DNA triple helix formation at (AT)n tracts.
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Adenine↗
SEARCH · PubMed Health
Results for “Magnesium Compounds”
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Distribution and properties of Na + ,K + -ATPase in human post-mortem kidney and the effects of diuretics.
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Adenosine Triphosphatases↗
The effect of cellulose phosphate on plasma and urinary magnesium at different levels of parathyroid function in man.
1. Previously published data obtained by magnesium infusion in man were found to conform to a Tm/glomerular filtration rate (GFR) model on the assumption of 80% diffusibility of plasma magnesium. The lower limit of Tm,Mg/GFR was 625 mumol/l. 2. Previously published data concerning the effect of cellulose phosphate on magnesium metabolism in normal subjects, patients with latent hypoparathyroidism and patients with primary hyperparathyroidism were found to conform to the same model, with the same limit for Tm,Mg/GFR for all three levels of parathyroid function. 3. The threshold for magnesium excretion is sharper with less 'splay' than for phosphate, but as for phosophate it is close to the normal blood concentration. 4. Because of the geometrical relationship between different methods of presentation of data, at a constant value for Tm,Mg/GFR changes in magnesium load or in GFR automatically produce changes in fractional magnesium clearance. This is the explanation for the increase in fractional magnesium clearance which occurs which with diminishing renal function. 5. Renal conservation of magnesium is a passive consequence of the fall in plasma magnesium. There was no evidence of augmented tubular reabsorption of magnesium in response to magnesium deprivation in any of the three groups of subjects. 6. The tubular reabsorption of magnesium was not altered detectably by a moderate deficiency or excess of parathyroid hormore. Changes in parathyroid hormone secretion are probably not concerned in normal magnesium homeostasis.
Biological Transport, Active↗
Optimization of submerged culture conditions for the mycelial growth and exo-biopolymer production by Cordyceps militaris.
AIMS: The objective of the present study was to determine the optimal culture conditions for exo-biopolymer production by Cordyceps militaris in shake flask culture. METHODS AND RESULTS: The optimal temperature and initial pH for both mycelial growth and exo-biopolymer production by Cordyceps militaris in shake flask culture were found to be 20 degrees C and 6.0, respectively. Sucrose (40 g x l(-1)) and corn steep powder (10 g x l(-1)) were the most suitable carbon and nitrogen source for both mycelial growth and exo-biopolymer production. CONCLUSION: Under optimal culture conditions, the maximum exo-biopolymer concentration in a 5-l jar fermenter indicated 10.3 g x l(-1), which was approximately three times higher than that in shake flask culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This process can have a significant impact on the industrial scale when sucrose and corn steep powder were used as carbon and nitrogen source.
Carbon↗
Lysosomal enzyme binding to mouse P388D1 macrophage membranes lacking the 215-kDa mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor.
Mouse P388D1 macrophages target newly synthesized acid hydrolases to lysosomes in spite of their lack of the 215-kDa mannose 6-phosphate (Man-6-P) receptor. We now report that these cells contain a membrane-associated Man-6-P receptor that is distinct from the previously described receptor. The new receptor binds lysosomal enzymes containing phosphomannosyl residues. This binding is inhibited by Man-6-P or by pretreatment of the lysosomal enzymes with alkaline phosphatase. Lysosomal enzyme binding occurs at neutral pH and dissociation of the bound ligand occurs at low pH values comparable to those found within endosomes or lysosomes. The new receptor differs from the 215-kDa Man-6-P receptor in two ways. It has an absolute requirement for divalent cations and is unable to bind Dictyostelium discoideum lysosomal enzymes, which contain methylphosphomannosyl residues rather than the usual phosphomannosyl monoesters. Based on the difference in cation requirement, we suggest that the 215-kDa receptor be referred to as Man-6-P receptor CI (cation independent) and the new receptor as Man-6-P receptor CD (cation dependent). We conclude that the Man-6-P receptor CD functions in the targeting of newly synthesized acid hydrolases to lysosomes in P388D1 macrophages.
Animals↗
DNA condensation in two dimensions.
We have found that divalent electrolyte counterions common in biological cells (Ca(2+), Mg(2+), and Mn(2+) ) can condense anionic DNA molecules confined to two-dimensional cationic surfaces. DNA-condensing agents in vivo include cationic histones and polyamines spermidine and spermine with sufficiently high valence (Z) 3 or larger. In vitro studies show that electrostatic forces between DNA chains in bulk aqueous solution containing divalent counterions remain purely repulsive, and DNA condensation requires counterion valence Z >/= 3. In striking contrast to bulk behavior, synchrotron x-ray diffraction and optical absorption experiments show that above a critical divalent counterion concentration the electrostatic forces between DNA chains adsorbed on surfaces of cationic membranes reverse from repulsive to attractive and lead to a chain collapse transition into a condensed phase of DNA tethered by divalent counterions. This demonstrates the importance of spatial dimensionality to intermolecular interactions where nonspecific counterion-induced electrostatic attractions between the like-charged polyelectrolytes overwhelm the electrostatic repulsions on a surface for Z = 2. This new phase, with a one-dimensional counterion liquid trapped between DNA chains at a density of 0.63 counterions per DNA bp, represents the most compact state of DNA on a surface in vitro and suggests applications in high-density storage of genetic information and organo-metallic materials processing.
Bacteriophage lambda↗
Identification of amino acids in the CD11a I-domain important for binding of the leukocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1)
Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhesion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I-domain of the LFA-1 alpha subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I-domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding. In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ in human and murine I-domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated. Substitution of alanine for proline at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this was due to conformational changes within the I-domain. Mutation of the aspartic acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1.
Animals↗
Cloning, purification, and properties of a novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic domain in MutT-like enzymes.
An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (M(r) = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 mumol min-1 mg-1 mM-1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Km for NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5'-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 microM.
Amino Acid Sequence↗
An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer.
Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the 3'-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved "DD35E" motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.
Amino Acid Sequence↗
Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity.
Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP. This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions. PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %). The specificity constant (Vmax/Km) for DGPP was 1. 3-fold higher than that of PA. DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 showed homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.
Amino Acid Sequence↗
The integrin alpha1 A-domain is a ligand binding site for collagens and laminin.
The integrin alpha1beta1 is a cell surface receptor for collagens and laminin. The alpha1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the alpha1 A-domain in ligand binding and the extent to which it reproduced the ligand binding activity and specificity of the parent molecule, we produced recombinant alpha1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from alpha1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The alpha2 A-domain, from the alpha2beta1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for alpha1. This is the first demonstration of laminin binding by A-domains. Specificity of A-domain-ligand binding was further investigated using the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4. alpha1 A-domain bound to all three fragments, while the alpha2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the first time identify a limited proteolytic fragment of a ligand that contains integrin A-domain binding activity. To gain insight into the contribution that the A-domain makes to ligand binding within the whole integrin heterodimer, we measured binding constants for A-domain-collagen interactions using surface plasmon resonance biosensor technology. The values obtained were similar to those reported for intact integrin binding, suggesting that the A-domain is the major collagen binding site in the alpha1beta1 and alpha2beta1 integrins.
Animals↗
A GTP-dependent vertebrate-type phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis.
This is the first report on a bacterial verterbrate-type GTP-dependent phosphoenolpyruvate carboxykinase (PCK). The pck gene of Mycobacterium smegmatis was cloned. The recombinant PCK was overexpressed in Escherichia coli in a soluble form and with high activity. The purified enzyme was found to be monomeric (72 kDa), thermophilic (optimum temperature, 70 degrees C), very stable upon storage at 4 degrees C, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by alpha-ketoglutarate. The requirement for a divalent cation for activity was fulfilled best by Mn(2+) and Co(2+) and poorly by Mg(2+). At 37 degrees C, the highest V(m) value (32.5 units/mg) was recorded with Mn(2+) and in the presence of 37 mm dithiothreitol (DTT). The presence of Mg(2+) (2 mm) greatly lowered the apparent K(m) values for Mn(2+) (by 144-fold in the presence of DTT and by 9.4-fold in the absence of DTT) and Co(2+) (by 230-fold). In the absence of DTT but in the presence of Mg(2+) (2 mm) as the co-divalent cation, Co(2+) was 21-fold more efficient than Mn(2+). For producing oxaloacetate, the enzyme utilized both GDP and IDP; ADP served very poorly. The apparent K(m) values for phosphoenolpyruvate, GDP, and bicarbonate were >100, 66, and 8300 micrometer, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 12 microm, respectively. Thus, this enzyme preferred the gluconeogenesis/glycerogenesis direction. This property fits the suggestion that in M. smegmatis, pyruvate carboxylase is not anaplerotic but rather gluconeogenic (Mukhopadhyay, B., and Purwantini, E. (2000) Biochim. Biophys. Acta. 1475, 191-206). Both in primary structure and kinetic properties, the mycobacterial PCK was very similar to its vertebrate-liver counterparts and thus could serve as a model for these enzymes; examples for several immediate targets are presented.
Amino Acid Sequence↗
Intermittent and continuous faecal markers in short-term metabolic balance studies in young women.
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Adult↗
The effect of calcium intake on bone composition and bone resorption in the young growing rat.
A low Ca intake by both rats and man increases bone resorption, decreases bone mass and increases the risk of osteoporosis. The skeletal effect of high Ca intakes is less clear, particularly during periods of bone mineral accrual. Twenty-four female 5-week-old rats, Wistar strain, were randomized by weight into three groups of eight rats each and fed ad libitum a semi-purified diet containing 2 (Ca-restricted), 5 (normal) or 20 (Ca-supplemented) g Ca/kg for 3 weeks. When compared with the normal Ca diet, urinary Ca excretion was unaffected by the dietary restriction of Ca for 3 weeks, but was greater (P<0.001) in Ca-supplemented rats. Urinary pyridinoline (Pyr) and deoxypyridinoline (Dpyr) levels were significantly greater during weeks 2 (Pyr P<0.05, Dpyr P<0.001) and 3 (Pyr P<0.01, Dpyr, P<0.001) of dietary Ca restriction, but were unaffected by Ca supplementation. Femoral dry weight and the concentration of Mg and P in femora were unaffected by dietary Ca concentration. Femoral Ca concentration was reduced (P<0.05) in the Ca-restricted group compared with the other two groups. In conclusion, these results suggest that increasing dietary Ca intake, well above the recommended level, had no effect on bone mineral composition or bone resorption (as assessed with urinary pyridinium crosslinks) in young growing female rats. In addition, these results confirm the findings of previous studies which have shown that bone Ca content in young growing rats was reduced by dietary Ca restriction and that this reduction results, at least in part, from an increased rate of bone resorption.
Amino Acids↗
Time courses of plasma magnesium concentrations and urinary magnesium excretion in cows subjected to acute changes in potassium intake.
Hypomagnesaemic tetany in cows develops occasionally after an acute increase in K intake such as can occur when cows are transferred to spring grass. There is evidence that under these conditions plasma Mg concentrations are only transiently decreased. In this study the questions addressed were whether the plasma Mg concentration, indeed adapts to a high K intake as only dietary variable, and whether urinary Mg excretion is associated with this adaptation. Dry cows were fed rations containing either 26 or 50 g K/kg dm, the extra K being in the form of KHCO3. When the cows were acutely transferred from the low to the high K ration, plasma Mg concentrations fell slightly, but significantly from 0.86 to 0.76 mmol/l within five days, but rose again to 0.80 mmol/l after another 23 days, this rise being also statistically significant. None of the animals developed tetany. The decrease in plasma Mg concentration in individual animals after five days on the high-K ration ranged from 6 to 21%. The time course of urinary Mg excretion resembled that of plasma Mg concentration; minimum Mg excretion was seen after four to six days on the high-K ration with a subsequent increase thereafter. To explain the transient lowering of plasma Mg concentration, it is suggested that the K-induced decrease in Mg status caused a delayed increase in the carrier-mediated component of Mg absorption, which in turn caused an increase in urinary Mg excretion. When the cows were acutely switched from the high to the low K ration, plasma Mg concentration and urinary Mg excretion rose, but no transient changes were seen.
Adaptation, Biological↗
Celecoxib-loaded poly(D,L-lactide-co-glycolide) nanoparticles prepared using a novel and controllable combination of diffusion and emulsification steps as part of the salting-out procedure.
A novel procedure for the manufacture of celecoxib-loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles is described that is based upon combining salting out and emulsion-evaporation steps. An entrapment efficiency, a measure of the actual to theoretical drug content, of 97.3% was achieved, being superior to that achieved when these popular techniques were used separately (emulsion evaporation, 40.1%; salting out, 10.0%). The ratio of a water miscible solvent (acetone) to a non water-miscible solvent (dichloromethane) was shown to be the primary determinants of size and drug loading. Once optimized, using an organic phase of 3 : 1 acetone : dichloromethane vol : vol ratio, further control on particle parameters could be exerted using modification of acetone diffusion by alterations in MgCl2 x 6H2O concentration. This step was shown to have a small effect on both the mean nanoparticle size and entrapment efficiency, but found to reduce the polydispersity considerably. Diffusion control using a 45% w/v MgCl2 x 6H2O solution produced nanoparticles with a mean size of 151.4 nm, a polydispersity index of 0.023 and 98.1% entrapment efficiency. Electron microscopy showed the particles to be smooth and spherical. Sheer homogenization during the emulsification step was shown to be not as effective as sonication, with the latter technique able to produce nanoparticles after 1 min of application. Drug release studies across a semi-permeable membrane demonstrated a reduction in the burst effect as the ratio of acetone in the organic phase was increased. Calorimetry studies suggested that celecoxib existed in the nanoparticle as a molecular dispersion, with additional evidence for a strong interaction between the PLGA and the absorbed poly(vinyl alcohol) stabilizer. Formation of a strong interaction between celecoxib and PLGA, together with the formation of a radial drug gradient give a release profile that does not possess the prevalent burst effect seen with other nanoparticulate drug-loaded systems.
Acetone↗
Mapping tRNA and 5S RNA tertiary structures by charge dependent Fe(II)-catalyzed cleavage.
Chemical and enzymatic footprinting experiments have made it possible to identify protein binding sites in DNA and RNA, and to localize structural differences within nucleic acids to a resolution of a single base pair. We show here that by combining three reagents, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, differential maps of sites in RNA that vary in their local conformation and/or charge can be constructed. Comparison of profiles with respect to controls in the absence of a counterion such as Mg2+ allows analysis of sites responsive to tertiary structure. A single site that is labile to metals such as Pb2+ exists in tRNA(Phe) and a number of other tRNA's; this site is hyper-reactive to Fe(II), but not to the other probes. Scission induced by the neutral complex, Fe(II).EDDA, offers the most general measure of surface accessibility, since its distribution about the target molecule is insensitive to charge. Enhanced cleavage by Fe(II) relative to the other agents is detected at several adjacent sites in 5S RNA, consistent with conformational mobility. Protection at a series of positions in the arm formed by loops E and D with helix IV suggests further that at low temperature this arm interacts with loop A and helix I.
Base Composition↗
Successful management of primary hypomagnesaemia with high-dose oral magnesium citrate: a case report.
UNLABELLED: We report a 40-d-old female infant who presented to our clinic with afebrile generalized convulsive episodes due to severe hypomagnesaemia with secondary hypocalcaemia. Laboratory investigations revealed mild hypoparathyroidism, which was to return normal following magnesium (Mg) treatment, and normal fractional renal excretion of Mg (0.8%). After the diagnosis of primary hypomagnesaemia was established, the patient was discharged with oral Mg subcarbonate and intramuscular Mg sulphate. On regular follow-up until the age of 4 y, the child was asymptomatic. Mean serum calcium levels remained normal, while mean serum Mg levels remained subnormal. During this 4-y period, oral Mg dose was gradually increased while the doses and frequency of administration of parenteral Mg were decreased. Additionally, oral Mg subcarbonate was switched to Mg citrate because of its side effects. Finally, parenteral Mg was discontinued as the dosage of oral Mg supplementation reached a level of 90 mg/kg/d elemental Mg citrate, without any gastrointestinal side effects. Some screening tests were performed to evaluate the complications of chronic hypomagnesaemia. Bone age, bone densitometry, ECG and renal sonography were all normal. Our patient is now 5 y old and symptom free. CONCLUSION: Treatment with high doses of oral Mg was successful in keeping our patient symptom free and normocalcaemic, but cannot fully normalize serum Mg concentrations. Thus, during the regular follow-up of patients with primary hypomagnesaemia, the main target must be to increase oral Mg supplementation to a dosage which can maintain normocalcaemia rather than normomagnesaemia.
Administration, Oral↗