Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid

Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals.     

Comparison of six rabbit liver cytochrome P-450 isozymes in formation of a reactive metabolite of acetaminophen

This laboratory has recently reported the isolation of an ethanol-inducible form of rabbit liver microsomal cytochrome P-450, designated isozyme 3a. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, all of which are present in significant amounts in the liver microsomes from rabbits chronically administered ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective, and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a (or a homologous enzyme in other species) may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.     

The postribosomal particle of rabbit liver contains protein-synthesis factors and serum albumin mRNA

As demonstrated by indirect immunoprecipitation and polyacrylamide gel electrophoresis, an 85S particle separated by sucrose density-gradient centrifugation from the postribosomal pellet of rabbit liver, is able to synthesize serum albumin if supplemented with both ribosomal subunits and sources of energy. It is retained on heparin bound to Sepharose 4B, contains translatable mRNA and apparently all protein factors required for translation. This particle may represent a highly organized protein synthesizing machinery, the combination of which with ribosomes results in formation of new protein molecules.     

Changes in composition and function of thylakoid membranes as a result of photosynthetic adaptation of chloroplasts from pea plants grown under different light conditions

The hypothesis that chloroplasts having different light-saturated rates of photosynthesis will have different proportions of the intrinsic thylakoid complexes engaged in light-harvesting and electron transport (Anderson, J.M. (1982) Mol. Cell. Biochem. 46, 161–172) has been tested. Peas were grown in light regimes which varied in light intensity, quality and time of irradiance, and ranged from sunlight through red to blue-enriched light of very low radiation. The electron-transport capacity at saturating light of Photosystem I and Photosystem II of chloroplasts isolated from light-adapted peas was 2-fold and 5–6-fold lower, respectively, in the lowest radiation compared to sunlight. There was a marked increase in the amount of total chlorophyll associated with the main chlorophyll $\text{a}\text{b-}\text{proteins}$ (LHCP¹, LHCP² and LHCP³) and a 2-fold decrease in the core reaction centre complex of Photosystem II (CP a) as the radiation decreased; the $\text{LHCP}{}^{\mathrm{1–3}}\text{CP}$ a ratio changed from 3.5 to 9.0. The amount of chlorophyll associated with Photosystem I varied from 34% in sunlight to 27% in the lowest radiation, but the antenna size of Photosystem I was not markedly different; there was a 2-fold decrease in the amount of cytochrome f on a chlorophyll basis, which partly accounted for the decreased electron-transport capacity of Photosystem I. Since the increases or decreases in the levels of each of the components correlated with decreasing radiation, it is clear that the light-adaptation of both light-harvesting and electron-transport components is indeed closely co-ordinated.     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Detection of beta-adrenergic receptors on rabbit mononuclear cells isolated free of significant contamination by other cell types

In order to study rabbit mononuclear cell surface receptors, it was necessary to develop a procedure to isolate mononuclear cell preparations that are free of significant contamination by other cell types, especially platelets. Centrifugation of dextran-sedimented, anti-coagulated whole blood through Hypaque (density 1.060) at 600 X g for 5 min at 22 degrees C eliminated greater than 93% of starting platelets. A second 5-min Hypaque centrifugation of Hypaque-Ficoll-isolated mononuclear cells (MNC) (approximately 80% lymphocytes) at 450 X g for 5 min at 22 degrees C reduced platelet contamination to less than one platelet per three MNC, and resulted in the overall removal of greater than 99.5% of starting platelets. These relatively pure MNC which were isolated in less than 2 hr were identified as having beta-adrenergic receptors by radioligand binding techniques using [125I]iodohydroxybenzylpindolol [( 125I]IHYP). Binding of [125I]IHYP to intact rabbit MNC was a saturable, stereospecific, and rapid process with a dissociation constant (KD) of 0.53 +/- 0.18 nM and a binding capacity of 3,461 +/- 235 sites/cell.     

Epicuticular waxes from Agropyron dasystachyum,agropyron riparium and Agropyron elongatum

Epicuticular waxes from whole plants of Agropyron dasystachyum var. psammophylum, A. riparium and A. elongatum contain hydrocarbons (5–8 %), long chain esters (12–15%) and free acids (2–5%). The major esters are C₃₄C₅₆ esters derived from C₁₆C₃₀ acids and alcohols (1-hexacosanol is the major alcohol) but C₃₁, C₃₃ and C₃₅ esters (3–11%) are also present. The latter esters are C₁₈ and C₂₀ acid esters of C₁₃ and C₁₅ 2-alkanols. A. dasystachyum wax contains 2% free alcohols, that of A. riparium contains 17% and that of A. elongatum 11% (1-hexacosanol is the major alcohol in each). Diesters (2%), C₈C₁₂ diols esterified by (E)-2-alkenoic acids, are present in A. riparium wax. Hentriacontane-14,16-dione is present: 29% in A. dasystachyum wax and 32% in A. riparium wax, but only 5% in A. elongatum wax. 25-Oxohentriacontane-14,16-dione forms 14% of A. dasystachyum wax and 27% of A. elongatum wax but the oxo β-diketones of A. riparium wax (5%) consist of both 10-oxo- and 25-oxohentriacontane-14,16-diones in the ratio 4:1. Hydroxy β-diketones of the waxes are 25- and 26-hydroxyhentriacontane-14,16-diones; in A. dasystachyum (20%) the ratio is 3:1, in A. elongatum (20%) the ratio is 9:1 but in A. riparium (5%) it is ca 1:2. The configuration of the hydroxyl group in the 26-hydroxy β-diketone is opposite to that in the 25-hydroxy derivative. The unusual composition of the oxygenated β-diketones of A. riparium confirms that this species should be regarded as separate from A. dasystachyum. Wax from A. elongatum also contains 4-hydroxy-25-oxohentriacontane-14,16-dione (4%) and an unusual oxo-β-ketol, 18-hydroxy-7,16-hentriacontanedione (2%), both these components are probably derived biosynthetically from the 25-oxo β-diketone which is the major component of this wax. Syntheses of racemic 18-hydroxy-7,16-hentriacontanedione and of a model β-ketol, 12-hydroxy-10-pentacosanone, are described.     

Factors affecting invertase activity in soils

Summary The rate of reducing sugars released through invertase activity exhibited a buffer pH optimum of 5.0. Generally, the decline in invertase activity in its pH-profile near the optimal pH range was due to a reversible reaction that involved ionization or deionization of the functional groups in the active centre of the protein, but under highly acidic or alkaline conditions (pH<4 to >9) the reduced activity appears to be due to irreversible inactivation of the enzyme. The dependence of the reaction on the amount of enzyme present was linear up to 3 g of soil. By varying the substrate concentration, it was found that the reaction rate of this enzyme approached zero-order kinetics when 145mM of sucrose solution was added to soils. Application of three linear transformations of the Michaelis-Menten equation indicated that the apparent K_m constants varied among the soils studied, but the results obtained by the three plots were similar. By using the Lineweaver-Burk plot, the K_m values in five soils ranged from 16.3 to 42.1 (avg.=24.5) mM and V_max values ranged from 1.98 to 7.37 mg of reducing sugars released/g of soil/24 h. The optimum temperature for invertase activity in soils was observed at 50°C and denaturation of the enzyme began at 55°C. The activation energy (E_a) and enthalpy of activation (H^*) values for invertase activity, expressed in kJ/mole, ranged from 6.1 to 43.1 and 3.5 to 40.5, respectively. The Q₁₀ values for the invertase reaction in soils with a temperature range to 10 to 50°C ranged from 1.08 to 1.96. Under standerd conditions, the accumulation of reducing sugars was linear with time up to 48 h. Among the various pretreatments that affected invertase activity in soils, toluene, TCA, and PMA inhibited the enzyme by an average of 19, 54, and 11%, respectively. Steam-sterilization essentially destroyed soil invertase.     

Substance P and enkephalin in transected axons of medulla and spinal cord

The accumulation of transported materials in cut axons is demonstrated by the light and electron microscopic immunocytochemical localization of substance P and enkephalin in the caudal medulla and cervical spinal cord of adult rat. Two days following unilateral knife-cuts in the caudal medulla or spinal (C2-C3) levels, substance P and enkephalin-like immunoreactivity (SPLI and ELI) are detected in lesioned axons located rostral and caudal to the transection. Rostrally, SPLI and ELI are detected in the lateral reticular region and ventrolateral fasciculus corresponding to the location of previously identified bulbospinal pathways. Caudally, previously unidentified, propriospinal pathways showing SPLI are detected in the dorsal columns and in the dorsolateral fasciculus. In contrast, ELI is found caudal to the transection only in the reticular region of the medulla. For both peptides, immunoreactivity is present throughout axons containing numerous large, dense core, and small clear vesicles. These results support the concept of both particulate and soluble modes of transport for substance P and enkephalin within axons of the central nervous system.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.