A kinetic study of gamma-glutamyltransferase (GGT)-mediated S-nitrosoglutathione catabolism.

S-Nitrosoglutathione (GSNO) is a nitric oxide (NO) donor compound which has been postulated to be involved in transport of NO in vivo. It is known that c-glutamyl transpeptidase (GGT) is one of the enzymes involved in the enzyme-mediated decomposition of GSNO, but no kinetics studies of the reaction GSNO-GGT are reported in literature. In this study we directly investigated the kinetics of GGT with respect to GSNO as a substrate and glycyl- glycine (GG) as acceptor co-substrate by spectrophotometry at 334 nm. GGT hydrolyses the c-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and c-glutamyl-GG. However, as both the substrate GSNO and the first product CGNO absorb at 334 nm, we optimized an ancillary reaction coupled to the enzymatic reaction, based on the copper-mediated decomposition of CGNO yielding oxidized cysteinyl-glycine and NO. The ancillary reaction allowed us to study directly the GSNO/GGT kinetics by following the decrease of the characteristic absorbance of nitrosothiols at 334 nm. A Km of GGT for GSNO of 0.398 ± 31 mM was thus found, comparable with Km values reported for other c-glutamyl substrates of GGT

A New MEN2 Syndrome with Clinical Features of Both MEN2A and MEN2B Associated with a New RET Germline Deletion

Background. Multiple endocrine neoplasia type 2 (MEN2) is a hereditary cancer syndrome caused by RET proto-oncogene mutation. Two different clinical variants of MEN2 are known (MEN2A and MEN2B): medullary thyroid carcinoma (MTC) almost always present and associated with pheochromocytoma (Pheo), and primary hyperparathyroidism (HPTH) in MEN2A and with Pheo and other nonendocrine diseases in MEN2B. Case Report. A 7-year-old girl, previously treated for a pelvic plexiform neurofibroma, arrived at our observation with a peculiar MEN2B syndrome and with HPTH. The neck ultrasound showed bilateral thyroid nodules, local lymph node lesions, and a suspicious left hyperplastic parathyroid. The CT scan showed a megacolon and described the persistence of the pelvic tumor. A new RET germline deletion in exon 11 (c.1892_1899delCGAGCT; p.Glu632_Leu633del) was found. She underwent total thyroidectomy, central compartment and latero-cervical lymph node dissection, and neck exploration for primary HPTH. The histology confirmed bilateral MTC, multiple lymph node metastases, a hyperplastic parathyroid, and a parathyroid adenoma. Conclusions. This is the first case of a complex syndrome characterized by peculiar features of MEN2B, without Pheo but with a pelvic plexiform neurofibroma and with HPTH, which is typical of MEN2A. A "de novo"new germline RET deletion located in exon 11 was found

After 20 Years, RET genetic screening still identifies new germline and somatic mutations

Objectives: In the last 20 years we performed RET genetic screening in more than 1000 MTC patients either hereditary or sporadic. Methods: RET genetic screening was performed in DNA extracted from blood and/or tissue by direct sequencing. TA cloning was performed to characterize new mutations and deletions. Site-directed mutagenesis, focus formation and soft agar assays were performed to test in vitro the activity of the new mutations. The Align GVGD program was employed for the in silico analysis. Results: in the last year we identified 3 MTC patients with new RET alterations. The first case had a 7bp “somatic” in frame deletion in exon 11 encompassing codon 629-631. The second case showed the simultaneous presence of a “somatic” E616Q mutation in exon 10 and a “somatic” C630G mutation in exon 11 on different alleles. Moreover, in the same patient, we found an alternative splicing causing the in frame skip of exon 10 in the allele carrying the C630G mutation. The third case harboured a new “germline” mutation(E632K in exon 11) although the MTC was apparently sporadic. According to the in vitro and the in silico tests, both E616Q and E632K RET mutations were not transforming while the C630G RET mutation showed a high transforming activity. Conclusions: 1) RET genetic screening should be performed by sequencing analysis in all MTC patients to detect also new RET mutations that would be missed when looking only at the “hot spot” mutations; 2) all new mutations must be evaluated by in silico and/or in vitro analysis to define their transforming ability since in some cases they may be inactive mutations

Clinical utility of genetic diagnosis for sporadic and hereditary medullary thyroid carcinoma

Medullary thyroid cancer (MTC) is a rare thyroid tumor whose prevalence is 3-5% among all thyroid tumors. The pathogenesis of MTC is mainly related to germline or somatic RET activating point mutations that are causative of hereditary and sporadic cases, respectively. Hereditary MTC can occur as multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B) and familial MTC (FMTC) that differ for the association with other endocrine neoplasia. Germline RET point mutations are prevalently localized in exons 5, 8, 10-11, 13-16 and a significant genotype-phenotype correlation has been observed. RET genetic screening is mandatory in all patients with a diagnosis of MTC regardless from their apparent sporadic origin. The identification of RET germline mutation in an apparently sporadic case is of great clinical utility because it allows the identification of those subjects who will develop the tumor. RET positive relatives must undergo clinical and biochemical tests to verify if the MTC is already present and, according to the type of RET mutation, they have to be screened for the presence of pheochromocytoma and/or hyperparathyroidism. If a MTC is already present patients must be surgically treated. If MTC is not yet present subjects will be followed up with basal and stimulated calcitonin serum measurement, which is the serum marker of MTC. Indeed, RET negative subjects can be reassured that they do not run any risk to develop the disease as well as their children. In conclusions RET genetic screening allows the identification of the hereditary/sporadic nature of MTC and of a relevant percentage of hidden familial MTC. Furthermore, it favors the early diagnosis of MTC in RET positive subjects. RET positive patients and no clinical evidence of MTC can be followed and surgical treatment can be delayed. Finally RET negative relatives do not need to be further monitored

Chromosome 10 and RET gene copy number alterations in hereditary and sporadic Medullary Thyroid Carcinoma.

About 30% of hereditary Medullary Thyroid Carcinoma (MTC) have been demonstrated to harbour imbalance between mutant and wild-type RET alleles. We studied the RET copy number alterations (RET CNA) in 65 MTC and their correlation with RET mutation and patients' outcome. Fluorescence in situ Hybridization and Real-time PCR revealed RET CNA in 27.7% MTC but only in a variable percentage of cells. In sporadic MTC, RET CNA were represented by chromosome 10 aneuploidy while in hereditary MTC by RET amplification. A significant higher prevalence of RET CNA was observed in RET mutated MTC (P=0.003). RET CNA was also associated to a poorer outcome (P=0.005). However, the multivariate analysis revealed that only RET mutation and advanced clinical stage correlated with the worst outcome. In conclusion, 30% MTC harbour RET CNA in variable percentage of cells suggesting cell heterogeneity. RET CNA can be considered a poor prognostic factor potentiating the poor prognostic role of RET mutation

Chromosome 10 and RET gene copy number alterations in hereditary and sporadic Medullary Thyroid Carcinoma.

About 30% of hereditary Medullary Thyroid Carcinoma (MTC) have been demonstrated to harbour imbalance between mutant and wild-type RET alleles. We studied the RET copy number alterations (RET CNA) in 65 MTC and their correlation with RET mutation and patients' outcome. Fluorescence in situ Hybridization and Real-time PCR revealed RET CNA in 27.7% MTC but only in a variable percentage of cells. In sporadic MTC, RET CNA were represented by chromosome 10 aneuploidy while in hereditary MTC by RET amplification. A significant higher prevalence of RET CNA was observed in RET mutated MTC (P = 0.003). RET CNA was also associated to a poorer outcome (P = 0.005). However, the multivariate analysis revealed that only RET mutation and advanced clinical stage correlated with the worst outcome. In conclusion, 30% MTC harbour RET CNA in variable percentage of cells suggesting cell heterogeneity. RET CNA can be considered a poor prognostic factor potentiating the poor prognostic role of RET mutation. (C) 2011 Elsevier Ireland Ltd. All rights reserved

CLINICAL IMPACT OF RET GENETIC SCREENING IN THE MANAGEMENT OF MEDULLARY THYROID CARCINOMA (MTC) PATIENTS: 20 YEARS OF EXPERIENCE

During the last 20 years, we performed RET genetic screening in 1556 subjects: 1007 arrived as affected by an apparently sporadic form of MTC and 95 were clearly affected by a hereditary form. The remaining 454 were relatives of RET positive MTC patients. An unsuspected germline RET mutation was found in 69/1007(6.7%) apparently sporadic MTC patients and were reclassified as hereditary: 60 FMTC and 8 MEN 2A syndromes were identified. The remaining 939 patients were RET negative thus their sporadic nature was confirmed. Ninety-five patients, 71 index cases and 24 relatives, arrived at our first clinical observation as affected by a hereditary MTC. A germline RET mutation was found in 69/71 index cases. The 71 families were classified in FMTC (n=33), MEN 2A (n=26), MEN 2B (n=12). A total of 140 MEN 2 families (94 FMTC, 34 MEN 2A, 12 MEN 2B) have been diagnosed. Following the identification of a germline RET mutations in the index cases, 454 relatives performed the RET screening and 137 were found to be gene carriers (GC). RET screening allowed us to identify 33 different mutations. The V804M (exon 14) is the most prevalent mutation (22.4%), but codon 634 (exon 11) is the most frequent (n = 34). MEN 2A syndromes are mainly associated to the C634R mutation while the MEN 2B to the M918T mutation. Among all mutations we found rare variants (R833C, A883T, M848T, M918V, E632K, S904F, T338I, V648I) whose biological significance has to be proven. On the basis of our experience, the clinical impact of the RET genetic screening can be summarized as follows: 1) identification of hereditary cases (7-8%) that would be clinically considered as sporadic; 2) identification of GC unaware of their condition thus allowing their early treatment; 3) identification of new RET mutations not necessarily pathogenetic for the disease

Trypanosoma cruzi infection in dogs and cats and household seroactivity to T. Cruzi in a rual community in Northeast Brazil

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-06-27T12:06:00Z No. of bitstreams: 1 Mott KE Trypanososma Cruzi Infection In Dogs And Cats......PDF: 6284767 bytes, checksum: 655952fb0c9fe4f2975d1f8482510822 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-06-27T12:27:09Z (GMT) No. of bitstreams: 1 Mott KE Trypanososma Cruzi Infection In Dogs And Cats......PDF: 6284767 bytes, checksum: 655952fb0c9fe4f2975d1f8482510822 (MD5)Made available in DSpace on 2018-06-27T12:27:09Z (GMT). No. of bitstreams: 1 Mott KE Trypanososma Cruzi Infection In Dogs And Cats......PDF: 6284767 bytes, checksum: 655952fb0c9fe4f2975d1f8482510822 (MD5) Previous issue date: 1978Wellcome Trust and its collaborative activities in Brazil are under the aegis of the Pan American Health OrganizationHarvard School of Public Health. Department of Tropical Public Health. Boston. Massachusetts, USAUniversidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilHarvard School of Public Health. Department of Tropical Public Health. Boston. Massachusetts, USAFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilLondon School of Hygiene & Tropical Medicine. London, UKThe prevalence of Trypanosoma cruzi parasitemia as determined by xenodiagnosis on domestic dogs and cats was correlated with household rates of seroreactivity to T. cruzi and household Panstrongylus megistus infestation in a rural area in northeast Brazil where P. megistus was the only domiciliary triatomine vector. T. cruzi infection was present in about 18% of domestic dogs and cats. Two-thirds of seroactive children below age 10 resided in houses with T. cruzi-infected animals. In houses with a T. cruzi-infected dog or cat, as well as at least one infected P. megistus, the household rate of seroactivity to T. cruzi was five times greather than in houses with non-infected domestic animals and no infected triatomine vectors. Domestic dogs and cats are important reservoirs of T. cruzi in an endemic area where P. megiustus is the only domiciliary triatomine vector