Soybean lipoxygenase-1 oxygenates synthetic polyenoic fatty acids with an altered positional specificity: evidence for inverse substrate alignment.

The positional specificity is the decisive enzyme property for classification of lipoxygenases and for the currently used lipoxygenase nomenclature. It has been reported before that soybean lipoxygenase-1, which oxygenates polyenoic fatty acids at alkaline pH to the corresponding n - 6 hydroperoxy derivative, exhibits a different positional specificity when either the reaction conditions or the substrate structure is altered. To investigate the impact of structural substrate modifications on the positional specificity of this enzyme and to force an inverse substrate binding, we synthesized arachidonic acid analogues modified at the omega-terminus. Care was taken that the double bond system remained unchanged so that hydrogen abstraction from all three bisallylic methylenes was theoretically possible. We found that omega-modification of arachidonic acid leads to an impaired substrate affinity and a reduced reaction rate, but we did not detect any 5-lipoxygenation products, suggesting that structural modification of the omega-end may not be sufficient to force an inverse substrate orientation. However, when both ends of the fatty acid chain (omega-terminus and free carboxylate) were modified simultaneously, a considerable share of 5-lipoxygenation products was detected. These results indicate that introduction of polar or bulky groups at the methyl terminus of polyenoic fatty acids was not sufficient to force an inverse substrate orientation. However, simultaneous introduction of an omega-OH group and methylation of the carboxylate led to formation of significant 5-lipoxygenation products, suggesting an inverse head to tail substrate orientation.

Phase I study of twice-weekly gemcitabine and concomitant external-beam radiotherapy in patients with adenocarcinoma of the pancreas.

PURPOSE: To determine the maximum tolerated dose and dose-limiting toxicity associated with twice-weekly gemcitabine and concomitant external-beam radiotherapy in patients with adenocarcinoma of the pancreas. METHODS AND MATERIALS: Twenty-one patients with biopsy-proven adenocarcinoma of the pancreas were treated with external-beam radiotherapy to a dose of 50.4 Gy in 28 fractions, concurrent with gemcitabine, infused over 30 min before irradiation on a Monday and Thursday schedule. The dose of gemcitabine was escalated in 5 cohorts of 3--6 patients each. Initial gemcitabine dose was 10 mg/m(2), with dose escalation until dose-limiting toxicity was observed. RESULTS: The maximum tolerated dose of gemcitabine was 50 mg/m(2), when given in a twice-weekly schedule with radiation. Dose-limiting toxicity was seen in 2 patients at 60 mg/m(2), and consisted of severe upper gastrointestinal bleeding approximately 1 month after completion of treatment. Six patients had radiographic evidence of response to treatment, and 5 of these underwent complete surgical resection. Three patients who underwent complete resection had been deemed to have unresectable tumors before enrollment on trial. Four patients are alive, including 2 without evidence of disease more than 1 year after resection. CONCLUSION: The combination of external-beam radiation and twice-weekly gemcitabine at a dose of 50 mg/m(2) is well tolerated and shows promising activity for the treatment of pancreatic cancer. Our data suggest a higher maximum tolerated dose and different dose-limiting toxicity than previously reported. Further investigation of this regimen is warranted.

Determinants of umbilical cord arterial 8-iso-prostaglandin F2alpha concentrations.

OBJECTIVE: To determine the concentration of 8-isoPGF(2alpha) in cord blood as a measure of oxidative stress during labour, and to compare them with other established parameters of in vivo lipid peroxidation and with the acid-base status of the newborn. METHOD: Umbilical cord arterial and venous blood samples were collected from 81 singleton term deliveries for determination of 8-isoPGF(2alpha), malondialdehyde and organic hydroperoxides. In addition, metabolites derived from the oxidative metabolism of purines during hypoxia-reoxygenation and routine cord blood of oxygen saturation, pH, pO2, pCO2, HCO3 and base excess were measured. RESULTS: Arterial concentrations of 8-isoPGF(2alpha) were significantly higher in cases with fetal distress, tight nuchal cord (P < 0.001), the umbilical coiling index, and male sex (P < 0.05) (R2 = 0.48). No correlation was found with any parameter of acid-base status. In arterial and venous blood the concentrations of organic hydroperoxides and hypoxanthine significantly correlated with the fetal nuchal cord (P < 0.001) (R2 = 0.26 and 0.16, respectively). CONCLUSION: Our findings indicate that 8-isoPGF2(alpha) in cord arterial blood is a suitable parameter to quantify a possible oxidative stress in the fetus during labour. Measurements of the F2-isoprostane concentrations in cord blood at labour provide a clinically useful method to assess the perinatal outcome.

The inhibition of mammalian 15-lipoxygenases by the anti-inflammatory drug ebselen: dual-type mechanism involving covalent linkage and alteration of the iron ligand sphere.

Mammalian lipoxygenases have been implicated in inflammation and atherosclerosis and, thus, lipoxygenase inhibitors may be of pharmacological interest. In cells, lipoxygenases occur in a catalytically silent ground state that requires activation to become active. We found that the seleno-organic drug ebselen [2-phenyl-1, 2-benzisoselenazol-3(2H)-one], which exhibits anti-inflammatory properties, irreversibly inhibited pure rabbit 15-lipoxygenase, with an IC50 in the nM range when preincubated with the enzyme in the absence of fatty acid substrates. Subsequent dialysis, gel filtration, or substrate addition did not restore the enzyme activity, and experiments with [14C]ebselen indicated a covalent linkage of the drug. The presence of sulfhydryl compounds in the incubation mixture prevented both enzyme labeling and inactivation, but we did not see any reactivation when sulfhydryl compounds were added afterward. X-ray absorption studies indicated that ebselen did alter the geometry of the iron ligand sphere, and the data are consistent with an iron complexation by the drug. When fatty acid substrate was present during lipoxygenase-ebselen interaction, the inhibitory potency was strongly reduced and a competitive mode of action was observed. These data suggest that ebselen inactivated the catalytically silent ground-state lipoxygenase irreversibly by covalent linkage and alteration of the iron ligand sphere. In contrast, it functions as a competitive inhibitor of the catalytically active enzyme species. The pharmacological relevance of ebselen as a potential in vivo lipoxygenase inhibitor will be discussed.

Daily paclitaxel and thoracic radiation therapy for non-small cell lung cancer: preliminary results.

Platinum-based combination chemotherapy plus thoracic radiation prolongs survival for patients with stage III non-small cell lung cancer. Paclitaxel demonstrates significant clinical antitumor activity in this disease and potentiates the effects of ionizing radiation by arresting cells at the sensitive G2/M cell cycle phase. The optimal schedule of paclitaxel administered concomitantly with thoracic radiation has not been established. The preliminary results of this phase I trial, which was designed to define the dose-limiting adverse event and the maximum tolerated dose of paclitaxel administered daily before each fraction of thoracic radiation, are being presented. Twenty-nine patients with inoperable clinical stage II to IIIB non-small cell lung cancer received two 21-day cycles of primary chemotherapy with carboplatin and paclitaxel. Six weeks from the initiation of therapy, daily paclitaxel was administered intravenously over 1 hour without premedication before 68 Gy of thoracic radiation in 34 fractions. Twenty-six patients completed concomitant daily paclitaxel with radiation and are evaluable for toxicity. Early radiation esophagitis was the dose-limiting toxicity at the 15 mg dose level. A daily paclitaxel dose of 10 mg or 6 mg/m2 and 68 Gy of thoracic radiotherapy are recommended for further study. Preliminary data from this dose-escalation trial suggest that this combined modality treatment with concurrent radiation and daily paclitaxel following primary induction therapy for stage II to III non-small cell lung cancer is feasible. The observed adverse effects within the radiation field suggest active radiosensitization by low-dose daily paclitaxel.

A kinetic model for the interaction of nitric oxide with a mammalian lipoxygenase.

Nitric oxide (NO) has been known for a long while to act as an inactivator of the soybean lipoxygenase-1 and cyclooxygenase. More recently, NO was shown to interact also with a mammalian 15-lipoxygenase [Wiesner, R., Rathmann, J., Holzhütter, H. G., Stosser, R., Mader, K., Nolting, H. & Kühn, H. (1996) Nitric oxide oxidises ferrous mammalian lipoxygenases to a pre-activated ferric species, FEBS Lett. 389, 229-232]. In this paper we present a detailed kinetic analysis of the interaction of NO with the 15-lipoxygenase from reticulocytes. Time courses of hydroperoxide formation were monitored after an anaerobic incubation of the enzyme with varying concentrations of NO and for varying length of the incubation period. Owing to the presence of O2 during the enzymatic reaction, the inhibitory effect of NO declines in a time-dependent manner since NO is converted into non-reactive NO2. This is manifested as a lag phase of the time course. The lag phase becomes more pronounced if the enzyme is incubated over a fixed period (15 s) with increasing concentrations of NO. In contrast, increasing the length of the incubation period at fixed concentrations of NO diminishes the duration of the lag phase. These experimental findings can be accounted for by a kinetic model which assumes (a) fast reversible binding of NO to the inactive ferrous Fe(II) form of the enzyme and (b) slow irreversible conversion of the Fe(II)-NO complex into an activated ferric form of the enzyme which is susceptible to peroxide activation. The rate constants for these two different kinetic modes of NO action were estimated by fitting the solutions of the model equations to the experimental time courses. The dissociation constant of the Fe(II)-NO complex amounts to 2.5 microM. Computer simulations performed on the basis of the model indicate that the response of the lipoxygenase to increasing intracellular NO concentrations depend upon the available peroxide tone: at low peroxide concentration the enzyme is initially trapped in the inactive ferrous form and thus may be activated by NO via the activated ferric species. On the other hand, at high peroxide concentrations, a partial inhibition of the initially active enzyme is expected.

Nitric oxide oxidises a ferrous mammalian lipoxygenase to a pre-activated ferric species.

Nitric oxide is known as an inhibitor of soybean lipoxygenase-1. Investigating the interaction of a mammalian 15-lipoxygenase with nitric oxide, we found that this enzyme is also inhibited reversibly when incubated with nitric oxide for a short time period (5 s) under anaerobic conditions. This inhibition may be due to the formation of a dissociable lipoxygenase-nitric oxide complex. With longer incubation periods the ferrous lipoxygenase is oxidised to a ferric form. This oxidation renders the enzyme more susceptible to peroxide activation, causing a time-dependent shortening of the kinetic lag phase.

[Strut fracture of a convex-concave 60 degree Björk-Shiley mitral valve prosthesis 5 years after implantation--metallurgic analysis of the prosthesis strut]. / Bügelbruch einer konvex-konkaven Mitralklappenprothese Björk-Shiley 60 degrees 5 Jahre nach Implantation--Metallurgische Analyse des Prothesenbügels.

Acute mechanical failure of prosthetic heart valves is rare, but associated with high mortality when occurring. For convexo-concave Björk-Shiley prostheses only fractures of the outlet strut are reported. We present a case of lethal mechanical complication 5 years after implantation. By additional metallurgic analysis we were able to identify a sequential course of the outlet strut fracture. This could lead to new approaches for early detection of this complication.

Formation of ketodienoic fatty acids by the pure pea lipoxygenase-1.

A pure lipoxygenase from dried green pea seeds (isoenzyme 1) oxygenates linoleic acid to 9(S/R)-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPODE) and 13(S/R)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE). Furthermore (10E,12Z)-9-keto-10,12-octadecadienoic acid (9-KODE) and (9Z,11E)-13-keto-9,11-octadecadienoic acid (13-KODE) in a ratio of 1:1 were formed. Uv-spectroscopic measurements and HPLC data indicated a hydroperoxy fatty acid: keto fatty acid ratio of about 2:1. The product mixture formed from arachidonic acid was even more complex. 15-, 11-, 9- and 5-H(P)ETE1 and their corresponding keto derivatives have been detected. The chemical structures of the compounds have been identified by HPLC analysis, by uv- and ir-spectroscopy and gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The data presented indicate that a pure lipoxygenase catalyzes the formation of both hydroperoxypolyenoic fatty acids and ketopolyenoic fatty acids from linoleic acid and arachidonic acid. The possible mechanism of the formation of the keto compounds is discussed.

Pyruvate utilization of rabbit reticulocytes--a compartmental study.

The utilization of [2-14C] pyruvate via the citric acid cycle of rabbit reticulocytes was studied to elucidate the importance of carboxylation- and decarboxylation reactions in this cell type. Unknown flux rates were determined by fitting the observed time-dependent labeling of CO2, alanine, glutamate and aspartate (all carbon atoms and C alpha) to the corresponding set of differential equations. The results reveal a high activity of the shuttle rate between mitochondrial pyruvate and the C4 pool catalysed by the pyruvate carboxylase (v = 19.1 nM/ml cells/min) and the malic enzyme (v = 57.5 mM/ml cells/min) which is comparable with the flux rate within the citrate cycle (v2 = 46.2 nM/ml cells/min). Moreover, our studies provide strong indications for the existence of a pyruvate utilizing metabolic pathway which has not been described so far.

Quantification of pathways of glucose utilization and balance of energy metabolism of rabbit reticulocytes.

In this work it is demonstrated that glucose constitutes the main substrate of energy metabolism of rabbit reticulocytes under aerobic conditions in the presence of 5 mM glucose. Amino acids and fatty acids are minor sources of energy. The shares of processes utilizing glucose in reticulocytes were estimated from tracer experiments. A new mathematical technique used permits the derivation of closed terms for the specific radioactivity of single positions of C atoms of the metabolites of the citrate cycle. By means of regression analysis, the undetermined flux rates in the citrate cycle were calculated. On the basis of the data an overall balance sheet of glucose utilization and of ATP generation is given. About 45% of the glucose of reticulocytes is catabolized via the citrate cycle, about the same percentage yields lactate. Only 2% of the glucose was oxidized in the oxidative pentose pathway whereas the remainder is used for the formation of serine and glycine required for hemoglobin synthesis. These results are related to knowledge about the main processes utilizing ATP in reticulocytes, i.e. the synthesis of hemoglobin and the energy-dependent proteolysis. Our approach to the investigation of metabolic relations in the reticulocytes can be applied to other tissues in which equilibria between large metabolite pools play a role.

Determination and characteristics of energy-dependent proteolysis in rabbit reticulocytes.

In reticulocytes exists an extensive and time-limited ATP-dependent proteolysis which has been measured by a new method. This enzyme system attacks exclusively the mitochondria during the maturation of reticulocytes. The proteolysis is inhibited by anaerobiosis and salicylhydroxamic acid which indicates that it is preceded by the attack of lipoxygenase. There is some evidence for the universality of the ATP-dependent proteolysis, since liver mitochondria are also subject to it.

Balance of glucose utilization in rabbit reticulocytes.

The shares of glucose utilizing processes in red blood cells under aerobic conditions were estimated in tracer experiments and with mathematical methods. A complex method of these pathways in reticulocytes is given. Our approach to the solution of the problem in reticulocytes can be transferred to other tissues. The new mathematical technique used is suitable for derivation of closed terms for the specific radioactivity of single positions of C-atoms of the metabolites of citrate cycle. Therefore, by means of regressions analysis, the calculation of unknown concentrations and flux rates is possible.

Determination and characteristics of energy-dependent proteolysis in rabbit reticulocytes.

1. A method is reported, based on the isotopic dilution of lysine, which is suitable for the measurement of the degradation of stroma proteins of reticulocytes. 2. Proteolysis is extensive and time-limited. It may reach 50% of the stroma in 2 h at pH 7.6 and 37 degrees C. It is strongly dependent on pH and temperature, being small at low pH and temperatures. Proteolysis is largely ATP-dependent. The extent of proteolysis is nearly proportional to the degree of reticulocytosis. 3. The proteolytic system may be reconstructed in hemolysates if ATP is produced or supplied. It is assumed that the system is identical and that studied by Goldberg et al. and Hershko et al. on abnormal or denatured proteins as substrates. The main significance of the proteolytic system appears to be the degradation of mitochondria during maturation of the red cell.

Nitrogen economy and the metabolism of serine and glycine in reticulocytes of rabbits.

In this work it is demonstrated the NH3 potentially available from the oxidation of amino acids by the reticulocyte is utilized for the new-synthesis of serine via transamination reactions with hydroxypyruvate and phosphohydroxypyruvate. These compounds are derived from glucose, which furnishes in this manner the carbon skeleton of serine. It was shown that serine is mainly degraded via glycine, while glycine is in the main formed from serine. Furthermore it was found that serine synthesis is localized in the cytosol, while the reversible transformation of serine to glycine takes place in the mitochondria. A transfer of methylenetetrahydrofolate occurs in both directions across the mitochondrial membrane. A concentrative uptake of serine by the mitochondria was found, while glycine is transported slowly in both directions. On the basis of the data an overall balance is given of the quantitative relations between protein breakdown and hemoglobin synthesis as well as for the new-formation and utilization of serine and glycine. The new-formation of serine may amount to about one tenth of the glucose utilized by the reticulocyte and furnishes about one-half of the serine and glycine moieties required for the synthesis of heme and globin. The remainder is provided by the energy-dependent protein breakdown.

[gamma-Glutamyl transpeptidase in human eccrine sweat (author's transl)]. / Uber die Ausscheidung der gamma-Glutamyl-Transpeptidase im menschlichen ekkrinen Schweiss.

UNLABELLED: The concentration of gamma-glutamyl transpeptidase was determined in eccrine, thermal sweat of 56 healthy men (ages 20--60 years) and 48 healthy women (ages 17--55 years). Samples of sweat were collected from the chest and back. RESULTS: The concentrations of gamma-glutamyl transpeptidase showed great individual variations. The sex specific comparison of the concentrations of gamma-glutamyl transpeptidase revealed that the women excreted in the sweat from the chest and the back nearly the double amount of this enzyme. The gamma-glutamyl transpeptidase concentrations determined in the sweat from the chest of the examined men and women were three times higher as compared with those excreted simultaneous from the back of the same persons. In the group of the women was this differences statistically significant.

[Nitrogen-economy and synthesis of serine and glycine in reticulocytes]. / Stickstoff-Okonomie und Synthese von Serin und Glyzin in Retikulozyten.

Amino acids constitute the main substrate of the reticulocyte. The amino acid pool of reticulocytes represents a characteristic selection. The composition of the amino acid pool is dependent on maturation. From the preferential oxidation of short-chain amino acids one would expect a ratio about of 5:1 between the oxygen consumption and ammonia formation. If one corrects for NH3-formation by the deamination of nucleotides the ratio between the oxygen consumption and NH3-formation is about an order of magnitude higher than the theoretical ratio. The small liberation of NH3 in energy production from amino acids results from the re-utilization of their alpha-NH2-group for the synthesis of serine and glycine, while the C-skeleton stems from glucose. Serine is formed via OH-pyruvate and OH-pyruvate. Serine and glycine serve preferentially for synthesis of hemoglobin. In reticulocytes there exists a compartmentation of glycine which accounts for differences between serine and glycine in isotopic experiments. From the time dependent change of the specific activities of pulse-labelled serine and glycine one may calculate that the serine synthesis amounts to 15--30% of the glucose utilization.