Comparison of rumen and abomasal infusions of an exogenous emulsifier on fatty acid digestibility of lactating dairy cows.

We evaluated the effects of infusing an exogenous emulsifier (polysorbates-C18:1) either into the rumen or abomasum on fatty acid (FA) digestibility and production responses of lactating dairy cows. Nine ruminally cannulated multiparous Holstein cows (170 ± 13.6 d in milk) were assigned to a treatment sequence in replicated 3 × 3 Latin squares with 18-d periods consisting of 7 d of washout and 11 d of infusion. Treatments were abomasal infusions of water carrier only into the rumen and abomasum (control, CON), 30 g/d polysorbate-C18:1 (T80) infused into the rumen (RUM), or 30 g/d T80 infused into the abomasum (ABO). Emulsifiers were dissolved in water and delivered at 6-h intervals (total daily infusion was divided into 4 equal infusions per day). Cows were fed the same diet that contained [% diet dry matter (DM)] 32.2% neutral detergent fiber (NDF), 16.1% crude protein, 26.5% starch, and 3.41% FA (including 1.96% FA from a saturated FA supplement containing 28.0% C16:0 and 54.6% C18:0). Two orthogonal contrasts were evaluated: (1) the overall effect of T80 {CON vs. average of the T80 infusions [1/2 (ABO + RUM)]}, and (2) the effect of ABO versus RUM infusion. Compared with CON, infusing T80 increased the digestibilities of NDF (2.85 percentage units), total (4.35 percentage units), 16-carbon (3.25 percentage units), and 18-carbon FA (4.60 percentage units), and tended to increase DM digestibility and total and 18-carbon FA absorption. Compared with RUM, ABO decreased the intakes of total (28 g/d), 16-carbon (7 g/d), and 18-carbon FA (19 g/d); tended to increase the digestibility of total and 18-carbon FA; and had no effect on the absorption of total, 16-carbon, or 18-carbon FA. Production responses did not change among our treatments. In conclusion, infusing 30 g/d polysorbates-C18:1 increased NDF and total, 16-carbon, and 18-carbon FA digestibility. Compared with RUM, ABO tended to increase the digestibilities of total and 18-carbon FA; however, this may be related to the fact that ABO reduced the intakes of total, 16-carbon, and 18-carbon FA, not necessarily due to better emulsifying action per se. In summary, ABO and RUM both improved FA absorption.

Clove Oil Endorsed Transdermal Flux of Dronedarone Hydrochloride Loaded Bilosomal Nanogel: Factorial Design, In vitro Evaluation and Ex vivo Permeation.

The goal of this study was to develop a bilosomal gel formulation to enhance transdermal permeability of dronedarone hyrdrochloride (DRN) which suffers from poor oral absorption and limited bioavailability. To overcome this obstacle, bilosomes were successfully prepared using 23 full-factorial design. Span®40, cholesterol, sodium deoxycholate (bile salt), clove oil (permeability enhancer), and either Tween® 60 or Tween® 80 (edge activator) were used in bilosome preparation by ethanol injection method. In this design, independent variables were X1, edge activator type; X2, edge activator amount (mg); and X3, permeability enhancer concentration (% w/v). Optimal formula (B2) of the highest desirability of (0.776) demonstrated minimum vesicle size (VS) of 312.4 ± 24.42 nm, maximum absolute value of zeta potential (ZP) - 36.17 ± 2.57 mV, maximum entrapment efficiency (EE %) of 80.95 ± 3.01%, maximum deformability Index (DI) of 8.24 ± 1.26 g and maximum drug flux after 12 h (J12) of 21.23 ± 1.54 µg/cm2 h upon ex vivo permeation study. After 12 h, 70.29 ± 6.46% of DRN was released from B2. TEM identification of B2 showed spherical shaped nanosized vesicles which were physically stable for 3 months at different temperatures. B2 was incorporated into carboxymethylcellulose gel base for easiness of dermal application. B2 gel demonstrated good physical properties, non-Newtonian psuedoplastic flow, and enhanced release (57.0 ± 8.68% of DRN compared to only 13.3 ± 1.2% released from drug suspension after 12 h) and enhanced skin permeation.

Extracellular production of azurin from Pseudomonas aeruginosa in the presence of Triton X-100 or Tween 80.

Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox protein supresses the tumor growth selectively. High-level secretion of proteins into the culture medium offers a significant advantage over periplasmic or cytoplasmic expression. The aim of this study was to investigate the effect of nonionic surfactants on the expression of the Pseudomonas aeruginosa azurin. Different concentrations of Triton X-100 and Tween 80 were used as supplements in growth media and extracellular azurin production was stimulated by both surfactants. According to western blot analysis results, in the presence of Triton X-100, maximum azurin expression level was achieved with 96 h of incubation at 1% concentration, and 48 h at 2% concentration. On the other hand, maximum azurin expression level was achieved in the presence of 1% Tween 80 at 72 h incubation. This study suggested for the first time a high level of azurin secretion from P. aeruginosa in the presence of Triton X-100 or Tween 80, which would be advantageous for the purification procedure.

Effects of Various Pharmaceutical Excipients on the Intestinal Transport and Absorption of Sulfasalazine, a Typical Substrate of Breast Cancer Resistance Protein Transporter.

Breast cancer resistance protein (BCRP) transporter is an efflux transporter that utilizes energy from adenosine triphosphate hydrolysis to push its substrates, regardless of the concentration gradient. Its presence on the apical membrane of the intestinal mucosa is a major obstacle for the intestinal absorption of its substrates. In this study, we examined the effects of various pharmaceutical excipients on the intestinal transport and absorption of sulfasalazine, a BCRP substrate. Four excipients, including 0.05% and 0.075% BL-9EX, 0.01% and 0.05% Brij 97, 0.075% Labrasol, and 0.05% and 0.1% Tween 20 decreased the secretory transport of sulfasalazine in an in vitro diffusion chamber. Further investigation in an in situ closed loop experiment in rats showed that 0.05% and 0.1% BL-9EX and 0.1% Brij 97 effectively enhanced the intestinal absorption of sulfasalazine while maintaining minimal toxicity to the intestinal mucosa. However, 0.1% Brij 97 also increased the intestinal absorption of 5(6)-carboxyfluorescein, a paracellular marker compound. These findings suggest that BL-9EX might effectively inhibit the BCRP-mediated efflux of sulfasalazine in vivo, indicating that BL-9EX could improve the intestinal absorption of sulfasalazine and other BCRP substrates.

Surfactant-enhanced biodegradation of crude oil by mixed bacterial consortium in contaminated soil.

This study evaluated the effects of two surfactants (i.e., Tween 80 and SDS) on biodegradation of crude oil by mixed bacterial consortium in soil-aqueous system. The mixed bacterial consortium was domesticated from the activated sludge of cooking plant through a progressive domestication process. High-throughput sequencing analysis revealed that Rhodanobacter sp. was the dominant bacteria. The higher CMCeff value for two surfactants was observed in soil-aqueous system compared with that in aqueous system, which was likely due to their adsorption onto soil particles. Either Tween 80 or SDS can be utilized as carbon source and promote the growth of mixed bacterial consortium. Further findings evidenced that the degradation of crude oil can be enhanced by adding either Tween 80 or SDS. The performance of Tween 80 was generally superior to SDS for the crude oil degradation. The highest crude oil degradation efficiency was 42.2 and 31.0% under the conditions of 5 CMCeff of Tween 80 and 2 CMCeff of SDS, respectively. Furthermore, the degradation efficiency of crude oil in remediation experiment (i.e., 77%) evidenced that the integration of adding Tween 80 and inoculating mixed bacterial consortium was effective for crude oil-contaminated soil decontamination.

Influence of emulsifier blends having different hydrophilic-lipophilic balance value on growth performance, nutrient digestibility, serum lipid profiles, and meat quality of broilers.

Energy is a major cost component in diets for poultry. We hypothesized that the supplementation of emulsifier blends in broiler diets may contribute to the efficient utilization of energy and in increasing fat digestibility, thereby improving performance. To test our hypothesis, an experiment was conducted to evaluate the effect of a blend of emulsifiers on growth performance, nutrient digestibility, serum lipid profiles, and meat quality of broilers. A total of 768 1-d-old Ross 308 male broiler chicks with an average initial body weight of 45.55 ± 0.34 g were used in a 35 days feeding trial. Broilers were sorted into 4 treatments, 12 replications per treatment, and 16 birds per pen. Dietary treatments consisted of corn-soybean meal based basal diet and the basal diet supplemented with 0.05%, 0.075%, and 0.10% emulsifier. As a result of this study, the inclusion of 0.05%, 0.075%, and 0.10% emulsifier blends (sodium stearoyl-2-lactylate (SSL) and Tween 20) in the basal diet linearly increased (P = 0.0001) body weight gain (BWG) and improved feed conversion ratio (FCR) (linear effect P = 0.0001) on d 7 to 21, d 21 to 35 as well as overall. Broilers fed with different levels of emulsifier blends also showed a linear increment (P < 0.05) in dry matter (DM) and fat digestibility. A trend of linear reduction (P = 0.051) in low density lipoprotein (LDL) in the serum of broilers fed emulsifier blend was observed. The lightness value of breast muscle color linearly increased (P = 0.001), the redness and yellowness values tended to increase (P = 0.072 and P = 0.094 respectively), and the water holding capacity (WHC) showed trends in reduction (P = 0.078) with an increase in the level of emulsifier blends. With regards to relative organ weight, spleen weight was linearly (P = 0.001) reduced with the increase in the supplemental levels of emulsifier blends. A positive correlation between emulsifier content in the diet and BWG, DM and fat digestibility, and breast muscle color indices, were also observed. In conclusion, emulsifier blend supplementation positively influenced growth performance and nutrient digestibility in broiler chickens.

Development of Cassette PAMPA for Permeability Screening.

The parallel artificial membrane permeability assay (PAMPA) is widely used in early-stage drug discovery to discriminate compounds by intestinal permeability. The purpose of the current study was to establish a cassette (n-in-1) PAMPA to enable permeability screening of lipophilic compounds. A double-sink PAMPA consisting of a pH gradient (i.e., pH 6.5 and 7.4 for the donor and receiver compartments, respectively) and a lipophilic sink (i.e., a surfactant in the receiver solution) was utilized with cassette incubation of 10 reference compounds. Sample analysis was conducted using selected reaction monitoring (SRM) with a triple quadrupole LC-MS/MS system. Correlation between PAMPA permeability and human intestinal absorption (HIA) of the reference compounds yielded two false negatives, namely propranolol (PPN) and verapamil (VER); these two compounds showed a substantially lower recovery (ca. 10%) than other reference compounds (>69%). This cassette PAMPA was repeated subsequently with polysorbate 80 added to the donor compartments, which resulted in a significant increase in both the recovery and the permeability of the false negatives. Accordingly, the permeability class of all reference compounds could be unambiguously differentiated using this cassette PAMPA. Also, a strong linear correlation (r=0.9845) was observed between the cassette and discrete permeability of all reference compounds.

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a.

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2(4) factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R (2) = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L(-1) and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL(-1)) occurred in the MRS broth supplemented with 5.5 g L(-1) glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL(-1).

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2⁴ factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R² = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L⁻¹ and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL⁻¹) occurred in the MRS broth supplemented with 5.5 g L⁻¹ glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL⁻¹.

Designing excipient emulsions to increase nutraceutical bioavailability: emulsifier type influences curcumin stability and bioaccessibility by altering gastrointestinal fate.

The influence of emulsifier type on the ability of excipient emulsions to improve the solubility, stability, and bioaccessibility of powdered curcumin was examined. Oil-in-water emulsions prepared using three different emulsifiers (whey protein isolate, caseinate, or Tween 80) were mixed with curcumin powder and then incubated at either 30 °C (to simulate applications of salad dressings) or 100 °C (to simulate applications of cooking sauces). The transfer of curcumin into the excipient emulsions was appreciably higher for excipient emulsions held at 100 °C than those held at 30 °C, and was appreciably higher for surfactant-stabilized emulsions than protein-stabilized emulsions. For example, the amounts of curcumin transferred into emulsions held at 30 and 100 °C were 66 and 280 µg mL(-1) for Tween 80, but only 17 and 208 µg mL(-1) for caseinate. The total curcumin concentration in the digesta and mixed micelle phases collected after excipient emulsions were exposed to a simulated gastrointestinal tract (mouth, stomach, and small intestine) depended on emulsifier type. The total amount of curcumin within the digesta was higher for protein-stabilized emulsions than surfactant-stabilized ones, which was attributed to the ability of the proteins to protect curcumin from chemical degradation. For example, the digesta contained 204 µg mL(-1) curcumin for caseinate emulsions, but only 111 µg mL(-1) for Tween 80 emulsions. This study shows the potential of designing excipient emulsions to increase the oral bioavailability of curcumin for food and pharmaceutical applications.

Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants.

Aflatoxin B1 (AFB1) is a highly toxic fungal metabolite having carcinogenic, mutagenic and teratogenic effects on human and animal health. Accidental feeding of aflatoxin-contaminated rice straw may be detrimental for ruminant livestock and can lead to transmission of this toxin or its metabolites into the milk of dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus produces ligninolytic enzymes like laccase and manganese peroxidase (MnP). These extracellular enzymes have been reported to degrade many environmentally hazardous compounds. The present study examines the ability of P. ostreatus strains to degrade AFB1 in rice straw in the presence of metal salts and surfactants. Laccase and MnP activities were determined spectrophotometrically. The efficiency of AFB1 degradation was evaluated by high performance liquid chromatography. Highest degradation was recorded for both P. ostreatus MTCC 142 (89.14 %) and P. ostreatus GHBBF10 (91.76 %) at 0.5 µg mL(-1) initial concentration of AFB1. Enhanced degradation was noted for P. ostreatus MTCC 142 in the presence of Cu(2+) and Triton X-100, at toxin concentration of 5 µg mL(-1). P. ostreatus GHBBF10 showed highest degradation in the presence of Zn(2+) and Tween 80. Liquid chromatography-mass spectrometric analysis revealed the formation of hydrated, decarbonylated and O-dealkylated products. The present findings suggested that supplementation of AFB1-contaminated rice straw by certain metal salts and surfactants can improve the enzymatic degradation of this mycotoxin by P. ostreatus strains.

Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

In vitro penetration properties of solid lipid nanoparticles in intact and barrier-impaired skin.

Treatment of skin diseases implies application of a drug to skin with an impaired epidermal barrier, which is likely to affect the penetration profile of the drug substance as well as the carrier into the skin. To elucidate this, the effect of skin barrier damage on the penetration profile of a corticosteroid applied in solid lipid nanoparticles (SLN) composed of different lipids, varying in polarity, was studied. The studies were carried out in vitro using impaired and intact porcine ear skin, and the SLN were compared with a conventional ointment. It was shown that a significantly higher amount of corticosteroid remained in the skin, intact as well as barrier impaired, when SLN was used as a vehicle. In general, the penetration profile of the drug substance into the skin was affected by the type of lipid used in the formulation and related to lipid polarity and drug substance solubility. When formulated in SLN and applied to intact skin, the permeation of the drug substance across the skin was significantly reduced, as compared to the ointment. Altogether, in both barrier-impaired and intact skin, a higher amount of drug substance remained in the skin during application of SLN for 6, 16, and 24h, as compared to the ointment. These results emphasize the applicability of SLN to create a drug reservoir in skin, with the drug localized distinctively in the stratum corneum.

A quantitative kinetic study of polysorbate autoxidation: the role of unsaturated fatty acid ester substituents.

PURPOSE: To study the role of unsaturated fatty acid ester substituents in the autoxidation of polysorbate 80 using quantitative kinetics. METHODS: Oxidation kinetics were monitored at 40 degrees C in aqueous solution by tracking head space oxygen consumption using a fiber optic oxygen sensor with phase shift fluorescence detection. Radical chain initiation was controlled using an azo-initiator and assessed by Hammond's inhibitor approach, allowing oxidizability constants (k(p)/(2k(t))(1/2)) to be isolated. Reaction orders were determined using modified van't Hoff plots and mixed polysorbate micelles. RESULTS: The oxidizability constant of polysorbate 80 ((1.07 +/- 0.19) x 10(-2) M(-1/2) s(-1/2)) was found to be 2.65 times greater than polysorbate 20 ((0.404 +/- 0.080) x 10(-2) M(-1/2) s(-1/2)). The additional reactivity of polysorbate 80 was isolated and was first-order in the unsaturated fatty acid ester substituents, indicating that the bulk of the autoxidative chain propagation is due to these groups. This data, and the observation of a half-order dependence on the azo-initiator, is consistent with the classical autoxidation rate law (-d[O(2)]/dt = k(p)[RH](R(i)/2k (t))(1/2)). CONCLUSIONS: Polysorbate 80 autoxidation follows the classical rate law and is largely dependent on the unsaturated fatty acid ester substituents. Clarification of the substituents' roles will aid formulators in the selection of appropriate polysorbates to minimize oxidative liabilities.

Investigation on bioremediation of oil-polluted wetland at Liaodong Bay in northeast China.

An investigation on the effect of various microbes on degradation was carried out as part of the study on bioremediation of oil-polluted wetland at LiaoDong Bay in northeast China. The method used involved direct inoculation of selected bacteria, which were capable of degrading oil, to the soil samples. The combination of various bacteria showed better results in terms of oil degradation than any single ones due to their synergetic effects. The operation conditions [pH 8.0, 25 degrees C, C/N/P (40:5.6:1)] for these bacteria to degrade the oil content in the soil samples were also studied and optimized. Addition of appropriate surfactants was helpful for bacteria growth, thus favoring the oil degradation. For instance, after adding Tween 80 (300 mg/kg) for 8 days, the number of bacteria was amplified 6.22 times and the rate of oil degradation increased by 20%. Adequate amount of H2O2 was also beneficial for microbes to decompose oil. However, overdosage may cause the death of the bacteria. The addition of 400 mg/l H2O2 each time was suitable. Seven thousand milligrams of H2O2 was added entirely in 11 days, and the rate of oil degradation increased significantly from 27% (without H2O2) up to 67%. The study clearly demonstrated that the direct soil inoculation was an effective method for environmental bioremediation.

S5 Lipase: an organic solvent tolerant enzyme.

In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.

Enhancing the intrinsic bioremediation of PAH-contaminated anoxic estuarine sediments with biostimulating agents.

Estuarine sediments are frequently polluted with hydrocarbons from fuel spills and industrial wastes. Polycyclic aromatic hydrocarbons (PAHs) are components of these contaminants that tend to accumulate in the sediment due to their low aqueous solubility, low volatility, and high affinity for particulate matter. The toxic, recalcitrant, mutagenic, and carcinogenic nature of these compounds may require aggressive treatment to remediate polluted sites effectively. In petroleum-contaminated sediments near a petrochemical industry in Gwangyang Bay, Korea, in situ PAH concentrations ranged from 10 to 2,900 microg/kg dry sediment. To enhance the biodegradation rate of PAHs under anaerobic conditions, sediment samples were amended with biostimulating agents alone or in combination: nitrogen and phosphorus in the form of slow-release fertilizer (SRF), lactate, yeast extract (YE), and Tween 80. When added to the sediment individually, all tested agents enhanced the degradation of PAHs, including naphthalene, acenaphthene, anthracene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene. Moreover, the combination of SRF, Tween 80, and lactate increased the PAH degradation rate 1.2-8.2 times above that of untreated sediment (0.01-10 microg PAH/kg dry sediment/day). Our results indicated that in situ contaminant PAHs in anoxic sediment, including high molecular weight PAHs, were degraded biologically and that the addition of stimulators increased the biodegradation potential of the intrinsic microbial populations. Our results will contribute to the development of new strategies for in situ treatment of PAH-contaminated anoxic sediments.

In vitro investigation on the impact of the surface-active excipients Cremophor EL, Tween 80 and Solutol HS 15 on the metabolism of midazolam.

The impact of the surface-active formulation ingredients Cremophor EL, Tween 80 and Solutol HS 15 on the intrinsic clearance (Clint) of midazolam (MDZ) was investigated in rat hepatocytes and microsomes. In rat hepatocytes with 0.003%, 0.03% and 0.3% (w/v) Solutol HS 15 already present in the incubation medium, the Clint was significantly reduced in a dose-dependent manner by about 25%, 30% and 50%, respectively. In the presence of Cremophor EL and Tween 80 a significant reduction in Clint by about 30% and 25%, respectively, was observed at 0.03% surfactant concentration. At 0.3% of Cremophor EL and Tween 80, Clint was reduced by about 50% and 20%, respectively. A reduction in Clint was also observed in experiments with rat liver microsomes. At surfactant concentrations up to 0.03%, cytotoxicity assays (lactate dehydrogenase release, adenosine triphosphate content) as well as light microscope investigations did not reveal any cytotoxic impact of the surfactants on the hepatocyte monolayer. A potential interaction of the surfactants with biological membranes was determined using phosphatidylcholine-cholesterol liposomes loaded with self-quenching concentrations of carboxyfluorescein. No marked release of carboxyfluorescein from the liposomes (that would be an indication for a surfactant-dependent disruption of membrane integrity) was observed up to concentrations of 0.03% of the different surfactants. It is concluded that cytochrome P450 3A mediated metabolism of MDZ seems to be prevented by all surfactants at concentrations above 0.03%. In our experiments the surfactants did not show toxic effects at concentrations that resulted in a decreased Clint of MDZ. Thus, a direct inhibition of the metabolizing enzymes, a molecular interaction with the microsomes as well as an alteration of membrane properties that did not yet result in a release of LDH have to be taken into consideration as reasons for the observed changes in the metabolism of MDZ.

Morphology and physiology of the dimorphic fungus Mucor circinelloides (syn. M. racemosus) during anaerobic growth.

The dimorphic Mucor circinelloides requires an anaerobic atmosphere and the presence of 30% CO2 to grow as a multipolar budding yeast, otherwise hyphal growth predominates. Establishing other means to control the morphology would be a distinct advantage in the development of a fermentation process for this organism for the production of heterologous proteins. Thus, conditions suppressing polarised growth while at the same time abolishing the CO2 requirement were investigated in submerged cultivations. It was found that supplementing cultures with mixtures of ergosterol and Tween 80 resulted in yeast-like growth under 100% N2. Their impact on growth and morphological development was assessed at a range of concentrations. Maximum biomass levels and the specific growth rate decreased at elevated levels of ergosterol and Tween 80. Possible effects of carbon dioxide and the added fatty acid/sterol mixture on supporting yeast growth by influencing the fluidity of the plasma membrane or affecting polarised growth are discussed.

Effect of different carbon sources and cold shock on protein synthesis by a psychrotrophic Acinetobacter sp.

The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80.