Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II colagen [3α₁(II)] in the absence of extracellular Ca²⁺ and Type Icollagen [2α₁?(I)·α₂] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca²⁺ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl₂. If added directly to the suspension culture medium containing no CaCl₂, calcitonin stimulates an active efflux of Ca²⁺ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Yeast RNA polymerase III: A zinc metalloenzyme

Atomic absorption spectroscopy has been used to demonstrate that zinc is associated with yeast RNA polymerase III. The enzyme purified by DNA-Sepharose chromatography gives a single predominant protein band in polyacrylamide gel electrophoresis and contains 0.7 gram-atoms of zinc per 100,000 grams of protein. The zinc is tightly associated with the enzyme and cannot be removed by passing the protein through a column of Chelex-100 resin under conditions where free zinc is quantitatively removed. Inhibition by the chelating agent 1,10-phenanthroline demonstrates that the zinc is essential to the catalytic process. The enzyme is inhibited 50% at 0.1 mM and 100% at 1 mM 1,10-phenanthroline.     

Theoretical considerations regarding the cell membrane function during cold-induced preservation of tissues and organs: new possibilities for optimizing the process

An analysis of the effects of temperature variation on the function of the mammalian cell membrane strongly suggests that during the cold-induced preservation of living tissues and organs the passive flow of micromolecules and ions across the cell membrane is the main factor in determining the state of the cellular biological system. Practically, the optimal organ (tissue) storage could be obtained at low temperatures (exceeding the freezing point) by using preservation fluids with constitutive substances in concentrations equal to those existing in the normal cytoplasm. Consequently, the formulae for adequate preservation fluids must be calculated according to the peculiar cytoplasmic constitution of the cells of each organ and tissue.     

Evidence of ubisemiquinone radicals in electron transfer at the cytochromes b and c1 region of the cardiac respiratory chain

A highly purified reduced ubiquinone-cytochrome c reductase preparation (the b-c₁III complex) has been made. The b-c₁III complex is not reconstitutively active with succinate dehydrogenase. When the complex at about 10 mg/ml is reduced by succinate in the presence of catalytic (nanomolar) amounts of SDH and a ubiquinone protein (required in the succinate dehydrogenase region i.e, OP-S), a ubisemiquinone radical(s) has been detected using EPR measurements. The formation of the radical(s) is concurrent with the reduction of cytochrome b after the complete reduction of cytochrome c₁. All these rates are dependent on the amounts of succinate dehydrogenase and QP-S used. The maximal concentration of the radical formed is independent of the amounts of succinate dehydrogenase and QP-S added but dependent on the amount of succinate present. The formation of the radical and the reduction of b and c₁ by succinate requires the presence of phospholipids. Addition of thenoyltrifluoroacetone not only prevents the formation of the ubisemiquinone but also abolishes the prior formed radical and causes the reoxidation of b. Antimycin A also diminishes the radical intensity but causes only slight reoxidation of prior reduced cytochrome b. Treatment of the b-c₁III complex with α-chymotrypsin results in the diminishing of the radical formation. Consideration of all these results presented collectively indicates the existence of a ubiquinone binding protein in the b-c₁III complex preparation.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

Primary photosynthetic processes: The problem of rapid irreversible redistribution of electronic energy

The general principles of a “device” aimed at the transformation of the energy of absorbed light quanta to the energy of a long-lived system with separated charges are analyzed. A set of conditions which must be fulfilled for efficient functioning of such a construction is formulated. A comparison of these results with the experimental data for the bacterial reaction centre is given. Some possibilities are discussed for the further experimental checking of the theory.     

Abnormal bovine erythrocyte membrane proteins and glycoproteins during and after infection with Anaplasma marginale

The erythrocyte membrane proteins from normal and Anaplasma-infected bovine blood have been compared. Two distinct new polypeptides were present in membranes from acutely infected cells. The glycoprotein pattern was also altered: in addition to the three main bands observed in normal cells, there were four new bands present which were glycosylated. The normally found membrane glycolypeptide (250000 D) was missing. The role of these protein alterations in relation to the infectious process is discussed.     

ω-Hydroxylation of acyclic monoterpene alcohols by rat lung microsomes

Rat lung microsomes were shown to ω-hydroxylate acyclic monoterpene alcohols in the presence of NADPH and O₂. NADH could neither support hydroxylation efficiently nor did it show synergistic effect. The hydroxylase activity was greater in microsomes prepared from β-naphthoflavone (BNF)-treated rats than from phenobarbital (PB)-treated or control microsomal preparations. Hydroxylation was specific to the C-8 position in geraniol and has a pH optimum of 7.8. The inhibition of the hydroxylase activity by SKF-525A, CO, N-ethylmaleimide, ellipticine, α-naphthoflavone, cyt. $\text{c}$ and p-CMB indicated the involvement of the cyt. P-450 system. However, NaN₃ stimulated the hydroxylase activity to a significant level. Rat kidney microsomes were also capable of ω-hydroxylating geraniol although the activity was lower than that observed with lungs.     

Neocarzinostatin: spectral characterization and separation of a non-protein chromophore.

Chromatographically purified neocarzinostatin exhibits absorption, fluorescence, magnetic circular dichroic and circular dichroic spectral characteristics above and below 300 nm atypical for a protein with its reported aminoacid composition, indicating the presence of a non-protein chromophore. The drug complex, stable at acidic pH, can be dissociated by treatment with reducing or denaturing agents at neutral or basic pH. Chromatography of the dissociated complex, or more conveniently, methanol extraction of the lyophilized drug, separates a protein with an amino-acid composition identical to neocarzinostatin and a highly fluorescent chromophore free of amino-acids.     

Poly(A)-protein interactions and transport of mRNA in isolated nuclei.

RNA-protein complexes (RNP), formed in a cell-free system using nuclei from GH cells, prelabelled, with [³H] leucine,were isolated from nucleoplasm and from postnuclear supernatant (PNS). Radioactive proteins, associated with newly synthesized HnRNP and mRNP-like material in the PNS were examined. On SDS gels, the [³H] protein pattern of poly(A)HnRNP was found to be more complex than that of PNS poly(A)-RNP. Only three radioactive proteins (78K, 100K and 120K) were associated with the poly(A) segments of cell-free formed HnRNP and PNS poly(A)-RNP. Cordycepintriphosphate and α-amanitin reduced the transport of cell-free formed poly(A) RNP associated [³H] proteins to the extent of 52% and 46% respectively. It may be concluded that the poly(A) segment of newly synthesized mRNA-like material is directly or indirectly (by providing binding sites for specific proteins) responsible for the transport of poly(A) containing mRNA.     

Mechanism of action of benzo(a)pyrene and nicotine on hormone production by rat pituitary tumor cells

The effects of the cyclic aromatic hydrocarbon, benzo(a)pyrene (BaP) and that of the tobacco alkaloid, nicotine, on prolactin (PRL) and growth hormone (GH) synthesis by rat pituitary tumor cells in culture (GH cells) have been studied. Treatment of GH cells with nicotine (0.1–300 μg/ml) neither affected the growth, nor significantly altered the general pattern of hormone production in these cells. BaP at concentrations greater than 5 μg/ml irreversively inhibited the growth of these cells. The sublethal concentrations of BaP, which did not affect either 1) cell growth, or 2) amino acid transport or 3) total protein synthesis or degradation, did however inhibit specifically, hormone synthesis by these cells. More interestingly concentrations of nicotine which did not affect either cell growth or hormone synthesis, modulated both of these cellular processes in the presence of BaP. A concentration dependent stimulation of microsomal BaP monooxygenase activity was observed in nicotine or BaP treated cells. The effects of these drugs on stimulation of BaP monooxygenase activity seems to be additive. Nicotine also enhanced the association of radioactivity (presumably [³H] BaP metabolites) with DNA in [³H] BaP treated cells. It is concluded that nicotine by itself did not demonstrate any cytotoxic effect nor influence hormone synthesis in GH cells. However, this constituent of tobacco smoke stimulated BaP monooxygenase activity and the interaction of [³H] BaP metabolites with cellular DNA and also modulated BaP induced inhibition of hormone synthesis in GH cells.     

Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

The effects of sulphur supplementation on cellulose digestion in vitro and on nutrient digestion,nitrogen metabolism and rumen characteristics of lambs fed on good quality fescue and tropical star grass hays

Experiments were conducted in vitro and in vivo to determine the effects of sulphur (S) supplementation of a good quality fescue hay containing 0.27% total S and a tropical star grass hay containing 0.20% total S. Addition of S was on an isosulphurous basis of either sodium sulphate or D,L-methionine. Cellulose digestion in vitro was improved (P < 0.001) by the addition of 1% urea. Supplementation of forage with 0.05, 0.10 or 0.15% S from either sodium sulphate or methionine also stimulated cellulose digestion in vitro. There were no differences between S sources. The addition of 0.4 or 0.8% nitrate-nitrogen (nitrate-N) (potassium nitrate) depressed (P < 0.05) cellulose digestion in vitro of both hays. No effect of animal adaptation to nitrate was evident. Addition of S partially counteracted the depression in cellulose digestion due to nitrate. Trials were conducted in vivo in which 12 crossbred wether lambs (fescue experiment) or 12 crossbred intact male lambs (star grass experiment) were randomly assigned to one of three treatments: control (forage with no addition of S); forage plus 0.15% S as sodium sulphate; and forage plus 0.15% S as D,L-methionine. Lambs were housed in metabolism crates and each experiment was replicated twice. Dry matter intakes were highest for methionine-supplemented fescue and for S-supplemented star grass, regardless of S source. Dry matter digestibility tended to increase with S addition (fescue experiment) and was significantly higher for S-supplemented star grass. There was a significant increase (P < 0.05) in neutral detergent fibre (NDF) and acid detergent fibre (ADF) digestibility due to supplemental S, regardless of S source. Nitrogen retention, ammonia-N and ruminal volatile fatty acids were unaffected by S supplementation.     

Synthetic routes to higher-carbon sugars. 1-C-Formylation of a 2-deoxyaldose via the anion of its diethyl dithioacetal

Diphenylmethylation of carbohydrate hydroxyl groups may be effected by the thermal reaction with diazo(diphenyl)methane in the absence of catalysts. Migration of the labile ester groups of methyl 2,3,4-tri-O-acetyl-α-d-glucopyranoside and 3-O-benzoyl-1,2-O-isopropylidene-α-d-glucofuranose does not occur during diphenylmethylation by this procedure. The diphenylmethyl group may be readily removed by catalytic hydrogenolysis, and is sufficiently acid-stable to enable the selective hydrolysis of acetal groups. Its use as an O-4 protecting-group and as a non-participating O-2 protecting-group in α-glycoside synthesis has been demonstrated in syntheses of methyl 2,3,6-tri-O-methyl-α-d-glucopyranoside and kojibiose octa-acetate, respectively.     

Reexamination of the interaction between ferredoxin:NADP reductase and NADP

A comparison was made of graphical and subtractive methods for the determination of the dissociation constant of a complex between ferredoxin:NADP reductase and NADP. The subtractive method gave K_d values near 10 μm which are consistent with recently determined values for K_m,NADP in assays of NADP photoreduction by chloroplast membranes. The graphical method gave values which were considerably higher. The difference between the two methods is due to the failure of the graphical method to correct for the amount of each component present in the complex at the low NADP/ flavoprotein ratios necessary for binding studies. A second NADP binding site of much lower affinity (K_d approx 1 mm) was also detected.     

Clottable and nonclottable components in products derived from single animal bovine plasmas

The distribution of clottable and nonclottable absorbance between product and discard was determined for each step of a standard four-step procedure. Analyses of step 1 and step 2 products reveal the presence of components which differ in solubility in ethanol or (NH₄)₂SO₄. Solubility differences among comparable products apparently are the result of variable distributions of native components and their early degradation products. At step 1 the product is precipitated at 4% ethanol and ?1.4 °C. Clottability is 0.75 ± 0.05. Studies of the dependence of precipitate yield on clottable absorbance concentration for individual plasmas reveal a precise relation. As the plasma clottable absorbance concentration increases, the fraction due to precipitating components is constant. However, the sum of the solubilities of precipitating components and the concentration due to completely soluble components both increase linearly. At step 2 the product is precipitated at ~ 4.9 AU/ml, pH 6.4, 0.12 g/ml (NH₄)₂SO₄, and 22 °C. Elimination of nonclottable absorbance precipitated and occluded at step 1 increases clottability to 0.967 ± 0.009. At step 3 the product is recovered after discarding components which precipitate over 16 h, ~33 AU/ml, pH 6.4, Γ/2 0.3, and 0 °C. Product clottability is not increased but fibrin, present at ~3%, and nonclottable components are distributed at this step so that they precipitate preferentially at step 4 where the product is recovered after discarding precipitate which forms over 2 h at 5 AU/ml, pH 6.6, Γ/2 0.12, and 0 °C. Step 4 products have a clottability of 0.980 ± 0.004 and fibrin contents of 0.4 to 0.8%.