin and another (larger) component, which was not ceruloplasmin. The latter, tentatively named transcuprein, had an apparent molecular weight of 270,000 and a high affinity for Cu2+, as judged by processing through Chelex-100, dilution, and exchange with albumin copper, in vitro and in vivo. It was capable of donating copper to tumor cells in serum-free medium. Analysis of "cold" plasma by furnace atomic absorption confirmed the presence of 10-15% of plasma copper in this peak. Plots of percent dose and 67Cu specific activity against time showed that copper followed a very specific pathway after binding to albumin and transcuprein, entering mainly the liver, then reappearing in the plasma on ceruloplasmin, and then achieving peak distribution in peripheral tissues (muscles, brain, etc.). 67Cu disappeared from liver and kidney with an apparent half-life of 4.5 days, the same exponential rate found for whole body turnover. Apparent turnover of ceruloplasmin copper was more rapid. Even after 7-12 days, tracer copper in plasma was still found exclusively with ceruloplasmin. The results indicate that copper follows a carefully prescribed path, on entering the blood and binding to a new transport protein.

Animals↗
PubMed1985

Distribution of copper among components of human serum.

We examined the distribution of copper among four components of human serum separated by chromatography on Sephadex G-150 and Affi-gel blue. Analysis of copper by furnace atomic absorption indicated that normal adults have copper at an average of 600 ng/ml associated with ceruloplasmin; at 120 ng/ml with transcuprein, a new copper transport protein; at 150 ng/ml with albumin; and at 90 ng/ml with one to three components of low molecular weight (less than 30,000). Cancer patients had more total copper but similar proportions in the four serum fractions. In both groups, some individuals had very high levels of copper in transcuprein, albumin, and/or one or more components of the low-molecular-weight fraction. The results showed that, contrary to earlier conclusions, ceruloplasmin copper only comprised about 60% of the total in human serum; and not just ceruloplasmin but also other forms of serum copper may be elevated in cancer patients.

Adult↗
PubMed1984

Cellular location of rat muscle ferritins and their preferential loss during cell isolation.

Heart and other muscles of the rat contain two forms of ferritin separable in polyacrylamide gel electrophoresis. The cellular location of the fast- and slow-migrating ferritins was investigated using primary cultures of hindlimb skeletal muscle, and isolated myocardial cell populations. Muscle and non-muscle cells were isolated in good yield from hearts of adult rats pretreated with large doses of iron to increase their ferritin content. In virtually all cases, the isolated muscle cells contained traces only of the fast-migrating species and the non-muscle cells contained small amounts of the slow-migrating ferritin. During cell isolation, 90-100% of both ferritins was lost and could be recovered in the perfusates and solutions employed, while one third of the total tissue protein, and a larger percentage of creatine phosphokinase, was recovered in the isolated cells. Primary cultures of thigh muscle from adult rats which had differentiated into multi-nucleated myotubes, were incubated for 1-3 days with chelated iron. These cells contained substantial amounts of the electrophoretically fast migrating ferritin, with its characteristic larger Stokes' radius (determined by quantitative polyacrylamide gel electrophoresis). None of the slow-migrating ferritin species was detected, although hindlimb muscle from iron-treated rats contained both forms. It is concluded that the fast-migrating ferritin of muscle, which is much larger and more asymmetric than other ferritins, is confined to the muscle cell population, while the other form is predominantly or exclusively in the non-muscle cells. Both ferritins are lost preferentially over other proteins during procedures which injure muscle tissue.

Animals↗
PubMed1983

Effects of CO2 exposure on distribution of various forms of iron and copper in guinea-pig tissues.

The effects on iron and copper distribution and metabolism of exposure to high levels of CO2 were studied in the guinea-pig. Mature, male animals were placed in an atmosphere of 15% CO2, 21% O2 (balance N2), and sacrificed from 1 h to 1 week thereafter. Total iron and copper concentrations of blood, liver, spleen and bone, as well as concentrations of heme and ferritin iron, were measured together with blood hematocrit, reticulocytes, plasma hemoglobin, plasma ceruloplasmin and copper concentrations. The results show clearly that rapid and sustained red cell damage or hemolysis ensued several h from the start of CO2 treatment. This resulted in loss of iron and copper from the blood, an influx of both elements into liver, spleen and bone, and a rise in plasma ceruloplasmin. Influx of iron into liver and spleen caused an accumulation of ferritin, the main site for iron storage in cells. Following the effect on red cells, there was an accumulation of heme iron, and a decreased hematocrit, best explained by a depressed activity of the reticuloendothelial and erythropoietic systems. A period of adaptation succeeded these events, in which all blood parameters and most tissue values returned to normal, despite the continuing presence of high CO2. The only changes not reversed were the elevations in liver, spleen and bone iron stores. These remained high, with a net accumulation of greater than 2 mg iron, or 3-4 times more than originally present. The results indicate that at least in the guinea-pig, high CO2 exposure results in red cell damage and other events leading to an accumulation of additional iron in the body; also, that iron accumulated as ferritin and hemosiderin in liver and spleen may not be readily available to restore blood hemoglobin concentrations on an acute basis.

Animals↗
PubMed1983

Interactions of pH and ascorbate in intestinal iron absorption.

Iron absorption is frequently studied experimentally in animals by placing iron directly into tied-off intestinal segments in vivo, usually in acid solutions. We have monitored the changes in pH that occur in the intestinal lumen when acid iron solutions are administered and have related this to the time course of iron absorption in iron-deficient rats. Within 5 minutes of giving of 5.6 micrograms Fe (FeCl3), in 0.50 ml 0.01 M HCl, 0.9% NaCl, the pH of the lumen had climbed above pH 4, where Fe3+ is insoluble; reached 6.8 by 10-15 minutes. In parallel with rising pH, mucosal Fe uptake ceased by 5 minutes after its administration. Addition of 2 mM ascorbate to the same solution prevented the cessation of iron uptake despite the usual pH rise. Intestinal fluid of fasted rats had a limited buffering capacity from pH 7 to 3. When added to this fluid, 59FeCl3 largely precipitated, but microgram quantities bound to components with apparent molecular weights greater than 25,000. This iron was available to desferrioxamine B. The results demonstrate the dramatic effects of pH on iron bioavailability, in the absence of chelators such as ascorbate, and the importance of considering iron solubility in measurements of iron absorption.

Animals↗
PubMed1982

Concentration, structure and iron saturation of ferritins from normal human lung and lung tumors with graded histopathology.

Samples of graded, human lung cancer and of normal lung were assayed for total iron, ferritin, and ferritin iron saturation. Both kinds of tissues contained highly variable amounts of total and ferritin iron and had a range of ferritin iron:protein ratios. No quantitative correlations were found between cancer histopathology and these parameters, in contrast to previous findings for transplantable rat hepatomas. Examination of pooled ferritins isolated from normal lung and lung tumors by quantitative polyacrylamide gel electrophoresis, isoelectric focusing before and after acid-urea dissociation, and SDS electrophoresis, revealed no structural differences. It is concluded that at least for the human lung, malignancy of the kind examined causes no change in ferritin gene expression, and that ferritin assays would not be useful in the grading or detection of human lung cancer.

Ferritins↗
PubMed1981

Circulating ceruloplasmin is an important source of copper for normal and malignant animal cells.

The relative uptake of copper from ceruloplasmin and non-ceruloplasmin plasma pools, by normal and malignant cells, was investigated in vivo and in vitro, using 64Cu and 67Cu. 1. Most of the copper administered intravenously to normal and tumor-bearing rats was removed within 1 h, a substantial portion entering the liver. There were differences in the apparent avidity of individual tissue for ceruloplasmin vs. ionic copper, but when calculated on the basis of actual microgram absorbed, all showed a preference for ceruloplasmin. 2. Appreciable amount of copper from either source were also absorbed by the tumors, and cultured Ehrlich ascites tumor cells showed a rapid uptake and marked preference for ceruloplasmin over non-ceruloplasmin copper, as did primary rat muscle cell cultures. 3. Ceruloplasmin protein was also absorbed by normal and neoplastic rat tissues, but less rapidly than ceruloplasmin copper, as determined by administration of pure [3H]leucine- or [125I]ceruloplasmin. Copper deficiency did not accelerate this process. 4. It is concluded that, at least in rat, ceruloplasmin is the preferred plasma source of copper for normal and malignant cells, and that the copper on ceruloplasmin turns over more rapidly than the protein moiety, a finding consistent with its role as a copper transport protein.

Animals↗