Biomedical subjects

Ritu Khurana

Publications and source records attributed to Ritu Khurana.

7 recordsLinked to original sources

PubMed2005

Mechanism of thioflavin T binding to amyloid fibrils.

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.

Amyloid↗

PubMed2005

Clinical aspects of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease of unknown etiology affecting both articular tissues and extraarticular organs. The disease is often progressive and results in pain, stiffness, and swelling of joints culminating in significant morbidity and increased mortality. This chapter discusses the epidemiology, possible etiology, clinical manifestations, diagnostic approach and treatment options of RA.

Journal Article↗

PubMed2004

Unraveling the amyloid associated with human medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is associated with amyloid deposition in the surrounding tissues. MTC-positive tumor thyroid tissues surgically removed from patients were used in our study to extract amyloid. We tested the MTC extracts for the presence of amyloid by measuring fold enhancement of thioflavin T fluorescence. Transmission electron microscopic study and atomic force microscopy of MTC patient extracts revealed typical amyloid fibrils. Matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated full-length calcitonin as the constituent of the MTC amyloid from seven patients. Our results unequivocally demonstrated that full-length calcitonin is the sole constituent of amyloid in MTC.

Amyloid↗

PubMed2003

A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy.

Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.

Amyloid↗

PubMed2003

A model for amyloid fibril formation in immunoglobulin light chains based on comparison of amyloidogenic and benign proteins and specific antibody binding.

In an attempt to understand the mechanism of amyloid fibril formation in light chain amyloidosis, the properties of amyloidogenic (SMA) and benign (LEN) immunoglobulin light chain variable domains (VL) were compared. The conformations of LEN and SMA were measured using secondary and tertiary structural probes over the pH range from 2 and 8. At all pH values, LEN was more stable than SMA. The CD spectra of LEN at pH 2 were comparable to those of SMA at pH 7.5, indicating that the low pH conformation of LEN closely resembles that of SMA at physiological pH. At low pH, a relatively unfolded intermediate conformation is populated for SMA and rapidly leads to amyloid fibrils. The lack of such an intermediate with LEN, is attributed to sequence differences and accounts for the lack of LEN fibrils in the absence of agitation. A kappa IV-specific monoclonal antibody that recognizes the N-terminal of SMA caused unraveling of the fibrils to the protofilaments and was observed to bind to one end of SMA protofilaments by atomic force microscopy. The antibody result indicates that each protofilament is asymmetric with different ends. A model for the formation of fibrils by SMA is proposed.

Amyloid↗

PubMed2002

Elucidation of the molecular mechanism during the early events in immunoglobulin light chain amyloid fibrillation. Evidence for an off-pathway oligomer at acidic pH.

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.

Biopolymers↗

PubMed2002

Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH.

Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivo fibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability, and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 m urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.

Amyloid↗