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PubMed1995

Laparoscopic detection of hepatic metastases in patients with residual or recurrent medullary thyroid cancer.

BACKGROUND: After initial operations for medullary thyroid cancer (MTC), reoperation with removal of metastatic disease confined to the neck may benefit some patients. The identification of distant metastases precludes the possibility of curative reoperation. METHODS: Forty-one patients with hypercalcitoninemia after initial surgical treatment for MTC underwent laparoscopic (n = 36) or open (n = 5) examination and biopsy of the liver. Thirty-seven of these patients underwent imaging by computed tomography (CT), magnetic resonance imaging (MRI) of the liver, or both, and 17 underwent selective venous catheterization (SVC) with measurement of hepatic and peripheral vein stimulated calcitonin levels. RESULTS: Liver metastases were found in eight patients, seven by laparoscopy and one by open examination. Seven of these patients had normal CT or MRI scans of the liver. Laparoscopy or open liver examination revealed metastases in 2 of 11 patients with elevated hepatic vein-peripheral vein stimulated calcitonin ratios (greater than 1.3). Metastases appeared as small (less than 5 mm), bright white nodules on the surface of the liver. CONCLUSIONS: Direct examination and biopsy of the liver by laparoscopy may show small deposits of metastatic MTC in patients with normal CT and MRI scanning.

Adolescent↗
PubMed1995

Presymptomatic DNA screening in families with multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma.

BACKGROUND: Missense mutations of the ret proto-oncogene on chromosome 10q11.2 are the underlying cause of hereditary medullary thyroid carcinoma (MTC), either as familial MTC only (FMTC) or as a part of multiple endocrine neoplasia type 2 syndrome (MEN 2). This study presents our experience with direct presymptomatic DNA screening in MEN 2 and FMTC kindreds. METHODS: Twenty one families with MEN 2 or FMTC were considered in the study. One hundred three individuals had been analyzed; 56 were at risk. The ret mutations were detected by DNA analysis of exons 10, 11, and 16 by using nonradioactive labeling method based on digoxigenin DNA sequencing technique. Serum calcitonin evaluation was carried out in all individuals at risk. Thyroidectomy was performed in those who had to undergo surgery. RESULTS: The ret mutations were identified in all 21 families. In MEN 2A and FMTC families mutations occurred in exons 10 and 11. MEN 2B families had mutations in exon 16. The most frequent mutation in MEN 2A and FMTC affected codon 634. Twenty one gene carriers were identified in unaffected individuals at risk. Ten of 21 gene carriers had elevated calcitonin levels, and 11 had normal levels. MTC or C-cell hyperplasia was found in six gene carriers with pathologic calcitonin values who underwent operation. In a 5-year-old gene carrier with normal calcitonin values C-cell hyperplasia was evident. CONCLUSIONS: Direct predictive DNA analysis allows us to identify MEN 2 or FMTC gene carriers and offer them prophylactic treatment.

Base Sequence↗
PubMed1997

Evaluation of fluorodeoxyglucose-positron emission tomographic scanning and its association with glucose transporter expression in medullary thyroid carcinoma and pheochromocytoma: a clinical and molecular study.

BACKGROUND: Imaging of metastatic sites of medullary thyroid carcinoma (MTC) is successful in less than 60% of cases of residual or recurrent disease. Positron emission tomography (PET) with [18F]fluoro-2-deoxy-D-glucose (FDG) takes advantage of the fact that malignant tumors are capable of increased uptake and use of glucose, which is mediated by the members of the glucose transporter family of proteins (GLUT 1 through GLUT 5). METHODS: FDG-PET images of 10 patients with recurrent or persistent MTC after primary operation were compared with images by computed tomography or magnetic resonance imaging. Identified metastatic lesions were assessed by intraoperative findings and pathology reports. Expression of GLUT 1 through GLUT 5 was examined by Western blot analysis of tumor tissue from eight of the patients evaluated and an additional panel of 10 MTCs and seven pheochromocytomas. RESULTS: FDG-PET identified 31 foci of FDG accumulation in 10 patients, and 16 of these metastatic sites were resected and confirmed by histologic analysis. Only 11 foci were demonstrated by computed tomographic or magnetic resonance imaging. None of the glucose transporters examined displayed significant expression. Two pheochromocytomas were successfully imaged by FDG-PET. CONCLUSIONS: FDG-PET imaging can be useful in the localization of cervicomediastinal MTC metastases and pheochromocytoma. The increased glucose uptake in these tumors, as evidenced by FDG-PET, does not appear to be attributable to the expression of the glucose transporter proteins GLUT 1 through GLUT 5.

Adolescent↗
PubMed1997

Improved selection of patients for reoperation for medullary thyroid cancer by imaging with radiolabeled anticarcinoembryonic antigen antibodies.

BACKGROUND: This study examined the utility of using radiolabeled anticarcinoembryonic antigen (CEA) monoclonal antibodies (MAbs) as a noninvasive imaging method to aid in the selection of patients for reoperation after operation for medullary thyroid carcinoma (MTC). METHODS: Sixteen patients with persistent or recurrent hypercalcitoninemia since operation or MTC, but had negative or equivocal conventional imaging study results, were given 99mTc-, 123I-, or 131I-labeled NP-4 or MN-14 anti-CEA MAbs. Scintigraphic images were then performed to determine detection of tumor lesions. RESULTS: The MAb scans were positive in 13 (81%) of the 16 patients studied. However, in only three of 13 patients was disease confined to the cervical or mediastinal nodes. In 10 patients, disease was additionally or solely found in distant organs such as liver (five patients), bone (four patients), and periaortic nodes (one patient). On the basis of our studies, only three of the 13 patients with positive scans would benefit from repeat neck exploration, another three would possibly benefit from neck or mediastinal exploration and hepatic resection, and the remaining seven patients would not benefit from any reoperation with curative intent. CONCLUSIONS: MTC imaging with anti-CEA MAbs could be very useful in determining the ideal candidates for repeat neck exploration.

Adult↗
PubMed1986

[Hemothorax disclosing hemorrhagic necrosis of a pheochromocytoma: circumstance for detection of multiple endocrine neoplasia type IIa. Detection of 5 familial cases].

Whilst investigating 26 members of the same family, we discovered 5 cases of multiple endocrine neoplasia type II a. The present report demonstrates the diagnostic value of basal plasma thyrocalcitonin (TCT) assays, before and after stimulation with pentagastrin, and of plasma carcinoembryonic antigen (CEA) assays. Some of the clinical features encountered were novel--e.g. in one patient the phaeochromocytoma was revealed by a haemothorax with cardiovascular collapse--and others were peculiar; thus, in 4 cases the medullary thyroid carcinoma (MTC) was less than 2 cm in diameter and without lymph node or visceral metastases, even in patients aged 87, 59 and 56. More classically, MTC, always multifocal, was clinically silent, as were the two cases of phaeochromocytoma and hyperparathyroidism. Phaeochromocytomas were difficult to diagnose. Ultrasonic tomography did not prove very helpful and the disease was transmitted as an autosomal dominant trait. Finally, MTC secreted a variety of substances (TCT, CEA, beta-endorphin, somatostatin), and HLA A2-B15 antigens were found in 3 patients.

Adrenal Gland Neoplasms↗
PubMed1981

Management of hereditary medullary thyroid carcinoma.

In this paper we have briefly outlined our views on the management of hereditary MTC. We have stressed the fact that although many patients with the usual form of disease have an indolent clinical course there exists a population of patients who may be at risk for widely disseminated virulent tumour. Also, some patients can have marked invasion of adjacent tissues in the cervical region from localized tumour. Hence it seems rational to attempt removal of the primary tumour prior to the development of regional or distant metastases. The role of the combined calcium-pentagastrin provocative test to identify patients with preclinical disease has been outlined. The only definitive treatment for the disease is total thyroidectomy with resection of adjacent cervical nodes. Many patients who are treated at a time when the disease has become clinically detectable will have residual tumour or recurrence as indicated by abnormal calcitonin levels in the blood. Since most of these patients seem to take an indolent clinical course, it is suggested that they should not be treated with radiation therapy and/or chemotherapy, even though residual MTC is known to be present. Even in patients with advanced stages of MTC, the use of chemotherapy and radiation therapy has not been encouraging. However, some aspects of the management of patients with aggressive disease are discussed. Finally, the lack of features to predict which patients may have a poor prognosis is outlined and some new approaches to defining such parameters are discussed.

Antineoplastic Agents↗
PubMed1999

Influence of laminin substratum on cell proliferation and CALC I gene expression in medullary thyroid carcinoma C cell lines.

Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT) and CT gene-related peptide (CGRP), the two splice peptide products of the CALC I gene. Normal and hyperplastic C cells are intrafollicular, in contact with the basement membrane (BM) that is maintained around the differentiated tumors. To investigate the relationships between MTC evolution and BM constituents, we examined the modifications induced by laminin-1 and -2 (merosin), two isoforms colocalized in the follicular BM, on three MTC cell lines: murine rMTC 6-23 and CA-77 cells, and human TT cells. Laminin exerted a mitogenic activity on rMTC 6-23 and on TT cells, causing a concurrent decrease in both CT and CGRP mRNA levels and production of the peptides. Conversely, laminin reduced the proliferation rate and enhanced CGRP synthesis and secretion in CA-77 cells. This antiproliferative response, which coincides with an increase in differentiation markers, is comparable to that reported in normal cells and also in the neoplastic Caco-2 cell line. This suggests that laminin could exert opposite effects depending on the stage of tumor evolution.

Animals↗
PubMed2001

Thyroid cancer.

Four types of thyroid cancer comprise more than 98% of all thyroid malignancies. Papillary thyroid carcinoma (PTC) may have a very benign course while undifferentiated thyroid carcinoma (UTC) belongs to the most aggressive human malignancies. A variety of genes have been identified to be involved in the pathogenesis of thyroid carcinoma. Somatic Ras mutations seem to be an early event and are frequently found in follicular thyroid carcinomas. Somatic rearrangements of RET and TRK are almost exclusively found in PTC and may be found in early stages. Germline RET missense mutations lead to hereditary medullary thyroid carcinoma (MTC). In contrast, the significance of somatic RET mutations in sporadic MTC is unknown. p53 seems to play a crucial role in the dedifferentiation process of thyroid carcinoma. The precise role of PTEN remains to be elucidated. The only clearly identified exogenous factor that may lead to thyroid carcinoma (mainly PTC) is radiation. Of interest, radiation is capable to induce RET rearrangements. In general, early diagnosis is mandatory to enable the chance of cure. Surgery is the treatment of choice. Depending on the tumour type, surgery in combination with either radioiodine, external radiation or chemotherapy often enables the control of local tumour burden. In MTC and UTC, once thyroid cancer is spread to distant organs, efficacious therapeutic agents are almost non-existing. However, our growing knowledge of genes involved in thyroidal oncogenesis may contribute to the development of more effective treatment modalities. Some preliminary data on gene therapy are quite promising.

Adenocarcinoma, Follicular↗
PubMed1999

The cost effectiveness of 3D conformal radiation therapy compared with conventional techniques for patients with clinically localized prostate cancer.

BACKGROUND: We previously demonstrated the advantages of three-dimensional conformal radiation therapy (3DCRT) in improved rates of biochemical (bNED) control in certain subsets of patients with clinically localized prostate cancer. However, in this era of cost consciousness and limited resources, the cost effectiveness of 3DCRT compared with conventional external beam irradiation (CRT) remains unexamined. METHODS AND MATERIALS: Between October 1, 1987 and November 30, 1991, 193 patients with clinically localized prostate cancer received definitive external beam irradiation at Fox Chase Cancer Center. The 1998 Medicare fee schedule was used to determine treatment charges and to provide a reference for a national comparison. Complete charges for pretreatment work-up, treatment, and follow-up were tabulated for each patient. The mean total charges (MTC) using the Lin method of estimating medical costs was used to analyze and compare costs between groups. A matched case/control analysis was performed to further evaluate the effect of cost between techniques. The median follow-up was 72 months (range 3-118). RESULTS: The overall 5-year actuarial rate of bNED control was 41% and 53%, respectively, for the CRT and 3DCRT patients (p = 0.03). The MTC for the CRT patients was $10,544.53. For the 3DCRT patients, the MTC was $8,955.48. The sample mean of the total costs from the observed deaths for the two patient groups by follow-up interval ranged from $9,800.63 to $59,635.01 for the CRT patients to $17,259.00 to $24,250.38 for the 3DCRT patients. No statistically significant difference in cost was observed between groups using the matched case/control analysis. CONCLUSION: Initial work-up and treatment costs were greater for patients treated with 3DCRT compared with patients treated with conventional techniques. However, with longer follow-up, the mean total cost of treatment was not statistically different between the two treatment groups. Because of improved rates of bNED control for these patients and the increased costs associated with the treatment of a greater fraction of patients with recurrent disease following CRT, 3DCRT was cost effective for patients with clinically localized prostate cancer.

Aged↗
PubMed1997

Cloning and sequencing of a new 15-hydroxyprostaglandin dehydrogenase related mRNA.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH) inactivates prostaglandins. We recently reported an mRNA sequence coding for a predicted isomer (PGDH(rI)) of this enzyme. The TT cell line, derived from medullary thyroid carcinoma (MTC), expresses mRNAs for both isomers. We report here the expression by TT cells and MTC of a third 15-PGDH related mRNA (PGDH(rII)), 241 nt shorter than type-I 15-PGDH. RNase protection assays confirmed that TT cells expressed this mRNA (PGDH(rII)). Thus different splicing patterns could be involved in the post-transcriptional regulation of type-I 15-PGDH gene in MTC.

Amino Acid Sequence↗
PubMed2003

Evaluation of the BDProbeTec ET system as screening tool in the direct detection of mycobacterium tuberculosis complex in respiratory specimens.

We evaluated the BDProbeTec ET System (Becton Dickinson) for the routine detection of Mycobacterium tuberculosis complex (MTC) in respiratory specimens and pleural fluids, comparing with microscopy (Ziehl Neelsen stain, ZN) and culture in liquid (BACTEC MGIT 960, MGIT) and solid (Löwenstein Jensen, LJ) media. Five hundred and two specimens, collected from 266 patients, of which 257 with suspected tuberculosis and 9 receiving anti-tuberculosis treatment, were investigated. Thirty-nine specimens were positive by any method, including false positives. Mycobacteria were isolated from 33 specimens (32 Mycobacterium tuberculosis and 1 Mycobacterium chelonae). Thirty-six specimens were BDProbeTec ET positive, 33 specimens were MGIT positive, 27 were LJ positive and 22 were ZN positive. With BDProbeTec ET, 2 specimens were false negative (culture positive), and 2 specimens from non-treated patients were false positive (culture negative). The overall sensitivity, specificity, and positive and negative predictive values for BDProbeTec ET compared to culture were 93.7, 98.7, 83.3, and 99.5%, respectively, while with smear-positive and smear-negative specimens the sensitivities were 100% and 81.5% respectively. In five treated patients the disappearance of MTC could be monitored using BDProbeTec ET in parallel with culture. The overall inhibition rate was 0.2%. BDProbeTec ET can be very useful for rapid detection of MTC, especially in smear-negative respiratory specimens.

Bacterial Typing Techniques↗
PubMed2001

Serotonin 5-HT1A receptor binding potential declines with age as measured by [11C]WAY-100635 and PET.

Positron emission tomography (PET) and [11C]WAY-100635 were used to examine the effect of age on serotonin-1A (5-HT1A) receptor binding potential (BP) in 19 healthy subjects. Regions of interest (ROI) were drawn on the co-registered magnetic resonance imaging (MRI) in orbitofrontal (OFC), dorsolateral prefrontal (DLPFC), anterior cingulate (ACC), lateral (LTC), and mediotemporal (MTC), parietal, occipital and cerebellar cortex, and the raphe nuclei. BP values were calculated using a simplified reference tissue method. In addition, a voxelwise analysis was performed using SPM99. Voxelwise analysis revealed a significant global decrease of 5-HT(1A) BP with age (set level <.001). ROI analysis revealed significant age-related 5-HT(1A) BP decreases in DLPFC (r = -0.56), ACC (r = -0.44), OFC (r = -0.42), LTC (r = -0.40), parietal (r = -0.65), and occipital cortex (r = -0.43), but not in MTC or raphe nuclei. Overall, cortical 5-HT(1A) BP declined by approximately 10% per decade, except for the MTC, where we did not find a significant age effect. Hence, careful age matching may be recommended for future studies using PET and [11C]WAY-100635 to examine 5-HT1A receptors.

Adult↗
PubMed2000

Multiple endocrine neoplasia type 2B--genetic basis and clinical expression.

Multiple endocrine neoplasia (MEN) type 2B is a heritable endocrine disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, multiple mucosal neuromas, and a marfanoid habitus. Intestinal ganglioneuromatosis, corneal nerve thickening and skeletal abnormalities are also often present. The disease is inherited in an autosomal dominant fashion and is caused by a single mutation in the RET proto-oncogene, with a methionine to threonine substitution at codon 918. The MTC in MEN 2B presents at an earlier age and tends to be more aggressive than the MTC in MEN 2A. It is multicentric and bilateral and occurs as young as age 3, with early lymph node metastases. Pheochromocytoma is also often bilateral but is rarely malignant. If pheochromocytoma is detected, adrenalectomy should precede thyroidectomy to avoid intraoperative catecholamine crisis. Patients at risk for MEN 2B should undergo genetic screening in infancy. Total thyroidectomy should be performed on all patients positive for RET mutations even prior to the onset of clinical symptoms.

Adrenal Gland Neoplasms↗
PubMed1998

Stability of the peritoneal membrane in long-term peritoneal dialysis patients.

One of main challenges of peritoneal dialysis (PD) is the functional and vital long-term stability of the peritoneal membrane. Few longitudinal and controlled studies on peritoneal function have been published, and the results are somewhat contradictory. We have performed a longitudinal study with 90 patients. The overall analysis has shown that creatinine mass transfer coefficient (MTC) significantly increases and ultrafiltration (UF) capacity decreases over time. Nevertheless, urea MTC remained unaltered and MTC ratios significantly decreased after the third year. Subsequently, we examined the clinical outcomes and identified 19 patients who required peritoneal resting periods for Type I UF failure and 71 patients who did not require such a procedure. The latter patients did not show any significant functional change over time, whereas the former 19 patients showed an increase of peritoneal creatinine transport and a loss of UF capacity. These data corroborate changes in long-term peritoneal function in approximately 20% of PD patients. These changes consist of an increase in effective exchange area, peritoneal permeability, or both, accompanied by signs suggestive of mesothelial regenerative capacity loss. Infectious peritoneal injuries, especially appearing during late PD periods, are deleterious to the peritoneum. The remainder of the functional-structural changes are related to the effects of currently used dialysate. Early diagnosis, preemptive, and therapeutic measures should permit better management of long-term PD patients. The particular response to these injuries has individual characteristics that when addressed permit PD to be used long-term.

Analysis of Variance↗
PubMed1997

Studies on the relationship between treatment condition and micronucleus induction in V79 cells exposed to silica and glass fibers.

Studies have been carried out to determine the relationship between treatment condition and frequencies of micronucleated cells (MNC) and multinucleated cells (MTC) in Chinese hamster lung fibroblasts (V79 cells) exposed to dusts and fibers. Cells were treated with Min-U-Sil 5 silica or Owens Corning AAA-10 glass fibers under three different conditions: 24-h exposure (24E), 24-h exposure followed by 24-h post-incubation in fresh medium (24E-24P), and 48-h exposure (48E). Results showed that the frequency of MNC increased in a concentration-related manner in silica-treated V79 cells only under the condition of 24E-24P. The increase in MNC frequency after 24-h exposure was not concentration-related. No significant increase in MNC was detected in cells sampled after 48-h treatment. The frequencies of MTC in the treatment groups were higher than that in the control group. However, the increase was not statistically significant. Compared with silica, glass fibers were more active for MTC and MNC induction on a mass basis. The highest response was also observed under the condition of 24E-24P. These results indicate that 24-h exposure followed by 24-h post-incubation is a suitable treatment condition for the micronucleus assay on mineral dusts and fibers.

Animals↗
PubMed2004

Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification.

Rapid, reliable diagnosis of tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. Conventional microbiological methods may not be an option if samples are formalin-fixed and paraffin-embedded (FFPE) for histopathological examination. With the demonstration of necrotizing granulomatous inflammation, tuberculosis becomes an important differential diagnosis, although it was not initially suspected. Following paraffin extraction, BDProbeTec ET strand displacement amplification for detection of Mycobacterium tuberculosis complex (MTC) was applied to 47 prospectively and 19 retrospectively collected FFPE samples from various sources with granulomatous inflammation and results were compared to tuberculosis notification. Of the prospective samples, 20 were from patients who were notified as having tuberculosis and the assay was positive in 18 (90%). Specificity was 100%. For 27 of the patients with prospectively collected FFPE specimens, culture was performed on a specimen collected at a later date from the same location. Culture revealed MTC in 14 and nontuberculous mycobacteria in four. BDProbeTec ET was positive in 13 (92.8%) of the patients with positive MTC culture and negative in the remaining. The sensitivity and specificity in 19 archival samples was 40% and 100%, respectively, compared to notification data. The assay provided rapid, correct diagnosis on different sources of FFPE samples collected prospectively and therefore offers an important supplementary method for patients where tuberculosis was not initially suspected.

Fixatives↗
PubMed1991

Mechanism of colchicine binding to tubulin. Tolerance of substituents in ring C' of biphenyl analogues.

The limits of structural variation of the substituent in position 4' of ring C' of biphenyl colchicine analogues (ring C in colchicine) were probed by the synthesis of a number of analogues and the examination of their binding to tubulin and its consequences. Binding was found to require the location in three-dimensional space of the oxygen in the 4'-substituent at a locus not far distant from those of the colchicine ring C oxygens. All those analogues that bind to the colchicine site of tubulin induced the GTPase activity and inhibited microtubule assembly, those containing a carbonyl group substoichiometrically and the others stoichiometrically. A similar relation was found for the induction of the abnormal polymerization of the colchicine analogue-tubulin complex, with methoxy-containing compounds requiring a higher temperature to induce the polymerization. A concerted analysis of the binding thermodynamics of colchicine and its various analogues has shown full consistency with the previously proposed two-step binding pathway that involves two nonidentical binding moieties in the ligand [Andreu, J. M., & Timasheff, S. N. (1982) Biochemistry 21, 534-543]. Comparison of the binding parameters of colchicine, its des(ring B) analogue (MTC), and ring A and C compounds individually with the thermodynamic parameters deduced for the first steps of the bindings of colchicine and MTC [Engelborghs, Y., & Fitzgerald, T. J. (1987) J. Biol. Chem. 262, 5204-5209] have led to the conclusion that binding can occur by two pathways leading to the identical product. In the first pathway, ring A binds first; this is followed by a rate-determining thermodynamically indifferent reaction (protein conformation change), and finally a rapid binding of ring C. In the second pathway, the events are the same except that the order of binding of the rings is reversed. Colchicine, due to the steric hindrance of ring B, can follow only the second pathway. For MTC, both kinetic pathways are open and binding may be initiated by random first contact of either ring A or ring C.

Animals↗
PubMed1996

Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin.

The kinetics of binding of R- and S-enantiomers were studied by the fluorescence stopped-flow technique. For the R-enantiomer, the time course of the increase in fluorescence is best fitted by a sum of two exponentials. In pseudo-first-order conditions, the first observed rate constant showed a linear concentration dependence whereas the second showed a hyperbolic one. The dissociation rate constants were determined independently by displacement experiments with 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). The two exponential phases were assumed to be due to a two-step binding mechanism: an initial binding followed by a conformational change. This is different from colchicine and MTC binding, where the two phases show a hyperbolic concentration dependence and are attributed to the parallel binding to different isoforms of tubulin [Banerjee, A., & Luduena, R. F. (1992) J. Biol. Chem. 267, 13335-13339]. R-isomer binding did not discriminate between the tubulin isoforms. The temperature dependence of all the rate constants were measured, and the entire thermodynamic reaction path was constructed. For the S-isomer, the direct fluorescence stopped-flow study showed that the signals were largely imputable to the fluorescence of the binding at low-affinity sites [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10674-10682]. Therefore, we exploited the competition between R- and S-isomers to determine the binding kinetics of the S-isomer to the R-site. The observed rate constants for competitive binding showed a linear concentration dependence, thus allowing us to calculate the association rate constant of the S-isomer to the R-site. The kinetics of displacement of the S-isomer by MTC allowed the dissociation rate constant for the S-isomer to be determined. The binding of both enantiomers to tubulin in presence of tropolone methyl ether (analog of the colchicine C ring) was decreased, indicating the involvement of the C subsite.

Animals↗