Changes in the synthesis of minor cartilage collagens after growth of chick chondrocytes in 5-bromo-2'-deoxyuridine or to senescence

Analyses were made of the minor collagens synthesized by cultures of chondrocytes derived from 14-day chick embryo sterna. Comparisons were made between control cultures, cultures grown for 9 days in 5-bromo-2'-deoxyuridine (BrdU) and clones of chondrocytes grown to senescence. Separation of minor collagens from interstitial collagens was achieved by differential salt precipitation in the presence of carrier collagens in acid conditions. The precipitate at 0.9 M NaCl 0.5 M acetic acid from control cultures was shown by CNBr peptide analysis to contain only the alpha 1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only alpha 1(I) and alpha 2(I) chains occurred. The synthesis of type III collagen was not detected. Analysis of the precipitate at 2.0 M NaCl, 0.5 M HAc from control cultures demonstrated the synthesis of 1 alpha, 2 alpha and 3 alpha chains together with the synthesis of short chain (SC) collagen of Mr 43000 after pepsin digestion. After BrdU treatment or growth to senescence alpha chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the alpha 1(V) and alpha 2(V) chains of type V collagen. In addition, for BrdU-treated but not for control cultures, intracellular immunofluorescent staining was observed with a monoclonal antibody which specifically recognizes an epitope present in the triple helix of type V collagen. Synthesis of short chain (SC) collagen was not detected after BrdU treatment or growth to senescence. These results suggest that chick chondrocytes grown in conditions known to cause switching of collagen synthesis from type II to type I collagen also undergo a switch from the synthesis of 1 alpha, 2 alpha and 3 alpha chains to the synthesis of the alpha 1(V) and alpha 2(V) chains of type V collagen. It appears that there are several cartilage-specific collagens which together undergo a regulatory control to the synthesis of collagens typical of other connective tissues.     

Maternal meal quality alters fetal rat brain serotonin concentrations

The quality of food eaten by pregnant animals may influence fetal neurotransmitter biosynthesis by altering the relative availability of precursor amino acids. Fetal brain concentrations of tryptophan and the neurotransmitter serotonin increase following the consumption of a carbohydrate-fat meal or after the injection of insulin in fasting animals; in contrast, the fetal concentrations of these compounds are reduced in animals whose mothers eat a meal containing casein protein or amino acids which compete with tryptophan for uptake from maternal blood into the placenta and fetus.     

Isolation of a low molecular weight zinc binding ligand from human milk

A low molecular weight zinc binding compound from human milk has been purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Evidence is provided that this compound is citrate. A higher amount of citrate-bound zinc was found in human milk than in cow's milk. It is suggested that the therapeutic value of human milk for patients with the genetic disorder of zinc metabolism acrodermatitis enteropathica (AE) derives from a greater content of bioavailable zinc citrate in human than in cow's milk.     

Developmental patterns of copper and zinc concentrations in mouse liver and brain: evidence that the gene crinkled (cr) is associated with an abnormality in copper metabolism

An abnormality in copper metabolism during both the prenatal and postnatal (preweaning) periods was found to be associated with the autosomal recessive gene ”crinkled“ (cr) in mice. Liver copper concentration was significantly lower in crinkled mice (cr/cr) than in littermate controls (+/?) from 18 days of gestation to 20 days after birth. Crinkled mice older than 20 days of age had liver copper concentrations similar to those of littermate controls. Liver zinc and brain copper and zinc were similar in crinkled and noncrinkled mice at all times tested. In both crinkled and noncrinkled mice, brain copper concentration increased during the suckling period, and liver copper concentration decreased.     

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.     

Equilibrium and structural studies of silicon(IV) and aluminium(III) in aqueous solution. 12. A potentiometric and 29Si-NMR study of silicon tropolonates

Complexation in the H⁺-Si(OH)₄-tropolone (HL) system was studied in 0.6 M (Na)Cl medium at 25° C. Speciation and formation constants were determined from potentiometric (glass electrode) and ²⁹Si-NMR data. Experimental data cover the ranges 1.5 ? - log[H⁺] ? 8.4, 0.002 ? B ? 0.012 M, and 0 ? C ? 0.060 M (B and C stand for the total concentration of Si and tropolone, respectively). In acid solutions (-log[H⁺] ? 3) a hexacoordinated cationic complex, SiL₃⁺, is formed with log K(Si(OH)₄ + 3HL + H⁺ XXX SiL₃⁺ + 4H₂O) = 7.08 ± 0.03. Furthermore, the formation of a disilicic acid was established from ²⁹Si-NMR data. The dimerization constant of Si(OH)₄ was found to be 10 exp (1.2 ± 0.1). In model calculations the solubility of quartz and amorphous SiO₂ in the presence of tropolone is demonstrated. Data were analyzed using the least-squares computer program LETAGROPVRID.     

Metal ion-biomolecule interactions. III. Versatility of metal ion binding to imidazoles. Synthesis and 1H nmr spectroscopic properties of N- and C-bound mercury(II) complexes

Methylmercury(II) and mercury(II) complexes of imidazole (1), 1-methylimidazole (2), and the 1,3-dimethylimidazolium ion (3) have been prepared in aqueous or ethanolic solution. Elemental analysis and ¹H nmr spectroscopy have been used to characterize the complexes. The MeHg (Me = methyl) binding sites have been identified as N₁, N₃ (1), N₃, C₂ (2), and C₂ (3). Reaction with HgO leads to the formation of Hg-bridged complexes of the type Im-Hg-Im, (Im = imidazole), where bonding occurs through N₁ (1) and C₂ (3); the latter is also formed as a result of symmetrization of the C₂-bound MeHg complex. The formation of the C₂-bound (carbene) complexes is discussed in terms of the increased acidity of the C₂ proton resulting from coordination of an electrophilic species at N₃. Based on electrostatic considerations, there appears to be a “minimum degree of activation” required before C₂ bonding can occur, which explains the lack of this coordination mode in 1. ¹⁹⁹Hg-¹H spin-spin coupling (⁴J) is observed for C-bound mercury, but not for N-bound mercury, which is interpreted in terms of a decreased ligand exchange rate in the former case, due to the greater stability of the Hg-C bond. ²J coupling constants measured in (CD₃)₂SO for a number of MeHg complexes of heterocyclic ligands (including the imidazoles of the present study) correlate well with the ligand pK_a (25°C, aqueous solution), according to ²J = ?3.88 pK_a + 248.5. Results in the present work are discussed in relation to our previous work with nucleosides. The significance of the results to biological systems is considered.     

Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.     

High affinity specific [3H] (+)PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart

The binding properties of the 1,4-dihydropyridine calcium channel antagonist, [³H](⁺)PN 200-110, were studied in rat cerebral cortical and cardiac homogenates (37°C, Krebs phosphate buffer). Specific binding of [³H](⁺)PN 200-110 was saturable, reversible, and of high affinity (K_d values are 35 and 64 pM for the cerebral cortex and heart, respectively). In parallel studies with [³H](⁺)PN 200-110, the dissociation constant of [³H]nitrendipine was 10–12 times higher. Substituted dihydropyridine calcium channel antagonists and agonists competitively inhibited specific [³H](⁺)PN 200-110 binding, but d-cis diltiazem enhanced and verapamil incompletely inhibited [³H](⁺)PN 200-110 binding in both the cerebral cortex and the heart. The effects of diltiazem and verapamil on [³H](⁺)PN 200-110 binding were due mainly to alterations in the dissociation constant (K_d), without alterations in the binding density (B_max). The new [³H](⁺)PN 200-110 receptor binding assay is remarkable for its low degree of nonspecific binding as compared to [³H]nitrendipine at physiological temperatures. [³H(⁺)PN 200-110 is a useful ligand for the further analysis of the dihydropyridine binding sites associated with calcium channels.     

Convulsant potencies of tetrazoles are highly correlated with actions on GABA/benzodiazepine/picrotoxin receptor complexes in brain

A series of tetrazole convulsants were examined for their potencies in displacing [35S]-t-butylbicyclophosphorothionate (TBPS) from the picrotoxin site on the benzodiazepine-GABA-chloride ionophore receptor complex. All of the tetrazole derivatives tested inhibited [35S]-TBPS binding from rat forebrain membranes, and except for one (undecamethylenetetrazole), had Hill coefficients near unity. Similar to other chemically unrelated convulsants the inhibition of [35S]-TBPS binding by the various tetrazole derivatives was unaffected by the addition of the bicucculine-like GABA antagonist, R 5135. To ascertain whether the inhibition of specific [35S]-TBPS binding by the tetrazole derivatives was related to their convulsant properties, we compared their in vitro potencies in displacing [35S]-TBPS binding with their minimum convulsant potencies in mice. A very good correlation was observed (r = 0.96, p less than 0.001) between their relative affinities for the [35S]-TBPS binding site and their convulsant potencies, indicating that pentamethylenetetrazol and related tetrazoles may produce their convulsant and anxiogenic actions via the GABA-benzodiazepine-chloride ionophore receptor complex.     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.     

Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with Mercury(II) and Methylmercury(II)

Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH₃Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by ¹H and ¹³C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH₃Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH₃Hg(II) bind to N₃ of thymidine in preference to N₁ of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.     

Ultraviolet difference spectroscopy of bovine carbonic anhydrase substituted with various divalent metals

The ultraviolet (uv) difference spectra of M(II)-apocarbonic anhydrase at pH 5–9 are reported. For Zn(II) at all pH's and Co(II) at pH ? 7.65 identical protein difference spectra are seen and a positive 300 nm feature is interpreted as consistent with interaction of a metal-bound hydroxyl with a Trp chromophore near the active site. Hg(II), Cu(II), and Cd(II) do not provoke a positive 300 nm band even at alkaline pH (although a Cd(II) spectral band at 300 nm becomes less negative, i.e., more like the holoenzyme with increasing pH) and the 280–292 nm spectral region is generally different from that of Zn(II) and high pH Co(II). A specific orientation of M-OH and, hence, an ordered solvent structure in the enzyme site is implied for enzyme activation. Ni(II) appears to bind to the vacated zinc site slowly, at low pH, in a manner similar to zinc. At higher pH's Ni(II) may be displaced toward a Tyr residue in the active site of apocarbonic anhydrase.     

An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.