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Endocrinology Vol. 145, No. 12 5465-5470
Copyright © 2004 by The Endocrine Society

BRIEF COMMUNICATION

Unraveling the Amyloid Associated with Human Medullary Thyroid Carcinoma

Ritu Khurana, Amit Agarwal, Virendra K. Bajpai, Nidhi Verma, Ashok K. Sharma, Ram P. Gupta and Kunnath P. Madhusudan

Molecular and Structural Biology Division (R.K., N.V.), Electron Microscopy Unit (V.K.B.), Sophisticated Analytical Instrument Facility (K.P.M.), Central Drug Research Institute, Chattar Manzil Building, Lucknow 226001; and Department of Endocrine Surgery (A.A.), Sanjay Gandhi Post Graduate Institute, Lucknow 226014; and Central Electronics and Engineering Research Institute (A.K.S., R.P.G.), Pilani 333031, India

Address all correspondence and requests for reprints to: Dr. Ritu Khurana, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. E-mail: rkhurana{at}ccmb.res.in.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AMYLOID IS A SELF-AGGREGATED protein/polypeptide forming long insoluble fibrils that are extracellularly deposited (1) in many protein-folding disorders such as prion disease, Alzheimer’s, and various systemic amyloidosis due to overexpression, proteolytic digestion, or mutations in protein. In addition to the primary and secondary amyloidosis that are associated with Ig light chains and serum amyloid A proteins, respectively, there are many other systemic amyloidosis associated with transthyretin, gelsolin, cystatin C, apolipoprotein A1, and lysozyme, etc. In addition to typical amyloid diseases, many common diseases such as type II diabetes, myocardial infarctions, and cancers are found to be associated with amyloid involving different polypeptides. Among tumor-associated amyloids, many nonneoplastic and malignant tumors of the breast and squamous cell carcinoma of the lungs are associated with amyloid (2). Several endocrine tumors have been demonstrated to be associated with amyloid wherein tumors secreting calcitonin, insulin, and GH very often produce amyloid, and those secreting gastrin, ACTH, and prolactin also sometimes demonstrate amyloid (3).

Medullary thyroid carcinoma (MTC) (4, 5) has been demonstrated to be associated with amyloid fibrils deposited in thyroid and adjacent enlarged lymph nodes (6). MTC is caused by transformation of the parafollicular C cells (7) and several thousand-fold increase in the blood calcitonin levels also resulting in amyloid deposition (8, 9). MTC is a feature of multiple endocrine neoplasia type 2a, which occurs along with pheocytochromas or adrenal medullary hyperplasia where many endocrine glands of a patient such as thyroid, adrenals and parathyroid are affected simultaneously and is a prime example of hereditary malignancy (10, 11). Vassar and Culling (12) demonstrated the presence of amyloid in the stroma and colloid of thyroidal acini within and adjacent to the MTC.

In this manuscript, we report identification of the component of amyloid from human MTC. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) analysis has been used after denaturation of amyloid to completely unravel the MTC amyloid. Association of calcitonin with MTC has been confirmed by immunoelectron microscopic studies using antibodies against calcitonin conjugated to colloidal gold particles (13). Sletton et al. (14) suggested involvement of an alternately processed prohormone of calcitonin in the amyloid in MTC. Our data directly test the component of MTC after denaturation of amyloid and MALDI-TOFMS analysis and challenges the suggestion made by Sletton et al. (14).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
MTC tissues
MTC tissues from patients were frozen soon after surgery and stored in –70 C freezer at Sanjay Gandhi Post Graduate Institute (Lucknow, India). No ethical committee consent was required for this study in our institutions. The tissues were transported in liquid nitrogen cryo canister.

Amyloid extraction from thyroid tissues
Extraction of amyloid was performed using the method described by Pras et al. (15), and the presence of amyloid in the extracts was monitored by enhanced thioflavin T fluorescence emission (16). Tissue (0.2–0.5 g) was thawed at 37 C for 20 min and then cut into small pieces with the help of a scalpel. The tissue pieces were washed three times for 20 min each with Tris-buffered saline (pH 7.4) in a 50-ml tube, and the tube was kept in a tube rotator to remove trapped blood cells. The buffer was decanted carefully. Tissue was then transferred into a glass homogenizer, and 20 ml fresh cold Tris-buffered saline (pH 7.4) were added and homogenized using a mechanized Teflon plunger in ice bath until no visible tissue pieces were observed. The homogenate was transferred to a centrifuge tube and centrifuged at 20,000 x g for 30 min at 5 C, and supernatant was separated. This process was repeated six times or until the absorbance of supernatant measured at 280 nm was less than 0.1. The pellet was then stored at 4 C in 2 ml triple-distilled water. Water extraction was performed five times by homogenization in 5 ml cold water and centrifugation at 10,000 x g for 1 h. The supernatant was saved in screw cap glass vials at 4 C.

Amyloid formation from synthetic calcitonin peptides
Synthetic human and salmon calcitonin peptides were purchased from Calbiochem (San Diego, CA) and set up to form amyloid under varying conditions in small screw cap glass vials and incubated at either 28 C and 37 C for human and salmon calcitonin peptides, respectively. Amyloid formation was monitored by fold enhancement of thioflavin T fluorescence emission at 482 nm upon excitation at 450 nm. We used the water extracts directly in our study without the ultracentrifugation step to concentrate the fibrils because we were unable to pellet all the amyloid fibrils in the extract by ultracentrifugation as described by Pras et al. (15).

Thioflavin T fluorescence measurement
A stock thioflavin T solution of 1 mM was prepared and concentration measured using extinction coefficient of 266, 20 M–1cm–1 at 416 nm. For both the Tris-buffered saline supernatants and water extracts, 20 µM thioflavin T (final concentration) was added to 0.5 ml supernatant, and fluorescence emission spectrum between 465 and 565 nm was measured upon excitation at 450 nm with excitation slit width at 5 nm and emission slit width at 10 nm and scan speed of 100 nm/min using PerkinElmer (Foster City, CA) spectrofluorimeter. Fluorescence emission at 482 nm was divided by the 20 mM thioflavin T fluorescence emission at 482 nm in TBS or water to calculate fold enhancement as the case may be.

Detection of extracted amyloid using enhanced thioflavin T fluorescence
Extraction of the amyloid from MTC tissues was performed using the method described by Pras et al. (15). Amyloid in the buffer and water extracts was tested by the fluorescence emission enhancement of thioflavin T dye (see Materials and Methods). Thioflavin T dye was initially identified by Vassar and Culling (16) to be very specific for amyloid in tissue sections, later Naiki et al. (17) and LeVine (18) designed a spectrofluorimetric assay for amyloid detection in solution using fluorescence excitation at 450 nm and emission at 482 nm and monitored enhanced fluorescence emission in the presence of amyloid.

Atomic force microscopy (AFM)
The MTC extracts were examined using AFM performed in contact mode using Nanoscope II (Digital Instruments, Santa Barbara, CA). Imaging was done in air using 0.7 µm AFM head. The sample is placed on xyz-piezo-translator and scanned by using a sharp diamond tip mounted on a gold-coated 200 µm triangular Si3N4 microfabricated cantilever (force constant = 0.6 N/m). The force between the tip and sample usually ranges from 10–7 to 10–9 N. Images consists of 400 scan of 400 pixels each. Typical image acquisition time was 150–200 sec/scan. Samples were prepared by placing a drop of fibril solution (~0.5 mg/ml) on freshly cleaved mica and dried. Samples containing buffers and salts were dried on mica and washed with water to remove salts.

Transmission electron microscopy (TEM)
A drop of aqueous solution of amyloid fibrils extracted from tissues was placed on the Parafilm (Greenwich, CT), and polystyrene-coated copper grid was floated film side down for 1–3 min and excess solution was absorbed using Whatman (Kent, UK) filter paper. Subsequently, sample contrast was enhanced by placing the grid film side down on a drop of the 1–2% uranyl acetate solution for 1–3 min. The grid was examined using FEI-Philips Technai-12 (Eindoven, The Netherlands) at 120 kV filament.

Amyloid sample preparation for MALDI and PAGE analysis
Amyloid samples were denatured in 7 M guanidine hydrochloride with 0.1% trifluoroacetic acid overnight. The denatured amyloid samples were bound to C-18 Zip-tip (Millipore, Bedford, MA) for concentrating the sample and removal of denaturant by pipetting every 10-µl sample five times to ensure complete binding. After binding of the sample, the C-18 Zip-tip was extensively washed with 0.1% trifluoroacetic acid 50 times for complete removal of all denaturant molecules. The sample was then eluted using 75% acetonitrile and 0.1% trifluoroacetic acid just before analysis using MALDI-TOF and SDS-PAGE.

SDS-PAGE
High-density (20% polyacrylamide gel containing 30% polyethylene glycol) polyacrylamide gel SDS-PAGE was performed using Phast gel system (Amersham Biosciences, Uppsala, Sweden). The samples of MTC amyloid were prepared by denaturing the amyloid using 7 M GdnHCl and using the C-18 zip-tips to remove denaturant molecules eluting the protein and adding sodium dodecyl sulfate (SDS)-sample buffer before loading the gel. The gels were either silver stained or stained with Coomassie brilliant blue.

MALDI-TOFMS
One microliter of the sample eluted from C-18 Zip-Tip with75% acetonitrile and 0.1% trifluoroacetic acid was mixed with 1 µl of {alpha}-cyano-4-hydroxycinnamic acid (1:1 methanol:acetonitrile) or sinapinic acid (in 60:40 0.1% TFA in water: acetonitrile) matrices. Analyses by MALDI-TOFMS were performed in the positive ion mode on a Micromass TofSpec 2E mass spectrometer (Micromass, Manchester, UK) equipped with a 337 nm nitrogen laser (4-nsec pulse) and time lag focusing. Analyses were carried out in the mass range 500–10000 with an accelerating voltage of 20 kV in both reflectron and linear modes. The data were accumulated over 40–50 laser shots. The instrument was calibrated with a mixture of angiotensin I, renin substrate, and ACTH (18–39 clip) mixed with appropriate matrix.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selection of MTC tissues
We used seven MTC tissues that were surgically removed by performing complete thyroidectomy in the endocrine surgery department in the Sanjay Gandhi Post-Graduate Research Institute as a part of the clinical procedure for patients. Pathological tests such as Congo red birefringence and immunohistochemistry using antibodies against calcitonin were performed on formalinized tissue sections to confirm that the tumor was MTC. All seven cases are listed in Table 1Go with their lab numbers and the results of diagnostic pathological tests. For one patient, a portion of thyroid tissue that was not affected by tumor was included in our study as normal control and is also listed in Table 1Go.


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  		TABLE 1. A list of seven MTC thyroid tissues and one normal tissue used in our study

 
Detection of amyloid in tissue extracts by thioflavin T dye
Buffer extracts did not show significant thioflavin T fluorescence fold enhancement in all tissue extracts. Varying amounts of amyloid was detected in the water extracts in most patients as judged by fold enhancement in thioflavin T fluorescence as summarized in Table 1Go. At least a 3-fold increase in thioflavin T fluorescence in presence of amyloid extracts was considered significant. Thioflavin T fluorescence emission fold enhancement varied from 5-fold to greater than 50-fold in different MTC patient water extracts, whereas it was less than 2-fold for normal thyroid extract (Table 1Go). A comparison of amyloid extracted from normal area of thyroid from a patient did not reveal amyloid, by either histopathological observations or by fluorescence emission fold enhancement of thioflavin T, or electron microscopy of water extracts (Table 1Go). One patient, 6 MTC, who was not conclusively proved MTC by histopathology, demonstrated significant amyloid by thioflavin T fold enhancement of the water extract.

In vitro amyloid formation from synthetic calcitonin peptides
Human calcitonin peptide readily formed amyloid with in a few hours when a 5 mg/ml solution was incubated at 28 C in PBS. A 5 mg/ml solution of salmon calcitonin peptide was incubated for 3–4 wk at 37 C before fibrils were observed by enhanced thioflavin T fluorescence or electron microscopy. In vitro amyloid formation from synthetic human and salmon calcitonin peptides has also been shown earlier by various groups (19, 20, 21).

Ultrastructural analysis of amyloid
TEM of amyloid fibrils extracted from tissues was performed by negative staining using uranyl acetate stain on polystyrene-coated copper grids and typical fibrils were observed in six MTC extracts tested and not in the normal thyroid extract (Table 1Go). The diameters of MTC extracted amyloid fibrils ranged from 2–8 nm. The variation in the diameters of fibrils arises due to the distance measurement on manually drawn lines on the computer monitor (using the software provided by FEI-Philips) across the fibrils on TEM images introducing various error factors. Figure 1Go, A and B, shows fibrils from two different patients 4 MTC and 1 MTC, respectively. Synthetic calcitonin fibrils were observed as negatively stained amyloid fibrils of 4–8 nm diameter by TEM as shown in Fig. 1CGo for human calcitonin fibrils and Fig. 1DGo for salmon calcitonin amyloid. The amyloid fibrils shown in Fig. 1Go, A and B, are characteristic intertwined mature amyloid fibrils also observed for several other protein and peptide amyloid (22) that we believe are formed from thinner protofilaments and protofibrils intertwining together (23, 24, 25).



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  		FIG. 1. TEM images of MTC and synthetic calcitonin amyloid. TEM images of MTC extracted amyloid fibrils of patient 4 MTC (A) and 1 MTC (B), human calcitonin amyloid (C), and salmon calcitonin amyloid (D) observed after loading the samples on polystyrene-coated copper grids and uranyl acetate staining.

 
AFM analysis of patient amyloid samples dried on freshly cleaved mica was also performed for all seven patient-extracted MTC samples, and all patient extracts revealed amyloid fibrils (Table 1Go). AFM images show amyloid fibrils of MTC water extracts placed on flat mica surface from two different patients 3 MTC and 1 MTC are shown in Fig. 2Go, A and B, respectively. The dark background is the mica surface and the lighter colored image is the fibril. The color grade in the z-axis is the height of the fibril scanned above the mica surface. The heights of MTC amyloid fibrils ranged from 2–4 nm corresponding to the diameter of amyloid fibrils. The height measured in AFM is translated from the z direction piezo movement and provides an accurate measurement of the diameter of amyloid fibrils compared with the diameters measured by electron microscopy. AFM images of MTC amyloid fibrils after high temperature (Fig. 2CGo) and synthetic human calcitonin amyloid after proteinase K treatment (Fig. 2DGo) showed no detectable morphological differences compared with the control fibrils (Fig. 2Go, A and B), indicating that both these treatments do not denature or unfold the amyloid conformation.



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  		FIG. 2. AFM images of MTC amyloid. The AFM pictures show amyloid fibrils imaged on the flat surface of mica. The dark background is the mica surface, and the lighter-colored image is the fibril. The color grade is the height of the fibril above the surface of the mica. The fibril height measured by AFM very accurately is indicative of the diameter of amyloid fibrils. Contact mode AFM images of 3 MTC (A) and 1 MTC (B) patient MTC amyloid with height measured between 2–4 nm. Patient 4 MTC treated to 80 C temperature (C) and synthetic human calcitonin treated with Proteinase K at 55 C (D), demonstrating no changes in the morphology of amyloid fibrils both by high temperature and proteinase K.

 
Because 6 MTC patient water extract demonstrated amyloid fibrils by thioflavin T fluorescence and TEM and AFM (Table 1Go), we included it in our study, believing that this patient may well be MTC at a molecular level even though it was not conclusively proved MTC by histopathology.

SDS-PAGE analysis of MTC extracted amyloid
Analyses of the MTC amyloid extract and synthetic calcitonin amyloid was performed on 20% polyacrylamide high-density gels obtained from Amersham Biosciences and especially designed to separate peptides ranging from 1–50 kDa. For synthetic calcitonin amyloid, we did not observe any band until the MTC amyloid was denatured. However, upon denaturation of synthetic calcitonin amyloid with 7 M GdnHCl and denaturant removed using C-18 Zip-tips we observed higher oligomeric bands corresponding to 6-, 12-, and 15-kDa bands under varying concentrations of SDS in the sample buffer (Fig. 3AGo, lane 3, shows a 15-kDa band). At higher concentrations of freshly prepared synthetic human calcitonin, we also observed a major band at 3 kDa and a 15-kDa band on SDS-PAGE with 2% SDS in the sample buffer (Fig. 3BGo, lane 2). All of this led us to conclude that disaggregated calcitonin monomer from MTC extract, and freshly prepared synthetic calcitonin can form SDS stable oligomers in the presence of SDS that shows up as oligomeric bands in 20% high-density SDS-PAGE. SDS-PAGE did not show bands for all the patient samples. Because SDS-PAGE gels bands were not always consistent, we decided to perform mass spectrometric analysis on these samples as native gels or other methods would not allow us determine the exact size of the constituent polypeptide of the MTC amyloid.



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  		FIG. 3. SDS-PAGE analysis of MTC extracted amyloid. A, Peptide markers—lane 1, salmon calcitonin; lane 2, MTC extracted amyloid from 4 MTC patient in lane 3; lane 4, low molecular weight protein markers and insulin on 20% high-density SDS-PAGE phast gel and stained with Coomassie brilliant blue. B, Peptide markers (lane 1), synthetic human calcitonin in 2% SDS sample buffer (lane 2) and was silver stained.

 
Calcitonin peaks observed in the MTC amyloid extracts using MALDI-TOFMS
MALDI-TOFMS analysis of MTC amyloid was performed by first denaturing the amyloid in 7 M guanidine hydrochloride followed by binding of peptides/proteins to C-18 zip-tips and removal of denaturant molecules. A major peak of mass 3419, corresponding to the mass of calcitonin, was observed in all seven patient MTC extracts and was not detected in the normal thyroid extract (Table 1Go). A major calcitonin peak and its dimer peak from three patients extracts (5 MTC, 4 MTC, and 7 MTC) were observed by linear mode upon mixing with {alpha}-matrix are shown in Fig. 4Go, A–C, respectively. The data observed for remaining patients had counts less than hundred for calcitonin peak and no calcitonin peak was observed for the normal thyroid tissue extract (Table 1Go). No calcitonin peaks were observed in any of the extracts until the amyloid was denatured indicating that the denaturation step was absolutely essential for detection of the component peaks from amyloid. Even when we used half a gram of tissue, we sometimes barely got enough material to perform MALDI analyses and ultrastructural studies.



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  		FIG. 4. MALDI-TOF spectra of denatured amyloid from MTC tissues. MALDI-TOF spectra of MTC extracts from three representative patients 5 MTC (A), 4 MTC (B), and 7 MTC (C) showing major peaks of mass 3420 (corresponding to monomeric calcitonin MH+) and a minor peak of 6839 (corresponding to dimeric calcitonin 2MH+) using linear mode with {alpha}-matrix after denaturation of amyloid in 7 M guanidine and removal of denaturant using C-18 Zip-tips.

 
We also mixed the MTC extracts with sinapinic acid matrix and explored the possibility of observing serum amyloid P protein (26) or other high molecular weight proteins possibly associated with amyloid, but were unable to detect any significant high molecular weight peaks. In addition no significant high molecular weight bands were observed in 8–25% SDS-PAGE gels (data not shown). This led us to conclude that amyloid-associated proteins are possibly separated during the extraction procedure and maybe found in salt extracts as suggested by Pras et al. (15) and demonstrated clearly by Sletton et al. (14) for MTC tissue extracts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Amyloid extraction procedure that we used to extract amyloid from MTC was described by Pras et al. in 1968 and has been used by many groups since then successfully. Amyloid is intact as shown by both the TEM analysis and AFM images in Figs. 1Go and 2Go, respectively. Westermark (27) also used the same method with slight modifications.

Our method of detecting component peaks of amyloid extracted from tissues by MALDI-TOFMS followed by denaturing the amyloid (in 7 M guanidine hydrochloride) and removal of denaturants using C-18 zip-tips has been reported here for the first time. This method can be applied to amyloid extracted from other tissues as well and is far superior to using SDS-PAGE because amyloid may not denature completely in SDS and also forms SDS stable oligomers. Observation of calcitonin peak in all patient amyloid extracts after denaturation and MALDI-TOFMS analysis led us to conclude that calcitonin forms amyloid in MTC. It would be much better to perform MS/MS analysis to say with complete authority that calcitonin is involved in amyloid formation in MTC tissue, but our attempts to perform MS/MS analysis did not give us satisfactory results most likely due to insufficient sample available. Because MALDI-TOFMS analysis gave the exact calcitonin size peaks for all seven patients in our hands and many other groups including Bulter and Khan (13) have clearly demonstrated that amyloid associated with MTC is recognized by the antibodies against calcitonin hormone, we conclude that it is calcitonin that is involved in amyloid formation in MTC.

Scrapie prion conformation is also insoluble in detergents (28) but can be denatured by guanidine hydrochloride (29). This matches with our results for calcitonin amyloid, which is not soluble in SDS but can be denatured in guanidine hydrochloride. It is possible that Sletton et al. (14) observed a band of approximately 6000 Daltons from the MTC extract on SDS-PAGE corresponding to a SDS stable dimer of calcitonin. Because our results also demonstrated a major 3.42-kDa peak with MALDI and either 6-, 12-, or 15-kDa bands on SDS-PAGE, we concluded that it is the full-length calcitonin that is the constituent of amyloid in MTC that has the tendency to form SDS stable oligomers.

Sletton et al. (14) concluded that it is the alternately processed prohormone of calcitonin that is involved in amyloid formation in MTC (14, 27). The results in the study conducted by Sletton et al. (14) were based on the observations of a major band corresponding to approximately 6000 Daltons from the extracted MTC amyloid on the SDS-PAGE. The actual prohormone of calcitonin is 9.9 kDa, and the alternately processed prohormone of calcitonin implicated in amyloid formation in MTC by Sletton et al. is 5.68 kDa. Because none of these are secreted extracellularly, they are very unlikely to be involved in the amyloid formation in MTC. In addition, Sletton et al. (14) attempted N-terminal sequencing of the MTC extracted amyloid, but no N-terminal sequence was detected most likely due to the N-terminal disulfide loop formed between cysteine 1 and cysteine 7 in calcitonin hormone. The amino acid composition analysis performed by Sletton et al. (14) of MTC amyloid was interpreted as a 53-amino acid polypeptide that was suggested to be an alternately processed prohormone of calcitonin of size 5680 Daltons. Because the actual prohormone of calcitonin has a size of 9908 Daltons. An alternate processing would require a different mechanism for processing of the calcitonin hormone in the transformed C cells that has not been reported so far. The data obtained by cyanogen bromide cleavage followed by N-terminal sequencing of the MTC amyloid by Sletton et al. (14) revealed region 9–19 amino acids of human calcitonin indicating involvement of calcitonin in MTC, but the authors (14) suggested an alternately processed prohormone of calcitonin.

Clear calcitonin peaks obtained by us using MALDI-TOFMS after denaturation of amyloid from MTC tissue extracts led us to conclude that it is the full-length calcitonin hormone and not an alternately processed prohormone of calcitonin that forms amyloid in MTC.


   Acknowledgments
 
We acknowledge the help and support provided by Dr. Uma Roy throughout the writing and completion of this work. Mr. Rit Vatsayan and Dr. Uma Roy from the Central Drug Research Institute (Lucknow, India) also helped with silver staining of SDS-PAGE. Ms. Madhuli and Ms. Abha are fondly acknowledged for their help in the electron microscopy laboratory at Central Drug Research Institute, Lucknow, India.


   Footnotes
 
The financial support for this work came from Central Drug Research Institute (CDRI) and Department of Science and Technology, New Delhi, India.

CDRI Communication No. 6492.

Abbreviations: AFM, Atomic force microscopy; MTC, medullary thyroid carcinoma; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometry; SDS, sodium dodecyl sulfate; TEM, transmission electron microscopy.

Received June 22, 2004.

Accepted for publication September 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sipe JD 1992 Amyloidosis. Annu Rev Biochem 64:947–975[CrossRef]
  2. Halliday BE, Silverman JF, Finley JL 1998 Fine-needle aspiration cytology of amyloid associated with non-neoplastic and malignant lesions. Diagn Cytopathol 18:270–275[CrossRef][Medline]
  3. Westermark P, Grimelius L, Polak JM, Larson L-I, van Noorden S, Wilander E, Pearse AGE 1977 Amyloid in polypeptide hormone-producing tumors. Lab Invest 37:212–215[Medline]
  4. Hazard JB, Hawk WA, Crille G 1959 Medullary (solid) carcinoma of the thyroid—a clinicopathologic entity. J Clin Endocrinol 19:152–161
  5. Williams ED 1966 Histogenesis of medullary carcinoma of the thyroid. J Clin Pathol 19:114–118[Abstract/Free Full Text]
  6. Scopsi L, Sampietro G, Boracchi P, DelBo R, Gullo M, Placucci M, Pilotti S 1996 Multivariate analysis of prognostic factors in sporadic medullary carcinoma of the thyroid: a retrospective study of 109 consecutive patients. Cancer 78:2173–2183[CrossRef][Medline]
  7. Williams ED, Brown CL, Doniach I 1966 Pathological and clinical findings in a series of 67 cases of medullary carcinoma of the thyroid. J Clin Pathol 19:103–113[Abstract/Free Full Text]
  8. Huang SN, McLeish WA 1968 Pheochromocytoma and medullary carcinoma of thyroid. Cancer 21:302–311[CrossRef][Medline]
  9. Meyer JJ 1968 Fine structure of two amyloid-forming medullary carcinomas of thyroid. Cancer 21:406–425[CrossRef][Medline]
  10. Shapiro MJ 1977 Medullary carcinoma of the thyroid gland. Am Surg 43:601–609[Medline]
  11. Sizemore GW, Health 3rd H, Carney JA 1980 Multiple endocrine neoplasia type 2. Clin Endocrinol Metab 9:299–315[Medline]
  12. Vassar PS, Culling CF 1961 The significance of amyloid in carcinoma of the thyroid gland. Am J Clin Pathol 36:244–247[Medline]
  13. Butler M, Khan S 1986 Immunoreactive calcitonin in amyloid fibrils of medullary carcinoma of the thyroid gland. Arch Pathol Lab Med 110:647–649[Medline]
  14. Sletton K, Westermark P, Natwig JB 1976 Characterization of amyloid fibril proteins from medullary carcinoma of thyroid. J Exp Med 143:993–998[Abstract/Free Full Text]
  15. Pras MD, Schubert M, Zucker-Franklin D 1968 The characterization of soluble amyloid prepared in water. J Clin Invest 47:924–933
  16. Vassar PS, Culling CF 1959 Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol 68:487–494[Medline]
  17. Naiki H, Higuchi K, Hosokawa M, Takeda T 1989 Fluorimetric determination of amyloid fibrils in vitro using fluorescent dye, thioflavin T. Anal Biochem 177:244–249[CrossRef][Medline]
  18. LeVine III H 1993 Thioflavine T interaction with synthetic Alzheimer’s disease ß-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci 2:404–410[Abstract]
  19. Arvinte T, Cudd A, Drake AF 1993 The structure and mechanism of formation of human calcitonin fibrils. J Biol Chem 268:6415–6422[Abstract/Free Full Text]
  20. Bauer HH, Aebi U, Häner M, Hermann R, Müller M, Arvinte T, Merkle HP 1995 Architecture and polymorphism of fibrillar supramolecular assemblies by in vitro aggregation of human calcitonin. J Struct Biol 115:1–15[CrossRef][Medline]
  21. Gilchrist PJ, Bradshaw JP 1993 Amyloid formation by salmon calcitonin. Biochim Biophs Acta 1182:111–114[Medline]
  22. Chamberlain AK, MacPhee CE, Zurdo J, Morozova-Roche LA, Hill HAO, Dobson CM, Davis JJ 2000 Ultrastructural organization of amyloid fibrils by atomic force microscopy. Biophys J 79:3282–3293[Abstract/Free Full Text]
  23. Ionescu-Zanetti C, Khurana R, Gillespie JR, Petrick JS, Trabachino LC, Minert LJ, Carter SA, Fink AL 1999 Monitoring the assembly of Ig light chain amyloid fibrils by atomic force microscopy. Proc Natl Acad Sci USA 96:13175–13179[Abstract/Free Full Text]
  24. Kad NM, Myers SL, Smith DP, Smith DA, Radford SE, Thomson NH 2003 Hierarchical assembly of ß2 microglobulin amyloid in vitro revealed by atomic force microscopy. J Mol Biol 333:785–797
  25. Khurana R, Ionescu-Zanetti C, Pope M, Neilson L, Li J, Ramirez-Alvarado M, Regan L, Fink AL, Carter SA 2003 A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy. Biophys J 85:1135–1144[Abstract/Free Full Text]
  26. Pepys MB, Rademacher TW, Amatayakul-Chantler S, Williams P, Noble GE, Hutchinson WL, Hawkins PN, Nelson SR, Gallimore JR, Herbert J, Hutton T, Dwek RA 1994 Human serum amyloid P component is an invariant constituent of amyloid deposits and has a uniquely homogenous glucostructure. Proc Natl Acad Sci USA 91:5602–5606[Abstract/Free Full Text]
  27. Westermark P 1975 Amyloid of medullary carcinoma of the thyroid; partial characterization. Ups J Med Sci 80:88–92[Medline]
  28. Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG 1983 Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35:349–358[CrossRef][Medline]
  29. Caughey B, Raymond GJ, Kocisko DA, Lansbury Jr PT 1997 Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies. J Virol 71:4107–4110[Abstract]



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