Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.     

Factors influencing uptake and retention of amino-containing drugs in large unilamellar vesicles exhibiting transmembrane pH gradients

The level of uptake and retention of amino-containing drugs in large unilamellar vesicles (LUVs) following uptake in response to a transmembrane pH gradient (DeltapH) can vary dramatically depending on the drug. For example, the anticancer drugs doxorubicin and epirubicin can be readily retained, whereas the anticancer drug vincristine and the antibiotic ciprofloxacin tend to leak out rapidly. In this investigation, we examine the influence of the hydrophobicity of the entrapped amines (that induce the DeltapH) and the anionic lipid content of the LUV on drug retention. It is shown that entrapment of increasingly hydrophobic monoamines (methylamine to amylamine) all lead to an induced DeltapH of 3 units and essentially complete drug uptake under the conditions employed, but do not lead to improved retention of vincristine and ciprofloxacin. However, significantly improved retention could be achieved by substitution of the anionic lipid distearoylphosphatidylglycerol (DSPG) for distearoylphosphatidylcholine (DSPC) in the LUV bilayer. Further, it is shown that if the induced DeltapH is reduced to 1.4 units (driven by entrapped diamine) nearly 100% accumulation of doxorubicin and epirubicin could be achieved, whereas only 25% loading for vincristine and ciprofloxacin was observed. Taken together these results provide methodology for improving (weak base) drug retention in liposomes and indicate that drugs that can partition into the lipid bilayer exhibit improved uptake and retention characteristics.     

Comparison of adriamycin and derivatives uptake into large unilamellar lipid vesicles in response to a membrane potential

The uptake of adriamycin (ADM) and several derivatives into large unilamellar vesicles (LUV) displaying a transmembrane potential and having a lipid composition close to that of the inner mitochondrial membrane has been measured. Drug association to neutral liposomes, made of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (70:30, w/w) was shown to be potential-dependent: in the absence of potential, accumulation of drug was almost undetectable, whereas between 11 and 50 nmol of drug/mumol phospholipid, depending on the anthracycline used, was associated to LUV exhibiting a membrane potential after 1 h incubation. Association of drugs to LUV with a lipid composition closer to that of the inner mitochondrial (cardiolipin, CL, 20%; PC 50%; PE, 30%, w/w) and displaying a membrane potential is higher than with neutral vesicles (between 40 and 76 nmol of anthracycline/mumol phospholipid after 1 h incubation). Since it is known that ADM and derivatives have a high affinity for CL, a fraction of the associated drug may bind to CL on the outer side of the vesicles. This was confirmed by the fact that, in the absence of potential, between 40 and 56 nmol of anthracycline/mumol phospholipid was still associated to LUV containing CL. In order to discriminate between drug adsorbed at the surface of the LUV and drug accumulated inside the LUV, an anthracycline fluorescence quencher (I-) was used. It was shown on neutral LUV displaying a membrane potential, that between 55 and 81% of the associated drug is actually entrapped inside the vesicles, inaccessible to the quencher. These percentages decreased to between 41 and 68%, respectively, in the presence of LUV containing CL and exhibiting a membrane potential, whereas for LUV of the same composition but displaying no membrane potential almost all the associated drug is adsorbed on the outer face of the LUV, accessible to the quencher, and likely bound to CL. This study brings evidence that antitumour anthracyclines despite important structural homologies do not accumulate to the same extent into vesicles mimicking the lipid composition and the membrane potential of mitoplasts. This ability to reach the matrix compartment of mitochondria could partly explain the differences of cardiotoxicities associated to anthracyclines with closely related molecular structure.     

Cholesterol mobilization and regression of atheroma in cholesterol-fed rabbits induced by large unilamellar vesicles

The antiatherogenic properties of repeated injections of egg phosphatidylcholine large unilamellar vesicles (LUVs) of 100 nm diameter were tested in an experimental model for atherosclerosis. Forty eight rabbits were divided into two diet groups fed standard rabbit chow or fed a cholesterol-enriched diet (0.5% by weight) to induce the formation of atherosclerotic lesions. Prior to the initiation of LUV therapy, the cholesterol diet was ceased and all animals were returned to standard rabbit chow. The treatment protocol consisted of a total of 10 bolus injections of vesicles, at a phospholipid dose of 300 mg/kg body weight or the equivalent volume of saline, with one injection given to each animal every 10 days. LUV injections brought about a large movement of cholesterol into the blood pool and resulted in a significant reduction in the cholesterol content as well as the degree of surface plaque involvement of aortic tissue in atherosclerotic animals. Most notably, the thoracic aorta of LUV-treated animals exhibited a 48% reduction in tissue cholesterol content per gram of protein compared to saline-treated controls. Histochemical analyses revealed that aortas from animals receiving the repeated injections of LUVs displayed less cholesterol deposits in lesions, and a moderate reduction in intimal-to-medial thickness. This regression of atheroma, induced by LUV therapy, was observed even though animals possessed persistent elevated plasma cholesterol levels after the cholesterol-enriched diet was ceased. These results suggest that repeated injections of LUVs, working with endogenous HDL, may be a useful therapy in the management of atherosclerosis.     

Poly(ethylene glycol)-induced fusion and rupture of dipalmitoylphosphatidylcholine large, unilamellar extruded vesicles

High concentrations (> or = 20 wt %) of poly(ethylene glycol) (PEG) induce large, unilamellar, dipalmitoylphosphatidylcholine model membrane vesicles to fuse when the bilayers contain small amounts of amphipathic peturbant molecules. In addition to fusion, similar concentrations of PEG induce these vesicles to leak their contents. In this paper, we have asked if fusion could occur independently of leakage or if fusion might be described as local bilayer rupture followed by resealing. By following the release of MW 10,000 fluoresceinated dextran trapped inside vesicles, it was determined that PEG-induced leakage was the result of major membrane disruption and not small-pore formation. Fusion of vesicles containing 0.5 mol % palmitic acid was clearly observed at 20 wt % PEG, while 25 wt % was needed to cause rupture. On the other hand, vesicles containing 0.5 mol % lysophosphatidylcholine ruptured at roughly the same concentration needed to induce rupture. Two methods were developed for removing PEG so that fusion products could be characterized. Quasi-elastic light scattering demonstrated that fusing vesicles grew in size and that nonfusing vesicles did not. Moreover, PEG concentrations that induced rupture led to the appearance of species with mean diameters much larger than those of fused vesicles. High-resolution nuclear magnetic resonance showed that the population of large vesicles that correlated with rupture was composed of multilamellar vesicles while the population resulting from fusion alone remained unilamellar. We conclude that, upon incubation with and subsequent removal of PEG, vesicles were either unaffected, or fused to form larger, unilamellar vesicles, or ruptured to form larger, nonunilamellar species.     

Ursodeoxycholate stabilizes phospholipid-rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles

Ichthyophis kohtaoensis, a member of the limbless Gymnophiona, has a specialized subterranean burrowing mode of life and a predominantly olfactory-guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 microm in diameter in adults), functional investigations of single retinal cells by electrophysiological methods have so far failed. Therefore, the content and distribution of retinal transmitters have been investigated as indications for a functioning sense organ in an animal that is supposed to be blind. In this study, the organization and development of the dopaminergic system have been examined in the retinae of embryonic, larval, and adult I. kohtaoensis, by using an antiserum against tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, and an antiserum against dopamine itself. Labeled somata are situated in the inner nuclear layer and in the ganglion cell layer. Dopamine-positive fibers form a dense diffuse plexus, that covers the whole inner plexiform layer, whereas tyrosine hydroxylase-immunoreactive processes show a tendency to arborize in a stratified manner. Tyrosine-hydroxylase-immunolabeled fibers can occasionally be observed in the optic nerve head of larval stages. During ontogenesis and larval development, the distribution of transmitter-expressing cells changes and their number decreases, but no general degeneration of the visual system is detectable. Adult Ichthyophis still have retinal transmitters, indicating that the eyes, although obviously playing a minor role in a subterranean ecological niche, retain all the elements of functioning sense organs.     

Kinetics of the interactions of a dicarboxylic porphyrin with unilamellar lipidic vesicles: interplay between bilayer thickness and pH in rate control

The transfer of a dicarboxylic porphyrin from phosphatidylcholine fluid-phase unilamellar vesicles towards albumin is studied focusing on bilayer thickness and pH effects. The kinetics of this process yield the rate constants for the porphyrin flip-flop from the inner to the outer hemileaflet and its exit towards aqueous medium. Phospholipids with monounsaturated 14-22 carbon chains are used. Interplay between bilayer thickness and pH for the control of the rate constants is observed. This results in the amplification, at physiological pH, of the effect of membrane thickness on the flip-flop and exit rates as compared to pH 8.5 and 6.5. These data are explained by the degree of porphyrin burying within the bilayer resulting from a compromise between favorable hydrophobic interactions with the hydrocarbon phase and unfavorable penetration of the polar carboxylic chains. The balance between the two effects depends particularly on the neutralization of one carboxylic chain. Considering the bilayer hydrophobicity profile and the porphyrin size, the optimization of hydrophobic interactions appears dependent on the bilayer thickness. The flip-flop and the exit are governed by neutralization and deprotonation of the carboxylic chains, respectively, the rate of these proton exchanges being dependent on the porphyrin location within the bilayer.     

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.     

Enrichment of presenilin 1 peptides in neuronal large dense-core and somatodendritic clathrin-coated vesicles

Presenilin 1 is an integral membrane protein specifically cleaved to yield an N-terminal and a C-terminal fragment, both membrane-associated. More than 40 presenilin 1 mutations have been linked to early-onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function (s) of the wild-type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear-core synaptic vesicles, the large dense-core vesicles, and the somatodendritic and nerve terminal clathrin-coated vesicles. Both the N- and C-terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense-core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense-core vesicle-mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin-coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear-core synaptic vesicle or in the nerve terminal clathrin-coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full-length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full-length protein may not have a vesicular function.     

Humoral response to hsp 65 in multiple sclerosis and other neurologic conditions

The expression of heat shock proteins (hsp) within the target organ is implicated in the pathogenesis of a number of diseases of suspected autoimmune etiology, including MS. To pursue the potential role of a humoral response to the hsp 60/65 kd family in MS, we studied serum and CSF by Western blotting using recombinant Mycobacterium bovis hsp 65 and human hsp 60 as antigens and compared the findings with samples from patients with other neurologic diseases (OND). Analysis of the IgG response in CSF from 18 patients with MS indicated moderate reactivity in 10 cases and no reactivity in eight. In the OND group, reactivity was found in the CSF from one of two patients with Parkinson's disease, four of four Alzheimer's disease patients, and two of two patients with amyotrophic lateral sclerosis. CSF samples from seven of seven patients with subacute sclerosing panencephalitis were negative, as were samples from two normal subjects. There was no reactivity in CSF from two Huntington's disease patients. We conclude that antibodies reactive with hsp 60/65 are present in CSF of some MS patients but are also present in a number of chronic neurodegenerative conditions. The findings indicate that a humoral response to hsp 60/65 in the CSF is not specific for MS.     

erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer

BACKGROUND: Overexpression of the erbB-2 protein by breast cancer cells has been suggested to be a predictor of response to doxorubicin. A retrospective study was designed to test this hypothesis. METHODS: In National Surgical Adjuvant Breast and Bowel Project protocol B-11, patients with axillary lymph node-positive, hormone receptor-negative breast cancer were randomly assigned to receive either L-phenylalanine mustard plus 5-fluorouracil (PF) or a combination of L-phenylalanine mustard, 5-fluorouracil, and doxorubicin (PAF). Tumor cell expression of erbB-2 was determined by immunohistochemistry for 638 of 682 eligible patients. Statistical analyses were performed to test for interaction between treatment and erbB-2 status (positive versus negative) with respect to disease-free survival (DFS), survival, recurrence-free survival (RFS), and distant disease-free survival (DDFS). Reported P values are two-sided. RESULTS: Overexpression of erbB-2 (i.e., positive immunohistochemical staining) was observed in 239 (37.5%) of the 638 tumors studied. Overexpression was associated with tumor size (P=.02), lack of estrogen receptors (P=.008), and the number of positive lymph nodes (P=.0001). After a mean time on study of 13.5 years, the clinical benefit from doxorubicin (PAF versus PF) was statistically significant for patients with erbB-2-positive tumors--DFS: relative risk of failure (RR)=0.60 (95% confidence interval [CI]=0.44-0.83), P=.001; survival: RR=0.66 (95% CI=0.47-0.92), P =.01; RFS: RR=0.58 (95% CI=0.42-0.82), P=.002; DDFS: RR=0.61 (95% CI=0.44-0.85), P=.003. However, it was not significant for patients with erbB-2-negative tumors-DFS: RR=0.96 (95% CI=0.75-1.23), P=.74; survival: RR =0.90 (95% CI=0.69-1.19), P=.47; RFS: RR=0.88 (95% CI=0.67-1.16), P=.37; DDFS: RR=1.03 (95% CI=0.79-1.35), P=.84. Interaction between doxorubicin treatment and erbB-2 overexpression was statistically significant for DFS (P=.02) and DDFS (P=.02) but not for survival (P= .15) or RFS (P=.06). CONCLUSIONS: These data support the hypothesis of a preferential benefit from doxorubicin in patients with erbB-2-positive breast cancer.     

Alterations of pH in response to increased dietary protein in cattle are unique to the uterus

This study was undertaken with two objectives: 1) to determine whether the effect of excess dietary protein on intrauterine pH in cattle is specific to the uterus or manifested in other bodily fluids and 2) to determine whether the effect of excess ruminally degradable protein on uterine pH can be ameliorated by substitution with a less-degradable protein source. Thirty-six Holstein cows in early lactation were fed isoenergetic total mixed rations that either 1) met undegradable intake protein (UIP) and degradable intake protein (DIP) requirements (Balanced), 2) met DIP requirements and exceeded UIP requirements by 25% (High UIP), or 3) met UIP requirements and exceeded DIP requirements by 25% (High DIP). After diets had been fed > or = 2 wk, uterine, blood, salivary, and urinary pH and plasma urea nitrogen were determined at estrus (d 0) and d 7. Plasma urea nitrogen (mg/dL) was not different between estrus and d 7 but was significantly affected by diet (Balanced, 16.1 +/- 2.3; High UIP, 19.2 +/- 1.6; High DIP, 22.3 +/- 2.6; P < .05). There was no effect of treatment on the pH of any fluid measured at estrus: intrauterine, blood, salivary, and urinary pH averaged 6.84 +/- .05, 7.39 +/- .01, 8.30 +/- .05, and 8.15 +/- .05, respectively. In contrast, on d 7, uterine pH was significantly lower in both high-protein groups, regardless of protein degradability (Balanced, 7.13 +/- .05; UIP, 6.95 +/- .04; DIP, 6.85 +/- .05; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies capable of releasing diphtheria toxin in response to the low pH found in endosomes

Diphtheria toxin (DT) undergoes a rapid conformational change in response to the acidity encountered within endosomes. That transition is integral to the passage of its catalytic domain into the cytosol and thus its lethal action. The importance of this translocation mechanism led us to develop several monoclonal antibodies that bind DT at neutral pH but spontaneously release the toxin when critical epitopes denature or unfold upon lowering the pH to 4.5-5.5. Hybridomas were selected using a microtiter plate assay that measured the pH-dependent detachment of antibody from immobilized toxin. The acid-sensitive epitopes involved were on the catalytic, transmembrane, and receptor binding domains of DT. This pH-induced disruption of the binding of toxin to these monoclonal antibodies was analyzed by sedimentation velocity ultracentrifugation. Antibody combining sites were fully occupied at pH 5.5, partially bound at pH 5.0, and totally empty at pH 4.5. It was estimated that the Ka for antibody-toxin binding was approximately 1000-fold lower at pH 5.0 than at neutral pH. This novel acid-triggered release mechanism provides a basis for delivery of antibody-bound toxin into cells accompanied by its immediate dissociation as the complex enters acidic vesicles.     

Kinetics and thermodynamics of emulsion delivery of lipophilic antioxidants to cells in culture

Oil-in-water emulsions are being used increasingly for the delivery of lipophilic drugs, but the fundamental physicochemical principles governing such delivery have not been explored. We determined the kinetics and thermodynamics of delivery from emulsions to cells in culture for two lipophilic compounds, U74006 and U74500. Two fundamental properties dominate the delivery, (a) the concentration of the compound in the lipid phase of the emulsion is directly proportional to the concentration of the compound in cells at equilibrium, and (b) the rate of transfer is directly proportional to the concentration of particles in contact with the cells. Thus, the transfer is consistent with direct partitioning from the lipid phase of the emulsion to cells and occurs by the direct collision of emulsion particles with cells. The details of the mechanism of delivery differ between the two compounds. Specifically, delivery of U74006 is first-order with respect to the drug accumulating in the cells. The transfer of U74500 is best described as a sum of two simultaneous pseudo first-order processes consistent with delivery from a single donor compartment to two receiver compartments. Furthermore, two molecules of U74500 appear to be involved in each transfer event. Our results show that relatively simple principles govern the delivery of compounds from oil-in-water emulsions to cells.     

The influence of dietary salt and energy on the response to low pH in juvenile rainbow trout

This study evaluated the role of diet, specifically the relative importance of salt content versus energy content, in the response of juvenile rainbow trout to environmental acid stress in soft water ([Ca2+] = 0.057 mmol L-1, [Na+] = 0.047 mmol L-1). Diets were formulated at two energy levels (regular, 16.3 MJ kg-1, and low, 9.8 MJ kg-1) and two levels of NaCl (regular, approximately 263 mmol kg-1, and low, approximately 64 mmol kg-1), yielding four treatment combinations, each fed at a ration of 0.6% body weight d-1. A fifth group of fish was not fed during the experiment. All groups were subjected to an initial acid challenge (24 h at pH 5.0 plus 12 h at pH 4.0), followed by 28 d of exposure to pH 5.2. Following the initial acid challenge, typical ionoregulatory disturbances were seen, but most effects had attenuated or disappeared by day 20 of chronic low-pH exposure. However, after 28 d, fish fed the regular-salt diets maintained the restored ionic homeostasis, whereas those fed low-salt diets did not, regardless of the energy content of the diet. Growth and food conversion efficiency were greatest in trout fed the regular-energy/regular-salt diet, negative in fish fed the low-energy/regular-salt diet, and intermediate in trout on the other diets; starved fish lost weight. Fish maintained on the regular-energy/low-salt diet exhibited the most deleterious effects, including elevated cortisol levels and a 4.1% d-1 mortality rate. Fish fed the low-energy/low-salt diet, those fed regular-salt diets, and starved fish were not as adversely affected by the acid stress. Following a regular-energy meal, fish tended to exhibit an elevated rate of oxygen consumption, but this did not occur after a low-energy meal, regardless of its salt content. Elevated oxygen consumption may be accompanied by a loss of ions via the osmorespiratory compromise. We hypothesize that fish fed the regular-energy/low-salt diets were most deleteriously affected in an acidified environment because they were unable to replace increased branchial ion losses with dietary salts. These results indicate that it is the salt content of the food rather than the energy content that is critical in protecting against the deleterious effects of low pH.     

Symmetry and pH dependency of the lactate/proton carrier in skeletal muscle studied with rat sarcolemmal giant vesicles

To study the pathogenesis of hyperlipoidemia and atheromatosis and the metabolism of lipoprotein, we have developed a colorimetric method for simultaneously determining the activities of post-heparinplasma lipoprotein lipase (LPL) and hepatic lipase (HL). The intralipid was kept for LPL and HL at 37 degrees C, pH8.3 for 30 min, with 100 microliters post-heparin plasma. The LPL and HL in the post-heparin plasma could hydrolyse the triglyceride in intralipid into glycerine and free fatty acid (FFA). Determining the amount of FFA by copper-reagent method, we could measure the activities of LPL and HL. The kinetics of LPL and HL in post-heparin plasma was observed. K(m) values for LPL and HL were 0.9 mumol/L and 2.4 mumol/L respectively. The C. V. for LPL and HL were 4.5% (n = 4), 2.9% (n = 6) and 6.4% (n = 6), 4.8% (n = 6) respectively.     

Accumulation of benzoic acid in suspension cultured cells of Pinus thunbergii Parl. in response to phenylacetic acid administration

The generation and accumulation of both benzoic acid (BA) and its conjugates were induced in suspension cultured cells of Pinus thunbergii by administering either phenylacetic acid (PA), a toxic metabolite of Bacillus cereus (strain HY-3) accompanying the pine wood nematode, or a lyophilized culture supernatant of this bacterium. BA conjugates reached their maximal levels in quantity two days after the administration and then decreased gradually until the 14th day, while BA increased significantly throughout this period. This pattern is similar to that in 3-year-old pine trees treated with PA, suggesting that the pathological reaction of pine tissues to the PA toxin might be involved in the pathogenesis mechanism for the pine wilt disease.     

Disruption of the transmembrane pH gradient--a possible mechanism for the antibacterial action of azelaic acid in Propionibacterium acnes and Staphylococcus epidermidis

The effect of the topical acne treatment azelaic acid on the transmembrane proton gradient (delta pH) of Propionibacterium acnes and Staphylococcus epidermidis was studied in vitro at external pH values found on human skin (pH 4.0-6.0). Bacteria were grown in defined media using continuous culture and delta pH was estimated by measuring the accumulation of [14C] benzoic by the cells using flow dialysis. In both P. acnes and S. epidermidis the addition of 30 mM azelaic acid and the membrane active inhibitors nigericin (150 microM) and CCCP (150 microM) resulted in a rapid release of [14C] label into the dialysate indicating the dissipation of delta pH between external pH values of 4.0-6.0. The addition of 60 mM NaCl as an iso-osmotic control and 150 microM valinomycin did not induce the release of [14C] label. The addition of 30 mM azelaic acid reduced the delta pH of P. acnes by 44% at external pH 4.0 and 28% at external pH 6.0. In S. epidermidis 30 mM azelaic acid reduced delta pH by 88% at external pH 5.0 and 20% at external pH 6.0. Rapid loss of viability occurred in suspensions of P. acnes and S. epidermidis containing 30 mM azelaic acid at pH 4.0 with no viable cells recovered after 60 min incubation. At pH 6.0 little change in viable numbers of P. acnes and S. epidermidis were observed over a 2 h incubation period. The results indicate that the antibacterial activity of azelaic acid is associated with the perturbation of intracellular pH.