Antiapoptotic activity of the Bowman-Birk inhibitor can be attributed to copurified phospholipids.

Previous studies have shown that extracts from soy possess potent antiapoptotic activity in in vitro and in vivo models. We recently reported that this antiapoptotic activity can be attributed to the presence of specific phospholipids. In this study, a conventional preparation of the soy-derived Bowman-Birk inhibitor (BBI) was tested for antiapoptotic activity in a C3H/10T1/2 cell serum deprivation assay. The BBI preparation was separated into lipid- or protein-containing fractions by organic extraction. The lipid fraction contained only antiapoptotic activity; the protein fraction contained only enzyme inhibition activity. We therefore conclude that the antiapoptotic activity of the BBI preparation is due to specific phospholipids that copurify with BBI. These phospholipids retain their antiapoptotic activity after autoclave treatment, whereas autoclave treatment of the protein fraction results in a loss of its enzyme inhibition activity.

Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid.

We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor. LPA activated the pheromone response pathway in S. cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene. LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product). This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway. The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ. LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation. These results indicate that Edg-2 encodes a highly specific LPA receptor.

The effect of a synthetic hexadentate iron chelator (CP130) and desferrioxamine on rabbit kidneys exposed to cold and warm ischaemia.

The effect of CP130 (a synthetic hexadentate pyridinone iron chelator) on the formation of two markers of lipid peroxidation (TBA-reactive material and Schiff's bases) in rabbit kidneys following a 72 h period of cold (0-4 degrees C) ischaemia was investigated by either adding CP130 to the flush/storage solution (hypertonic citrate solution) or by administering the agent intravenously 15 min before removal of the organs. In both cases, CP130 blocked the adverse rises in lipid peroxidation caused by ischaemia and subsequent reoxygenation of the homogenates in vitro. Both CP130 and desferrioxamine (DFX) (administered intravenously 15 min before ischaemia and 5 min before reperfusion) were also found to significantly reduce post-ischaemic rates of in vitro lipid peroxidation in kidneys rendered warm ischaemic for 90 min followed by reperfusion for 5 or 60 min in situ. Kidneys exposed to warm ischaemia and reperfusion developed interstitial and intracellular oedema, congestion and haemorrhage. DFX administration had little effect on the histological outcome, whereas CP130 significantly reduced interstitial oedema (at 5 min reperfusion compared to the DFX-treated group), intracellular oedema (at 60 min reperfusion compared to the DFX-treated group) and congestion (at 5 min reperfusion compared with a control group not given any agent). It is concluded that while CP130 and DFX exhibited similar antioxidant properties, CP130 provided better protection from ischaemia/reperfusion injury at the histological level. Synthetic iron chelators may therefore be of benefit in clinical organ transplantation by protecting against tissue damage caused by prolonged ischaemia.

Reoxygenation following hypoxia stimulates lipid peroxidation and phosphatidylinositol breakdown in kidney cortical slices.

Reoxygenation of hypoxic (120 min at 37 degrees) rabbit kidney cortical slices in vitro resulted in a rapid increase in lipid peroxidation and phosphatidylinositol hydrolysis. No changes in phosphatidylinositol breakdown occurred during hypoxia or upon reoxygenation in the absence of calcium. Incubation of renal slices with carbon tetrachloride resulted in increased lipid peroxidation but had no effect on phosphatidylinositol breakdown. It is concluded that altered intracellular calcium homeostasis during reoxygenation is involved in mediating increased phosphatidylinositol hydrolysis through activation of a specific phospholipase C, but that oxidative stress per se does not have a significant effect on the inositol phosphate secondary messenger response in this model system.

Ebselen. Antioxidant capacity in renal preservation.

Ebselen (PZ51) was tested for its ability to inhibit oxidative membrane damage and improve outcome of rabbit kidneys rendered cold ischaemic for 72 hr. In view of the rapid metabolism of ebselen, the antioxidant capacities of its two principal metabolites were first compared with that of the parent drug in an in vitro hepatic microsomal lipid peroxidation system initiated by NADPH/Fe(3+)-ADP. The potent antioxidant activity of ebselen was confirmed but metabolite I (2-glucuronylselenobenzanilide) exhibited no antioxidant potential up to a concentration of 50 microM; metabolite II (4-hydroxy-2-methyl-selenobenzanilide) did inhibit lipid peroxidation but was about 80 times less effective than the parent compound. The storage of rabbit kidneys in hypertonic citrate solution at 0 degrees for 72 hr of cold ischaemia resulted in greatly increased susceptibility to oxidative membrane damage in both the cortex and medulla as determined by the subsequent in vitro formation of two markers of lipid peroxidation (Schiff's bases and thiobarbituric acid-reactive material). Inclusion of ebselen (50 microM) in the flush and storage solution led to a highly significant reduction in these oxidative markers in both regions of the kidney. Intracellular and interstitial oedema was noted in organs subjected to 72 hr cold ischaemia and was reduced by ebselen (50 microM in the flush/storage solution). The rate of post-ischaemic lipid peroxidation was found to correlate well with the extent of oedema in the renal medulla (r = 0.84, P less than 0.001) but no such correlation was found in the cortex. Administration of ebselen (5.5 mg/kg i.v. and 100 microM in the flush/storage solution) did not improve the long-term survival of rabbits following autotransplantation of a single kidney stored for 48 or 72 hr. No protective effect of ebselen could be demonstrated either in terms of graded physiological function or histological outcome.

A chelator is required for microsomal lipid peroxidation following reductive ferritin-iron mobilisation.

In the past, antioxidant and chelator studies have implicated a role for iron-dependent oxidative damage in tissues subjected to ischaemia followed by reperfusion. As ferritin is a major source of iron in non-muscular organs and therefore a potential source of the iron required for oxygen radical chemistry, we have determined conditions under which ferritin iron reduction leads to the formation of a pool of iron which is capable of catalysing lipid peroxidation. Under anaerobic conditions and in the presence of rat liver microsomes, flavin mononucleotide (FMN) catalysed the reduction of ferritin iron as shown by both continuous spectrophotometric measurements of tris ferrozine-Fe(II) complex formation and post-reaction Fe(II) determination. The presence of either ferrozine or citrate was not found to alter the time course or extent of ferritin reduction. In contrast, the addition of air to the reactants after a 20 min period of anaerobic reduction resulted in peroxidation of the microsome suspension (as determined with the 2-thiobarbituric acid test) only in the presence of a chelator such as citrate, ADP or nitrilotriacetic acid. These results support the concept that reduced ferritin iron can mediate oxidative damage during reperfusion of previously ischaemic tissues, provided that chelating agents such as citrate or ADP are present.

Long-term trial with the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1). I. Iron chelation and metabolic studies.

A long-term clinical trial of 1-15 months has been carried out with the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in 13 transfusion-dependent iron-loaded patients. Urinary iron excretion was greatest in patients with thalassaemia major and was related to the number of previous transfusions but not to the serum ferritin level. Substantial increases of urinary iron were observed in all the patients when the frequency of the daily dose was doubled and in response to 2 x 3 g L1 daily 11 of 12 patients tested excreted greater than 25 mg iron daily, the mean daily intake of iron from transfusion. Serum ferritin levels have fluctuated but overall have remained unchanged. Pharmacological studies in five patients have indicated rapid absorption probably from the stomach and variable plasma half life of 77 +/- 35 min (X +/- SD). Glucuronation was identified as a major route of L1 metabolism. Short-term intensive chelation studies using repeated administration of L1 resulted in further increases of urinary iron excretion by comparison to a single dose. In one case 325 mg of iron were excreted in the urine following the administration of 16 g (5 x 2 g + 2 x 3 g) within 24 h. Iron excretion studies were carried out in six transfusional iron-loaded patients who were maintained on a low iron diet before and during chelation. No significant increases of faecal iron excretion were observed with L1 using daily doses of up to 3 x 3 g and 4 x 2 g. The high level of compliance during treatment with L1 and the levels of urine iron excretion that can be achieved increase the prospects for oral chelation in transfusional iron-loaded patients.

Pharmacokinetic studies in humans with the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one.

Pharmacokinetic studies have been carried out with the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1). HPLC analysis of serum of a normal volunteer and seven transfusional iron loaded patients who ingested a 3 gm dose of L1 revealed that L1 was most probably absorbed from the stomach and was transferred to the blood with a half-life of 0.7 to 32 minutes. L1 reached maximum concentration in the serum 12 to 120 minutes after administration with 85% to 90% elimination within the first 5 to 6 hours, with a half-life of 47 to 134 minutes. L1 and its glucuronide metabolite were identified in serum and urine but not in feces. In most cases hydrolysis of 24-hour urine samples with use of beta-glucuronidase resulted in almost complete recovery of the administered dose. Urinary iron excretion was proportional to the iron load but not to the serum or urine concentration of L1. The therapeutic efficiency of L1 can therefore be improved by repeated administration of 2 to 3 gm doses at least every 6 hours.

Development of an HPLC method for measuring orally administered 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in biological fluids.

"High-performance" liquid-chromatographic (HPLC) methods have been developed for identifying 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in serum and urine. Ion pairing with heptane- or octanesulfonic acid in pH 2.0-2.2 phosphate buffer and reversed-phase chromatography were required to separate these compounds from endogenous compounds in both biological fluids. In both the 2-methyl and 2-ethyl series of 1-substituted compounds (H, methyl, ethyl, or propyl) the elution times increased in accordance with the n-octanol/water partition coefficients (propyl greater than ethyl greater than H greater than methyl). Urine samples were filtered (0.4 microns pore size) and injected either undiluted or after dilution with elution buffer. After the addition of internal standard, the plasma or serum samples were deproteinized by treatment with HCIO4, 0.5 mol/L, centrifuged, and the supernates were injected directly onto the HPLC. Using these procedures, we could identify 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in the serum and urine of a thalassemic patient who had received a 3-g dose of the drug and in the urine of other patients who had received the same dose. One or more possible metabolites were also observed in the chromatograms of both urine and serum. The 24-h urinary output of L1 (0.22-2.37 g) and iron (10.6-71.5 mg) varied but there was no correlation between the two with respect to quantity or concentration. Instead, urinary iron output was higher in patients with a greater number of transfused units of erythrocytes. This is the first study in humans to show that L1 is absorbed from the gut, enters the circulation, and is excreted in the urine.

Delayed, ferrous iron-dependent peroxidation of rat liver microsomes.

Measurement of both chemiluminescence (CL) and the formation of 2-thiobarbituric acid-reacting substances (TBAR) has been used to study the delayed, nonenzymatic lipid peroxidation (LP) initiated in rat liver microsomes by ferrous chloride. Following Fe2+ addition, the CL technique revealed a burst of light emission (peak, Phase II) which was preceded by a period of little or no detectable photon production (delay, Phase I) and succeeded by an increased emission (Phase III). Analysis of TBAR indicated a low rate of LP during the delay which increased more than fivefold during a 1-min period and which corresponded to the CL peak. The delay length depended on both the Fe2+ concentration and the microsome concentration; increased Fe2+ yielded longer delays while increased microsome concentration decreased the delay. As reported by others [J. R. Bucher, M. Tien, and S. D. Aust (1983) Biochem. Biophys. Res. Commun. 111, 777-784; J. M. Braughler, L. A. Duncan, and R. L. Chase (1986) J. Biol. Chem. 261, 10282-10289], Fe3+ also decreased the delay. The ferric-nitrilotriacetate (Fe3+-NTA) complex was found to be more efficient than "free" Fe3+ [Fe(NO3)3]; a 100 microM concentration of the 1:1 Fe3+-NTA complex eliminated the delay due to 100 microM Fe2+, whereas 400 microM Fe(NO3)3 reduced the delay from 17.5 to 2.5 min. Incubation under reduced O2 tension demonstrated a requirement for O2 during the delay. The use of antioxidants [butylated hydroxytoluene, (+)-catechin, promethazine, and uric acid] and inhibitors of the Haber-Weiss reaction (mannitol, Tris buffer, dimethyl sulfoxide, catalase, and superoxide dismutase) indicated that the initiating species has characteristics of a weak oxidizing radical capable of either hydrogen or electron abstraction from suitable target molecules. We hypothesize that the delay that is sensitive to the Fe2+:microsome ratio is due to reductive elimination of the initiating species by "free" Fe2+. The nature of the initiating species has yet to be determined; however, the argument is presented that the perferryl ion (Fe3+-O2-.) may possess the characteristics required for the initiator.

Lipid peroxidation stimulated by iron nitrilotriacetate in rat liver.

The complex of ferric iron with nitrilotriacetate (iron-NTA) given i.p. is an unusually potent stimulus for lipid peroxidation (LP) in vivo, as monitored by exhaled alkanes. Localization of 59Fe-labeled NTA radioactivity in mouse liver and accumulation of thiobarbituric acid (TBA)-reacting material in liver after i.p. injection suggested that the effect of i.p. iron-NTA could be primarily hepatic. It was found that 100 microM iron-NTA added to a hepatocyte suspension gassed with air stimulated ethane production (3 +/- 1 pmoles/10(6) cells/min) versus an undetectable control, and at a sensitivity of 0.083 pmole/10(6) cells/min. Under similar conditions, hepatocytes stimulated by iron-NTA generated low level chemiluminescence (CL) in parallel with formation of TBA-reactants; the generation of CL was concentration related. Liver was homogenized and fractionated by ultracentrifugation: iron-NTA stimulated CL in whole liver homogenate as in intact cells. The greater part of this activity localized to the microsomal and mitochondrial fractions where NADH or NADPH was required. Using rat liver microsomes, it was shown that iron-NTA in the presence of NADPH stimulated two phases of CL with an initial phase maximum in 1-2 min (phase 1) which decreased abruptly to be followed by a prolonged rise (phase 2); NADH could replace NADPH. Ferrous iron (as chloride) caused a burst of CL, whereas ferric iron was inactive. However, complex differences exist between CL stimulated by Fe(II) and by iron-NTA in the presence of reducing equivalents. Under conditions resulting in the production of CL, a microsomal system with iron-NTA and reducing equivalent accumulated TBA-reactants in parallel with the stimulated CL and rapid increase in oxygen consumption. Both desferrioxamine and butylated hydroxyanisole were able to strongly inhibit the CL stimulated by iron-NTA. When iron-NTA and iron-ADP were compared in the microsomal system, similar responses were obtained but major differences characterized the effects of these iron chelates on whole cells with the ADP complex being relatively inactive. We conclude that iron-NTA stimulated free radical reactions in liver by undergoing cyclic oxidation and reduction and that these reactions utilized oxygen, generated CL, and formed TBA-reactants and ethane. At a subcellular level, the reactions of iron-NTA resembled those reported for iron-ADP.

Structure of Moloney murine leukemia viral DNA: nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences.

Some unintegrated and all integrated forms of murine leukemia viral DNA contain long terminal repeats (LTRs). The entire nucleotide sequence of the LTR and adjacent cellular sequences at the 5' end of a cloned integrated proviral DNA obtained from BALB/Mo mouse has been determined. It was compared to the nucleotide sequence of the LTR at the 3' end. The results indicate: (i) a direct 517-nucleotide repeat at the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end of the primer tRNA (strong-stop DNA); (iii) an 11-nucleotide inverted repeat is present at the ends of the 5'-LTR and a total of 17 out of 21 nucleotides at the termini are inverted repeats; (iv) sequences CAATAAAAG (at positions -24 to -31) and CAATAAAC (at positions +46 to +53) resembling the hypothetical DNA-dependent RNA polymerase II promoter site can be identified in the 5'-LTR; (v) the sequence GAAA appears to be repeated on both sides of the junction of viral and cellular sequences; and (vi) in analogy with the bacterial transposons, the presence of an inverted repeat sequence at the termini of 5'-LTR suggests that M-MLV also has the integration properties of a transposon.

Repair and mutagenesis of herpes simplex virus in UV-irradiated monkey cells.

Mutagenic repair in mammalian cells was investigated by determining the mutagenesis of UV-irradiated or unirradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cells. These results were compared with the results for UV-enhanced virus reactivation (UVER) in the same experimental situation. High and low multiplicities of infection were used to determine the effects of multiplicity reactivation (MR). UVER and MR were readily demonstrable and were approximately equal in amount in an infectious center assay. For this study, a forward-mutation assay was developed to detect virus mutants resistant to iododeoxycytidine (ICdR), probably an indication of the mutant virus being defective at its thymidine kinase locus. ICpR-resistant mutants did not have a growth advantage over wild-type virus in irraidated or unirradiated cells. Thus, higher fractions of mutant virus indicated greater mutagenesis during virus repair and/or replication. The data showed that: (1) unirradiated virus was mutated in unirradiated cells, providing a background level of mutagenesis; (2) unirradiated virus was mutated about 40% more in irradiated cells, indicating that virus replication (DNA synthesis?) became mutagenic as a result of cell irradiation; (3) irradiated virus was mutated much more (about 6-fold) than unirradiated virus, even in unirradiated cells; (4) cell irradiation did not change the mutagenesis of irradiated virus except at high multiplicity of infection. High multiplicity of infection did not lead to higher mutagenesis in unirradiated cells. Thus the data did not demonstrate UVER or MR alone to be either error-free or error-prone. When the two processes were present simultaneously, they were mutagenic.