Nonimmunogenic Hydrogel-Mediated Delivery of Antibiotics Outperforms Clinically Used Formulations in Mitigating Wound Infections.

Treatment of chronic wound infections caused by Gram-positive bacteria such as Staphylococcus aureus is highly challenging due to the low efficacy of existing formulations, thereby leading to drug resistance. Herein, we present the synthesis of a nonimmunogenic cholic acid-glycine-glycine conjugate (A6) that self-assembles into a supramolecular viscoelastic hydrogel (A6 gel) suitable for topical applications. The A6 hydrogel can entrap different antibiotics with high efficacy without compromising its viscoelastic behavior. Activities against different bacterial species using a disc diffusion assay demonstrated the antimicrobial effect of the ciprofloxacin-loaded A6 hydrogel (CPF-Gel). Immune profiling and gene expression studies after the application of the A6 gel to mice confirmed its nonimmunogenic nature to host tissues. We further demonstrated that topical application of CPF-Gel clears S. aureus-mediated wound infections more effectively than clinically used formulations. Therefore, cholic acid-derived hydrogels are an efficacious matrix for topical delivery of antibiotics and should be explored further.

Evaluation of sodium deoxycholate as solubilization buffer for oil palm proteomics analysis.

Protein solubility is a critical prerequisite to any proteomics analysis. Combination of urea/thiourea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) have been routinely used to enhance protein solubilization for oil palm proteomics studies in recent years. The goals of these proteomics analysis are essentially to complement the knowledge regarding the regulation networks and mechanisms of the oil palm fatty acid biosynthesis. Through omics integration, the information is able to build a regulatory model to support efforts in improving the economic value and sustainability of palm oil in the global oil and vegetable market. Our study evaluated the utilization of sodium deoxycholate as an alternative solubilization buffer/additive to urea/thiourea and CHAPS. Efficiency of urea/thiourea/CHAPS, urea/CHAPS, urea/sodium deoxycholate and sodium deoxycholate buffers in solubilizing the oil palm (Elaeis guineensis var. Tenera) mesocarp proteins were compared. Based on the protein yields and electrophoretic profile, combination of urea/thiourea/CHAPS were shown to remain a better solubilization buffer and additive, but the differences with sodium deoxycholate buffer was insignificant. A deeper mass spectrometric and statistical analyses on the identified proteins and peptides from all the evaluated solubilization buffers revealed that sodium deoxycholate had increased the number of identified proteins from oil palm mesocarps, enriched their gene ontologies and reduced the number of carbamylated lysine residues by more than 67.0%, compared to urea/thiourea/CHAPS buffer. Although only 62.0% of the total identified proteins were shared between the urea/thiourea/CHAPS and sodium deoxycholate buffers, the importance of the remaining 38.0% proteins depends on the applications. The only observed limitations to the application of sodium deoxycholate in protein solubilization were the interference with protein quantitation and but it could be easily rectified through a 4-fold dilution. All the proteomics data are available via ProteomeXchange with identifier PXD013255. In conclusion, sodium deoxycholate is applicable in the solubilization of proteins extracted from oil palm mesocarps with higher efficiency compared to urea/thiourea/CHAPS buffer. The sodium deoxycholate buffer is more favorable for proteomics analysis due to its proven advantages over urea/thiourea/CHAPS buffer.

Stability of Vegetal Diamine Oxidase in Simulated Intestinal Media: Protective Role of Cholic Acids.

Food biogenic amines, in particular, histamine, are often responsible for various enteric and vascular dysfunctions. Several years ago, the oral administration of copper-containing diamine oxidase (DAO), also called histaminase, able to oxidatively deaminate biogenic amines, had been suggested as a food supplement to control food allergy and enteric dysfunctions. This report is aimed to generate a global image on the behavior of orally administrated DAO dosage forms in the intestinal tract. The catalytic stability of DAO from Lathyrus sativus seedlings in various simulated intestinal media with different pH and containing different association of cholic acids, pancreatic proteases, bicarbonate, lipids, or alcohol was investigated. Cholic acids and lipids protected the enzyme in the simulated intestinal fluids. However, they were not able to protect against the inhibitory effect of 24-36% (v/v) ethanol. These observations may be relevant for oral administration of enzymes as food supplements or therapeutic bioactive agents.

Optimization of washing conditions with biogenic mobilizing agents for marine fuel-contaminated beach sands.

Washing is a rapid and effective treatment to remediate contaminated sands impacted by oil spills, although synthetic additives used to increase extraction efficiency may cause additional pollution issues due to their intrinsic toxicity and very often low biodegradability. In this study, different biogenic mobilizing agents (soybean lecithins, cyclodextrins, cholic acids, plant-derived cleaners, rhamnolipids and sophorolipids) were tested in the washing of beach sands artificially contaminated with the Intermediate Fuel Oil IFO-180. Among these, a de-oiled soybean lecithin (SL-1), hydroxypropyl-ß-cyclodextrins (HPB-CD) and sophorolipids (SR) achieved hydrocarbon removals close to those attained with the synthetic surfactant Triton™ X-100 (TX) in preliminary washing tests carried out at constant mixing rate, water/sand ratio and IFO-180 contamination level using agents concentrations close to their critical micelle concentration (0.1% and 1% w/v for microbial and non-microbial agents, respectively). The effects of agent concentration, water/sand ratio, mixing rate and IFO-180 contamination on hydrocarbons removal were modelled using face-centred central composite design and ANOVA. Optimal washing parameters for sand contamination levels in the range 0.5-20 g/kg were identified with response surface methodology. While HPB-CD and SR performed equally to TX only at low sand contaminations, SL-1 attained hydrocarbon removal higher or equal to that of TX at any IFO-180 contamination and at lower application rates. SL-1 also outperformed TX when minimizing the water/sand ratio, i.e., the volume of water used. Considering its lower toxicity, higher biodegradability and higher hydrocarbon removal efficiencies, SL-1 is an effective and environmentally sustainable alternative to synthetic surfactants in washing treatments for marine fuel-contaminated sands.

Bile acids and their oxo derivatives: Potential inhibitors of carbonic anhydrase I and II, androgen receptor antagonists and CYP3A4 substrates.

Some biological properties of bile acids and their oxo derivatives have not been sufficiently investigated, although the interest in bile acids as signaling molecules is rising. The aim of this work was to evaluate physico-chemical parametar b (slope) that represents the lipophilicity of the examined molecules and to investigate interactions of bile acids with carbonic anhydrase I, II, androgen receptor and CYP450s. Thirteen candidates were investigated using normal-phase thin-layer chromatography in two solvent systems. Retention parameters were used in further quantitative structure-activity relationship analysis and docking studies to predict interactions and binding affinities of examined molecules with enzymes and receptors. Prediction of activity on androgen receptor showed that compounds 3α-hydroxy-12-oxo-5ß-cholanoic and 3α-hydroxy-7-oxo-5ß-cholanoic acid have stronger antiandrogen activity than natural bile acids. The inhibitory potential for carbonic anhydrase I and II was tested and it was concluded that molecules 3α-hydroxy-12-oxo-5ß-cholanoic, 3α-hydroxy-7-oxo-5ß-cholanoic, 3,7,12-trioxo-5ß-cholanoic acid and hyodeoxycholic acid show the best results. Substrate behavior for CYP3A4 was confirmed for all investigated compounds. Oxo derivatives of bile acids show stronger interactions with enzymes and receptors as classical bile acids and lower membranolytic activity compared with them. These significant observations could be valuable in consideration of oxo derivatives as building blocks in medicinal chemistry.

Targeting the Eph-ephrin System with Protein-Protein Interaction (PPI) Inhibitors.

Eph-ephrin system is emerging as a new potential target in several diseases including cancer, diabetes, neurodegenerative diseases and inflammation. In the last decade, several efforts have been made to develop small molecule antagonists of Eph receptors. Both natural and synthetic compounds were discovered with (poly) phenol and steroidal derivatives on one side and the α1 agonist doxazosin, 2,5-dimethylpyrrol- 1-yl-benzoic acids and amino acid conjugates of lithocholic acid on the other. In the present paper we critically present available data for these compounds and discuss their potential usefulness as pharmacological tools or as candidates for a lead-optimization program.

Plasma membrane as the target site of cholic acid analogs.

Although the mechanism is unknown, Calculus Bovis and its active components, cholic acid analogs (CAAs), have been used in China to treat a wide range of diseases. Based on the previous finding that the potency of CAA is strongly dependent on the intrinsic surface activity, this paper aimed to investigate the role of the plasma membrane in the pharmacological activity of CAAs. First, CAAs (0.1 mM) caused a surface activity-dependent depression on ATPase activity in the cell membrane extract, but it had no effects on other cellular extracts, suggesting an indispensable role of the membrane environment for pharmacological activity. Second, CAAs lowered the membrane fluidity of cultured Caco-2 cells with the same rank-order of potency sequence. Third, the hypothesis that any functional protein located on the membrane is influenced by changes in cellular membrane fluidity was supported by: ileal contraction that was induced by acetylcholine and mediated by the muscarinic receptor (M-receptor) or the relaxation induced by adrenaline and mediated by the ß-adrenergic receptor (ß-receptor) was inhibited by CAAs. They also had similar rank-order of potency and the effects on the plasma membrane. Collectively, the plasma membrane may be a target for the CAAs to exert the multiple pharmacological effects which are mediated by the alteration of the membrane mobility and the function of integral membrane proteins.

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield.

Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

In vitro stabilization and minimum active component of polygalacturonic acid synthase involved in pectin biosynthesis.

Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an alpha1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.

Biochemical and physiological properties of a novel series of long-acting insulin analogs obtained by acylation with cholic acid derivatives.

PURPOSE: This study was conducted to assess the suitability of insulin analogs acylated by various cholic acid derivatives for use as basal insulin, and to test the most promising of these, LysB29(Nepsilon-lithocholyl-gamma-Glu) des(B30) human insulin (NN344) in pigs. METHODS: Circular dichroism spectroscopy and size-exclusion chromatography were used to explore the physicochemical properties of the analogs, and affinities for albumin and insulin receptors were determined. After subcutaneous injection in pigs, disappearance half-times were measured, and the plasma profile and glucose-lowering effect in a euglycemic clamp were assessed for NN344. RESULTS: NN344 showed glucose-lowering activity lasting more than 24 h. Glucose infusion rate was essentially constant from 5 to 19 h after injection. NN344 seemed to be a dodecamer in the presence of zinc ions and phenol. Without phenol, the apparent molecular mass was >5000 kDa. Formation of such a self-assembly at the site of s.c. injection and its subsequent slow decomposition might explain the long duration of action of NN344. A measurable affinity for albumin of the lithocholic acid ligand may also contribute to the prolonged action. CONCLUSIONS: NN344 is a candidate for a neutral soluble basal insulin that might offer people with diabetes a prolonged duration, smooth, and predictable basal insulin supplement.

MHC II molecules and invariant chain reside in membranes distinct from conventional lipid rafts.

Major histocompatibility complex class II (MHC II) peptide complexes can associate with lipid rafts, and this is a prerequisite for their recruitment to the immunological synapse and for efficient T cell stimulation. One of the most often used criterion for raft association is the resistance to extraction by the detergent Triton X-100 (TX-100) at low temperature. For MHC II, a variety of detergents have been used under different conditions, leading to variable and often conflicting conclusions about the association of MHC II with detergent-resistant membranes (DRMs). To clarify whether these inconsistencies were caused by variations in the isolation protocols or reflect different biochemical properties of MHC II lipid complexes, we used two standardized procedures for the isolation of membranes resistant to TX-100, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Brij 98. Our results suggest that some of the reported variations in the association of MHC II with DRMs are caused by differences in the methods. We also show that in our hands, specific and efficient flotation of MHC II and the MHC II-associated invariant chain from mouse B-lymphoma cells was only achieved with Brij 98, but not with TX-100 and CHAPS. We furthermore used DRMs prepared from hen egg lysozyme-fed B-lymphoma cells to activate the T cell hybridoma 3A9. In agreement with our biochemical data, T cell activation could only be achieved with Brij 98- but not with TX-100-resistant membranes. Thus, MHC II and also the invariant chain belong to a set of proteins comprising the T cell receptor, prominin, and the prion protein, which reside in membrane environments distinct from conventional lipid rafts.

Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane.

Glycosphingolipids and cholesterol form lateral assemblies, or lipid 'rafts', within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergent-insoluble complexes (in non-ionic detergents at 4 degrees C; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4 degrees C or (ii) 37 degrees C, or at 4 degrees C after pre-extraction with (iii) saponin or (iv) methyl-beta-cyclodextrin (MbetaCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbetaCD. Furthermore, pre-extraction with MbetaCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbetaCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.

Transformation of cholanic acid derivatives into pharmacologically active esters of phenolic acids by heterogeneous Wittig reaction.

Steroid esters of cynnamic acid derivatives have been synthesized by a heterogeneous Wittig reaction under sonochemical conditions from the corresponding triphenylphosphonium bromides and unprotected phenolic aldehyds using K2CO3 as a base. 5 beta-Cholan-3 alpha, 7 alpha, 12 alpha, 24-E-ferulate (11') exhibited a marked inhibitory effect on influenza virus A. The synthetic 3 alpha, 24-E-diferulates of 5 beta-cholan-3 alpha, 24- diol, 5 beta-cholan-3 alpha, 12 alpha, 24-triol and 5 beta-cholan-3 alpha, 7 alpha, 12 alpha, 24-tetrol (8, 9 and 12) showed antitumor activity on leukemia P-388 in mice.

Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems.

Using complementary physical-chemical techniques we defined five different crystallization pathways as functions of time (30 days) and increasing lecithin (egg yolk) content in pathophysiologically relevant model biles super-saturated (cholesterol saturation indices, 1.2 - 2.7) by dilution of approximately equal to 29 g/dl bile salt-lecithin-cholesterol micellar solutions. As evidenced by quasi-elastic light-scattering spectroscopy, supersaturation was heralded by the appearance of unilamellar vesicles. With the lowest lecithin contents, arc-like crystals with habit and density (d 1.030 g/mL) consistent with anhydrous cholesterol appeared first and evolved via helical and tubular crystals to form plate-like cholesterol monohydrate crystals (d 1.045 g/mL). With higher lecithin fractions, cholesterol monohydrate crystals appeared earlier than arc and other transitional crystals. With typical physiological lecithin contents, early liquid crystals (d 1.020 g/mL) were followed by cholesterol monohydrate crystals and subsequent appearances of arc and other intermediate crystals. With higher lecithin contents, liquid crystals were followed by cholesterol monohydrate crystals only, and at the highest lecithin mole fractions, liquid crystals appeared that did not generate solid crystals. Added calcium increased solid crystal number in proportion to its concentration (5 - 20 mM) but did not influence appearance times, crystallization pathways, or micellar cholesterol solubilities. Decreases in temperature (37 degrees --> 4 degrees C), total lipid concentration (7.3 --> 2.4 g/dL), and bile salt hydrophobicity (3 alpha, 12 alpha --> 3 alpha, 7 alpha, 12 alpha --> 3 alpha, 7 beta hydroxylated taurine conjugates) progressively shifted all crystallization pathways to lower lecithin contents, retarded crystallization, and decreased micellar cholesterol solubilities. The lecithin content of mother biles decreased markedly during crystallization especially where liquid crystals were a coexisting phase at equilibrium. This systematic study provides a framework for understanding cholesterol crystallization in human and animal biles and for examining factors that influence the kinetics of phase separation.