Inhibition in vitro linoleic acid peroxidation and haemolysis by caffeoyltryptophan

Antioxidant activities of caffeoyltryptophan were investigated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging system, the superoxide anion generation system and the superoxide anion-mediated linoleic acid peroxidation system. At 10 microM, caffeoyltryptophan showed greater scavenging activity on DPPH than dl-alpha-tocopherol or ascorbic acid. DPPH radical scavenging activity of caffeoyltryptophan increased dose-dependently at concentrations ranging from 1 to 50 microM; 1 mol of caffeoyltryptophan reacted with ca 4 mol of radical. Caffeoyltryptophan caused 80% inhibition of superoxide anion generation at 50 microM. The inhibitory activity of caffeoyltryptophan was as strong as that of 5-caffeoylquinic acid. Caffeoyltryptophan inhibited the formation of conjugated diene from linoleic acid. The inhibitory activity increased in the order caffeic acid < 5-caffeoylquinic acid < caffeoyltryptophan < dl-alpha-tocopherol. Effects on the in vitro haemolysis and peroxidation of mouse erythrocytes induced by H2O2 were also examined. Caffeoyltryptophan exhibited strong inhibitory activities; Tryptophan was ineffective in these systems. These data suggest that caffeoyltryptophan may be a natural antioxidant in the human diet and, as such, may intervene in toxicological processes that are mediated by radical mechanisms.     

Proanthocyanidins from the bark of Hamamelis virginiana exhibit antimutagenic properties against nitroaromatic compounds

The antimutagenic activity of Hamamelis virginiana bark was examined in the Ames assay. A commercial tincture and a methanolic extract showed dose-dependent inhibitory effects on mutagenicity induced by 2-nitrofluorene. Tannin-free samples did not display any inhibition. Bioassay-guided fractionation resulted in the isolation of two active fractions which were shown to contain oligomeric, proanthocyanidins. They were capable of inhibiting the mutagenicity of selected nitroaromatic compounds. The mechanism of antimutagenic action was also studied. The proanthocyanidins did not act as bioantimutagens, but rather as direct-acting desmutagens. The antimutagenic effect increased with an increasing degree of polymerisation in the proanthocyanidins. The most active fraction consisted of catechin and gallocatechin oligomers with an average polymerisation degree of 9.2.     

Effect of quercetin on chemiluminescence of human platelets induced by arachidonic acid

Arachidonic acid (AA)-induced platelet chemiluminescence (CL) was measured with a lumiphotometer. Quercetin remarkably inhibited the CL, the IC50 of quercetin was 3 mumol.L-1. When quercetin plus aspirin, which inhibits only cyclooxygenase, was added, the inhibitory rate of platelet-CL obviously increased (P < 0.01). On the other hand, the quercetin had a scavenging effect on superoxide anion radical using alkaline sodium dithionite solution generation. The IC50 was 20.9 mumol.L-1. In addition, superoxide dismutase of 0.1 mg.ml-1 inhibited the platelet-CL by 97.8%, while mannitol, a hydroxyl radical scavenger, only by 43.3% at a concentration of 80 mg.ml-1. These results suggest that the mechanism of the inhibiting AA-induced platelet-CL by quercetin was associated with scavenging the superoxide anion radical directly and with inhibiting the cyclooxygenase.     

Use of on-flow LC/1H NMR for the study of an antioxidant fraction from Orophea enneandra and isolation of a polyacetylene, lignans, and a tocopherol derivative

The CH2Cl2 extract of the leaves of Orophea enneandra displayed antifungal, antioxidant, and radical scavenging properties in bioautographic TLC assays. To obtain rapid information on the active compounds, on-flow LC/1H NMR and LC/UV/MS analyses of the antioxidant fraction were performed. The on-line information led rapidly to the partial identification of three closely related lignans, one tocopherol derivative, and one polyacetylene. This approach necessitated, however, large quantities to be injected to obtain satisfactory on-flow LC/1H NMR spectra, and isolation of the compounds was necessary to obtain complete NMR data. These compounds were isolated and identified as (-)-phylligenin (1), (-)-eudesmin (2), (-)-epieudesmin (3), polycerasoidol (4), and oropheic acid (5), a new polyacetylene. Their activities against the 2, 2-diphenyl-1-picrylhydrazyl radical and the fungus Cladosporium cucumerinum were investigated. This paper indicates the possibilities and limits of on-flow LC/1H NMR in this type of study.     

Thermal and antiradical properties of indirect moxibustion

The thermal and antiradical properties of indirect moxibustion stimulation were investigated by thermal qualitative and spectroscopic methods. The thermal effect of indirect moxibustion was mainly dependent on the spacing distance between the moxa and skin, and not on the moxa weight. The radical scavenging activities of moxa and moxa-tar were measured by a photometric absorbance method, chemical reaction with 1,1-diphenyl-2-picrylhydrazyl. The obtained results indicate that the inhibitory effects of moxa and moxa-tar on superoxide production are due to the radical scavenging mechanism.     

Protective effect of FK506 on ischemia/reperfusion-induced myocardial damage in canine heart

We investigated the cardioprotective effect of FK506, a newly developed immunosuppressive agent, on ischemia-reperfusion-induced myocardial damage and the inhibitory effect of FK506 on superoxide radical formation by neutrophils. Open-chest anesthetized dogs were divided into two groups: group 1, 2-h occlusion of the coronary artery followed by 1-h reperfusion; and group 2, 2-h occlusion followed by 1-h reperfusion with preadministration of FK506 (0.5 mg/kg). After reperfusion, heart mitochondria were prepared from the normal and reperfused areas and mitochondrial function and mitochondrial GSH (the reduced form of glutathione) and GSSG (the oxidized form of glutathione) concentrations were measured. In addition, neutrophils were collected from normal healthy dogs, and the inhibitory effect of FK506 on superoxide radical formation by neutrophils was also investigated. One-hour reperfusion after 2-h coronary occlusion induced significant mitochondrial dysfunction associated with a marked depletion of mitochondrial GSH concentration. FK506 reduced mitochondrial dysfunction, depletion of mitochondrial GSH concentration, and development of reperfusion arrhythmias. FK506 also reduced stimulant-induced superoxide radical formation by normal neutrophils dose dependently. Radical scavenging activity decreased in association with reperfusion, and FK506 reduced superoxide radical formation by neutrophils, which might contribute to lessening ischemia-reperfusion damage.     

Hepatoprotective, superoxide scavenging, and antioxidative activities of aromatic constituents from the bark of Betula platyphylla var. japonica

The 50% aqueous methanolic extract from the bark of Betula platyphylla SUKATCHEV var. japonica (MIQ). HARA was found to show potent inhibitory activity on the liver-injury induced by CCl4 or D-galactosamine (D-GalN)/lipopolysaccharide as well as O2- scavenging and antioxidative activities. From the 50% aqueous methanolic extract, two new diarylheptanoids named betulaplatosides Ia (1) and Ib (2) and a new arylbutanoid named betulaplatoside II (3) were isolated together with seventeen known aromatic constituents. 1, 2, and two known diarylheptanoids [(5S)-5-hydroxy-1,7-bis-(4-hydroxyphenyl)-3-heptanone 5-O-beta-D-apiofurano-syl-(1-->6)-beta-D-glucopyranoside (6) and aceroside VIII (7)] showed protective activity against D-GalN-induced cytotoxicity in primary cultured rat hepatocytes. Furthermore, several aromatic constituents exhibited potent O2- scavenging and antioxidative activities.     

Antioxidative effects of a processed grain food

Antioxidant biofactor: AOB is a unique processed grain food. It is a yellow-green powder. It contains the following extracts: germ extracts, soybean, rice bran, tear grass, sesame, wheat, citron, green tea, green leaf extract, and malted rice. These materials were slowly roasted under a powdered oure at less than 60 degrees C and fermented with Aspergillus oryzae over 3 days to transform each ingredient into low molecular weight substances. These conditions were different by each material, environmental humidity and temperature. It probably contains a variety of substances having antioxidant activity including flavonoids, alpha-tocopherol, vitamin C, and tannins. We investigated its antioxidative properties using electron spin resonance (ESR) and autoxidation of rat brain homogenates. The superoxide, hydroxyl radical, and the stable free radical, diphenyl-p-picrylhydrazyl (DPPH) radical scavenging activity of AOB was investigated using ESR spectrometry. In an in vitro study, a suspension of AOB was added directly to a superoxide generating system (hypoxanthine-xanthine oxidase; HX/XO) and investigated using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. At final concentrations of 0.01, 0.05, and 0.1 mg/ml, AOB dose-dependent scavenging activity was observed as 0.103, 0.619, and 1.369 U/ml, respectively. A concentration of 1.0 mg/ml completely scavenged DMPO-OOH signals; 1.0 mg/ml of AOB inhibited the DMPO-OH signal generated by Fenton's reaction, but its inhibitory effect was not competitive, and was inhibition of the Fenton's reaction. 1.0, 3.0, and 5.0 mg/ml of AOB were significantly inhibited the DPPH radical. In an in vivo study, rats were fed AOB orally at doses of 1 or 5 g/day for 24 h or for 3 days and the superoxide scavenging activity was measured in plasma. With the administration of 1 g/day for 3 days, the superoxide scavenging activity was about 1.8 times that of the control group fed a basal diet; 1.5 times the control with 5 g/day for 1 day, and 2.6 times the control with 5 g/day for 3 days, all of which represented significant increases in superoxide scavenging activity. AOB strongly inhibited the autoxidation of rat brain homogenates in vitro in a dose-dependent manner. However, each ingredient before roast and fermentation little inhibited lipid peroxidation. Roasting and fermentation with A. oryzae way be important to transform each ingredient into low molecular weight substances. Therefore, it was suggested that AOB possesses strong antioxidant and free radical scavenging activities.     

Protective action of epidermal growth factor and a fraction from Triticum vulgare extract in mouse tail necrosis

Several peptide growth factors, including EGF, are known to protect endothelium from oxygen-related damage or ischemia-reperfusion, in vitro experiments show that such protective effect involves endogenous endothelium-related factors like nitric oxide and prostanoids. However, in vivo demonstrations of a possible role in related vascular diseases are lacking. In our experiments, human EGF and fraction C, a 3-10 kDa oligosaccharidic fraction from an aqueous extract of Triticum vulgare, known as growth promoters for several cell types including endothelial cells, were found protective against ischemic necrosis of the mouse tail induced by i.v. k-carrageenin plus endothelin-1. After i.p. injection, peak activities were observed at 10 micrograms/kg EGF and 2 mg/kg fraction C. Pretreatment with L-NAME reduced protection in a dose-dependent manner. Addition of indomethacin increased the effect of L-NAME, suggesting that both nitric oxide and eicosanoids are involved in the protective effect of EGF and fraction C.     

Eugenol--an inhibitor of lipoxygenase-dependent lipid peroxidation

The effect of eugenol on enzymatic lipid peroxidation catalyzed by soybean lipoxygenase was studied in an in vitro system. Lipid peroxidation was inhibited by eugenol in a concentration-dependent manner. The half-maximal inhibition (IC50) was found to be 380 microM eugenol. Enzyme kinetic studies showed that eugenol non-competitively inhibited lipid peroxidation by altering the maximum velocity (Vmax) and without any change in Michaelis-Menten constant (Km) values. The inhibitory mechanism implies that eugenol does not inactivate the enzyme directly but may interfere with fatty acid radical intermediates due to its hydroxy radical scavenging ability and thus play a role in inhibiting the propagation of lipid peroxidation.     

The effect of Kampo formulae on bone resorption in vitro and in vivo. I. Active constituents of Tsu-kan-gan

Four water extracts of Kampo formulae (Yi-kkan-sen, Dai-ho-in-gan, Ni-chi-gan, Tsu-kan-gan) were screened for their inhibitory activities on bone resorption induced by parathyroid hormone (PTH) in organ culture using neonatal mouse parietal bones. Among the Kampo formulae, Tsu-kan-gan (TKG) showed the most potent inhibitory activity. We further fractionated the TKG water extract by monitoring the inhibitory activity on bone resorption stimulated by PTH in vitro. The MeOH fraction of the water extract inhibited PTH-stimulated bone resorption, and its inhibitory activity was more potent than those of other fractions. The MeOH fraction was then subjected to Sephadex LH-20 column chromatography to give fractions I, II and III, which were examined for bone resorption activity. Fraction I inhibited PTH-stimulated bone resorption, and its inhibitory activity was more potent than those of the other fractions. Upon oral administration of the three fractions (100 mg/kg/d) to ovariectomized (OVX) mice, fractions I and III prevented the decrease of bone mineral density (BMD) of the lumbar vertebra. Eleven compounds isolated from the MeOH fraction were examined for their inhibitory effect on PTH-stimulated bone resorption. Among them, berberine (1), syringin (3), limonin (4) and mangiferin (10) showed a significant inhibitory effect on bone resorption. In the formation assay of osteoclast-like cells, these compounds decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). The inhibitory effect of TKG on bone resorption may be at least partly due to the inhibitory action of these compounds.     

Selective inactivation of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid with 4-hydroxy-2-nonenal

Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate dehydrogenase (PDH), while other NADH-linked dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.     

A case report of extensive vitiligo

Minimum inhibitory concentrations of doxycycline and oxytetracycline were determined against 55 Pasteurella multocida strains, 59 Actinobacillus pleuropneumoniae strains and 26 Mycoplasma hyopneumoniae strains isolated from the respiratory tract of pigs. An additional set of 76 P multocida strains isolated from pneumonic pigs was tested for their minimum inhibitory concentrations of doxycycline. The P multocida and A pleuropneumoniae strains were isolated in France and the minimum inhibitory concentrations were determined by an agar dilution method. The M hyopneumoniae strains were isolated in the United Kingdom and minimum inhibitory concentrations were determined by a serial broth dilution method. All the strains tested were susceptible to doxycycline whereas 15 per cent of the P multocida strains and 22 per cent of the A pleuropneumoniae strains were resistant to oxytetracycline. Doxycycline concentrations inhibiting 90 per cent of strains were 1 microgram/ml for P multocida and 2 micrograms/ml for A pleuropneumoniae. The ratio of the minimum inhibitory concentrations of doxycycline and oxytetracycline ranged between 1/1 and 1/4 for the oxytetracycline-susceptible strains and between 1/16 and 1/64 for the oxytetracycline-resistant strains. All the M hyopneumoniae strains were susceptible to doxycycline and oxytetracycline, the concentrations inhibiting 90 per cent of strains being 1 microgram/ml and 2 micrograms/ml, respectively. These data confirm that doxycycline has a higher in vitro activity against pig respiratory pathogens than oxytetracycline.     

Elevation of antioxidant potency in the brain of mice by low-dose gamma-ray irradiation and its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain damage

The elevation of endogenous thiol-related antioxidants and free radical scavenging enzymes in the brain of C57BL/6 female mice after low-dose gamma-ray irradiation and its inhibitory effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain damage were investigated. The brain level of the reduced form of glutathione (GSH) increased soon after irradiation with 50 cGy of gamma-rays, reached a maximum at 3 h post-treatment, and remained elevated until 12 h. Thioredoxin (TRX) was also transiently increased after irradiation. The activities of free radical scavenging enzymes, including Cu/Zn-superoxide dismutase, catalase and glutathione peroxidase, were significantly induced after irradiation as well. Cerebral malondialdehyde was remarkably elevated by MPTP treatment, and this elevation was suppressed by pre-irradiation (50 cGy). The contents of GSH and TRX were significantly decreased by MPTP treatment in comparison with those of the control group. These reductions both seemed to be attenuated by pre-irradiation with gamma-rays. These results suggest that low-dose gamma-ray irradiation induces endogenous antioxidative potency in the brain of mice and might be effective for the prevention and/or therapy of various reactive oxygen species-related neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

Interactions of the human class II major histocompatibility complex protein HLA-DR4 with a peptide ligand demonstrated by affinity capillary electrophoresis

The comparative mechanisms and relative rates of nitrogen dioxide (NO2.), thiyl (RS.) and sulphonyl (RSO2.) radical scavenging by the carotenoid antioxidants lycopene, lutein, zeaxanthin, astaxanthin and canthaxanthin have been determined by pulse radiolysis. All the carotenoids under study react with the NO2. radical via electron transfer to generate the carotenoid radical cation (Car.+). In marked contrast the glutathione and 2-mercaptoethanol thiyl radicals react via a radical addition process to generate carotenoid-thiyl radical adducts [RS-Car].. The RSO2. radical undergoes both radical addition, [RSO2-Car]. and electron abstraction, Car.+. Both carotenoid adduct radicals and radical cations decay bimolecularly. Absolute rate constants for radical scavenging were in the order of approximately 10(7)-10(9) M(-1) s(-1) and follow the sequence HO(CH2)2S. > RSO2. > GS. > NO2.. Although there were some discernible trends in carotenoid reactivity for individual radicals, rate constants varied by no greater than a factor of 2.5. The mechanism and rate of scavenging is strongly dependent on the nature of the oxidising radical species but much less dependent on the carotenoid structure.     

Anti-free radical mechanisms in captopril protection against reperfusion injury in isolated rat hearts

OBJECTIVE: Captopril, an angiotensin-converting enzyme (ACE) inhibitor, is known to modulate ischemia-reperfusion injury in the isolated hearts. This study was designed to examine the involvement of anti-free radical mechanisms in this protection. METHODS: Isolated perfused rat hearts were subjected to 60 mins of global ischemia and 30 mins of reperfusion with or without captopril (100 mumol/L). Myocardial resting tension and contractile force were recorded. At the end of reperfusion, hearts were analyzed for the activities of antioxidant enzymes, superoxide dismutase, glutathione peroxidase and catalase, as well as for the extent of lipid peroxidation. Another potent ACE inhibitor, enalapril (100 mumol/L) was used for comparison. RESULTS: Captopril significantly improved the recovery of contractile function as well as attenuated the rise in resting tension in the ischemic-reperfused hearts as compared to the control. Captopril-exposed ischemic-reperfused hearts showed an increase in the activity of superoxide dismutase with no change in glutathione peroxidase and catalase enzyme activities. Lipid peroxidation at the end of reperfusion was significantly attenuated in the captopril-exposed hearts compared to the control. Enalapril had no protective effect against ischemia-reperfusion induced contractile failure or rise in resting force. CONCLUSIONS: These results suggest that cardioprotection by captopril, against ischemia-reperfusion injury, may involve an anti-free radical mechanism independent of its ACE inhibition property.     

Isolation and characterization of maturation inhibiting compound in bovine follicular fluid

The protein fraction which is responsible for the inhibition of maturation of bovine oocytes in vitro was isolated from cow follicular fluid by means of column chromatography on a Sephadex G-200 and a Sepharose 4B, both in 0.1 M ammonium acetate, pH 6.7. The molecular weight of the maturation inhibiting protein fraction is approximately 60 kDa. At a concentration of 2.0 mg/mL in cultivation medium, 100% of the oocytes were arrested at the germinal vesicle stage. At a concentration of 0.25 mg/mL, the protein fraction still had some meiosis inhibiting effects, but 56% of the oocytes were capable of maturing to the metaphase of the second meiosis (MII). Without compact cumulus the inhibiting fraction had no meiosis retarding effect on the oocytes. Cow follicular fluid also exhibited this inhibitory effect on oocyte maturation in vitro. However, the follicular fluid from follicles of 2.5-5.0 mm diameter showed higher meiosis inhibiting effects than the follicular fluid from follicles of 5-10 mm diameter.     

A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide

Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid beta-peptide (A beta) can promote free radical production, we tested the hypothesis that HNE mediates A beta 25-35-induced disruption of neuronal ion homeostasis and cell death. A beta induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na+, K(+)-ATPase activity and induced an increase of neuronal intracellular free Ca2+ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both A beta and HNE. The antioxidant propyl gallate protected neurons against A beta toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates A beta-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

Inhibitory action of a truncated derivative of the amphibian skin peptide dermaseptin s3 on Saccharomyces cerevisiae

The inhibitory activity of a truncated derivative of the natural amphibian skin peptide dermaseptin s3-(1-16)-NH2 [DS s3 (1-16)] against Saccharomyces cerevisiae was studied. Significant growth inhibition was observed after exposure to 3.45 microgram of the peptide per ml at pH 6.0 and 7.0, with complete growth inhibition occurring at 8.63 microgram of peptide per ml for all pH values tested. Using confocal scanning laser microscopy, we have shown that DS s3 (1-16) disrupted the yeast cell membrane resulting in the gross permeabilization of the cell to the nuclear stain ethidium bromide. However, the principal inhibitory action of the peptide was not due to disruption of intracellular pH homeostasis. Instead, growth inhibition by the peptide correlated with the efflux of important cellular constituents such as ADP, ATP, RNA, and DNA into the surrounding medium. The combination of DS s3 (1-16) with mild heating temperatures as low as 35 degreesC significantly enhanced the inhibitory effect of the peptide (8.63 microgram/ml), and at 45 degreesC greater than 99% of the population was killed in 10 min. In summary, a derivative of a natural antimicrobial peptide has potential, either alone or in combination with mild heating, to prevent the growth of or kill spoilage yeast.