Inhibition of protohaem ferro-lyase in experimental porphyria. Isolation and partial characterization of a modified porphyrin inhibitor.

Rat adrenal 105,000 g supernatant contains two lipid moieties, 'lipid-I' and 'lipid-II' which contain non-esterified cholesterol and stimulate cholesterol side-chain cleavage in soluble or mitochondrial enzyme systems. Lipid-I contains relatively large low-density heat-stable particles, whereas lipid-II particles are smaller, more dense and heat-labile. Lipid-I and lipid-II can be separated from clear cytosol by ultracentrifugation and gel filtration respectively. Corticotropin plus cycloheximide treatment increases the non-esterified cholesterol concentrations in the lipid fractions, and stimulatory effects of lipids on cholesterol side-chain cleavage appear to correlate with non-esterified cholesterol concentrations therein. On addition of saturating amounts of cholesterol-rich lipid, pregnenolone synthesis and cholesterol binding to cytochrome P-450 are stimulated more in mitochondria from corticotropin-stimulated adrenals than in mitochondria from control or corticotropin-plus cycloheximide-stimulated adrenals. These results support the contention that the corticotropin-induced increase in mitochondrial cholesterol side-chain cleavage involves an increase in cholesterol utilization as well as an increase in cholesterol availability.     

Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca²⁺-ATPase activities with high-affinity sites for Ca²⁺ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca²⁺, the substrate specificity of Ca²⁺-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca²⁺-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca²⁺-dependent ATP-hydrolysis was greater than that of ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ at Ca²⁺ concentrations below 25 μM. At 0.2 mM Ca²⁺ the rates of ATP, ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca²⁺, but at 0.2 mM Ca²⁺, Ca²⁺-induced hydrolysis of ADP and AMP was greater than either ATP or $\text{p-}\text{nitrophenylphosphate}$. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca²⁺-stimulated ATP hydrolysis at 1 μM Ca²⁺ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca²⁺, Ca²⁺-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca²⁺-stimulated ATP hydrolysis, either at low or at high Ca²⁺ concentrations. Chlorpromazine fully inhibited Ca²⁺-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca²⁺, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca²⁺-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca²⁺-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca²⁺ concentrations, but this Ca²⁺-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.     

Use of fluorescent probes in the study of phospholipid--sterol bilayers.

1. The transfer of excitation energy between the fluorescent probes 1,6-diphenylhexa-1,3,5-triene and 12-(9-anthroyl)stearic acid and the cholesterol analogue cholesta-4,6-dien-3-one in phosphatidylcholine liposomes has been investigated. 2. The results indicate that probes and steroid are randomly distributed in the bilayer at steroid concentrations up to 35 mol%. 3. The degree of polarization of diphenylhexatriene fluorescence increases with increasing cholesterol content. Other sterols, differing in structure in the region of the polar group or in the side chain at position-17, produce similar but not identical effects. 4. the results are consistent with the proposal that diphenylhexatriene gives a general picture of the state of the bilayer and that there is no segregation of sterols in liquid-crystalline phosphatidylcholine bilayers.     

Inhibition of protohaem ferro-lyase by N-substituted porphyrins. Structural requirements for the inhibitory effect.

N-Methyl mesoporphyrin was a powerful inhibitor of protohaem ferro-lyase in vitro, whereas N-ethyl mesoporphyrin and N-methyl coproporphyrin were not and neither was the newly described green pigment produced by giving rats ethylene. This suggests that the size of the substituent at a pyrrole nitrogen and also the number of carboxylic acid side chains of the substituted porphyrin are important for the inhibitory effect. Evidence that N-methyl mesoporphyrin inhibited the enzyme, whereas the ethylene-derived pigment did not, was also obtained in vivo.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Idiopathic paraproteinemia. II. Transplantation of the paraprotein-producing clone from old to young C57BL/KaLwRij mice.

Transplantation experiments in the C57BL/KaLwRij mouse model of idiopathic paraproteinemia (IP) showed that an IP-producing clone can be further propagated in young, lethally irradiated mice and also equally as well in nonirradiated recipients by a bone marrow and/or spleen cell transfer. The latency period before the original paraprotein was detected in the sera of recipients varied in different experiments between 1 and 9 months after transplantation. With subsequent transplantations, the "take" frequency gradually decreased. Propagation of IP for three to four generations seems to be the final limit. In comparison to age-matched seems to be the final limit. In comparison to age-matched control groups, no substantial influence of the transplanted IP on the survival of the recipients was observed. In contrast, transplantation of cells from mice with a B cell lymphoma or a myeloma led to continuous propagation of the malignancy, with a high "take" frequency, progressive development of the paraproteinemia, and a shortened survival time of the recipients. These findings indicate that IP represents in its final stage in the aging C57BL mice an intrinsic cellular defect within the affected B cell clone, which is, however, different from that found in B cell malignancies.     

Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.

Spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells) displayed an increased adhesion when cultured in the presence of 10(-6) M all-trans retinol and acquired morphological characteristics of the normal phenotype. Thus it was of interest to investigate the metabolism of [15-(14)C]retinol in this system. Within 24 hours of culture, approximately 4.25% of the [(14)C]retinol was taken up by the cells. The hydrocarbon [(14)C]anhydroretinol was a major metabolic product and was identified by gas-liquid chromatography and by its typical ultraviolet absorption spectrum with maxima at 386, 364, and 346 nm. At 24 and 40 hours anhydroretinol represented 27% and 55%, respectively, of the total nonpolar metabolites or approximately 16% and 30% of the total radioactive products. Formalin-fixed fibroblasts or cultured intestinal mucosal cells did not convert retinol into anhydroretinol. A more polar product with a UV absorption maximum at 310 nm was also found. The time course of the synthesis of this product by 3T12 cells suggested a precursor-product relationship with anhydroretinol. A microsomal preparation from 3T12 cells was also active in synthesizing [(14)C]anhydroretinol and [(14)C]metabolite-310 from [(14)C]retinol. Moreover incubation of metabolite-310 with the 3T12 microsomes yielded anhydroretinol (40% conversion in 30 minutes), suggesting that metabolite-310 is an intermediate in the synthesis of anhydroretinol by these cells. Anhydroretinol appears to be an end product of the metabolism of retinol in 3T12-3 cells, as suggested by the finding that over 90% of [(14)C]anhydroretinol incubated for 30 hours with 3T12-3 cells was recovered unaltered, without the formation of detectable retroretinol, retinol, or retinoic acid.-Bhat, P. V., L. M. De Luca, S. Adamo, I. Akalovsky, C. S. Silverman-Jones, and G. L. Peck. Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.     

Frost-weathering on Mars: Experimental evidence for peroxide formation

Summary A laboratory study of the interaction of H₂O frost with samples of the minerals olivine (Mg,Fe)₂SiO₄ and pyroxene (Mg,Fe)SiO₃ at –11°C to –22°C revealed that an acidic oxidant was produced. Exposure of the frost-treated minerals to liquid H₂O produced a sudden drop in pH and resulted in the production of copious O_2(g) (as much as ~ 10²⁰ molecules g^–1). Exposure of frost-treated samples to 5 ml of 0.1M HCOONa solution resulted in the rapid oxidation of up to 43% of the formate to CO_2(g). These reactions were qualitatively similar to the chemical activity observed during the active cycles of the Viking lander Gas Exchange and Labeled Release Biology experiments. Attempts to identify the oxidant by chemical indicators were inconclusive, but they tentatively suggested that chemisorbed hydrogen peroxide may have formed. The formation of chemisorbed peroxide could be explained as a byproduct of the chemical reduction of the mineral. The following model was proposed. H^c was incorporated into the mineral from surface frost. This would have left behind a residual of excess OH^– _(ads) (relative to surface H⁺). Electrons were then stripped from the surface OH^– _(ads) (due to the large repulsive potential between neighboring OH^– _(ads)) and incorporated into the crystal to restore charge balance and produce a chemical reduction of the mineral. The resultant surface hydroxyl radicals could then have combined to form the more stable chemisorbed hydrogen peroxide species. While the chemisorbed peroxide should be relatively stable at low temperatures, it should tend to decay to O_(ads) + H₂O_(g) at higher temperatures with an activation energy of 34 kcal mole^–1. This is consistent with the long-term storage and sterilization behavior of the Viking soil oxidants. It is possible that as little as 0.1–1% frost-weathered material in the Martian soil could have produced the unusual chemical activity that occurred during the Viking Gas Exchange and Labeled Release experiments.This paper contains the material given in invited presentations at the COSPAR Meeting, Innsbruck, Austria, 5–7 June 1978 and at the Second Conference on Simulation of Mars Surface Properties, NASA Ames Research Center, 17–18 August 1978