The effect of coenzyme A and structurally related thiols on the mammalian fatty acid synthetase

A comparison was made of the structural features of thiol compounds which can interact with the mammalian fatty acid synthetase. Three functional characteristics were examined: (i) the ability of the free thiols, at low concentrations, to satisfy the essential thiol requirement of the enzyme, (ii) the ability of the free thiols, at higher concentrations, to inhibit enzyme activity, and (iii) the ability of the malonyl esters of these thiol compounds to act as substrates for fatty acid synthesis. The relative effectiveness of the various thiols studied was identical in all three roles. Coenzyme A and N-hexanoylcysteamine were the most effective, pantetheine and N-butyrylcysteamine were less effective, and N-acetylcysteamine was totally ineffective. These results lend strong support to our hypothesis (A. Stern, B. Sedgwick, and S. Smith, 1982, J. Biol. Chem.257, 799–803) that the various effects of CoA and structurally related thiols are localized at one and the same site, namely, the site of transfer of substrates between coenzyme A ester form and enzyme-bound form.     

Gelation of liposome interior. A novel method for drug encapsulation.

Liposomes can be loaded with weak acids and bases, which exist in solutions in equilibrium with membrane permeable uncharged form, using various gradients across their membranes. Because in some cases the estimated drug concentration in the loaded liposomes exceeds their aqueous solubility we investigated the physical state of the liposome encapsulated anticancer drug Doxorubicin. X-Ray diffraction, electron microscopy, and test tube solubility experiments have shown that upon encapsulation the drug molecules form a gel-like phase.     

Fecundity of Euphausia lucens (Hansen) - laboratory evidence for multiple broods

There has been much controversy in the past as to how many broods euphausiids are capable of producing per season. Several authors have suggested that multiple broods are possible, although this has not been demonstrated in the laboratory. In the present study, two Euphausia lucens females were shown to spawn continuously over a period of at least 2.5 months. The euphausiids produced ≈ 33–40 broods during this period, representing 134–192% of their body carbon. Daily egg production rates varied from 0 to 86 eggs, representing a mean of 3–4% of their body carbon d⁻¹. Egg production rates were often severely depressed during moulting, and were also somewhat lower during experimental manipulation (changing food conditions). The results suggest that egg production by E. lucens, and perhaps by many other euphausiid species, are considerably higher than was previously thought.     

Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.     

Inhibition and killing of fungi by the polyamine oxidase-polyamine system

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.     

Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin

Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H₂O₂ was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type lb Cu(II).     

Parallel activities of fatty acid methyl esters and analogous phorbol diesters toward mouse lymphocytes

Linear saturated fatty acid methyl esters were comitogenic with lectins for mouse lymphocytes, the degree of comitogenicity being strongly dependent on the length of the acyl group, and maximal for methyl tetradecanoate. Lesser effects were found for analogs with 10, 12 or 16 acyl carbon atoms, whereas those with fewer than 10 or more than 16 were inactive. Analogous structure-function relationships have been described for various membrane-active and tumor-promoting phorbol diesters, where there is a similar dependence on ester acyl group length for many activities. The fatty acid esters may therefore represent simple model compounds for studying mechanistic aspects of phorbol diester activity.