Familial parathyroid tumours-comparison of clinical profiles between syndromes.

INTRODUCTION: Primary hyperparathyroidism (PHPT) caused by parathyroid tumours is mostly sporadic, with a genetic cause identified in 5-10% of cases. Familial parathyroid tumours can be included in complex syndromes, such as multiple endocrine neoplasia (MEN) type 1, 2A and 4 or hyperparathyroidism-jaw tumour syndrome (HPT-JT). OBJECTIVE: Characterisation of the familial parathyroid tumours followed-up at our centre and comparison of the different clinicopathological manifestations between the syndromes. METHODS: Retrospective analysis of 48 patients with familial parathyroid tumours harbouring RET (n = 11), CDC73 (n = 20) and MEN1 (n = 17) germline mutations was performed. RESULTS: Cases of PHPT in MEN2A syndrome presented with lower serum PTH (sPTH) and serum calcium (sCa) levels at diagnosis (sPTH = 108.0 (IQR 53.3) pg/mL, sCa = 10.6 ± 1.1 mg/dL) than MEN1 (sPTH = 196.9 (IQR 210.5) pg/mL, sCa = 11.7 ± 1.2 mg/dL) (p = 0.01, p = 0.03, respectively) or HPT-JT cases (sPTH = 383.5 (IQR 775.8) pg/mL, sCa = 12.9 ± 1.8 mg/dL) (p = 0.01; p < 0.001, respectively). There was a statistical difference in sCa levels between MEN1 and HPT-JT (p = 0.02), but not between sPTH (p = 0.07). The predominant first manifestation of the syndrome in MEN1 was gastroenteropancreatic neuroendocrine tumour (GEP-NET) in 47.1% of the cases, in MEN2A was medullary thyroid cancer (90.9%) and in HPT-JT was PHPT in 85% patients. In MEN1 syndrome, the number of affected parathyroid glands was significantly higher than in MEN2A (p < 0.001) and HPT-JT (p = 0.01). CONCLUSION: The first manifestation of the syndrome in MEN1 cases was GEP-NET and not PHPT. Although presenting at similar ages, patients with MEN2A exhibit less severe biochemical and clinical PHPT at diagnosis than the other familial syndromes.

Correlation of molecular data with histopathological and clinical features in a series of 66 patients with medullary thyroid carcinoma.

PURPOSE: Medullary thyroid carcinoma (MTC) displays a wide variety of histopathological features, and several histological variants have been described. In follicular cell-derived thyroid carcinomas, there is a good correlation between genotype and phenotype. In this study, we investigated whether such a correlation is also present in MTC. METHODS: The histopathological features were evaluated in a series of 66 molecularly characterised tumours and correlated with the clinical characteristics. RESULTS: Most MTC exhibited the classical variant (83.3%). Other variants included spindle cell (6.1%), pseudopapillary (4.5%), paraganglioma-like (3.0%), angiosarcoma-like (1.5%), and oncocytic follicular (1.5%). Tumours were classified into four groups: group 1, with somatic p.Met918Thr and p.Ala883Phe RET mutations; group 2, with other RET mutations; group 3, with RAS mutations; and group 4, without RET or RAS mutations. Tumours from groups 1 and 4 were typically associated with the classical variant, with abundant fibrosis, lymphovascular invasion, extrathyroidal extension, and more advanced stages of disease, whereas group 2 included histological variants other than the classical variant (namely, pseudopapillary and paraganglioma-like), with tumours that were highly cellular, less invasive, and with a better overall prognosis. In tumours from group 4, amyloid deposition was characteristically absent or low. The spindle cell variant appeared only in tumours from group 3, which had high cellularity and a degree of invasion and prognosis intermediate between groups 1 and 2, but better than group 4. The grade of fibrosis correlated directly with the clinical outcome. CONCLUSION: Our results support the idea that a genotype-phenotype correlation does, indeed, exist in MTC. However, further studies are warranted to confirm these findings in a larger sample size.

The role of EIF1AX in thyroid cancer tumourigenesis and progression.

PURPOSE: The EIF1AX gene was recently described as a new thyroid cancer-related gene. Its mutations were mainly reported in poorly differentiated (PDTC) and anaplastic thyroid cancers (ATC), but also in well-differentiated thyroid cancer (WDTC) and in benign thyroid lesions, although less frequently. Our aim was to address whether EIF1AX mutations are present in the different stages of thyroid tumourigenesis (from hyperplasia to well-differentiated and to poorly differentiated/undifferentiated lesions), and to clarify its role in this process. METHODS: We analysed the EIF1AX gene in a series of 16 PDTC and ATC cases with coexistent well-differentiated regions and/or benign lesions. In EIF1AX mutant cases we also assessed the presence of RAS genes mutations. RESULTS: We identified the mutation p.Ala113_splice in the EIF1AX gene in two PDTCs (neither present in the well-differentiated counterparts nor in the benign areas). One of these tumours also evidenced the mutation p.Glu61Arg in NRAS in both poorly and well-differentiated regions, further suggesting that the EIF1AX p.Ala113_splice mutation could be associated with tumoural progression. In another patient we did not find any EIF1AX alteration in the PDTC component, but we detected the EIF1AX p.Gly6_splice mutation in the PTC area (both regions were RAS wild-type). This mutation did not seem to be related with dedifferentiation. CONCLUSIONS: According to our results, distinct mutations on EIF1AX may be related to different phenotypes/behaviours. Despite being a small series, which reflects the difficulty in retrieving PDTC and ATC surgical samples with well-differentiated and/or benign areas, our study may provide new insights into thyroid cancer tumourigenesis and dedifferentiation.

The efficacy of HRAS and CDK4/6 inhibitors in anaplastic thyroid cancer cell lines.

PURPOSE: Anaplastic thyroid carcinomas (ATCs) are non-responsive to multimodal therapy, representing one of the major challenges in thyroid cancer. Previously, our group has shown that genes involved in cell cycle are deregulated in ATCs, and the most common mutations in these tumours occurred in cell proliferation and cell cycle related genes, namely TP53, RAS, CDKN2A and CDKN2B, making these genes potential targets for ATCs treatment. Here, we investigated the inhibition of HRAS by tipifarnib (TIP) and cyclin D-cyclin-dependent kinase 4/6 (CDK4/6) by palbociclib (PD), in ATC cells. METHODS: ATC cell lines, mutated or wild type for HRAS, CDKN2A and CDKN2B genes, were used and the cytotoxic effects of PD and TIP in each cell line were evaluated. Half maximal inhibitory concentration (IC50) values were determined for these drugs and its effects on cell cycle, cell death and cell proliferation were subsequently analysed. RESULTS: Cell culture studies demonstrated that 0.1 µM TIP induced cell cycle arrest in the G2/M phase (50%, p < 0.01), cell death, and inhibition of cell viability (p < 0.001), only in the HRAS mutated cell line. PD lowest concentration (0.1 µM) increased significantly cell cycle arrest in the G0/G1 phase (80%, p < 0.05), but only in ATC cell lines with alterations in CDKN2A/CDKN2B genes; additionally, 0.5 µM PD induced cell death. The inhibition of cell viability by PD was more pronounced in cells with alterations in CDKN2A/CDKN2B genes (p < 0.05) and/or cyclin D1 overexpression. CONCLUSIONS: This study suggests that TIP and PD, which are currently in clinical trials for other types of cancer, may play a relevant role in ATC treatment, depending on the specific tumour molecular profile.

Assistive devices: an effective strategy in non-pharmacological treatment for hand osteoarthritis-randomized clinical trial.

This study aims to evaluate the use of assistive devices as a strategy in non-pharmacological treatment for hand osteoarthritis (HOA). This is a randomized, prospective, parallel, assessor-blinded clinical trial, in which patients with a diagnosis of HOA were randomly allocated to an intervention group (IG), where they received assistive devices for daily life activities, or to a control group (CG), where they received a guideline leaflet with information on joint protection and disease features. The primary outcomes considered were occupational performance, measured by the Canadian Occupational Performance Measure (COPM), and hand function was evaluated through the Score for the Assessment and Quantification of Chronic Rheumatoid Affections of the Hands (SACRAH). The secondary outcomes were pain, measured by the visual analog scale (VAS), and quality of life, measured by the World Health Organization Quality of Life Instrument, Short Form (WHOQOL-BREF). We compared both outcomes before and after interventions and outcomes between groups. Participants from the two groups were assessed at the time of inclusion in the study, 30, and 90 days after initial evaluation. Out of the 39 patients included, 19 were allocated to the IG and 20 to the CG. Only two patients from the CG did not complete the follow-up period. The patients' hand function and occupational performance improved after intervention (30 days-SACRAH-p < 0.05; COPM-p < 0.05; VAS-p < 0.05). When comparing results between the groups, there was a statistical difference in COPM (performance-p < 0.001; and satisfaction-p < 0.001), in the first reevaluation carried out. The use of assistive devices has proved to be an effective alternative in non-pharmacological treatment for HOA. CLINICAL TRIAL REGISTRATION: NCT02667145.

Effects of Maytenus ilicifolia on reproduction and embryo-fetal development in Wistar rats.

Maytenus ilicifolia (Celastraceae), popularly known as espinheira-santa, is a native plant from the Atlantic forest and is commonly used in popular medicine to treat inflammation and as an abortifacient. To evaluate the effects of M. ilicifolia on pregnant rats during the organogenic period (T1) or throughout the gestational period (T2), an extract obtained using an acetone-water mixture at a 70:30 ratio was administered via gavage at a dose of 15.11 mg·kg(-1)·day(-1) over 2 treatment periods (T1 and T2). No clinical signs of maternal toxicity were observed. Term fetuses did not present malformations or anomalies as the number of implantations, reabsorptions, live, and dead fetuses were similar to the control group. In conclusion, M. ilicifolia hydroacetonic extract is non-toxic to pregnant rats and appears to not interfere with the progress of embryo-fetal development.

The characterization of a thermostable and cambialistic superoxide dismutase from Thermus filiformis.

UNLABELLED: The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS-PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0·41, 0·56 and 13·73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3-dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript describes the expression and characterization of a superoxide dismutase (SOD) from Thermus filiformis with thermophilic and cambialistic characteristics. The SODs are among the most potent antioxidants known in nature, and their stability and pharmacokinetics can vary widely in accordance to their biological source. Although the currently clinical research work has been focused on human and bovine SODs, alternative sources may become more biotechnological attractive in the near future. Our study brings new insights for the research field of antioxidant enzymes with potential application on pharmaceutical, cosmetics and food formulations.

Calcium transport in gill cells of Ucides cordatus, a mangrove crab living in variable salinity environments.

Crustaceans show discontinuous growth and have been used as a model system for studying cellular mechanisms of calcium transport, which is the main mineral found in their exoskeleton. Ucides cordatus, a mangrove crab, is naturally exposed to fluctuations in calcium and salinity. To study calcium transport in this species during isosmotic conditions, dissociated gill cells were marked with fluo-3 and intracellular Ca(2+) change was followed by adding extracellular Ca(2+) as CaCl2 (0, 0.1, 0.25, 0.50, 1.0 and 5mM), together with different inhibitors. For control gill cells, Ca(2+) transport followed Michaelis-Menten kinetics with Vmax=0.137±0.001 ∆Ca(2+)i (µM×22.10(4)cells(-1)×180s(-1); N=4; r(2)=0.99); Km=0.989±0.027mM. The use of different inhibitors for gill cells showed that amiloride (Na(+)/Ca(2+) exchange inhibitor) inhibited 80% of Ca(2+) transport in gill cells (Vmax). KB-R, an inhibitor of Ca influx in vertebrates, similarly caused a decrease in Ca(2+) transport and verapamil (Ca(2+) channel inhibitor) had no effect on Ca(2+) transport, while nifedipine (another Ca(2+) channel inhibitor) caused a 20% decrease in Ca(2+) affinity compared to control values. Ouabain, on the other hand, caused no change in Ca(2+) transport, while vanadate increased the concentration of intracellular calcium through inhibition of Ca(2+) efflux probably through the plasma membrane Ca(2+)-ATPase. Results show that transport kinetics for Ca(2+) in these crabs under isosmotic conditions is lower compared to a hyper-regulator freshwater crab Dilocarcinus pagei studied earlier using fluorescent Ca(2+) probes. These kinds of studies will help understanding the comparative mechanisms underlying the evolution of Ca transport in crabs living in different environments.

Papillary thyroid carcinoma of thyroglossal duct cyst: a retrospective analysis.

BACKGROUND: The incidence of thyroglossal duct cyst (TDC) carcinoma is uncommon (approximately 1%) and rarely reported in the literature. Treatment modalities have included tumourectomy, Sistrunk's procedure and/or total thyroidectomy. AIM: To try and determine the most adequate surgical approach for papillary thyroid carcinoma (PTC) arising in TDC. RESULTS: We reviewed the clinical charts of 22 patients with PTC of TDC treated between January 1974 and December 2008 (0.63% of the total of 3458 patients with PTC treated over that period). All patients underwent the Sistrunk's procedure. Fourteen (64%) were submitted to total thyroidectomy and 11 of these patients were ablated with lodine131. Seven (50%) of the 14 patients treated with total thyroidectomy had tumour both in the thyroid gland and in TDC but lymph node metastases were present only in four. None of the patients died of the disease and all of them are still alive without recurrence with a mean follow-up of 8 years (range: 2-27 years). The mean survival rate of the patients submitted to total thyroidectomy (n= 14) was not statistically different from that of patients treated with the Sistrunk's procedure alone (9.23 +/- 7.65 vs. 8.95 +/- 6.22, p= 0.940). CONCLUSION: Papillary thyroid carcinoma arising in thyroglossal duct cysts is a very rare malignant tumour. In spite of the multifocal character of several of our cases, their analysis showed that the prognosis in the vast majority of patients with TDC carcinoma is excellent. Moreover, we found no relation between outcome and surgical procedure.

Histopathology of experimental myiasis in mice as a result of infestation and experimental implantation of Dermatobia hominis larvae.

A laboratory model of myiasis as a result of Dermatobia hominis (L.) larvae was developed using mice as hosts. Mice in three groups were each infested with one newly hatched larva and skin biopsies processed for histopathology at 4, 12, and 20 d postinfestation (dpi). Mice in three other groups were each subjected to implantation of one larva collected from an infested (donor) mouse at 4, 12, and 20 dpi. Skin lesions of these receptor mice were then assessed at 10, 14, and 6 d postimplantation (dpimp), respectively. The inflammatory process in infested mice at 4 dpi was discrete, consisting of a thin necrotic layer around the larva, edema, many neutrophils, few eosinophils, mast cells, and proliferation of fibroblasts. At 12 dpi, there was a thicker necrotic layer, edema, many neutrophils and eosinophils, few mast cells, neoformation of capillaries, proliferation of the endothelium and fibroblasts, and early stages of fibrosis. These histopathological characteristics together with fibrosis were observed over a large area of the lesion at 20 dpi. Mice submitted to larval implantations demonstrated similar skin histopathology to that seen in the infested rodents, 10 dpimp corresponding to 12 dpi and 6 or 14 dpimp to 20 dpi. In all mice, the progressive acute inflammatory process followed a sequence linked to factors such as size of larvae and presence of secretory-excretory products. Both infested mice and those implanted experimentally with D. hominis larvae were shown to be suitable models for the study of the parasite-host relationship in this important zoonotic myiasis.

Baffled primary facultative ponds with inlets and outlets set at different levels treating domestic wastewater in northeast Brazil.

This study evaluates the performance of four 2.3 m deep pilot-scale, independently loaded, primary facultative ponds treating predominantly domestic sewage in northeast Brazil. The ponds contained longitudinal baffles giving different length to width ratios from 3.55 to 32.4. The ponds had mean hydraulic retention times of ~15 days, and mean surface organic loadings of 330 kg BOD(5).ha.d(-1) during the first experimental phase and 375 kg BOD(5).ha.d(-1) in the second. The vertical inlets and outlets pipes were positioned at 1.8 m and 5 cm respectively below the pond surface in the first phase and at 50 cm and 1.8 m respectively in the second. All the ponds functioned as efficient primary facultative ponds but statistical analysis demonstrated no differences in effluent quality for most of the parameters measured for the various configurations of baffles and inlet and outlet depths. All behaved similarly to the unbaffled pond. The only exceptions were suspended solids and chlorophyll a concentrations which tended to be lower for all combinations of baffles with the outlets set 1.8 m below the surface. This study suggested that the longitudinal baffling of primary facultative ponds when using vertical inlets and outlets may well not significantly improve pond performance.

The removal of ammonia from sanitary landfill leachate using a series of shallow waste stabilization ponds.

This study evaluated the efficiency of a shallow (0.5 m deep) waste stabilization pond series to remove high concentrations of ammonia from sanitary landfill leachate. The pond system was located at EXTRABES, Campina Grande, Paraiba, Northeast Brazil. The pond series was fed with sanitary landfill leachate transported by road tanker to the experimental site from the sanitary landfill of the City of Joao Pessoa, Paraiba. The ammoniacal-N surface loading on the first pond of the series was equivalent to 364 kg ha(-1) d(-1) and the COD surface loading equivalent to 3,690 kg ha(-1) d(-1). The maximum mean ammonia removal efficiency was 99.5% achieved by the third pond in the series which had an effluent concentration of 5.3 mg L(-1) ammoniacal-N for an accumulative HRT of 39.5 days. The removal process was mainly attributed to ammonia volatilization (stripping) from the pond surfaces as a result of high surface pH values and water temperatures of 22-26°C. Shallow pond systems would appear to be a promising technology for stripping ammonia from landfill leachate under tropical conditions.

Soluble inflammatory markers as predictors of liver histological changes in patients with chronic hepatitis C virus infection.

Host immune response seems to be mainly responsible for the progression of liver disease among patients with hepatitis C virus (HCV) infection. Immune activation involves the release of cytokines and their receptors that can be measured in plasma samples. The study aimed to evaluate the association between plasma levels of chemokines and soluble tumor necrosis factor receptors (sTNFR) and liver histological changes among patients with chronic HCV infection. Seventy-one treatment-naive patients were included. Plasma levels of CCL2, CCL3, CCL11, CCL24, CXCL9, CXCL10, sTNFR1, and sTNFR2 were measured and liver histological findings were reviewed. Plasma levels of CXCL9, sTNFR1, and sTNFR2 were significantly associated with liver fibrosis, with higher median levels found among patients with moderate/severe fibrosis (F >or= 2) if compared to those with no or mild fibrosis (p = 0.014; p = 0.012; p = 0.009, respectively). Plasma sTNFR2 levels were significantly associated with necroinflammatory activity, with higher median levels among patients with moderate/severe activity (A >or= 2) if compared to those with no or mild activity (2.34 ng/mL vs. 1.99 ng/mL; p = 0.019). In conclusion, plasma levels of CXCL9, sTNFR1, and sTNFR2 were independently associated with liver histological changes, suggesting a role of TNF activation and Th1-type cell-mediated immune response in the pathogenesis of HCV infection.

Gene expression profiling associated with the progression to poorly differentiated thyroid carcinomas.

BACKGROUND: Poorly differentiated thyroid carcinomas (PDTC) represent a heterogeneous, aggressive entity, presenting features that suggest a progression from well-differentiated carcinomas. To elucidate the mechanisms underlying such progression and identify novel therapeutic targets, we assessed the genome-wide expression in normal and tumour thyroid tissues. METHODS: Microarray analyses of 24 thyroid carcinomas - 7 classic papillary, 8 follicular variants of papillary (fvPTC), 4 follicular (FTC) and 5 PDTC - were performed and correlated with RAS, BRAF, RET/PTC and PAX8-PPARG alterations. Selected genes were validated by quantitative RT-PCR in an independent set of 28 thyroid tumours. RESULTS: Unsupervised analyses showed that gene expression similarity was higher between PDTC and fvPTC, particularly for tumours harbouring RAS mutations. Poorly differentiated thyroid carcinomas presented molecular signatures related to cell proliferation, poor prognosis, spindle assembly checkpoint and cell adhesion. Compared with normal tissues, PTC had 307 out of 494 (60%) genes over-expressed, FTC had 137 out of 171 (80%) genes under-expressed, whereas PDTC had 92 out of 107 (86%) genes under-expressed, suggesting that gene downregulation is involved in tumour dedifferentiation. Significant UHRF1 and ITIH5 deregulated gene expression in PDTC, relatively to normal tissues, was confirmed by quantitative RT-PCR. CONCLUSION: Our findings suggest that fvPTC are possible precursors of PDTC. Furthermore, UHRF1 and ITIH5 have a potential therapeutic/prognostic value for aggressive thyroid tumours.

Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas.

Screening of REarranged during Transfection (RET) gene mutations has been carried out in different series of sporadic medullary thyroid carcinomas (MTC). RET-positive tumours seem to be associated to a worse clinical outcome. However, the correlation between the type of RET mutation and the patients' clinicopathological data has not been evaluated yet. We analysed RET exons 5, 8, 10-16 in fifty-one sporadic MTC, and found somatic mutations in thirty-three (64.7%) tumours. Among the RET-positive cases, exon 16 was the most frequently affected (60.6%). Two novel somatic mutations (Cys630Gly, c.1881del18) were identified. MTC patients were divided into three groups: group 1, with mutations in RET exons 15 and 16; group 2, with other RET mutations; group 3, having no RET mutations. Group 1 had higher prevalence (P=0.0051) and number of lymph node metastases (P=0.0017), and presented more often multifocal tumours (P=0.037) and persistent disease at last control (P=0.0242) than group 2. Detectable serum calcitonin levels at last screening (P=0.0119) and stage IV disease (P=0.0145) were more frequent in group 1, than in the other groups. Our results suggest that, among the sporadic MTC, cases with RET mutations in exons 15 and 16 are associated with the worst prognosis. Cases with other RET mutations have the most indolent course, and those with no RET mutations have an intermediate risk.

Lymphadenopathy and expression of nodal mast cells and eosinophils in the myiasis by human bot fly.

Wistar rats (Rattus norvegicus) infested with Dermatobia hominis (L. Jr., 1781) had their axillary lymph nodes removed and histopathologically processed. Follicular hyperplasia in the germinal center was noted from 2 d postinfestation (dpi), exhibiting a high number of centerblasts, mitotic and apoptotic cells, and a thin parafollicular area. The paracortex showed hyperplasia rich in dendritic cells, immunoblasts, and endothelial venules, with diapedesis seen from 4 dpi onward. Hyperplasia of the medullar sinus also was first observed at this point, as well as dilated lymphatic sinus, lymph, macrophages, neutrophils, mast cells, and eosinophils. Medullar strings were expanded and filled with immunoblasts, mitotic cells, and plasmocytes. Lymphadenitis was not observed. The expression of mast cells was similar for both myiasis-affected and control rats but increased significantly (mastocytosis) at 7 and 15 d postlarval emergence (dple). Eosinophilia was observed at 4, 10, 15, 20, and 28 dpi as well as at 2, 7, and 15 dple, particularly on the last three observations of dpi and the earliest dple. This experimental approach allowed progressive tissue reactions in the lymph nodes to be monitored during myiasis, particularly those involving mast cells and eosinophils. These reactions abated and complete repair was observed at 60 dple.

Presence of the anther gland is a key feature in pollination of the early-branching papilionoids Dipteryx alata and Pterodon pubescens (Leguminosae).

The presence of glandular appendages in the anthers is a rare condition in angiosperms. In Leguminosae it occurs in species of the Mimosoid clade and in early-branching clades of papilionoids such as Dipterygeae. In Dipterygeae such appendages surprisingly exhibit a secretory cavity instead of secretory emergences as is the case for the Mimosoid clade. Thus, the objective of this study was to elucidate the function of anther glands in Dipteryx alata and Pterodon pubescens, species in the Dipterygeae clade that exhibit a pollen release mechanism that is intermediate between the explosive and valvular types. Flower buds and flowers were processed for surface, anatomical, histochemical and ultrastructural analyses. Anther glands consist of a cavity secreting sticky substances (oleoresins and polysaccharides) that play a key role during the flower's lifespan by aggregating pollen grains and attaching them to the floral visitor's body. Other floral features that are important for understanding the pollen release mechanism that is intermediate between the valvular and the explosive types are: (i) keel petals intertwined with tector trichomes; (ii) glandular appendages in the abaxial and lateral sepals and in petals composed of secretory ducts; and (iii) a continuous secretion process of the anther glands followed by an asynchronous dehiscence of anthers. The well-adapted papilionoid flag blossom with anther glands and keel petals intertwined with trichomes provided the foundation for a successful canalisation toward a pollen release mechanism intermediate between the explosive and valvular types inside early-branching papilionoids.

Identification and characterization of two novel germline RET variants associated with medullary thyroid carcinoma.

Activating germline mutations in the RET proto-oncogene are responsible for about 98 % of the familial forms of medullary thyroid carcinoma (MTC), which represent 25 % of all MTC cases. The search for germline mutations in this gene is important for the recognition of hereditary forms of MTC and further identification of at-risk relatives who may benefit from early clinical intervention. Genotype-phenotype correlations are well established for most disease-causing RET mutations, allowing risk stratification. The association of a new RET variant with the MTC phenotype and familial predisposition requires the assessment of its functional and clinical significance. The aim of this study was to evaluate the oncogenic potential of two newly identified RET germline variants associated with late-onset MTC. In vitro functional assays were designed to address the transforming potential of novel RET variants, through their expression in non-transformed cells, and comparing their effect with wild-type RET. The new variants were identified in codons 515 (p.C515W) and 636 (p.T636M) located, respectively, in exons 8 and 11, thus resulting in amino acid substitutions in the extracellular region of the tyrosine kinase receptor RET. Through functional assays, we observed increased cell growth and proliferation, loss of contact inhibition, and a stimulation of cell migration, suggesting that these new RET variants hold some relevant transforming potential. The transforming potential of these novel RET variants was of low-grade, when compared to that of RET MEN2A-causing mutation p.C634R, probably explaining the mild phenotype characterized by late onset and low clinical aggressiveness.

Some forms of big big prolactin behave as a complex of monomeric prolactin with an immunoglobulin G in patients with macroprolactinemia or prolactinoma.

The purpose of this study was to characterize the structure of big big PRL (bb-PRL) in patients with macroprolactinemia or prolactinomas. Serum samples from these patients were fractionated by Sephadex G-100 chromatography, and PRL was measured in the eluate by an immunoradiometric assay. The fractions containing bb-PRL were subjected to affinity chromatography with an antihuman immunoglobulin G (IgG) agarose column. PRL and IgG were assayed in the fractions obtained after affinity chromatography by immunoradiometric assay and radial immunodiffusion, respectively. bb-PRL was also immunoprecipitated with an antihuman PRL antibody, followed by polyacrylamide gel electrophoresis and Western blotting. We found that an average of 60% (range, 27-87%) of bb-PRL from patients with macroprolactinemia was retained by the agarose, indicating that this form of PRL contains an IgG. In one of the patients with prolactinoma, the big big form constituted 76% of the total PRL immunoreactivity. Most (75%) of the bb-PRL from this patient behaved as an IgG after affinity chromatography. In the two other patients with prolactinoma, we found that 22% and 25% of the bb-PRL, which represented only 2% and 3% of the total PRL in the serum, reacted as an IgG. In both groups of patients, the 23-kilodalton form of PRL was detected after the immunoprecipitation of bb-PRL. These results show that bb-PRL is in part a complex of 23-kilodalton PRL with an IgG and not an antibody mimicking the actions of PRL, as has been demonstrated for some large forms of growth-hormone.

A possible role for D8/PSF-A-like sequences in lactotroph versus somatotroph expression of the human prolactin gene.

The transcription factor GHF-1/Pit-1 is essential for the expression of GH and prolactin (PRL) by somatotrophs and lactotrophs respectively. However, PRL is not expressed in mature somatotrophs despite the presence of GHF-1/Pit-1. A possible mechanism is the presence of a somatotroph-specific repressor in the 5'-flanking sequences of the PRL gene. The region -3500/-1750 of the human (h) PRL gene is associated with negative regulatory activity and contains an element, designated D8, that resembles repressor PSF-A sequences which are located in the distal upstream region of placental members of the human GH family. An internal deletion of D8 sequences resulted in a significant stimulation of promoter activity in somatotroph GC (P < 0.005) and somatolactotroph-like GH3 and GH4C1 cells (P < 0.05), but not lactotroph-like 235-1 cells after gene transfer. However, D8 binding was observed by nuclease protection with lactotroph- as well as somatotroph-like cell nuclear protein. Although proteins that bind to the D8 element appear ubiquitous, this element does yield tissue-specific complexes in mobility shift assays. Further, competition studies do not suggest an interaction between GHF-1/Pit-1 and D8 proteins. The hPRL D8 element was inserted upstream of a thymidine kinase promoter and used to transfect pituitary and non-pituitary HeLa cells, to assess intrinsic repressor activity and/or promoter specificity. Although no repression was observed, a significant ninefold increase in expression was observed in HeLa cells (P < 0.001) which was at least twofold greater than observed in any of the pituitary cell lines tested. These results implicate D8 in the somatotroph-specific repression of hPRL; however, they also suggest that D8 can act as a stimulator as well as a repressor, depending on the interaction of a ubiquitous D8 factor forming promoter and cell-specific complexes with other elements/factors.