Studies of the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one in thalassemia patients.
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Administration, Oral↗
Biomedical subjects
D M Templeton
Publications and source records attributed to D M Templeton.
Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry. I. Preliminary considerations and analysis of rat liver and serum.
Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.
Animals↗
Retention of glomerular basement membrane-proteoglycans accompanying loss of anionic site staining in experimental diabetes.
The occurrence of heparan sulfate proteoglycan anionic site staining in the glomerular basement membranes of healthy and streptozotocin-induced diabetic rats was investigated. After 12 weeks diabetes, a marked loss of staining by ruthenium red was observed, compared with the age-matched controls. However, extraction and quantitation of total proteoglycan from the glomerular basement membrane revealed no significant differences between the two groups, in terms of absolute yield of glycosaminoglycan, when the latter is expressed per glomerulus. Glomerular basement membranes from both groups become thicker in an age-dependent manner, although they do so at a faster rate in the diabetic group, and a higher collagen content is found in the diabetic membrane at 12 weeks. Thus when proteoglycan content is expressed per mass protein, relative decreases are observed in diabetes. Similarly, a relative but not absolute decrease in proteoglycan content was observed in glomerular basement membranes from spontaneously diabetic BB rats with diabetes of 12 weeks duration. Glycosaminoglycans isolated from control and diabetic glomerular basement membrane have similar characteristics on ion exchange chromatography, to each other, and to newly sulfated, intact glomerular proteoglycans labelled in vitro. We conclude that loss of anionic site staining with ruthenium red in early diabetes is not a consequence of absolute loss or undersulfation of heparan sulfate proteoglycan but reflects structural or biochemical changes in the diabetic matrix.
Animals↗
Comparative studies of glutathione reductase and lipoamide dehydrogenase.
1. Glutathione reductase and lipoamide dehydrogenase are structurally and mechanistically related flavoenzymes catalyzing various one and two electron transfer reactions between NAD(P)H and substrates with different structures. 2. The two enzymes differ in their coenzyme and functional specificities. Lipoamide dehydrogenase shows higher coenzyme preference while glutathione reductase displays greater functional specificity. 3. Binding preference of the two flavoenzymes for nicotinamide coenzymes is demonstrated by 31P-NMR spectroscopy. 4. The presence of arginines in glutathione reductase which is inactivated by phenyl glyoxal, is likely to be responsible for the NADPH-activity of glutathione reductase. 5. The substrate binding sites of the two enzymes are similar, though their functional details differ. 6. The active-site histidine of glutathione reductase functions primarily as the proton donor during catalysis. While the active-site histidine of lipoamide dehydrogenase stabilizes the thiolate anion intermediate and relays a proton in the catalytic process.
Animals↗
Synthesis of heparan sulfate proteoglycans by the isolated glomerulus.
Incorporation of [35S]sulfate into newly synthesized macromolecules was studied in the isolated rat glomerulus and found to be linear between 6 and 24 h. When whole glomeruli were treated under conditions that dissociate proteoglycan aggregates, greater than 90% of incorporated label was extracted. Of this, 80-90% was found to be the heparan sulfate proteoglycan. Similarly, a linear incorporation of [35S]sulfate into a glomerular basement membrane-enriched fraction was due almost entirely to proteoheparan sulfate. This predominance of heparan sulfate among the newly sulfated glycosaminoglycans has previously been observed in vivo and in the perfused kidney, but different patterns have hitherto been described in vitro. The present results suggest that under certain conditions, the isolated glomerulus is a suitable in vitro model for the study of proteoglycan synthesis. The pattern of incorporation of proteoglycans into the glomerular basement membrane reflects the time course and distribution of their synthesis by the whole glomerulus.
Animals↗
The basis and applicability of the dimethylmethylene blue binding assay for sulfated glycosaminoglycans.
A rapid spectrophotometric assay for sulfated glycosaminoglycans, based on reaction of these compounds with the dye 1,9-dimethylmethylene blue (DMMB), is gaining widespread acceptance. The limitations of the assay have not been explored, and we have investigated the absorption spectra of the dye in the presence of a variety of compounds in order to understand the mechanism of colour formation. The dye exhibits classical metachromasia in the presence of sulfated glycosaminoglycans, but appears to do so by a much simpler mechanism than other thiazine dyes such as Azure A. Evidence is offered to support the conclusion that dye dimer reacts with the polyanion to produce a single metachromatic species by ionic perturbation of the chromophore. As predicted, factors which disrupt dimerization, such as pyridine and pyridine nucleotides, preclude use of the assay in their presence, and high salt concentrations can give false positive readings by inducing metachromasia in the absence of polyanion. When these factors are understood, the assay can be used with convenience. In particular, it allows direct measurement of the glycosaminoglycan content of a few mg of alkaline-solubilized basement membrane.
Animals↗
Metal-binding properties of the isolated glomerular basement membrane.
The nature of binding of metal cations to the glomerular basement membrane has been investigated using isolated bovine glomerular basement membrane. Highest-affinity binding for a number of ions is attributable to the glycosaminoglycans (mostly heparan sulfate) of the membrane. Some ions, such as divalent Mn, Ca and Ni, have specific binding sites on these polymers, while for others the ion-polyelectrolyte interaction is of a non-specific nature. Both structural and binding data indicate a linear charge density of close to unity for the heparan sulfate of the glomerular basement membrane, which at the ionic composition of the plasma filtrate corresponds to a polymer surface potential of about -45 mV. Several independent observations are better explained by a model of counter-ion condensation about the glycosaminoglycans than by conventional double layer theories. These include the valence dependence of ion binding, the sharp ejection of divalent ions at a critical concentration of La3+, and the relative insensitivity of 63Ni2+ binding to NaCl concentration in the neighbourhood of physiological ionic strength. In its interactions with metal ions, the glomerular basement membrane behaves like a dilute solution of polyelectrolytes. This conclusion has important consequences for the extent of charge reduction of the filtration barrier of the kidney, bathed as it is in an electrolyte solution of mainly monovalent salts.
Animals↗
Biosynthesis and metabolism of metallothionein in rat during perinatal development.
Although high concentrations of zinc (Zn) and metallothionein (MT) have been detected in the livers of many newborn mammals, the factors affecting the biosynthesis and metabolism of MT are not yet clearly defined. In rat liver, MT levels rise sharply at the 18th day of gestation and continue to increase into the neonatal period. In late gestation, MT serves to bind Cu and Zn from the pre-existing pools of these metals, as well as to accumulate additional amounts of Zn. The high levels of Zn and MT are maintained for about two weeks in neonates and then decreased to adult levels at weaning. Perinatally, the Zn/MT ratio remains constant for several days, suggesting a carefully regulated process. Injection of ZnSO4 into newborn rats results in increased accumulation of Zn and induced synthesis of MT in liver, while injection of CdCl2 has no effect. When pregnant rats are fed with a Zn-D diet, there are marked reductions in the levels of both hepatic Zn and MT in neonates. Similar studies with copper and iron deficient diets have no effect on high hepatic MT and Zn levels in newborn rats. Immunohistochemical localization of MT using a specific antibody to MT shows a progressive increase in cytoplasmic MT with the appearance of nuclear MT by day 20 of gestation in fetal rat liver. The presence of MT in the nucleus of hepatocytes is maintained in neonates for several days and later MT is detected in the cytoplasm during postnatal development.
Animals↗
Interaction of toxic cations with the glomerulus: binding of Ni to purified glomerular basement membrane.
Basement membrane was prepared from the glomeruli of bovine kidneys using either detergent extraction, sonication or trichloroacetic acid (TCA) treatment. An assay was developed to measure the binding of radiolabelled metal salts to particulate suspensions of the membrane. 63Ni bound to the anionic glycosaminoglycan (GAG) sites of the membrane. This binding could be blocked by the cationic dye ruthenium red, and was sensitive to treatment with heparitinase but not to mild collagenase digestion. At pH 7.4 at low ionic strength (5 mM Tris-HCl), a high affinity (Ka = 4.5 X 10(6) M-1) binding site was distinguished. It was insensitive to increasing salt concentration, but was abolished by desulfation of the basement membrane preparation. 54Mn showed a similar binding pattern to Ni, while 65Zn and 109Cd lacked the high affinity site. In all cases the bulk of binding was of lower affinity and was of a non-specific electrostatic nature. Most, but not all, was abolished by salt concentrations comparable to those of the plasma filtrate (140 mM NaCl). These results are discussed in the context of sensitivity of the glomerular charge barrier to toxic divalent ions.
Amino Acids↗
Nickel binding to the C-terminal tryptic fragment of a peptide from human kidney.
In kidney the nickel ion exists primarily as soluble cytoplasmic complexes. We have recently identified a major component of these complexes in the human kidney as a Ni(II) complex of a low molecular weight anionic peptide (Templeton, D.M. and Sarkar, B. (1985) Biochem. J. 230, 35-42). We have now purified a small amount of this peptide to homogeneity and developed an HPLC technique to study its metal-binding properties on sub-nanomole quantities. We are able to demonstrate a binding stoichiometry of one Ni atom per molecule of peptide, with an apparent dissociation constant of 1.1 X 10(-5) M. A similar site exists for Cd. The site for Ni persists after trypsinization, and is localized in the 20-residue C-terminal tryptic fragment of the peptide.
Amino Acids↗
The reaction of metallothionein with mercuribenzoate. A dialysis and 113Cd-n.m.r. study.
Reaction of rat liver cadmium-metallothionein-II(Cd-MT-II) with p-hydroxymercuribenzoate(pHOHgBzO-) causes displacement of bound Cd. When pHOHgBzO- -induced displacement of 109Cd is observed after dialysis of the reaction mixture, the stoichiometry is consistent with stepwise displacement of tetraco-ordinate Cd atoms by non-random entry of reagent into the polynuclear clusters. 113Cd n.m.r. allows direct observation of the effects on bound Cd of stepwise titration of 113Cd-MT-II with pHOHgBzO-. The first equivalent reduces all resonances approximately equally. Subsequently differential reactivity of the protein thiolates towards the reagent gives rise to differential decreases in the 113Cd signal intensities. Resonances previously attributed to a three-metal cluster are lost before those arising from the four-metal cluster. These results are interpreted in terms of current models of the MT structure. They are distinct from the results of reaction of MT with 5,5'-dithiobis-(2-nitrobenzoic acid), which distinguishes between only two classes of thiolates, terminal and bridging. Such different patterns of reactivity of the protein thiolates may underlie a biological activity of this protein.
Cadmium↗
Low molecular weight targets of metals in human kidney.
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Animals↗
Peptide and carbohydrate complexes of nickel in human kidney.
The predominant renal and urinary forms of nickel consist of low-Mr complexes. Similarities in the nature of these complexes have been found in kidneys of rats exposed parenterally to NiCl2 and in rat kidneys treated with NiCl2 in vitro. Similar complexes have also been identified after treatment of bovine and human renal soluble fractions with NiCl2. The bulk of nickel in all cases is associated with sulphated oligosaccharide fractions containing uronic acids and neutral sugars. This binding is non-specific, and nickel is readily displaced from these fractions by copper. Smaller amounts of nickel are bound to an acidic peptide, which was purified from human kidneys and partially characterized. Nickel was not displaced from this material by copper at physiological pH. These nickel complexes have not been found in plasma, suggesting that ligand exchange occurs during or after glomerular filtration of the metal.
Amino Acids↗
Kinetic and spectroscopic studies of transhydrogenase activity and nucleotide site of lipoamide dehydrogenase.
The kinetic behavior and spectroscopic characteristics of the nucleotide site(s) of lipoamide dehydrogenase have been investigated. Both subunits of the dimeric enzyme interact with NAD+. The binding of NAD+ is associated with a negative trough around 420-450 nm and a positive peak at 507 nm of the difference spectrum. The transhydrogenation between NADH and thionicotinamide nucleotide or acetylpyridine nucleotide is shown to proceed via a Ping Pong or an ordered Bi Bi mechanism, respectively, at pH above 7.0. Lowering pH or acetamidation lose the spectral characteristic of the positive peak of the enzyme-NAD+ complex with a concurrent change in the kinetic mechanism in the NADH+-acetylpyridine nucleotide transhydrogenation.
Animals↗
Chemical modifications of metallothionein, II. Metabolic fate of cadmium bound to metallothionein polymers.
The factors which determine the deposition of cadmium (Cd) were investigated. While intravenously administered Cd2+ salts result in Cd2+ binding loosely to plasma proteins, and subsequently being deposited in the liver, Cd complexed with metallothionein remains tightly bound during glomerular filtration, and most of the dose of Cd is deposited in the kidney. 2 Different chemically crosslinked polymers of MT have been prepared. Cadmium bound to these polymers remains in circulation longer than any other known form of the metal, and the reticuloendothelial system plays a part in its clearance. Using these species, mechanisms of distribution and metabolism of Cd-protein complexes are discussed.
Animals↗
Metallothionein synthesis and localization in relation to metal storage in rat liver during gestation.
The metallothionein (MT) content of fetal rat liver was measured daily during the final week of gestation, by both cadmium saturation and polarographic methods. MT levels rise sharply at day 18 of gestation and continue to increase into the neonatal period. In late gestation, MT serves to bind Cu and Zn from the preexisting hepatic pools of these metals, as well as to accumulate additional amounts of both metals. The fetal MT is similar to the adult rat protein both in terms of its protein composition and metal-binding properties. Perinatally the Zn/MT ratio remains constant for several days suggesting a carefully regulated process. At birth, most of the hepatic Zn and a significant amount of hepatic Cu are bound to MT. Immunohistochemical localization MT shows a progressive increase in cytoplasmic MT with the appearance of nuclear MT by day 20 of gestation in fetal rat liver. The results are discussed in terms of a model for regulation of MT synthesis and for the metal storage role of MT in perinatal development.
Animals↗
Chemical modifications of metallothionein. Preparation and characterization of polymers.
Reaction of rat liver metallothionein-II with two bifunctional cross-linking reagents, glutaraldehyde and dimethyl suberimidate, produces high yields of polymeric species. It is argued that cross-linking is trapping preformed aggregates of the protein, which therefore represent a stabilized quaternary structure of metallothionein. The two polymeric species differ in a number of respects. With dimethyl suberimidate, the polymer retains all metal-binding sites of the monomer, and has an unaltered isoelectric point. Reaction with glutaraldehyde causes loss of one or two Cd2+/Zn2+-binding sites and elevates the pI. Both species are nearly spherical aggregates, in contrast with the highly asymmetrical metallothionein. Both polymers are linked through lysine residues, and the thiol groups remain reduced. The biological significance of these aggregates is discussed.
Amino Acids↗
Effects of zinc deficiency on pre-existing cadmium-metallothionein in the pancreas.
Although the injection of cadmium salts increases the metallothionein (MT) concentration in a number of organs, including pancreas, the induced synthesis of pancreatic metallothionein was specifically decreased in zinc deficiency. The fate of Cd-induced metallothionein and the change in tissue distribution of Zn and Cd were studied with the onset of Zn deficiency. Rats were repeatedly injected with CdCl2 to induce MT synthesis, and were subsequently made Zn deficient. The lability of the pancreatic Zn pool, as compared with that of liver and kidney, was demonstrated. During Zn deficiency, there was little change in Cd concentration or MT level in either liver or kidney. When Zn deficiency led to a decrease in pre-existing pancreatic MT, Cd was not eliminated from the pancreas, but was transferred to a high molecular weight fraction. On the basis of these results we suggest that the presence of Cd-Zn-MT has little effect on the eventual pancreatic Zn and MT contents, during subsequent Zn deficiency.
Animals↗