Characterization Of Amyloid Fibrils and Protective Effects Of Silibinin

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Amyloid fibrils are abnormal, fibrous protein deposits that grow on the outer membrane of the cells. They are insoluble and do not function to provide structural support or motility in humans. Amyloids are known to show major impact on diseases like Alzheimer’s and type II diabetes which progress over a period of time and are associated with high mortality (1).

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There are no effective treatments known for amyloid-related diseases, therefore, searching for compounds that can effectively inhibit the formation of amyloid fibrils and/or disaggregate the preformed amyloid fibrils will act as a more promising treatment (2, 3). Silibinin are extracted from the medicinal plant Silybum marianum, also known as milk thistle, and have traditionally been used for the treatment of liver diseases (4). In a research, it was shown that silibinin could interrupt the complex structure of the amyloid protein, and transform the fibrils into shapeless aggregates hence decreasing its effects on the amyloid-related diseases (5). However, there are various forms of amyloid-fibril proteins. This paper focuses on amyloid-fibril formation and their characterization in various different amyloid-fibril proteins.

Further, this paper will explore cytotoxicity induced by amyloid fibrils affecting patients who take insulin. The term ‘amyloid’ was coined initially by Schleiden and then by Virchow in the mid-19th century to describe the iodine stained deposits seen in the liver at an autopsy. Initially, the deposits were thought to be high in carbohydrate until their high nitrogen content was later established. However, the inaccurate name persisted despite the discovery of its highly proteinaceous composition (7). Through microscopic studies and transmission electron micrographs, it was confirmed that amyloid carried a fibrillar or thread-like structure. Further advances in the biomedical and biophysical arena helped to isolate amyloid fibrils from the tissues and use X-ray fiber diffraction to exhibit the cross-?? structure (8, 9). Amyloid fibrils are highly stable and insoluble which makes them very useful in a large number of naturally occurring bionanotechnology. However, fibrils can also be destructive as they have the ability to accumulate in the tissue and form basis of diseases or aggravate a given disease (1). In order to understand more about amyloid fibrils, it was necessary to isolate them without completely dissociating them. The highly stable structure of amyloid fibrils made it hard to isolate them from the tissues without affecting its structural integrity of the fibrils.

Therefore it became necessary to devise an effective method of isolation. Cohen and Calkins in their paper, The Isolation of amyloid fibrils and a study of the effect of collagenase and hyaluronidase, provided an effective method to isolate the amyloid fibrils without destroying their structural integrity. Hepatic tissues with excessive amyloid fibrils were extracted from post-mortem patients and was extracted using subsequent centrifugation and washing. The extracted fibrils and normal liver tissue as a control were then treated with the enzymes collagenase and hyaluronidase and then centrifuged. On observing it in the electron microscope, it was noted that only the amyloid fibril rich liver tissues showed fibrils, hence a new technique to isolate fibrils was discovered (9). Cohen and Calkin’s work was also one of the primary works to identify that fibril, a major component of amyloid, is not a collagen. It was also identified that fibrils do not contain high amounts of hyaluronic acid, making it less soluble in water (1,9). In order to identify if collagen is the primary component of the fibrils, the fibrils were treated with a buffer and collagenase solution as the control and experimental reactions. The collagenase based reaction was incubated at different time periods to identify if there as more growth in the collagenase based solutions.

The quantity of the fibrils was found to be roughly the same in the control and the experimental preparations at different time intervals. Ineffectiveness of collagenase indicated that there was no collagen present in the fibrils as in the presence of collagenase, the fibrils would have grown if they had collagen in them (9). Isolation of amyloid fibrils sparked a growth in the research based on amyloid fibrils and the diseases associated with amyloid fibrils. In a study, Westermark isolated amyloid from a lymph node of a medullary thyroid carcinoma, using the method devised by Cohen and Calkin. It was found in this study that amyloid fibrils carried major protein unit. The presence of a major subunit brought about a major advancement in understanding the structure of amyloid fibrils which later helped in finding an inhibition mechanism for amyloid formation, that affects diseases like Alzheimer’s and diabetes type I (9, 10, 11). The structure of the fibril isolated from the thyroid carcinoma was studied by isolating a tumor tissue from a patient having medullary thyroid carcinoma with metastases. The amyloid was isolated using collagenase enzyme, as shown by Cohen and Calkins. After the centrifugation in the presence of required buffers, it was run using an SDS page to separate the different protein components. The acquired protein was then purified using gel filtration and observed under an electron microscope. The amyloid fibrils were observed in the form of small clumps.

On performing spectrophotometry, it was confirmed that no peaks were shown in the range where amyloid fibril would normally be observed. Knowledge of the structure of amyloid fibrils helps in understanding the process of pathology of the amyloidoses and provides a structure of rational design for drugs to inhibit or reverse amyloid formation (11). This can be beneficial in various diseases like diabetes and Alzheimer’s. It has been suggested that nearly all proteins have the ability to form amyloid under certain conditions, which has implications for the understanding of protein folding (12). Amyloid precursor proteins do not share a common size, sequence or secondary structure, yet the mature fibrils appear to share similar highly organized multimolecular morphology and mechanisms of toxicity (13). This indicated that there is a possible connection in the formation of different types of amyloid fibrils. Sletten et al in their study characterizing amyloid fibril proteins further elaborated that human amyloid fibrils contain two different components: one soluble and the more heterogeneous component. The soluble component is called the P component and is immunologically homogeneous meaning they are made of same kind of proteins. The other component, the fibril, is more heterogeneous meaning that it is made of different kind of proteins. Amyloid fibrils were first acquired from lymph nodes of the medullary carcinoma of the thyroid and isolated using the Cohen and Calkin’s method of enzyme degradation.

Antisera against amyloid proteins prepared and treated to the fibrils. Finally gel filtration and SDS page were conducted in order to look at the various band indicating proteins of different sizes and charges (14). As discussed earlier, two different components were found. It was also observed that the fibrils mainly consisted of a low molecular weight protein which was immunologically distinct and did not react with various antisera against known amyloid fibril proteins (15). In a study it was shown that the influence of the number of non-fibrillar or the P-component on amyloid related diseases is well known. However the authors noted that non-fibrillar proteins increase protein strength and density of the local interactions of fibrils, causing them to forma compact, and localized structure, as seen in different amyloid related diseases. Therefore, the soluble component, or the P-component play an important role in forming aggregation state and solubility of the amyloid fibrils, hence affecting amyloidoses or amyloid deposits (19) Amyloid deposits have varied origins and chemical compositions, however they still have common systematic forms. The common systemic forms as seen in Sletten et al., are related to the serum amyloid protein or immunoglobulin chains but in amyloidosis the fibril proteins may contain hormone-like peptides such as procalcitonin in medullary carcinoma of thyroid and islet polypeptide in pancreatic islets, especially in Type 2 diabetes (15, 16, 17).

Insulin, a polypeptide, has long been known to be capable of conversion in vitro to a fibrillar, amyloid-like, form (16). Fibrillary insulin has recently been demonstrated clinically significant localised amyloidosis in microscopic quantities around the needle tip after prolonged subcutaneous infusions of insulin in rats, and in a human diabetic subject (18). In a study done in 1988, major amyloid fibril protein was extracted and, by means of its amino acid composition and amino acid sequence, it was shown to contain intact insulin molecules (15). Diche et al. in their study conducted in 1988 described the amyloid deposits occurring in an insulin-dependent diabetic patient and give the analytical finding of extracted amyloid fibril protein. A young man with type 1 diabetes mellitus developed a localized amyloidosis at the sites of the injections of insulin. This amyloid fibril was extracted and histopathology was conducted on the tissues. This was followed by immunohistochemistry, by staining proinsulin and an antiserum to insulin. The tissue was also homogenized to obtain amyloid fibrils and purify them (15). It was found that amyloids form by partial proteolysis of larger precursor molecules, which cases of localized amyloidosis, as seen in the diabetic patient, is expressed close to the site of deposition. The lower molecular weight fragment molecules spontaneously polymerize into fibrils. Amyloid P-component was also demonstrable in the deposits, as it has been in every other amyloid (15).

Katebi et al. look at the potential of silibinin to interact and inhibit the amyloid formation in bovine insulin (insulin derived from a cow). It looks at the ability of insulin to attach to silibinin. It further looks at the protective effects of silibinin in the cell against the toxicity caused by the amyloid fibrils on neuroblastoma cells– cancer formed in early forms of nerve cells (3, 6). It looks at the ability of insulin to attach to silibinin. It further looks at the protective effects of silibinin in the cell against the toxicity caused by the amyloid fibrils on neuroblastoma cells– cancer formed in early forms of nerve cells (3, 6). The specific effect of silibinin on the fibrillation of bovine insulin was studied by using specific methods of amyloid detection using color assays. These assays are methods that use a dye to color the amyloid fibrils and observe the changes in it using a highly efficient microscope. The assays help in quantifying the change in the amyloid fibril formation under various conditions such as the different concentration of silibinin, or temperature difference (20, 21). It was found that higher doses of silibinin caused the inhibition of the amyloid fibril formation, in a given time. It was measured that the time taken for the formation of the first phase of the new structure or interaction increased from 2 hours to 4 hours in the presence of silibinin.

This indicated that silibinin interferes in the early formation of the insulin complex, which increases the reliability of silibinin as an effective treatment. Impact of silibinin on the structural changes cause in insulin due to amyloid fibrils was studies using color assays. It was concluded that the presence of silibinin lowers the intensity of the color, which indicates that silibinin has inhibiting effects on structural changes in insulin due to the amyloid formation (3, 22). In the second part of the study, the protective effects of silibinin on insulin amyloid fibril that causes toxicity in cancer cells in nerves was analyzed. This was analyzed using an assay that acts a sensitive and reliable indicator of the cell metabolism. This assay analyzes the shift of a yellow based dye to purple based on the activity of a mitochondrial enzyme that helps in the removal of hydrogen from the cells. The assay was conducted as a cell viability test for the effect of silibinin on the amyloid. It was concluded that cells exposed to silibinin at various concentrations did not show any evidence of toxicity in a 24 hour period (3, 23).

The study gives important insights into the mechanism of amyloid fibril-induced neuronal cell death and the action of silibinin. It also emphasizes the potential application of silibinin to prevent the treatment of amyloid-related diseases like type II diabetes and Alzheimer’s. Amyloid fibrils are extensively known for its affect on major diseases like Alzheimer’s and diabetes, hence it is very important to understand their structure, and formation. Structure and formation not only provide a way to derive methods to stop their growth. With the help of isolation technique devised by Cohen and Calkins, scientist were able to further study structure and formation of amyloid fibrils. This further enabled them to understand in what ways do fibrils affect diabetes. Such studies help not only to provide a way for other scientists to replicate what has been already done but also pursue research t a higher level. By knowing the structure and characterization of the amyloid fibrils, Katebi et al. were able to understand the protective effects of silibinin on insulin amyloid fibrils.

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