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Biomedical subjects

R Wiesner

Publications and source records attributed to R Wiesner.

At least 73 records · Page 4Linked to original sources

Anticarcinogenic action of protease inhibitors.

Protease inhibitors are synthesized in biological systems and play a critical role in controlling a number of diverse physiological functions. They participate in blood clotting and lysis of clots, in growth processes by modulation of proteolytic digestion of proteins and thus availability of amino acids, and in the induction of selective DNA amplification. When incorporated into the diet, protease inhibitors appear to suppress many types of cancer. In vitro, they suppress neoplastic transformation caused by chemical carcinogens, ionizing radiation, and oncogenes. These observations offer the hope that judiciously applied protease inhibitors in small concentrations may prevent a wide range of human cancers. This hope is further supported by epidemiological studies which show that populations consuming relatively large amounts of protease inhibitors have a lower occurrence of cancer. The tasks remaining are to determine the kind and the level of protease inhibitors that are most effective in preventing cancer without also having toxic side effects and to incorporate them into our diet. Perhaps the most encouraging investigations are those using small nontoxic protease inhibitors available in pure form (epsilon-aminocaproic acid, a trypsin plasminogen activator inhibitor, and nicotinamide, a chymotrypsin inhibitor and known vitamin). Both agents have been shown to be preventive agents of cancer in animals and in vitro models. Further studies with natural protease inhibitors may yield even more effective agents which when incorporated into our diet will prevent the development of many types of cancer.

Antineoplastic Agents↗

Chymotrypsin-specific protease inhibitors decrease H2O2 formation by activated human polymorphonuclear leukocytes.

Stimulated phagocytic cells generate active oxygen species which are known to contribute to inflammatory diseases, necrosis of surrounding tissues, mutagenicity and carcinogenicity. Until now, it was not certain whether protease inhibitors are capable of decreasing the production of those oxygen species, and if they are, what type of protease inhibitor is the most active. In this work we monitored formation of H2O2 by 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated polymorphonuclear leukocytes (PMNs) because H2O2 is the immediate precursor of the actual damaging species. These determinations were carried out in the absence or presence of protease inhibitors and/or superoxide dismutase (SOD). The protease inhibitors tested were: potato inhibitors 1 (PtI-1) and 2 (PtI-2), a chymotrypsin-inhibitory fragment of PtI-2 (PCI-2), chicken ovoinhibitor (COI), turkey ovomucoid ovoinhibitor (TOOI), Bowman-Birk inhibitor (BBI), lima bean inhibitor (LBI) and soybean (Kunitz) trypsin inhibitor (SBTI). The order of activity, as measured by inhibition of H2O2 formation by TPA-activated PMNs during incubation at 37 degrees C for 30 min, was (in descending order): PtI-1 greater than or equal to PCI-2 greater than PtI-2 greater than COI greater than BBI greater than or equal to TOOI greater than LBI greater than SBTI. Thus, the most effective were the chymotrypsin-specific inhibitors PtI-1 and PCI-2, followed by the bifunctional inhibitors recognizing both chymotrypsin and trypsin, and the least active was SBTI, a predominantly trypsin inhibitor. At the higher concentrations of protease inhibitors tested, the inhibitory activity was similar in both the absence and presence of SOD. These results show that protease inhibitors specific for chymotrypsin but not those that are trypsin-specific are capable of inhibiting formation of active oxygen species during the oxidative burst of stimulated human PMNs.

Chymotrypsin↗

Formation of lipoxin B by the pure reticulocyte lipoxygenase.

The pure reticulocyte lipoxygenase converts 5,15-DiHETE via a lipoxygenase reaction to 5,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid (a lipoxin B isomer) as shown by GC/MS analysis of its trimethylsilyl ether. With arachidonic acid, 15-HETE and 15-HETE methyl ester this lipoxin B isomer was also formed. The results presented here indicate that pure mammalian lipoxygenases are able to form lipoxins via sequential multiple oxygenation of arachidonic acid or its hydroxy derivatives.

Gas Chromatography-Mass Spectrometry↗

Requirement of monohydroperoxy fatty acids for the oxygenation of 15LS-HETE by reticulocyte lipoxygenase.

The lipoxygenase from reticulocytes oxygenates 15LS-HETE to 8-hydroperoxy-15-hydroxy-5,9,11,13-eicosatetraenoic acid and 5-hydroperoxy-15-hydroxy-6,8,11,13-eicosatetraenoic acid only in the presence of catalytic concentrations of monohydroperoxy fatty acids. During this reaction the hydroperoxy fatty acids are converted to more polar products including hydroxy fatty acids. From kinetic measurements of 15LS-HETE oxygenation it was calculated that 1 mol monohydroperoxy fatty acid is consumed during the oxygenation of about 9 mol 15LS-HETE.

Fatty Acids↗

The occurrence of a lipoxygenase pathway in reticulocytes of various species.

Reticulocytes from various species (rat, mouse, rhesus monkey) obtained by phenylhydrazine treatment of the animals metabolized polyenoic fatty acids via a lipoxygenase pathway. Linoleic acid was converted to 13-hydro(pero)xy-9,11(Z,E)octadecadienoic acid [13-H(P)ODE] and 9-hydro(pero)xy-10,12(E,Z)octadecadienoic acid [9-H(P)ODE], whereas arachidonic acid was oxygenated to 15-hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid (15-HETE) as shown by straight-phase high-pressure liquid chromatography (SP-HPLC). Addition of calcium and ionophore A 23,187 strongly enhanced the formation of lipoxygenase products, whereas 5,8,11,14eicosatetraenoic acid (ETYA) completely inhibited their formation. Estimates of the specific radioactivities of the lipoxygenase products indicate differences in the metabolization of externally added and endogenously released polyenoic fatty acids. These results strongly suggest that lipoxygenases generally occur in immature red blood cells.

5,8,11,14-Eicosatetraynoic Acid↗

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes.

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.

Animals↗

Metabolism of [1-14C]-arachidonic acid by cultured calf aortic endothelial cells: evidence for the presence of a lipoxygenase pathway.

Intact cultured calf aortic endothelial cells from a 10th-14th subculture rapidly metabolize exogenous [1-14C]-arachidonic acid by three different routes: i) incorporation into triglycerides and phosphilipids in a ratio of about 2:1, ii) formation of lipoxygenase metabolites (12-hydroxy-5,8,10,14-eicosatetraenoic acid) and iii) formation of cyclooxygenase metabolites (6-keto-PG F1 alpha and PG F2 alpha). From analyses by thin-layer chromatography and high-pressure liquid chromatography it was established that the main lipoxygenase metabolites in intact cells are 12-hydroxy-5,8,10,14-eicosatetraenoic acid and a compound proposed to be (a) dihydroxyeicosatetraenoic acid(s). In frozen and thawed cells the incorporation of arachidonic acid into cellular lipids is abolished, whereas the lipoxygenase pathway is strongly enhanced. Under these conditions the cells produce predominantly 15-hydroxy-5,8,11,13-eicosatetraenoic acid in addition to 12-hydroxy-5,8,10,14-eicosatetraenoic acid. The formation of lipoxygenase products was inhibited by heating the cells or by preincubation with nordihydroguaiaretic acid or BW 755 degrees C, whereas indomethacin was without effect. The formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid by intact cells was inhibited by 5,8,11-eicosatriynoic acid. Indomethacin and acetylsalicylic acid inhibited the formation of cyclooxygenase metabolites. 15Ls-hydroxy-5,8,11,13-eicosatetraenoic acid was incorporated into cellular lipids, but not dihydroxyeicosatetraenoic acids. Exogenous [3H]-labelled prostacyclin and TxB2 were not incorporated but were metabolized to less polar products.

Animals↗

The formation of products containing a conjugated tetraenoic system by pure reticulocyte lipoxygenase.

The pure lipoxygenase from reticulocytes converts 5,15-di-HETE at 2 degrees C to product(s) showing a characteristic UV spectrum with maxima of strong absorbance at 300 and 316 nm and shoulders at 285 and 350 nm. Their formation was completely prevented by the lipoxygenase inhibitors, ETYA and NDGA, by heating of the enzyme and by anaerobiosis. At 35 degrees C the products were formed only initially and in small amounts. The reaction products were purified by SP-HPLC and shown to migrate in the region of tri-HETEs in thin-layer chromatograms. The lipoxygenase from soybeans also converts 5,15-di-HETE to these product(s) with a comparable initial rate but different kinetics. These data suggest that 5,15-di-HETE is converted via a lipoxygenase reaction to 5,6,15-trihydroxy-7,9,11,13-(e,e,c,e)- and/or 5,14,15-trihydroxy-6,8,10,12-(e,c,e,e)-eicosatetraenoic acid, both of which contain a conjugated tetraene system.

Anaerobiosis↗

Reevaluation of the presence of the major antigen Ca++ complex in Bernard-Soulier syndrome platelets. Elastase degradation of the complex in Bernard-Soulier syndrome platelet preparations.

Crossed immunoelectrophoresis of human platelet membranes reveals a major antigen GPIIb-GPIIIa-Ca++ complex which is absent to decreased in Glanzmann's thrombasthenia. In our initial report, we also noted its absence in two patients with Bernard-Soulier syndrome associated with the presence of two neoantigens, and postulated that this may be due to endogenous proteolysis. It now appears that this observation is due to exogenous proteolysis from granulocyte-elastase contamination in the platelet membrane preparation. Purified elastase is capable of degrading the major antigen-Ca++ complex, particularly in its dissociated form, to the pattern initially reported for Bernard-Soulier platelets. Of particular interest is the observation that the elastase-degraded dissociated complex can be reassociated in the presence of Ca++. This indicates that the elastase-sensitive antigenic determinants recognized by the rabbit anti-human platelet membrane antibody are not required for the bonds which reassociate the complex.

Blood Coagulation Disorders↗

Reticulocyte lipoxygenase exhibits both n-6 and n-9 activities.

Purified reticulocyte lipoxygenase converts arachidonic acid to both 15- and 12-hydroxyperoxyeico-satetraenoic acids. The proportion of the two reaction products does not change during the purification procedure as shown by HPLC analysis. By means of isoelectric focusing it was not possible to separate the n-6 and n-9 activities. Reticulocyte lipoxygenase was completely inactivated by both 5,8,11-eicosatriynoic and 5,8,11,14-eicosatetraynoic acids in contrast to soybean lipoxygenase-1 which was inactivated only by 5,8,11,14-eicosatetraynoic acid. These results indicate that reticulocyte lipoxygenase exhibits both n-6 and n-9 activities. A contamination of the enzyme preparation with other lipoxygenases, e.g., the n-9 lipoxygenase from thrombocytes appears to be excluded.

Animals↗

Protease inhibitors: possible anticarcinogens in edible seeds.

Protease inhibitors, which are common constituents of seeds (rice, beans, and maize) have been shown to inhibit breast, colon, and skin cancers in animal experiments. Epidemiological studies have shown that diets rich in these components (ie seed proteins) decrease the occurrence of prostatic, breast, and colon cancers in man. We have demonstrated that a typical purified inhibitor from soybeans, the Bowman-Birk inhibitor, is excreted in the feces complexed with digestive proteases from the duodenum. This results in greater excretion of proteins, thus effectively decreasing the amount of protein available for digestion. Excess protein in the diet in addition to fats may contribute to increased cancer. Thus, the addition of seeds (chick-peas, beans, etc) to the diet rather than removal of meat or fat offers the opportunity of preventing these cancers, because the protease inhibitors present would effectively remove a portion of the proteins being consumed. Protease inhibitors also act directly as antioxidants in relation to tumor promoters and ionizing radiation. To what extent this contributes to the nutritional effects of eating seeds containing protease inhibitors remains to be determined.

Animals↗

Lipoxygenase from rabbit reticulocytes: iron content, amino acid composition and C-terminal heterogeneity.

Purified lipoxygenase from rabbit reticulocytes contains 1 g atom iron per molecule. The amino acid composition shows that the enzyme contains 11 cysteine residues but no disulfide bridges and contains a relatively high content of hydrophobic amino acids. The enzyme shows heterogeneity in the C-terminus. With the tritium labeling method the amino acids His, Asn and Ile were found in the C-terminal region. The absorption co-efficient was found to be 1.68 (E0.1% 280 1 cm) in the native state and 1.77 in the unfolded state.

Amino Acid Sequence↗