Enzymatic Activity Of Lysozyme Research Articles

Oxidative Folding of Conopeptides Modified by Conus Protein Disulfide Isomerase.

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis-trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis-trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.

Cypermethrin Formulation (Ustad-10 EC) Induces Genotoxicity via Apoptosis, Affects Nutritional Physiology, and Modulates Immune Response in Silkworm Philosamia ricini (Lepidoptera: Saturniidae).

Cypermethrin is a pyrethroid insecticide with high insecticidal activity, low mammalian toxicity, and biodegradability. The present study aimed to determine the acute toxicity and evaluate the secondary toxic effects of a commercial formulation of cypermethrin on silkworm Philosamia ricini Hutt of Northeast India. The potential genotoxicity of cypermethrin on silkworm hemocyte was examined by comet assay, caspase activation, and annexin V affinity assay. Alteration in nutritional physiology and histoarchitecture of the gut region was evaluated. Additionally, immunotoxicological effect of cypermethrin was studied by phenoloxidase (PO), lysozyme assay, and abundance of circulating hemocytes. The LC50 value at 24-, 48-, 72-, and 96-h exposure period was recorded as 185.96, 105.34, 72.42, and 58.41 µg/liter, respectively. Approximately sevenfold increase in mean comet tail length was observed at 24 h posttreatment with sublethal concentrations of cypermethrin. Cypermethrin also induced apoptosis and activated caspase reaction in silkworm hemocytes. Moreover, a significant decrease in digestive enzyme activity was observed at higher concentrations of cypermethrin. In cypermethrin-exposed groups, alteration in histoarchitecture was also observed in the form of ruptured microvilli and thin, deformed, fused mucous layer. The PO enzyme and lysozyme enzyme activity was also altered with sublethal concentration of cypermethrin. Total hemocyte count was reduced to 10587.10, 10052.30, 9234.30, and 8842.60 per mm3 with 10, 20, 30, and 40 µg/liter, respectively. The results offer new insights into the negative consequences of very low concentrations of cypermethrin formulations on nonmulberry silkworm of Northeast India.

Synthesis of novel muramic acid derivatives and their interaction with lysozyme: Action of lysozyme revisited.

The interaction of lysozyme with the N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) unit of peptidoglycan (PGN) polymer of the bacterial cell wall is of immense importance to understand the mechanism of lysozyme on PGN. The synthesis of three novel NAM derivatives containing fused oxazinone ring to the NAM moiety has been achieved. The synthesized compounds were evaluated for their potential as a glycomimetic acceptor of lysozyme using different biophysical and computational methods such as 1H NMR, STD NMR, DOSY and Molecular docking. Novel modified muramic acid derivatives have been synthesized in excellent yield containing fused cyclooxazine ring embedded on the muramic acid moiety using a newly developed hydrazinolysis reaction condition. From various biophysical studies, it has been established that the compound containing endo modified muramic acid moiety (compound 1) shows significant binding property for the lysozyme while the other isomer (compound 2) did not bind to the lysozyme. The catalytic residues Glu35 and Asp52 were found to be in the close proximity for the active molecule which justifies the selectivity of this molecule in conjunction to lysozyme enzymatic activity.

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.

Albumin-lysozyme interactions: Cooperative adsorption on titanium and enzymatic activity

The interplay of albumin (BSA) and lysozyme (LYZ) adsorbed simultaneously on titanium was analyzed by gel electrophoresis and BCA assay. It was found that BSA and lysozyme adsorb cooperatively. Additionally, the isoelectric point of the respective protein influences the adsorption. Also, the enzymatic activity of lysozyme and amylase (AMY) in mixtures with BSA was considered with respect to a possible influence of protein-protein interaction on enzyme activity. Indeed, an increase of lysozyme activity in the presence of BSA could be observed. In contrast, BSA does not influence the activity of amylase.

Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme

In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed. The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant impact of ethanol; however, the lysozyme enzymatic activity was ca. 25% lower compared to the starting material. In conclusion, the addition of ethanol as a co-solvent in the spray drying feed for lysozyme did not compromise the conformation of the protein after drying, while it improved the inhaled aerosol performance.

Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth's cells in a model of ileitis in mice.

In this study, we showed the beneficial effects of donkey milk (DM) on inflammatory damages, endogenous antimicrobial peptides levels and fecal microbiota profile in a mice model of Crohn's disease. Nowadays, new strategies of microbiome manipulations are on the light involving specific diets to induce and/or to maintain clinical remission. Interest of DM is explained by its high levels of antimicrobial peptides which confer it anti-inflammatory properties. C57BL/6 mice were orally administered with or without indomethacin for 5days and co-treated with vehicle, DM or heated DM during 7days. Intestinal length and macroscopic damage scores (MDSs) were determined; ileal samples were taken off for microscopic damage (MD), lysozyme immunostaining and mRNA α-defensin assessments. Ileal luminal content and fecal pellets were collected for lysozyme enzymatic activity and lipocalin-2 (LCN-2) evaluations. Fecal microbiota profiles were compared using a real-time quantitative PCR-based analysis. Administration of indomethacin caused an ileitis in mice characterized by (1) a decrease in body weight and intestinal length, (2) a significant increase in MDS, MD and LCN-2, (3) a reduction in both α-defensin mRNA expression and lysozyme levels in Paneth's cells reflected by a decrease in lysozyme activity in feces, and (4) a global change in relative abundance of targeted microbial communities. DM treatment significantly reduced almost of all these ileitis damages, whereas heated DM has no impact on ileitis. DM consumption exerts anti-inflammatory properties in mice by restoring the endogenous levels of antimicrobial peptides which contribute in turn to reduce microbiota imbalance.

Properties of lysozyme/sodium alginate complexes for the development of antimicrobial films

Complexation study of lysozyme (0.714g/L) by sodium alginate at pH7 showed that aggregates formation was a two-phase process. The first phase (from 0 to 0.1g/L sodium alginate) corresponded to the combination of individual complexes to form aggregates which caused an increase of turbidity and average size and a rapid sedimentation. Charge neutralization estimated by ζ-potential measurements occurred at 0.1g/L sodium alginate concentration. The second phase (from 0.1 to 4g/L of sodium alginate) was characterized by the formation of aggregates having a less dense structure with higher average size despite the drop in turbidity and the high dispersion in the medium. Lysozyme enzymatic activity decreased upon complexation with sodium alginate but was fully recovered after calcium chloride addition. In order to check whether lysozyme reversible inactivation was only due to substrate diffusion limitation or to conformational changes upon complexation, fluorescence and UV–Vis absorption measurements were performed. Moreover, lysozyme/sodium alginate complexes were used to manufacture an edible antimicrobial film to target lysozyme sensitive microorganisms.

Modulatory effects of levamisole and garlic oil on the immune response of Wistar rats: Biochemical, immunohistochemical, molecular and immunological study.

Levamisole (LEVA) and garlic are prevalent immunomodulators in humans and animals. Therefore, the present study aimed to examine the immunomodulatory effects of LEVA and garlic oil (GO) alone or in combination on the immune response of Wistar rats. A total of 24male Wistar rats were allocated into four equal groups: Control group, which was given adlibitum access to food and water; and groups2‑4, which were orally administered LEVA [2.5mg/kg body weight (BW) every 2days], GO, (5ml/kg BW daily), or LEVA plus GO, respectively for 4consecutive weeks. Serum immunoglobulin (Ig)G and IgM levels were measured using a radial immunodiffusion assay. Serum cytokine levels, including interferon (IFN)-γ, interleukin (IL)-5 and tumor necrosis factor (TNF)-α, were measured using enzyme‑linked immunosorbent assay kits. Total blood counts were measured automatically using a cell counter. Serum lysozyme enzymatic activity was determined by measuring the diameters of the zones of clearance relative to lysozyme. Immunohistochemical detection of CD4 and CD8 was carried out using the streptavidin-biotin-peroxidase method. Furthermore, the mRNA expression levels of IL‑4, IL‑5 and IL‑12 were measured in the leukocytes and thymus gland by semi-quantitative polymerase chain reaction. The results revealed that LEVA increased serum levels of IFN‑γ, IL‑5 and TNF‑α cytokines, whereas co‑administration of LEVA and GO decreased the stimulatory action of LEVA alone. LEVA and GO alone increased the serum levels of IgG, IgM and total blood cell counts, and co‑administration of GO and LEVA inhibited the effects of LEVA. At the cellular level, in the spleen, LEVA increased immunoreactivity of CD4 and CD8, whereas co‑administration of GO with LEVA decreased this strong expression. At the molecular level, in leukocytes, LEVA upregulated the mRNA expression levels of IL‑2, IL‑4 and IL‑5, whereas GO alone downregulated mRNA expression. Co‑administration of GO with LEVA inhibited the LEVA‑induced upregulation of IL‑2, IL‑4 and IL‑5 mRNA expression. In the thymus, both LEVA and GO upregulated the mRNA expression levels of IL‑4 and IL‑5, whereas LEVA alone did not affect IL‑12mRNA expression. Co‑administration of GO with LEVA inhibited LEVA‑induced upregulation of IL‑4 and GO‑induced upregulation of IL‑12 expression, and had an additive upregulatory effect on IL‑5 expression. In conclusion, LEVA stimulated T‑helper(Th)1 cytokines, whereas GO stimulated a Th2 response, and co‑administration of GO with LEVA inhibited the stimulatory effects of LEVA and balanced the Th1/Th2 response.

Characterization and pathogenicity assessment of gut-associated microbes of muga silkworm Antheraea assamensis Helfer (Lepidoptera: Saturniidae)

Antheraea assamensis Helfer (muga silkworm) is an economically important endemic insect species of North Eastern Region of India. The silkworm is often susceptible to infection by pathogenic bacteria, leads to a disease commonly known as flacherie which causes 40% loss per annum to the silk industry. In this study, we have reported isolation, characterization and pathogenicity assessment of gut-associated bacteria of healthy and diseased muga silkworms. Thirty five bacterial isolates were screened from the gut of healthy and diseased silkworms by morphological observation and biochemical tests. 11 and 5 strains from healthy and diseased silkworm respectively were identified by 16S rRNA gene sequencing and analyzed. Pseudomonas aeruginosa (DRK1), Ornithinibacillus bavariensis (DRK2), Achromobacter xylosoxidans (KH3) and Staphylococcus aureus (FLG1) strains were commonly found in healthy as well as diseased larvae whereas, Bacillus thuringiensis (MK1) was found only in diseased larvae. Survivability analysis was performed with the identified strains by injection and oral administration (104CFU/ml). The immune response of the silkworm against the pathogen was also studied by phenoloxidase and lysozyme enzyme activity assay, total and differential hemocyte count and phagocytic activity of hemocytes. It was observed that S. aureus, P. aeruginosa and B. thuringiensis significantly reduced the survivability of silkworm (p<0.001) hence found highly pathogenic. The lethal concentrations (LC50) values of the pathogenic strains were calculated at different time intervals (24, 48, 72 and 96h) within the range from 1.38×102 to 3.63×107CFU/ml. The pathogenic groups demonstrated inhibition of phenoloxidase activity and decreased in total hemocyte count after 48h of infection. However, the lysozyme activity increased significantly in the pathogenic groups compared to the control (p<0.05). Granulocytes and plasmatocytes showed phagocytosis whereas; other types of cells did not show any phagocytic activity. Increasing granulocytes and plasmatocytes counts corroborates the results of phagocytic activity. The present study might be helpful in understanding the disease prognosis and colonization of bacteria causing the disease in muga silkworm.

Ameliorative Effect of Oregano Essential Oil on Mycotoxins-Induced Immune Impairments in Growing Japanese Quail

Mycotoxins contaminated feed is one of the most serious problems in poultry and animal production sectors. Therefore, the current study aimed to investigate the effects of dietary oregano essential oil (OEO) for 4 weeks against the adverse effect of aflatoxins (AFs) on immunity, oxidative stress, Total and differential leucocytes counts (TLC and DLC), interleukin 1 (IL-1), nitric oxide (NO), lysozyme enzyme activity, serum malondialdehyde (MDA), catalase, superoxide dismutase (SOD) and reduced glutathione (GSH). Beside growth performance [Weekly body weight gain and feed conversion ratio (FCR)] were also determined. A total of 132 male, 2 weeks old Japanese quails chicks (Coturnix japonica) were used in this study and equally divided into 4 groups. Group I is considered as negative control at which birds fed only basal diet, group II served as positive control and were fed AFs 2.5 mg /Kg supplemented basal diet, Groups III and IV were fed basal diet with 2.5 mg AFs /Kg and treated with OEO at a rate of 200 and 400 mg/Kg , respectively. Regarding growth performance, there was a significant (P<0.05) reduction in weight gain and FCR at 4th week of the experiment in group II, additionally different pattern was noticed in group IV which represented a significant (P<0.05) improvement as compared to the other AFs treated groups and was nearly similar to that of group I. Concerning Lipid peroxidation, oxidative stress enzyme and immune parameters at 4th week of the experiment, MDA was significantly (P<0.05) increased in AFs treated quails (II and III) but when AFs co-administered with 400 mg OEO, MDA was nearly similar to that of group I. The SOD, catalase, and GSH were decreased significantly (P<0.05) in groups (II and III) and was remarkably improved in group IV. The NO level was increased significantly (P<0.05) while lysozyme activity was decreased significantly (P<0.05) in group II, while they were decreased and increased in group IV, respectively. IL-1 and globulin as well as TLC were declined significantly (P<0.05) in groups II and III and improved nearly similar to control one in group IV. In conclusion, OEO has a potential and a protective effect against AFs B1 immuno-deteriorating effect in Japanese quails especially at 400 mg/ kg dose.

Production of lysozyme microparticles to be used in functional foods, using an expanded liquid antisolvent process

Expanded liquid antisolvent (ELAS) precipitation is a modification of the well-known supercritical antisolvent (SAS) process. Different from SAS, ELAS allows the micronization of water-soluble compounds using an antisolvent formed by supercritical carbon dioxide (scCO2) and an organic solvent, used to improve water solubility in carbon dioxide.In this work, ELAS was proposed for the micronization of lysozyme, an enzyme characterized by an intense antibacterial activity, using as antisolvents mixtures formed by scCO2+acetone and scCO2+isopropyl alcohol.Microparticles of lysozyme, with a mean diameter ranging between 2.8 and 13.8μm, varying the kind of co-antisolvent, the concentration of the enzyme in the liquid solution and the position of the operating point in the ternary diagram formed by water, carbon dioxide and the process co-antisolvent were obtained.The obtained powders were characterized using XRD and FT-IR. These analyses showed that ELAS process did not influence solute integrity. UV–vis analysis revealed that lysozyme enzymatic activity was not significantly altered. The best results were obtained using acetone, measuring a biological activity of 95% with respect to the native protein.

Stability and enzyme activity of lysozyme in the presence of Fe3O4 nanoparticles

The goal of this work was investigation the effect of adsorption the Fe3O4 as magnetic nanoparticle on lysozyme activity and thermal stability. The turbidimetric assay (activity) of lysozyme was followed by the decreased optical density of a turbid cell suspension (about 0.3 mg/cm3 Micrococcus lysodeikticus) photometrically at 450 nm. In this study, the effect of nano-Fe3O4 on lysozyme activity was investigated by UV–Vis spectrophotometry at pH = 7.25 at 35 °C using sodium phosphate buffer. Measurements were carried out using 6 × 10−8 M (0.1 mg/cm3) concentration of lysozyme and a range of nano-Fe3O4 concentrations between 0 and 0.06 mg/cm3. It was found that by increasing nano-Fe3O4, the rate of M. lysodeikticus lyses (lysozyme activity) increases. The thermal stability of lysozyme was investigated in the presence of nano-Fe3O4 over the temperature range of 293–363 K in sodium phosphate buffer and pH = 7.25. Results indicated that by increasing nano-Fe3O4, the thermal stability of lysozyme increases.

Effect of PEAMA-g-PEG on the conformation of lysozyme by circular dichroism and florescence spectroscopy

Conjugation of biological (peptides/enzymes) and synthetic polymers has potential applications in both medicine and biotechnology. We have shown in our previous article that the enzymatic activity of lysozyme was suppressed by the complex formation with a cationic smart copolymer, poly(N,N-diethylaminoethyl methacrylate)-graft-poly(ethylene glycol) (PEAMA-g-PEG). In addition, the secondary structure of lysozyme was maintained. In the present study, tertiary structure of lysozyme were investigated using circular dichrosm (CD) and fluorescence spectroscopy. Near-UV CD spectral analysis clearly indicates that the complexation of lysozyme with PEAMA-g-PEG has almost no influence on the tertiary structure of lysozyme. Moreover, fluorescence spectrum of lysozyme and lysozyme complexed with PEAMA-g-PEG indicates the conformations of lysozyme were not disrupted.

The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities

ObjectiveTo investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. DesignXylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. ResultsWhile xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. ConclusionsXylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities.

Synthesis of lysozyme-metallacarborane conjugates and the effect of boron cluster modification on protein structure and function.

Two complementary methods, "in solution" and "in solid state", for the synthesis of lysozyme modified with metallacarborane (cobalt bis(dicarbollide), Co(C2 B9 H11 )2 (2-) ) were developed. As metallacarborane donors, oxonium adducts of cobalt bis(dicarbollide) and 1,4-dioxane or tetrahydropyran were used. The physicochemical and biochemical properties of the obtained lysozyme-metallacarborane conjugates were studied for changes in secondary and tertiary structure, aggregation behavior, and biological activity. Only minor changes in primary, secondary, and tertiary protein structure were observed, caused by the single substitution of metallacarborane on lysozyme. However, the modification produced significant changes in lysozyme enzymatic activity and a tendency toward time- and temperature-dependent aggregation.

Design and Evaluation of Hydrophobic Ion-Pairing Complexation of Lysozyme with Sodium Dodecyl Sulfate for Improved Encapsulation of Hydrophilic Peptides/Proteins by Lipid-Polymer Hybrid Nanoparticles

Hydrophobic ion-pairing (HIP) complexation-based approach has been employed to reduce aqueous solubilities of peptide and protein drugs. The solubility of a protein molecule is due to presence of charged amino acids present on the surface. HIP complexation is a technique to complex ionizable functional groups of protein and peptide molecules with oppositely charged functional groups of a complex forming agent. The main objective of this study was to formulate and evaluate HIP complexes of lysozyme with sodium dodecyl sulfate (SDS) as an ion paring agent. Results of % binding effciency shown that the formation of HIP complexes were dependent on pH of lysozyme solution and molar ratio of lysozyme to SDS. Aqueous solubilities of HIP complexes were very low compared to lysozyme alone. The e¬ffect of HIP complexation on enzymatic activity of lysozyme was also studied. Further, lipid-polymer hybrid nanoparticles (LPNs) loaded with lysozyme and lysozyme:SDS HIP complex were prepared by using a solid-in-oil-in-water (s/o/w) emulsion solvent evaporation method and characterised with respect to morphology, size and encapsulation efficiency. We observed significant improvement in encapsulation efficiency for lysozyme:SDS HIP complex-loaded LPNs. This study demonstrates a novel approach of formulating protein-loaded nanoparticles which can also be employed for delivery of proteins.

Inhibition of Lysozyme by Taurine Dibromamine

Hypobromous acid (HOBr) is a powerful oxidant produced by stimulated neutrophils and eosinophils. Taurine, a non-protein amino acid present in high amounts in the leukocytes, reacts instantaneously with HOBr leading to their haloamine derivative taurine dibromamine (Tau-NBr2). Lysozyme is a bactericidal enzyme also present in leukocytes and in secretory fluids. The inhibition of lysozyme is a pathway for bacterial proliferation in inflammatory sites. Here, we investigated the inhibition of the enzymatic activity of lysozyme when it was submitted to oxidation by Tau-NBr2. We found that the oxidation of lysozyme by Tau-NBr2 decreased its enzymatic activity in 80%, which was significant higher compared to the effect of its precursor HOBr (30%). The study and comparison of Tau-NBr2 and HOBr regarding the alterations provoked in the intrinsic fluorescence, synchronous fluorescence, resonance light scattering and near and far-UV circular dichroism spectra of lysozyme and oxidized lysozyme revealed that tryptophan residues in the active site of the protein were the main target for Tau-NBr2 and could explain its efficacy as inhibitor of lysozyme enzymatic activity. This property of Tau-NBr2 may have pathological significance, since it can be easily produced in the inflammatory sites.

Design and evaluation of a novel nanoparticulate-based formulation encapsulating a HIP complex of lysozyme

Formulation development of protein therapeutics using polymeric nanoparticles has found very little success in recent years. Major formulation challenges include rapid denaturation, susceptibility to lose bioactivity in presence of organic solvents and poor encapsulation in polymeric matrix. In the present study, we have prepared hydrophobic ion pairing (HIP) complex of lysozyme, a model protein, using dextran sulfate (DS) as a complexing polymer. We have optimized the process of formation and dissociation of HIP complex between lysozyme and DS. The effect of HIP complexation on enzymatic activity of lysozyme was also studied. Nanoparticles were prepared and characterized using spontaneous emulsion solvent diffusion method. Furthermore, we have also investigated release of lysozyme from nanoparticles along with its enzymatic activity. Results of this study indicate that nanoparticles can sustain the release of lysozyme without compromising its enzymatic activity. HIP complexation using a polymer may also be employed to formulate sustained release dosage forms of other macromolecules with enhanced encapsulation efficiency.

Indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804) is a potent modulator of LPS-stimulated macrophage functions

Indirubin is a deep-red bis-indole isomer of indigo blue, both of which are biologically active ingredients in Danggui Longhui Wan, an ancient Chinese herbal tea mixture used to treat neoplasia and chronic inflammation and to enhance detoxification of xenobiotics. Multiple indirubin derivatives have been synthesized and shown to inhibit cyclin-dependent kinases (CDKs) and glycogen-synthase kinase (GSK-3β) with varying degrees of potency. Several indirubins are also aryl hydrocarbon receptor (AhR) agonists, with AhR-associated activities covering a wide range of potencies, depending on molecular structure. This study examined the effects of indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), a novel indirubin with potent STAT3 inhibitory properties, on basal and LPS-inducible activities in murine RAW264.7 macrophages. Using a focused commercial qRT-PCR array platform (SuperArray®), the effects of E804 on expression of a suite of genes associated with stress and toxicity were determined. Most genes up-regulated by LPS treatment were suppressed by E804; including LPS-induced expression of pro-inflammatory cytokines and receptors, apoptosis control genes, and oxidative stress response genes. Using qRT-PCR as a follow up to the commercial arrays, E804 treatment suppressed LPS-induced COX-2, iNOS, IL-6 and IL-10 gene expression, though the effects on iNOS and COX-2 protein expression were less dramatic. E804 also inhibited LPS-induced secretion of IL-6 and IL-10. Functional endpoints, including iNOS and lysozyme enzymatic activity, phagocytosis of fluorescent latex beads, and intracellular killing of bacteria, were also examined, and in each experimental condition E804 suppressed activities. Collectively, these results indicate that E804 is a potent modulator of pro-inflammatory profiles in LPS-treated macrophages.