Investigating the effects of erythrosine B on amyloid fibril formation derived from lysozyme.

Formation of amyloid fibrils has been associated with at least 30 different protein aggregation diseases. The 129-residue polypeptide hen lysozyme, which is structurally homologous to human lysozyme, has been demonstrated to exhibit amyloid fibril-forming propensity in vitro. This study is aimed at exploring the influence of erythrosine B on the in vitro amyloid fibril formation of hen lysozyme at pH 2.0 and 55°C using ThT binding assay, transmission electron microscopy, far-UV circular dichroism absorption spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and synchronous fluorescence study. We found that lysozyme fibrillogenesis was dose-dependently suppressed by erythrosine B. In addition, our far-UV CD and ANS fluorescence data showed that, as compared with the untreated lysozyme control, the α-to-ß transition and exposure of hydrophobic clusters in lysozyme were reduced upon treatment with erythrosine B. Moreover, it could be inferred that the binding of erythrosine B occurred in the vicinity of the tryptophan residues. Finally, molecular docking and molecular dynamics simulations were further employed to gain some insights into the possible binding site(s) and interactions between lysozyme and erythrosine B. We believe the results obtained here may contribute to the development of potential strategies/approaches for the suppression of amyloid fibrillogenesis, which is implicated in amyloid pathology.

Comparative analysis of human γD-crystallin aggregation under physiological and low pH conditions.

Cataract, a major cause of visual impairment worldwide, is the opacification of the eye's crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

Carnosine's effect on amyloid fibril formation and induced cytotoxicity of lysozyme.

Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.

Curcumin's pre-incubation temperature affects its inhibitory potency toward amyloid fibrillation and fibril-induced cytotoxicity of lysozyme.

BACKGROUND: More than twenty-seven human proteins can fold abnormally to form amyloid deposits associated with a number of degenerative diseases. The research reported here is aimed at exploring the connection between curcumin's thermostability and its inhibitory activity toward the amyloid fibrillation of hen egg-white lysozyme (HEWL). METHODS: ThT fluorescence spectroscopy, equilibrium thermal denaturation analysis, and transmission electron microscopy were employed for structural characterization. MTT reduction and flow cytometric analyses were used to examine cell viability. RESULTS AND CONCLUSION: The addition of thermally pre-treated curcumin was found to attenuate the formation of HEWL fibrils and the observed fibrillation inhibition was dependent upon the pre-incubation temperature of curcumin. Our results also demonstrated that the cytotoxic effects of fibrillar HEWL species on PC 12 and SH-SY5Y cells were decreased and negatively correlated with curcumin's thermostability. Next, an enhanced stability of HEWL was perceived upon the addition of curcumin pre-incubated at lower temperature. Furthermore, we found that the alteration of curcumin's thermostability was associated with its inhibitory potency against HEWL fibrillation. GENERAL SIGNIFICANCE: We believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.

Chaperon solvent plug design in size-exclusion chromatography protein refolding process.

Although the chaperon solvent plug was reported as a strategy to reduce aggregation before the column inlet in SEC (size-exclusion chromatography) protein refolding process, the appropriate position at which sample injected and the volume of the chaperon solvent plug have not been elucidated. Therefore, the detail of chaperon solvent plug design was investigated in this work. Our results indicated that, to ensure good performances in the SEC refolding process, the appropriate front and tail volumes of chaperon solvent plug should be slightly larger than the optimal values, which depend on the flow dispersion from the injector to the column inlet. However, with the front volume more than the optimum, it could have an adverse effect on activity recovery but not the mass recovery, while no effect at all if the tail volume exceeded the optimum. Furthermore, it might be economical to replace the eluent (refolding buffer) after the tail of chaperon solvent plug with a cheaper one.

Deactivation of isoamylase and ß-amylase in the agitated reactor under supercritical carbon dioxide.

The current research examines the impact of agitation on deactivation of isoamylase and ß-amylase under supercritical carbon dioxide (SC-CO2). Our experimental results showed that the activity of either enzyme decreased with increasing pressure or speed of agitation. The degree of enzymatic deactivation caused by pressure became more prominent in the presence of agitation, suggesting that the agitation plays an important role in enzymatic deactivation in SC-CO2 environment. Moreover, the enzymatic deactivation behavior associated with agitation and pressure was further quantitatively analyzed using a proposed inactivation kinetic model. Our analysis indicated that isoamylase and ß-amylase exhibited significantly different relationships between the inverse of percentage residual activity and the product of number of revolution per time and time elapsed under pressurized carbon dioxide. We believe that the outcome from this work may provide a better understanding of the effects of agitation and pressure in enzyme deactivation behavior under SC-CO2.

Stability of hen egg white lysozyme during denaturation is enhanced by pretreatment with supercritical carbon dioxide.

Supercritical carbon dioxide (SC-CO(2)) has been successfully employed in a variety of applications due to its numerous advantages. Despite extensive investigations on the relationship between the activity of enzymes treated with supercritical fluids and supercritical operating conditions, there are no experimental studies that have addressed the effects of supercritical pretreatment on enzyme denaturation. In this study, we have explored the impact of SC-CO(2) pretreatment on the activity and stability of hen egg-white lysozyme during its course of denaturation. Our data indicated no noticeable enhancement in enzyme activity and stability in the presence of SC-CO(2) pretreatment for lysozyme samples denatured in 8 M urea at 50 degrees C and pH 6.2. However, SC-CO(2) pretreated lysozyme samples in 0.067 M phosphate buffer containing dithiothreitol (DTT) (0.1 M DTT, pH 6.2, 25 degrees C or 0.01 M DTT, pH 6.2, 50 degrees C) at 2500 psi and 50 degrees C had better residual activity relative to samples that were not pretreated. In addition, when denaturing at 65 degrees C and pH 9.0, the pretreatment in SC-CO(2) at 2500 psi and 50 degrees C resulted in the best stability of lysozyme. The result of this study may provide supporting evidence that supercritical fluids serve as potential media for enhancing the activity of enzymes used in a variety of biochemical applications.

Volatile organic compounds absorption in a cross-flow rotating packed bed.

A cross-flow rotating packed bed (RPB) process was evaluated for its absorption of some volatile organic compounds (VOCs) into water, including isopropyl alcohol, acetone, and ethyl acetate. The experimental results showed that the mass transfer coefficient (KGa) increased with increasing rotational speed, liquid rate, and gas rate, and thus an empirical correlation of KGa was proposed for the cross-flow RPB for the first time. It was found thatthis correlation could reasonably estimate our experimental KGa data as well as those reported in literatures. Although the mass transfer coefficient was lower than that in a countercurrent-flow RPB, a cross-flow RPB is believed to be capable of handling a higher gas rate because of its flow pattern.

Diseases of protein aggregation and the hunt for potential pharmacological agents.

Protein aggregation is a ubiquitous phenomenon significant to all aspects of science. Notably, the formation of protein aggregates is frequently encountered in biochemical research and biopharmaceutical industry. Formation of protein aggregates is generally regarded to be associated with partially folded intermediate species that are susceptible to self-association due to the exposure of hydrophobic core. Evidence supports the concept that the formation of aggregates in vitro is a generic property of proteins. In human etiology, more than 20 different devastating human diseases have been reported to be associated with protein aggregation. Although protein aggregation diseases have been the center of intense research, much remains to be learned regarding the underlying molecular mechanisms. In this review, the general background information on protein aggregation is first provided. Next, we summarize the properties, characteristics and causes of protein aggregates. Finally, from the perspectives of epidemiology, pathogenesis, existing mechanisms, relevant hypotheses, and current as well as potential therapeutic approaches, two examples of protein aggregation diseases, Alzheimer's disease and cataract, are briefly discussed. Importantly, while a variety of molecules have been suggested, the effective therapeutic drugs for curing the diseases involving protein aggregation have yet to be identified. We believe that a better understanding of the mechanisms of protein aggregation process and an extensive investigation into the drug penetration, efficacy, and side effects will certainly aid in developing the successful pharmacological agents for these diseases.

Effect of sample loop dimension on lysozyme refolding in size-exclusion chromatography.

The formation of misfolded protein aggregates, in particular inclusion bodies, has been widely considered as the major hindrance of good yield in refolding processes. To enhance the performance of protein refolding, extensive efforts were directed toward seeking out methods or means to reduce the aggregate production during the refolding process. Since simultaneous refolding and separation can be feasibly achieved within the packing matrices, size-exclusion chromatography (SEC) has been regarded as an efficient buffer exchange method to enhance protein refolding performance As of now, the effect of the process or operating parameters has yet to be thoroughly investigated. The present work is aimed at understanding how aggregate formation, as well as renaturation yield, varied with the diameter or length of sample loop in size-exclusion chromatography refolding process. Our results showed that not much difference was found in the patterns of aggregate formation for the contraction and the control cases. However, the formation of an additional peak was observed in the expansion cases. In addition, the amount of aggregates was not dependent on the sample loop diameter or length, but instead, influenced by injection volume and protein concentration. It was further concluded that a sample with large volume and low concentration was preferable for refolding process. We believe that the outcome from this work may shed light on the development of a more effective strategy for refolding processes.

Alkane utilization by Rhodococcus strain NTU-1 alone and in its natural association with Bacillus fusiformis L-1 and Ochrobactrum sp.

Linear (n-hexadecane) and branched (pristane) alkanes were degraded by a mixed culture isolated from an oil-contaminated field. The degradation was accompanied by formation of biofloccules. The culture was composed of Rhodococcus strain NTU-1, Bacillus fusiformis L-1, and Ochrobactrum sp. Rhodococcus strain NTU-1 carried out the degradation of the alkane via a hydroxylase. Bacillus fusiformis L-1 and Ochrobactrum sp. did not degrade the alkanes but aided the flocculation by forming more rigid bacterial aggregates that enhanced the trapping of alkanes. In batch cultures, transformation and removal of the linear and branched alkanes was achieved within 66 h with more than 95% efficiency.

Molecular dynamics simulations to determine the effect of supercritical carbon dioxide on the structural integrity of hen egg white lysozyme.

In this study, various molecular dynamics simulations were conducted to investigate the effect of supercritical carbon dioxide on the structural integrity of hen egg white lysozyme. The analyses of backbone root-mean-square deviation, radius of gyration, and secondary structure stability all show that supercritical CO(2) exhibits the ability to increase the stability of this protein, probably as a result of the solvent with less polarity, where hydrophobic interactions stabilizing the native structure are weakened and simultaneously the local hydrogen bonds are strengthened, resulting in stabilization of the secondary structures. The hydrophobic cores in the alpha- and beta-domains also play an important role in preventing this protein from thermal unfolding. As supercritical CO(2) has been attractive for biomedical applications because of the advantages of mild critical condition, nonflammability, nontoxity, and the purity of the resulting products, the structural stabilizing effect found in this study strongly suggests that it is possible to increase the thermostability of hen egg white lysozyme by pretreatment with supercritical CO(2), leading to better industrial applications of this protein.

Loofa sponge as a scaffold for the culture of human hepatocyte cell line.

Loofa sponge was investigated as a three-dimensional scaffold for stationary and perfusion culture of human hepatoblastoma cell line C3A/HepG2. In stationary culture, C3A/HepG2 cells in loofa cubes showed higher alpha-fetoprotein and albumin secretion rates than those in polyurethane foam (PU). To use loofa cylinders in a packed-bed reactor, immobilization of C3A/HepG2 cells by recirculating medium at 26 mL/min (superficial velocity = 51.7 cm/min) resulted in a cell loading density of 5.15 x 10(7) cells/cm(3)-loofa. This cell loading density is higher than values reported in the literature for packed-bed reactor intended for bioartificial liver. During 9 days of perfusion culture in the reactor, immobilized C3A/HepG2 showed steady synthesis of albumin with an average synthesis rate at 42.2 microg/10(6) cells/day. These experimental results and observations by SEM suggested that loofa sponge is a suitable scaffold for high-density culture of human hepatocyte cell line and the immobilized cells could express high levels of liver-specific functions.

Surface Equation of State of Nonionic CmEn Surfactants.

The surface equation of state (γ vs Γ/Γref), based on the experimental data from rapid expansion of a pendant bubble, has been used on studying the adsorption kinetics for C12E6 (Pan et al. J. Colloid Interface Sci. 1998, 205, 213) and C14E8 (Lin et al. J. Colloid Interface Sci. 2001, 244, 372). In those studies, the relationship between surface tension γ and relative surface concentration Γ/Γref was applied, and the γ(Γ/Γref) data profiles were found very useful for the determination of the adsorption isotherm and its parameters. In this work, the value of Γref was evaluated from the equilibrium surface tension data γ(C) and the Gibbs adsorption equation. It was found that the dependence of γ vs Γ is more sensitive than γ(Γ/Γref) and brings more help to studying the adsorption kinetics. The role of γ(Γ) data was examined using the equilibrium γ(C) and dynamic γ(t) data of six nonionic polyoxyethylene surfactants CmEn and two alcohols. The γ(Γ) data play an important role on the determination of the adsorption isotherm, model parameters, and controlling mechanism, and also on the evaluation of surfactant diffusivity. Applying γ(Γ) data with γ(ln C) data does bring a much better result on studying the adsorption behavior. According to the γ(Γ) data, (i) the Langmuir isotherm clearly fails to describe the above nonionic surfactants, and (ii) the interaction between the adsorbed surfactant molecules is significant.