Several techniques, such as X-ray crystallography, light scattering, fluorescence spectrometry, size exclusion chromatography, atomic force microscopy, and transmission electron microscopy, have been employed in studies of structural intermediates of fibril formation and fibrillar assembly of amyloid proteins. Electron microscopy, with a resolution of approximately 2 nm, offers a useful technique for the ultrastructural characterization of preprotofilaments, protofilaments, and mature fibrils formed during in vitro fibrillogenesis. For contrast enhancement of specimens, negative staining is applied. This chapter outlines the methodology used in laboratories for electron microscopic examination of negatively stained prefibrillar structures and amyloid fibrils. The Aβ-peptide used influences the kinetics of the fibril formation. This chapter uses Aβ1–42 (Bachem, Bubendorf, Switzerland), which forms fibrils within a few hours of incubation at 37°. In order to decelerate the fibril formation enabling it to investigate early intermediates and prefibrillar structures, this chapter has performed the in vitro studies at low concentrations of Aβ1–42 (170 μg/ml), as the kinetics of fibril formation is highly concentration dependent.
"The presence of disordered fibers (observed mainly in controlled pH) could be due to globular protein elements forming complex macro-aggregates. Similar fibers structures have been observed previously [78,79]. In addition, the differences in IBs size probably are due to differences in nucleation and IB growth properties during formation, as well the host proteins that interact with them. "
[Show abstract][Hide abstract] ABSTRACT: Background
Inclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold(DE3) by evaluating two pH culture strategies.ResultsUncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and ß-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented ß-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding.Conclusions
The manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.
"All reagents used in peptide synthesis pentafluorophenol (Spectrochem Pvt. Ltd., Mumbai), N,N-dicyclohexyl carbodiimide (Sigma-Aldrich), Fmoc-AA (Novabiochem), HOBt (Sigma-Aldrich) and TFA (Sigma-Aldrich) were of the purest analytical grade. The Aβ peptide fibrils were formed by solubilizing the peptide in sterile double distilled (DD) water (1 mg/ml) and incubated for 24 h at 37 °C (Nielsen et al., 1999). The protofibrillar form thus obtained was used fresh for the present study. "
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress is one of the hypothesized pathogenic mechanisms for neurodegenerative diseases, including Alzheimer's disease (AD); numerous studies suggest that Abeta is toxic to neurons by free radical mediated mechanism. A constant feature in AD brain is selective neuronal loss, accompanied by dysfunction of several neurotransmitter systems, such as cholinergic, serotoninergic and noradrenergic systems. In the present study, we studied the neuroprotective role of melatonin against amyloid protofibrils and the toxicity of protofibrils on serotoninergic and noradrenergic systems. Mice were divided into four groups (n=8 each), control, Scrambles Abeta(35-25) treated, Abeta(25-35) injected, and melatonin treated. A single dose of Abeta(25-35) (25 microg) was administered to mice via intraperitoneal injection. Melatonin (50 mg/kg body weight) was administered intraperitoneally for 3 days to the Abeta(25-35) injected mice. Control mice received only physiological saline and Scrambles receives Abeta(35-25) single intraperitoneal injection of 25 microg of Abeta(35-25). Our study showed that melatonin significantly reduces reactive oxygen species (ROS) production in the astrocytes, lymphocytes and hepatocytes of Abeta injected mice by increasing the levels of scavenging enzymes, SOD, catalase and GSH when compared to the untreated group. Immunohistochemistry study reveals that melatonin prevents the activation of GFAP in neocortex and transcription factor NF-kappaB in liver and neocortex of Abeta injected mice. It also prevents the elevation of dopamine depletion and its degradation products. Thus, while melatonin may be a potential therapeutic agent in the prevention of oxidative stress associated with Abeta and AD, it can also prevent dopamine turnover induced by Abeta.
Life Sciences 08/2008; 83(3-4):96-102. DOI:10.1016/j.lfs.2008.05.011 · 2.70 Impact Factor
"All reagents used in peptide synthesis pentafluorophenol (purchased from Spectrochem Pvt. Ltd., Mumbai), N,N-dicyclohexyl carbodiimide (Sigma-Aldrich), Fmoc-AA (Novabiochem ), HOBt (Sigma-Aldrich), TFA (Sigma-Aldrich) were of the purest analytical grade. The Ab peptide fibrils were formed by solubilizing the peptide in sterile double distilled (DD) water (1 mg/ml) and incubated 24 h at 378C . The protofibrillar form thus obtained was used fresh for the present study. "
[Show abstract][Hide abstract] ABSTRACT: The major pathological ramification of Alzheimer's disease (AD) is accumulation of beta-Amyloid (Abeta) peptides in the brain. An emerging therapeutic approach for AD is elimination of excessive Ass peptides and preventing its re-accumulation. Immunization is the most effective strategy in removing preexisting cerebral Abetas and improving the cognitive capacity as shown in transgenic mice model of AD. However, active immunization is associated with adverse effect such as encephalitis with perivascular inflammation and hemorrhage. Details about the mechanistic aspects of propagation of these toxic effects are matter of intense enquiry as this knowledge is essential for the understanding of the AD pathophysiology. The present work aimed to study the oxidative vulnerability in the plasma, liver and brain of the inflammation-induced rats subjected to Ass immunization. Induction of inflammation was performed by subcutaneous injection of 0.5?ml of 2% silver nitrate. Our present result shows that the proinflammatory cytokines such as IL1alpha and TNFalpha are increased significantly in the inflammation-induced, Abeta1-42, Abeta25-35 treated groups and inflammation with Abeta25-35 treated group when compared to control, complete Freund's adjuvant and Abeta35-25 treated groups. These increased proinflammatory cytokines concurrently releases significant amount of free radicals in the astrocytes of respected groups. The present result shows that nitric oxide (NO) level was significantly higher (P<0.001) in plasma, liver and brain of the rat subjected to inflammation, Abeta1-42, Abeta25-35 and inflammation with Abeta25-35 injected groups when compared to control. The increased level of lipid peroxides (LPO) (P<0.001) and decreased antioxidant status (P<0.05) were observed in the plasma, liver and brain of inflammation-induced group when compared to control. Our result shows that significant oxidative vulnerability was observed in the inflammation with Ass treated rats when compared to other groups. Based on our results, we suggest that immunization of AD patients with Ass should be done with caution as the increase in Ass could trigger the brain inflammation in uncontrollable level.
Free Radical Research 06/2005; 39(6):603-12. DOI:10.1080/10715760500117373 · 2.98 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.