Lipophilic Oxidants Research Articles

High concentrations of casein proteins exacerbate radical chain reactions and increase the extent of oxidative damage

The co-existence of proteins, lipids and riboflavin (RF) in milk together with the harsh conditions encountered during processing (e.g. high temperatures, light exposure) results in oxidative damage. Proteins represent ~30 % of the dry mass of milk, with caseins accounting for ~80 % (28 g L−1). Due to their high abundance and amphiphilic nature, caseins are targets for both hydrophilic and lipophilic oxidants. Although caseins are key milk components, and highly abundant, most previous work has employed non-biological dilute solutions. In this work we have investigated oxidative modification of αs-, β- and κ-caseins elicited by AAPH-derived oxygenated radicals, or RF-mediated photo-oxidation, at both low and high protein concentrations, to determine whether and how oxidative damage and resulting structural modifications are modulated by the protein concentration. The data obtained demonstrate that the pathways leading to casein modification are dependent on both the protein concentration and the oxidant employed. AAPH-mediated oxidation was more efficient than RF-induced photo-oxidation, in respect to the number of moles of amino acid side-chains consumed per mole of oxidant generated, the extent of damage detected by SDS-PAGE, and immunoblot detection of oxidation products. Quantification of amino acid consumption and product generation, using UPLC and LC/MS, demonstrates the occurrence of short chain reactions, with the chain-length dependent on the protein concentration. LC/MS peptide mass mapping analyses provide data on the sites of modification. Molecular crowding, arising from high casein concentrations and casein-casein interactions, therefore favors the occurrence of radical chain events that enhance the extent of protein oxidative damage.

Oxidative degradation of norfloxacin by a lipophilic oxidant, cetyltrimethylammonium permanganate in water–acetonitrile medium: A kinetic and mechanistic study

The present study reports the oxidative metabolism of an established antibacterial drug norfloxacin (NRF) by a lipid compatible lipophilic Mn(VII) oxidant, cetyltrimethylammonium permanganate (CTAP) in acetonitrile–water binary mixture in the presence of acetic acid. The metabolized products are identified as 7-amino-1-ethyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid, formaldehyde, and ammonia. The kinetics of the reaction is studied in aqueous acetonitrile media in the presence of acetic acid by UV–vis spectroscopic method by monitoring the absorbance of Mn(VII) at 530nm under pseudo first-order condition. The reaction is found to be first-order with respect to CTAP and fractional order with respect to norfloxacin and acetic acid. Occurrence of Michaelis–Menten type kinetics with respect to norfloxacin confirmed the binding of oxidant and substrate to form a complex before the rate determining step. A suitable ionic mechanism is proposed based on the experimental findings. The proposed reaction mechanism is supported by the effect of solvent polarity and effect of temperature on the reaction rate. High negative entropy of activation (ΔS≠=−259 to −158JK−1mol−1) supported the existence of a forced, more ordered and extensively solvated transition state. Further, solvent polarity–reactivity relationship revealed (i) the presence of less polar transition state compared to the reactants, (ii) differential contribution from dipolar aprotic (acetonitrile) and polar protic (water) solvents toward the reaction process through specific and nonspecific solute–solvent interaction and (iii) presence of intramolecular H-bonding in oxidant–substrate complex in acetonitrile rich domain and intermolecular H-bonding between oxidant–substrate complex and water in water rich domain.

A Subpopulation of Blasts with Attenuated p38MAPK Response Is Seen in Virtually All AML Patients and AML Cell Lines and Is Defined By Cells with Augmented Lipid-Associated Anti-Oxidant Defense

The serine/ threonine kinase, p38MAPK is activated by phosphorylation in response to a variety of cellular stresses including oxidative stress. Prolonged p38MAPK activation drives cell-cycle arrest and apoptosis; and in HSC activation of p38MAPK leads to a loss of reconstituting capacity (Ito et al, Nat.Med. 2006;12:446-451). In cancer, p38MAPK responses are often attenuated and cancer models suggest that this is a necessary adaptation for transformation (Dolado et al, Cancer Cell 2007;11:191-205). Previously we have shown that 60% of acute myeloid leukemia (AML) patients constitutively generate significantly more extracellular reactive oxygen species (ROS) than normal hematopoietic CD34+ cells (Hole et al, Blood 2013;122:3322-3330). Despite this, AML blasts showed low or absent p38MAPK phosphorylation; even in patients generating high levels of ROS. Here we examine p38MAPK activation at the single cell level in primary AML blasts using flow cytometry.We challenged AML blasts with a dose of hydrogen peroxide (H2O2) sufficient to completely activate p38MAPK in normal CD34+ cells (1 mM for 30 min), where the threshold for activation was defined as the 95th percentile of basal p38MAPK activation in unstimulated cells. Attenuated responses to H2O2 were seen in 14/15 (93%) of patients; where 16-95% of the total blast population failed to activate p38MAPK. These non-responding cells are hereafter termed “Δpp38MAPK cells” and were absent in normal CD34+ cells (p < 0.01; Figure 1). Examination of a panel of 6 AML cell lines showed that each of the lines contained Δpp38MAPK cells at different frequencies: MV4-11 (10%); HL60 (10%); KG-1 (15%), U937 (30%), NB4 (50%), THP-1 (65%). Further analysis showed that Δpp38MAPK cells were not distinguished by cell cycle phase, immunophenotype or reduced viability in either cell lines or AML blasts. These data suggest that nearly all AML patients harbor a population of blasts which have developed resistance to p38MAPK activation. We reasoned that failure to respond could arise either through defective p38MAPK signaling or because of enhanced anti-oxidative protection in a subpopulation of cells. To investigate the latter, we labelled cells with the lipophilic oxidation probe, C11 -BODIPY or the cytosolic oxidant probe, CM-DCFDA and monitored the oxidative response to H2O2 at the single cell level in the AML cell lines: KG-1, MV4-11 and THP-1. In each case C11 -BODIPY oxidation exactly matched the heterogeneous profile of p38MAPK activation in these cells, whereas CM-DCFDA showed only homogeneous responses to H2O2 induction. These data show that Δpp38MAPK cells are defined by an enhanced membrane-associated anti-oxidant capacity and we are currently analyzing this resistant subpopulation to identify the molecules responsible.To examine whether p38MAPK responsiveness influenced responsiveness to pro-oxidant drugs, we selected KG-1 and THP-1 cells (as representative examples of strong and weak p38MAPK responses respectively) and tested their sensitivity to the pro-oxidant drugs, phenethyl isothiocyanate (PEITC) and buthionine sulfoximine (BSO). We found that the IC50 was higher for THP-1 for both PEITC (KG-1 = 0.6µM; THP-1 = 7.5µM) and BSO (KG-1 = 50µM; THP-1 = 70µM), indicating that the p38MAPK responsiveness limits the effectiveness of pro-oxidant drugs. We next examined whether promoting p38MAPK activation could augment the effects of these pro-oxidants. We used the p38MAPK activator 2-benzylidene-3-(cyclohexylamino)-1-indanone (BCI), which promotes activation of p38MAPK via inhibition of a p38MAPK phosphatase, DUSP1. This compound weakly promoted phosphorylation of p38MAPK in THP-1 cells and consistent with this, had no effect on the efficacy of these compounds in these cells. However, BCI potently activated p38MAPK in KG-1 cells and showed synergy with BSO in this context (CI = 0.3; Figure 2) indicating that where BCI is effective in activating p38MAPK it can promote the effectiveness of pro-oxidant drugs.In summary, we show for the first time that AML patients almost universally display attenuated p38MAPK responses in all or part of the blast population and we suggest that this trait may be selected for to maintain self-renewing potential under the pro-oxidative conditions found in the leukemic marrow. Further we show that by manipulating p38MAPK activity, we can augment the potency of the pro-oxidant compound BSO. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intermolecular interactions of CTAB and potential oxidation inhibitors: physico-chemical controlled approach for food/pharmaceutical function

We report the impact of lipophilic oxidation inhibitors i.e. butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on cationic surfactant, cetyltrimethylammonium bromide (CTAB) properties in short chain alcohols and their hydroalcoholic solutions. Considering the extensive employment of BHA and BHT in human products, controlled physicochemical approaches were taken into consideration. Principally, density (ρ), ultrasonic sound velocity (u) and viscosity (η) measurements were carried out at specific temperatures T = (298.15, 303.15 and 308.15) K. The physico-chemical parameters apparent molar volume (Vϕ) and apparent molar adiabatic compression (Kϕ,S) were calculated and revealed the existence of hydrophobic interactions whereas, viscosity measurements depicted the region of micellization. Additionally, 1H NMR spectroscopic study was employed to interpret the intermolecular interaction of antioxidants within the micellar structure. 1H NMR analysis suggested the intermolecular interaction especially in the hydrophilic head and interface region of the surfactant with regard to shifting obtained within spectrum.

Analysis of esterified and nonesterified fatty acids in serum from obese individuals after intake of breakfasts prepared with oils heated at frying temperature

In this study, levels of esterified and nonesterified fatty acids (EFAs and NEFAs, respectively) were compared in obese individuals (body mass index between 30 and 47 kg m(-2)) in basal state and after intake of four different breakfasts prepared with oils heated at frying temperature. The target oils were three sunflower oils--pure, enriched with dimethylsiloxane (400 μg mL(-1)) as lipophilic oxidation inhibitor, and enriched with phenolic compounds (400 μg mL(-1)) as hydrophilic oxidation inhibitors--and virgin olive oil with a natural content of phenolic compounds of 400 μg mL(-1). The intake of breakfasts was randomized to avoid trends associated to this variability source. EFAs and NEFAs were subjected to a sequential derivatization step for independent gas chromatography-mass spectrometry analysis of both fractions of metabolites in human serum. Derivatization was assisted by ultrasonic energy to accelerate the reaction kinetics, as required for high-throughput analysis. Statistical analysis supported on univariate (multifactor ANOVA) and multivariate approaches (principal component analysis and partial least squares-discriminant analysis) allowed identification of the main variability sources and also discriminating between individuals after intake of each breakfast. Individuals' samples after intake of breakfasts prepared with virgin olive oil were clearly separated from those who ingested the remaining breakfasts. The main compounds contributing to discrimination were omega-3 and omega-6 EFAs with special emphasis on arachidonic acid and eicosapentaenoic acid. These two polyunsaturated fatty acids are the precursors of eicosanoid metabolites, which are of vital importance as they play important roles in inflammation and in the pathogenesis of vascular and malignant diseases as cancer.

Physiological Antioxidative Network of the Bilirubin System in Aging and Age-Related Diseases

Oxidative stress is detrimental to life process and is particularly responsible for aging and age-related diseases. Thus, most organisms are well equipped with a spectrum of biological defense mechanisms against oxidative stress. The major efficient antioxidative mechanism is the glutathione system, operating a redox cycling mechanism for glutathione utilization, which consists of glutathione and its peroxidase and reductase. However, this system is mainly effective for hydrophilic oxidants, while lipophilic oxidants require another scavenging system. Since many age-related pathological conditions are related to lipid peroxidation, especially in association with the aging process, the physiological role of the scavenging system for lipophilic oxidants should be considered. In this regard, the biliverdin to bilirubin conversion pathway, via biliverdin reductase (BVR), is suggested to be another major protective mechanism that scavenges lipophilic oxidants because of the lipophilic nature of bilirubin. The efficiency of this bilirubin system might be potentiated by operation of the intertwined bicyclic systems of the suggested redox metabolic cycle of biliverdin and bilirubin and the interactive control cycle of BVR and heme oxygenase. In order to combat oxidative stress, both antioxidative systems against hydrophilic and lipophilic oxidants are required to work cooperatively. In this regard, the roles of the bilirubin system in aging and age-related diseases are reassessed in this review, and their interacting networks are evaluated.

Oxidative Inactivation of the Proteasome in Retinal Pigment Epithelial Cells: A POTENTIAL LINK BETWEEN OXIDATIVE STRESS AND UP-REGULATION OF INTERLEUKIN-8

Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. Stress-induced overproduction of inflammatory cytokines, such as interleukin-8 (IL-8), is one of the early events of inflammation. The objective of this study was to elucidate mechanistic links between oxidative stress and overproduction of IL-8 in retinal pigment epithelial (RPE) cells. We found that exposure of RPE cells to H(2)O(2), paraquat, or A2E-mediated photooxidation resulted in increased expression and secretion of IL-8. All of these oxidative stressors also inactivated the proteasome in RPE cells. In contrast, tert-butylhydroperoxide (TBH), a lipophilic oxidant that did not stimulate IL-8 production, also did not inactivate the proteasome. Moreover, prolonged treatment of RPE cells with proteasome-specific inhibitors recapitulated the stimulation of IL-8 production. These data suggest that oxidative inactivation of the proteasome is a potential mechanistic link between oxidative stress and up-regulation of the proinflammatory IL-8. The downstream signaling pathways that govern the production of IL-8 include NF-kappaB and p38 MAPK. Proteasome inhibition both attenuated the activation and delayed the turnoff of NF-kappaB, resulting in biphasic effects on the production of IL-8. Prolonged proteasome inhibition (>2 h) resulted in activation of p38 MAPK via activation of MKK3/6 and increased the production of IL-8. Chemically inhibiting the p38 MAPK blocked the proteasome inhibition-induced up-regulation of IL-8. Together, these data indicate that oxidative inactivation of the proteasome and the related activation of the p38 MAPK pathway provide a potential link between oxidative stress and overproduction of proinflammatory cytokines, such as IL-8.

Nitrotyrosine and protein carbonyls are equally distributed in HT22 cells after nitrosative stress

The generation of reactive oxygen and nitrogen species is an inevitable result of cellular metabolism and environmental influence. Such oxidation processes are always combined with the formation of various protein oxidation products. Environmental oxidants might either be activated inside the cell or act by themselves. Therefore, differences in the localization of oxidant formation might change the major compartment of oxidant action. Therefore, we employed NO donors (SNOC, DETA/NO, and Spe/NO) alone or in combination with the redox-cycling bipyridinium compound paraquat, the superoxide- and NO-releasing compound SIN-1, the relatively more lipophilic oxidants tert-butyl and cumene hydroperoxide, and peroxynitrite itself to test the ability of these compounds to generate oxidized and nitrated proteins in various cellular compartments. Combined treatment with oxidants and nitrating compounds led to the formation of protein carbonyls and nitrotyrosine with a severalfold higher concentration in the cytosol, compared to the nucleus. In fluorescence microscopy studies, the resulting protein modifications show a similar distribution of oxidized proteins and nitrotyrosine with highest concentrations in the perinuclear area. Studying the time- and concentration-dependent formation and degradation of protein carbonyls and nitrated proteins large similarities could be measured. Therefore, it can be concluded that formation, localization, and kinetics of protein carbonyl and nitrotyrosine formation parallel each other depending on the stress-inducing agent.

Procyanidins protect Caco-2 cells from bile acid- and oxidant-induced damage

Procyanidins can exert cytoprotective, anti-inflammatory, and anticarcinogenic actions in the gastrointestinal tract. Previous evidence has shown that procyanidins can interact with synthetic membranes and protect them from oxidation and disruption. Thus, in this study we investigated the capacity of a hexameric procyanidin fraction (Hex) isolated from cocoa to protect Caco-2 cells from deoxycholic (DOC)-induced cytotoxicity, cell oxidant increase, and loss of monolayer integrity. Hex interacted with the cell membranes without affecting their integrity, as evidenced by a Hex-mediated increase in the transepithelial electrical resistance, and inhibition of DOC-induced cytotoxicity. DOC induced an increase in cell oxidants, alterations in the paracellular transport, and redistribution of the protein ZO-1 from cell-cell contacts into the cytoplasm. Hex partially inhibited all these events at concentrations ranging from 2.5 to 20 μM. Similarly, Hex (5–10 μM) inhibited the increase in cell oxidants, and the loss of integrity of polarized Caco-2 cell monolayers induced by a lipophilic oxidant (2,2′-azobis (2,4-dimethylvaleronitrile). Results show that the assayed procyanidin fraction can interact with cell membranes and protect Caco-2 cells from DOC-induced cytotoxicity, oxidant generation, and loss of monolayer integrity. At the gastrointestinal tract, large procyanidins may exert beneficial effects in pathologies such us inflammatory diseases, alterations in intestinal barrier permeability, and cancer.

Mitochondrial Transcription Factor A Induction by Redox Activation of Nuclear Respiratory Factor 1

The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondria-rich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network.

Flux of a Nickel(II) Salt versus a Nickel(II) Soap across Human Skin in vitro

Background: Contrary to in vitro data on skin diffusivity of nickel salts indicating slow rates of diffusion, contact allergy to nickel among the general population is frequent, indicating facile skin penetration. Objective: To demonstrate that the preferred diffusion of a lipophilic oxidation product of nickel formed in contact with the skin follows the multilamellar lipid region which envelops the corneocytes. Method: Diffusion of nickel chloride was compared with that of the lipophilic nickel soap, the dioctanoate. Results: The dioctanoate soap showed a significantly lower rate of diffusion (K_p = 1.4 × 10^–3 ± 5.7 × 10^–4 cm/h) than the chloride salt (K_p = 9.8 × 10^–3 ± 4.8 × 10^–4 cm/h), with a relatively high level of nickel retained in the tissue. Conclusions: (a) The molecular volume of the nickel dioctanoate overrides the effect of compound polarity; (b) the rates of diffusion measured through dermatomed skin are higher than those heretofore measured through full-thickness skin, epidermal tissue or stratum corneum; (c) both the nickel salt and soap appear to diffuse primarily via appendages; (d) the cause for the pronounced immune response to nickel in contact with the skin appears to lie in amounts retained in the viable skin strata; (e) skin from a young donor appears more permeable to nickel compounds than skin from an older source stemming from the same anatomical site.

Activation of extracellular signal-regulated kinases potentiates hemin toxicity in astrocyte cultures.

Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme-mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal-regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin-treated cultures at 30-120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10-phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z-VAD-FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z-VAD-FMK on cell survival was transient, the effect of MEK inhibitors was long-lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin-mediated oxidative injury.

New phenolic antioxidants of PYA and PYE class increase the resistance S. cerevisiae strain SP4, its SOD- and catalase-deficient mutants to lipophilic oxidants.

Was demonstrate the protection ability against reactive oxygen species afforded to S. cerevisiae (wild-type strain SP4 and its mutants deficient in major antioxidant enzymes--catalase T and A, CuZnSOD) by PYA and PYE, new groups of phenolic antioxidants (quaternary ammonium salts of dihydrocinnamic acid amino esters with different alkyl chains; synthesized in this laboratory). The survival of strains exposed to the lipophilic oxidation inducers tert-butyl hydroperoxide (TBHP) and 1,1'-azobis(4-cyclohexane carbonitrile) (ACCN) with or without antioxidant pretreatment was determined by plating. S. cerevisiae mutant deficient in SOD was found to be hypersensitive to TBHP and ACCN while the sensitivity of the strain deficient in catalase T and A was about the same as in the wild-type strain. A 1-h preincubation of cells of both the wild-type and the mutant strains with the phenolic antioxidants prior to exposure to TBHP or ACCN substantially increased the cell survival. The magnitude of protection depended on the strain and the length of the alkyl chain of the antioxidant; the best average protection against TBHP was provided by PYE and PYA compounds with 12- and 16-membered alkyl chains whereas PYE-8 and PYA-12 derivatives afforded the best average protection against ACCN.

Minimally oxidized LDL is a potent inhibitor of lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. As a variety of highly reactive lipid peroxidation products can transfer from oxidized LDL to HDL, we evaluated the potential deleterious effects of LDL oxidation on HDL-cholesterol metabolism. To address this issue, we exposed the HDL-containing d > 1.063 g/ml fraction of human plasma to copperoxidized LDL and assessed lecithin:cholesterol acyltransferase (LCAT) activity and apolipoproteinA-I (apoA-I) structure. To determine whether LCAT was directly affected by oxidized LDL, independent of crosslinking of apoA-I, we used an exogenous, [14C]cholesterol-labeled proteoliposome substrate to measure plasma LCAT activity. We observed an inhibition of LCAT activity where copper-oxidized LDL possessing only 2.3 +/- 0.1 and 7.3 +/- 1.4 TBARS produced 24 +/- 3% and 47 +/- 10% reductions in [14C]cholesterol esterification by 1 h, respectively. Copper-oxidized LDL that had been passed through a GF-5 desalting column, while retaining only one-third of its original TBARS, possessed nearly all of its LCAT inhibitory capacity suggesting that the LCAT inhibitory factor(s) was a lipophilic oxidation product. Analysis of polarlipids isolated from copper-oxidized LDL indicated that phospholipid and sterol fractions effectively inhibited LCAT. Copper-oxidized LDL, with as little as 6.3 TBARS, also produced intermolecular crosslinking of apoA-I molecules. Taken together, these data suggest that products of LDL oxidation may adversely affect HDL-cholesterol metabolism by two separate mechanisms: 1) a direct inhibitory effect on LCAT activity and 2) through crosslinking of apoA-I. If occurring in vivo, minimally oxidized LDL may impair cholesteryl ester formation on HDL thereby limiting the ability of HDL to function efficiently in the putative antiatherogenic reverse cholesterol transport pathway.

Killing ofCoccidioides immitisby hypochlorous acid or monochloramine

To identify possible explanations for the resistance of Coccidioides immitis to killing by human neutrophils, its susceptibility to typical oxidants generated during the neutrophil respiratory burst was compared to the sensitivity of other microbes. When microbial suspensions were exposed to hypochlorous acid, arthroconidia or spherules of C. immitis were killed more slowly than yeast cells of Candida (Torulopsis) glabrata or Staphylococcus aureus. In contrast, exposure to the more lipophilic oxidant, monochloramine, produced equivalent rates of killing for spherules and yeast cells of C. glabrata, and arthroconidia were killed more rapidly. When larger microbial concentrations were used, significantly greater concentrations of hypochlorous acid were required to kill equivalent percentages of spherules as compared to yeast cells of C. glabrata. Mixing studies with either whole spherules or extracts of spherules demonstrated that these substances could also block hypochlorous acid killing of S. aureus. These studies suggest possible mechanisms whereby C. immitis might resist oxidative phagocytic killing.

The role of thiols in nucleotide uptake into synaptic vesicles from Torpedo marmorata.

We have employed sulfhydryl group reagents in an attempt to determine the mechanism by which the transport of nucleotides into synaptic vesicles is controlled. Transport proved to be sensitive to N-ethylmaleimide; radiolabelled N-ethylmaleimide was used to locate the sulfhydryl group to the translocase-associated molecule previously identified as a polypeptide of Mr 34,000 [Lee and Witzemann (1983) Biochemistry 22, 6123-6130]. The nucleotide uptake was 75% inhibited by the mercurials rho-hydroxymercuribenzoate and rho-chloromercuriphenylsulfonate. Uptake was also sensitive to the reagents phenylarsine oxide and iodosobenzoic acid, which are specific for dithiols. These results indicate that a readily accessible dithiol is critical for nucleotide transport. Using the lipophilic oxidants iodosobenzoic acid and plumbagin, we demonstrated that nucleotide uptake was inhibited upon oxidation of the dithiol but that this did not involve an alteration in the affinity of the translocase for its substrate.

Immobilization of photosynthetically active intact chloroplasts in a crosslinked albumin matrix

Isolated higher plant chloroplasts with intact envelope membranes and bovine serum albumin were co-immobilized by treating the mixture with glutaraldehyde and then subjecting it to a freeze-thaw cycle. The immobilized chloroplasts are capable of photoinduced electron transport to lipophilic oxidants, but become compatible also with ionic oxidants after a transient hyposmotic shock.

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO(2) or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP(+) is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O(2) to H(2)O(2). Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.

Studies on the energy coupling sites of photophosphorylation. III. The different effects of methylamine and ADP plus phosphate on electron transport through coupling sites I and II in isolated chloroplasts

1. 1. The reduction of lipophilic (Class III) oxidants such as oxidized p-phenylenediamine consists of two components. One component requires both Photosystem II and Photosystem I and includes both sites of energy coupling associated with noncyclic electron transport. The second component requires only Photosystem II and includes only the site of energy coupling located before plastoquinone (Site II). When oxidized p-phenylenediamine is being reduced by both pathways, the overall rate of electron transport is stimulated by the addition of ADP plus phosphate or the uncoupler methylamine. However, if the Photosystem I component of oxidized p-phenylenediamine reduction is eliminated by a low concentration of the plastoquinone-antagonist dibromothymoquinone, the stimulation of electron transport by ADP plus phosphate or methylamine is also abolished, although the remaining Photosystem II-dependent electron transport remains firmly coupled to phosphorylation ( via coupling Site II). These results indicate that coupling Site II, unlike the well-known rate-limiting coupling site between plastoquinone and cytochrome f (Site I), does not exert any control over the rate of associated electron transport. 2. 2. When substituted p-benzoquinones ( e.g. 2,5-dimethyl- p-benzoquinone) or quinonediimides ( e.g. p-phenylenediimine) are used as Class III acceptors in conjunction with dibromothymoquinone, a small but significant stimultation of electron transport by ADP plus phosphate is observed. However, it can be shown that this stimulation does not arise from coupling Site II but rather is due to a low rate of electron flux through coupling Site I even in the presence of dibromothymoquinone. Apparently the p-benzoquinones can catalyze an electron “bypass” around the dibromothymoquinone-induced block at plastoquinone, possibly by substituting partially for the natural electron carrier. If this bypass electron flow is blocked at plastocyanin by KCN treatment, the stimulation of electron transport by ADP plus phosphate is eliminated, although a high rate of phosphorylation (from Site II only) remains. 3. 3. These results provide strong evidence that a profound difference exists between the two sites of energy coupling associated with non-cyclic electron transport in isolated chloroplasts. That is, the rate of electron flow through coupling Site I, which is the rate-determining step in the Hill reaction, is strictly regulated by phosphorylating conditions, whereas the rate of electron flux through coupling Site II is independent of phosphorylating conditions. 4. 4. A model is presented which accounts for the lack of control over electron transport exhibited by coupling Site II. It is postulated that Site II is coupled to an essentially irreversible electron transport step, so that conditions which affect the phosphorylation reaction would have no effect on the rate of electron transport through the coupling site. Two essentially irreversible reactions, closely associated with Photosystem II—the water-splitting reaction and the System II photoact itself—are discussed as possible locations for coupling Site II.

Photosystem-II electron transport and phosphorylation with dibromothymoquinone as the electron acceptor.

Dibromothymoquinone has two effects on isolated chloroplasts. At very low concentrations it inhibits the reduction of conventional hydrophilic electron acceptors, probably by acting as a plastoquinone antagonist. At higher concentrations it acts as an electron acceptor, intercepting electrons either before or at the site of its inhibitory activity. Reduced dibromothymoquinone can be readily reoxidized by excess ferricyanide in the reaction mixture or by molecular oxygen. The transfer of electrons to this substance from water is coupled to phosphorylation. The pH optima for this reduction and associated phosphorylation are both at 7.3, considerably lower than the pH optimum of 8.4 observed with the normal Hill reaction. The ratio of molecules of ATP formed to pairs of electrons transported is relatively constant (0.3 –0.4) between pH 6 and 9. The rate of reduction is independent of the presence or absence of ADP and phosphate or uncouplers.The reduction of dibromothymoquinone resembles the reduction of other lipid‐soluble oxi‐dants such as oxidized p‐phenylenediamines and 2,5‐dimethylquinone in several respects. Both reactions are insensitive to the plastocyanin inhibitors KCN and polylysine, but are sensitive to 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. They support phosphorylation with a similar efficieney. However, dibromothymoquinone reduction differs from the reduction of other lipophilic oxidants in that its sensitivity to dichlorophenyl‐dimethylurea decreases with increasing light intensities. This implies that it may be reduced via a pool (plastoquinone?) which is a common electron acceptor for independent photosystem II units whereas oxidized p‐phenylenediamines and 2,5‐dimethylquinone are reduced directly by these independent units.