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Medical palpation is a key skill for clinicians. It is typically trained using animal and synthetic models, which however raise ethical concerns and produce high volumes of consumables. An alternative could be visuo-haptic simulations, despite their training efficacy has not been proved yet. The assessment of palpatory skills requires objective methods, that can be achieved by combining performance metrics with electroencephalography (EEG). The goals of this study were to: (i) develop a visuo-haptic system to train nodule detection, combining a Geomagic Touch haptic device with a visuo-haptic simulation of a skin patch and a nodule, implemented using SOFA framework; (ii) assess whether this system could be used for training and evaluation. To do so, we collected performance and EEG data of 19 subjects performing multiple repetitions of a nodule detection task. Results revealed that participants could be divided in low and high performers; the former applied a greater pressure when looking for the nodule and showed a higher EEG alpha (8.5 - 13 Hz) peak at rest; The latter explored the skin remaining on its surface and were characterized by low alpha power. Furthermore, alpha power positively correlated with error and negatively with palpation depth. Altogether, these results suggest that alpha power might be an indicator of performance, denoting an increase in vigilance, attention, information processing, cognitive processes, and engagement, ultimately affecting strategy and performance. Also, the combination of EEG with performance data can provide an objective measure of the user's palpation ability.

A task force of the United Italian society of Endocrine Surgery (SIUEC) was commissioned to review the position statement on diagnostic, therapeutic and health‑care management protocol in parathyroid surgery published in 2014, at the light of new technologies, recent oncological concepts, and tailored approaches. The objective of this publication was to support surgeons with modern rational protocols of treatment that can be shared by health-care professionals, taking into account important clinical, healthcare and therapeutic aspects, as well as potential sequelae and complications. The task force consists of 12 members of the SIUEC highly trained and experienced in thyroid and parathyroid surgery. The main topics concern diagnostic test and localization studies, mode of admission and waiting time, therapeutic pathway (patient preparation for surgery, surgical treatment, postoperative management, management of major complications), hospital discharge and patient information, outpatient care and follow-up, outpatient initial management of patients with pHPT., (© 2024. The Author(s).)

Background: Since its outbreak in early 2020, the COVID-19 pandemic has diverted resources from non-urgent and elective procedures, leading to diagnosis and treatment delays, with an increased number of neoplasms at advanced stages worldwide. The aims of this study were to quantify the reduction in surgical activity for indeterminate thyroid nodules during the COVID-19 pandemic; and to evaluate whether delays in surgery led to an increased occurrence of aggressive tumours., Methods: In this retrospective, international, cross-sectional study, centres were invited to participate in June 22, 2022; each centre joining the study was asked to provide data from medical records on all surgical thyroidectomies consecutively performed from Jan 1, 2019, to Dec 31, 2021. Patients with indeterminate thyroid nodules were divided into three groups according to when they underwent surgery: from Jan 1, 2019, to Feb 29, 2020 (global prepandemic phase), from March 1, 2020, to May 31, 2021 (pandemic escalation phase), and from June 1 to Dec 31, 2021 (pandemic decrease phase). The main outcomes were, for each phase, the number of surgeries for indeterminate thyroid nodules, and in patients with a postoperative diagnosis of thyroid cancers, the occurrence of tumours larger than 10 mm, extrathyroidal extension, lymph node metastases, vascular invasion, distant metastases, and tumours at high risk of structural disease recurrence. Univariate analysis was used to compare the probability of aggressive thyroid features between the first and third study phases. The study was registered on ClinicalTrials.gov, NCT05178186., Findings: Data from 157 centres (n=49 countries) on 87 467 patients who underwent surgery for benign and malignant thyroid disease were collected, of whom 22 974 patients (18 052 [78·6%] female patients and 4922 [21·4%] male patients) received surgery for indeterminate thyroid nodules. We observed a significant reduction in surgery for indeterminate thyroid nodules during the pandemic escalation phase (median monthly surgeries per centre, 1·4 [IQR 0·6-3·4]) compared with the prepandemic phase (2·0 [0·9-3·7]; p<0·0001) and pandemic decrease phase (2·3 [1·0-5·0]; p<0·0001). Compared with the prepandemic phase, in the pandemic decrease phase we observed an increased occurrence of thyroid tumours larger than 10 mm (2554 [69·0%] of 3704 vs 1515 [71·5%] of 2119; OR 1·1 [95% CI 1·0-1·3]; p=0·042), lymph node metastases (343 [9·3%] vs 264 [12·5%]; OR 1·4 [1·2-1·7]; p=0·0001), and tumours at high risk of structural disease recurrence (203 [5·7%] of 3584 vs 155 [7·7%] of 2006; OR 1·4 [1·1-1·7]; p=0·0039)., Interpretation: Our study suggests that the reduction in surgical activity for indeterminate thyroid nodules during the COVID-19 pandemic period could have led to an increased occurrence of aggressive thyroid tumours. However, other compelling hypotheses, including increased selection of patients with aggressive malignancies during this period, should be considered. We suggest that surgery for indeterminate thyroid nodules should no longer be postponed even in future instances of pandemic escalation., Funding: None., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Up to 25% of patients with acute diverticulitis develop complicated disease. Colocutaneous fistula with lower limb fasciitis secondary to complicated diverticulitis is a rare event. A 71-year-old woman with Class 3 obesity and Type 2 diabetes was admitted to the hospital because of left lower limb fasciitis associated with acute sigmoid diverticulitis complicated by covered perforation. The fasciitis was treated with multiple fasciotomies, antibiotics and hyperbaric oxygen therapy. The patient was readmitted 25 days after discharge because of the formation of a left leg colocutaneous fistula associated with an enterocolic fistula. Patient underwent sigmoid resection with primary anastomosis and ileal loop repair. Three-month follow-up showed fistula healing and absence of symptoms. Fasciitis secondary to acute diverticulitis is a rare clinical scenario. Although our therapeutic strategy was successful, the optimal treatment timing and surgical technique for fasciotomy and colon resection remain to be assessed., (Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2020.)

Background and Objectives: The increasing use of arm totally implantable vascular access devices for breast cancer patients who require chemotherapy has led to a greater risk of complications and failures and, in particular, to upper extremity deep vein thrombosis. This study aims to investigate the outcomes of the arm peripherally inserted central catheter-PORT technique in breast cancer patients., Methods: The peripherally inserted central catheter-PORT technique is an evolution of the standard arm-totally implantable vascular access device implant based on guided ultrasound venous access in the proximal third of the upper limb with subsequent placement of the reservoir at the middle third of the arm. A prospective study was conducted on 418 adult female breast cancer patients undergoing chemotherapy. The primary study outcome was peripherally inserted central catheter-PORT failure., Results: Median follow-up was 215 days. Complications occurred in 29 patients (6.9%) and failure resulting in removal of the device in 11 patients (2.6%). The main complication we observed was upper extremity deep vein thrombosis, 10 (2.4%); all patients were rescued by anticoagulant treatment without peripherally inserted central catheter-PORT removal. The main reason for removal was reservoir pocket infection: 4 (0.9%) with an infection rate of 0.012 per 1000 catheter days. Cumulative 1-year risk of failure was 3.6% (95% confidence interval, 1.3%-7.1%). With regard to the patients' characteristics, body mass index <22.5 was the only significant risk for failure ( p = 0.027)., Conclusion: The peripherally inserted central catheter-PORT is a safe vascular device for chemotherapy delivery that achieves similar clinical results as traditional long-term vascular access devices (peripherally inserted central catheter and arm totally implantable vascular access device, in particular) in breast cancer patients.

Nerve biopsy represents the conclusive step in the diagnostic work-up of peripheral neuropathies, and its diagnostic yield is still debated. The aim of this study is to consider the impact of nerve biopsy on reaching a useful diagnosis in different peripheral neuropathies and its changing over time. We retrospectively analyzed 1,179 sural nerve biopsies performed in the period 1981-2017 at Neurological Clinic of Policlinico San Martino (Genoa). We relied on medical records and collected both clinical and pathological data in a database. Biopsy provided univocal diagnoses in 53% of cases (with an increase over time), multiple diagnostic options in 14%, while diagnosis was undetermined in 33% (undetermined reports decreased during the years). In 57% of patients, the pre-biopsy suspicion was confirmed, while in 43% sural biopsy modified the clinical diagnosis. The highest yield was in axonal neuropathies (29% undetermined reports vs. 40% in demyelinating and 48% in mixed neuropathies). In 68% of patients with vasculitic neuropathy, this etiology was already suspected, whereas in 32% nerve biopsy modified the clinical diagnosis. During the years, the number of annually performed biopsies decreased significantly ( p = 0.007), with an increase in the mean age of patients ( p < 0.0001). The percentage of hereditary neuropathies had a significant decrease ( p = 0.016), while the rate of vasculitic and chronic inflammatory neuropathies increased ( p < 0.0001). This is the largest Italian study addressing the yield of sural nerve biopsy. During the years, we observed a progressive refinement of the indication of this procedure, which confirms its utility for interstitial neuropathies, particularly if non-systemic vasculitic neuropathy is suspected., (Copyright © 2019 Prada, Massucco, Venturi, Geroldi, Bellone, Mandich, Minuto, Varaldo, Mancardi, Grandis and Schenone.)

Background: This study investigates the incidence of post-operative complications and risk factors of sentinel lymph node biopsy (SLNB) in melanoma patients., Methods: A retrospective cohort study was conducted at a single cancer institution on 408 consecutive SLNBs., Results: Fifty-five post-operative complications occurred in 39 (9.5%) patients and included: wound infection in 24 (5.9%), seroma and lymphorrhea in 15 (3.7%), wound dehiscence in seven (1.7%), lymphocele in six (1.5%) and others in three (0.7%). Univariate analysis failed to identify possible risk factors (i.e. gender, age, lymph node region, number of excised lymph nodes, Breslow index, pT levels, comorbidities, length of surgery and hospital stay). Metastatic sentinel nodes occurred in four of 135 (3%) patients with thin melanoma (Breslow <1 mm) and in 68 of 262 (25.9%) patients with Breslow >1 mm., Conclusion: For patients with thin melanoma in whom the incidence of lymph node metastasis is low, the reported post-operative morbidity of almost 10% of SLNB highlights the need for careful patient selection., (© 2019 Royal Australasian College of Surgeons.)

We report a case of an elderly woman presenting with a huge cervical mass invading the tracheal lumen. Diagnosed as invasive poorly differentiated thyroid cancer, after an endotracheal biopsy, stenting and radiotherapy, it was judged eligible for total thyroidectomy, but surgery was delayed due to pulmonary thromboembolism. The patient was therefore treated with lenvatinib with a neoadjuvant intent until hemodynamic stability was obtained. Thyroidectomy and radioiodine therapy were then performed and the postdose scan revealed an area of modest uptake in the anterior part of the neck. The patient is now in a good clinical status and she continues her follow-up program without any adjuvant therapy.

Medullary thyroid carcinomas (MTCs) are rare thyroid tumors occurring in both sporadic and hereditary forms, whose pathogenesis is related to RET proto-oncogene alterations. MTCs originate from parafollicular cells, which produce calcitonin that represents the biochemical activity of MTC. Total thyroidectomy is the main treatment for MTC and often cures patients with confined diseases. In the presence of metastasis, the therapeutic approach depends on the rate of disease progression. We report a case of a 54-year-old female with a single, incidentally discovered, thyroid nodule of 1 cm, classified as suspicious MTC after a stimulation test with intravenous (iv) calcium. After surgery, we examined the nodule using immunohistochemistry, immunofluorescence, and electron microscopy. In addition to calcitonin, we found that it expressed intracellular positivity for the tyrosine kinase RTK receptors ERBB1 and ERBB2. Consistently with MTC features, the ultrastructural examination of the tumor displayed heterogeneous spindle-shaped cells containing two groups of secretory granules. Because of the significant correlation found between high ERBB1/ERBB2 levels in MTCs and extrathyroidal growth, the detection of ERBB1 and ERBB2 expression suggests that the two oncoproteins may be involved in the tumor proliferative responses and/or in the differentiation of parafollicular C-cells. The biological, prognostic, and therapeutic significance of these patterns would merit further investigations., Competing Interests: The authors declare no conflict of interest.

The optimal method for BRAF mutation detection remains to be determined despite advances in molecular detection techniques. The aim of this study was to compare, against classical Sanger sequencing, the diagnostic performance of two of the most recently developed, highly sensitive methods: BRAF V600E immunohistochemistry (IHC) and peptide nucleic-acid (PNA)-clamp qPCR. BRAF exon 15 mutations were searched in formalin-fixed paraffin-embedded tissues from 86 papillary thyroid carcinoma using the three methods. The limits of detection of Sanger sequencing in borderline or discordant cases were quantified by next generation sequencing. BRAF mutations were found in 74.4 % of cases by PNA, in 71 % of cases by IHC, and in 64 % of cases by Sanger sequencing. Complete concordance for the three methods was observed in 80 % of samples. Better concordance was observed with the combination of two methods, particularly PNA and IHC (59/64) (92 %), while the combination of PNA and Sanger was concordant in 55 cases (86 %). Sensitivity of the three methods was 99 % for PNA, 94.2 % for IHC, and 89.5 % for Sanger. Our data show that IHC could be used as a cost-effective, first-line method for BRAF V600E detection in daily practice, followed by PNA analysis in negative or uninterpretable cases, as the most efficient method. PNA-clamp quantitative PCR is highly sensitive and complementary to IHC as it also recognizes other mutations besides V600E and it is suitable for diagnostic purposes.

This retrospective comparative multicenter study aims to analyze the impact on patient outcomes of total thyroidectomy (TT) performed by resident surgeons (RS) with close supervision and assistance of attending surgeons (AS).All patients who underwent TT between 2009 and 2013 in 10 Units of endocrine surgery (8 in Italy, 1 in France, and 1 in UK) were evaluated. Demographic data, preoperative diagnosis, extension of goiter, type of surgical access, surgical approach, operative time, use and duration of drain, length of hospitalization, histology, and postoperative complications were recorded. Patients were divided into 3 groups: A, when treated by an AS assisted by an RS; B and C, when treated by a junior and a senior RS, respectively, assisted by an AS.The 8908 patients (mean age 51.1 ± 13.6 years), with 6602 (74.1%) females were enrolled. Group A counted 7092 (79.6%) patients, Group B 261 (2.9%) and Group C 1555 (17.5%). Operative time was significantly greater (P < 0.001) in B (101.3 ± 43.0 min) vs A (71.8 ± 27.6 min) and C (81.2 ± 29.9 min). Duration of drain was significantly lower (P < 0.001) in A (47.4 ± 13.2 h) vs C (56.4 ± 16.5 h), and in B (42.8 ± 14.9 h) vs A and C. Length of hospitalization was significantly longer (P < 0.001) in C (3.8 ± 1.8 days) vs B (2.4 ± 1.0 days) and A (2.6 ± 1.5 days). No mortality occurred. Overall postoperative morbidity was 22.3%: it was significantly higher in B vs A (29.5% vs 22.3%; odds ratio [OR] 1.46, 95% confidence interval [CI] 1.11-1.92, P = 0.006) and C (21.3%; OR 1.55, 95% CI 1.15-2.07, P = 0.003). No differences were found for recurrent laryngeal nerve palsy, hypoparathyroidism, hemorrhage, and wound infection. The adjusted ORs in multivariate analysis showed that overall morbidity remained significantly associated with Group B vs A (OR 1.48, 95% CI 1.12-1.96, P = 0.005) and vs C (OR 1.60, 95% CI 1.19-2.17, P = 0.002), while no difference was observed in Group A vs B + C.TT can be safely performed by residents correctly supervised. Innovative gradual training in dedicated high-volume hospitals should be proposed in order to allow adequate autonomy for the RS and safeguard patient outcome., Competing Interests: The authors have no funding and conflicts of interest to disclose.

Purpose: The simultaneous management of primary colorectal cancer and synchronous liver metastases has been reported extensively in open surgery. Data regarding feasibility, safety, and outcomes of the laparoscopic procedure is emerging from the experience of a few surgical centers. This paper aims at discussing the technique and results of a one-step laparoscopic approach for colorectal cancer and liver metastases resection on a series of 35 patients., Methods: Between January 2008 and December 2013, 18 males and 17 females (median age 71 years) underwent colorectal and hepatic laparoscopic resection for colorectal metastatic cancer., Results: Thirty-five colorectal resections and 66 liver resections were performed; no conversion to open surgery has been indicated. Median blood loss was 200 ml, median operative time 240 min, and median hospital stay was 8 days (range 4-30). According to Clavien-Dindo classification, two class II complications, two class IIIb complications, and one class IV complication were recorded. Two high-risk patients died within 30 days from surgery., Conclusions: This series confirms the feasibility of synchronous laparoscopic colorectal and hepatic resections. To ensure the best outcomes, a careful selection of patients is needed. However, most patients can benefit from this surgical approach.

Parathyroid surgery underwent significant innovations in the past 20 years, after both the improvement of the imaging techniques used to localize abnormal parathyroids (ultrasonography and MIBI scintiscan) and the possibility of the intraoperative PTH assay. These two tools, in experienced hands, can correctly differentiate uniglandular (more than 85% of the cases of primary hyperparathyroidism) from multiglandular disease. These technological improvements led to the possibility of limiting the surgical exploration to the single parathyroid responsible for the hyperparathyroidism in the majority of cases, avoiding unnecessary bilateral exploration that might increase both the morbidity of the surgery and its global costs. Furthermore, from a technical point of view, the philosophy of a minimally invasive surgery has also been applied to parathyroidectomy and several techniques have been described which are commonly considered minimally invasive. In this paper, the authors want to summarize their indications to perform a focused parathyroidectomy vs. a more traditional bilateral exploration, their definition of a minimally invasive parathyroidectomy and finally, it is given an overview of the techniques currently used for a parathyroidectomy.

Context and Objective: We have studied the antitumor activity of a novel cyclic amide, CLM94, with anti-vascular endothelial growth factor (VEGF) receptor-2 and antiangiogenic activity in primary anaplastic thyroid cancer (ATC) cells in vitro and in vivo., Design and Main Outcome Measures: CLM94 was tested: 1) in two human cell lines (HMVEC-d, dermal microvascular endothelial cells; and 8305C, undifferentiated thyroid cancer) at 0.001-100 μm; 2) in ATC cells at the concentrations of 10, 30, and 50 μm; and 3) in an ATC cell line (AF) in CD nu/nu mice., Results: CLM94 significantly inhibited VEGF receptor-2 and epidermal growth factor receptor phosphorylation in HMVEC-d and proliferation in HMVEC-d and 8305C cells. A significant reduction of proliferation with CLM94 in ATC cells (P < 0.01, ANOVA) and a slight but significant reduction of proliferation with CLM94 30 and 50 μm in normal thyroid follicular cells (P < 0.01, ANOVA) were shown. CLM94 increased the percentage of apoptotic ATC cells dose-dependently (P < 0.001, ANOVA) and inhibited migration (P < 0.01) and invasion (P < 0.001). AF cell line was injected sc in CD nu/nu mice, and tumor masses became detectable 25 d afterward. CLM94 (40 mg/kg · d) significantly inhibited tumor growth (starting 10 d after the beginning of treatment). CLM94 significantly decreased the VEGF-A gene expression in the AF cell line and the VEGF-A protein and microvessel density in AF tumor tissues., Conclusions: The antitumor and antiangiogenic activity of a new "cyclic amide" compound, CLM94, is very promising in ATC, opening the way to a future clinical evaluation.

Peroxisome proliferator-activated receptors (PPAR)α have been shown to exert immunomodulatory effects in autoimmune disorders; no study evaluated the effect of PPARα activation in Graves' ophthalmopathy (GO). We show the presence of PPARα, δ and γ in GO fibroblasts and preadipocytes. PPARα activators have a potent inhibitory action on the secretion of CXCL9 and CXCL11 chemokines (induced by IFNγ and TNFα) in fibroblasts and preadipocytes. The potency of the used PPARα agonists was maximum on the secretion of CXCL11 (67% inhibition by fenofibrate) in fibroblasts. The relative potency of the compounds in GO fibroblasts was different with each chemokine. PPARα agonists were stronger inhibitors of CXCL9 and CXCL11 (in GO fibroblasts and preadipocytes) than PPARγ activators. This study first shows that PPARα activators inhibit CXCL9 and CXCL11 chemokines in normal and GO fibroblasts and preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Anaplastic thyroid cancer (ATC) is often incurable because it doesn't respond to radioiodine, radiotherapy or chemotherapy, and new therapeutic approaches are needed. Peroxisome proliferator-activated receptor-gamma (PPARg) gene and protein are present in ATC cells, and PPARg ligands inhibit cell proliferation, induce apoptosis, and also down regulate the invasive potential of ATC cells. Also, inhibitors of the Aurora serine/threonine kinases have antineoplastic effect on ATC cells in vitro and on ATC xenografts. Tyrosine kinases inhibitors are actually under evaluation for the treatment of ATC, for example imanitib or sorafenib. Other studies have focused on evaluating antiangiogenic agents for treatment of ATC. These agents include: combretastatin A4 phosphate, aplidin, PTK787/ZK222584, and human VEGF monoclonal antibodies (bevacizumab, cetuximab). Small-molecule adenosine triphosphate (ATP) competitive inhibitors directed intracellularly at epidermal growth factor receptor (EGFR)'s tyrosine kinase, such as erlotinib, or gefitinib are also under evaluation. The development of drugs that have multiple therapeutic targets and the utilization of multiple cancer-targeting agents are both emerging strategies for ATC treatment. For example, a preclinical study evaluated the activity of a dual inhibitor of EGFR and vascular endothelial growth factor (VEGF), NVP-AEE788, alone and in combination with paclitaxel for the treatment of ATC. Even if new therapeutic approaches against ATC are under development, more research is needed to finally identify therapies able to control and to cure this disease. The possibility of testing the sensitivity of primary ATC cells from each subject to different drugs could increase the effectiveness of the treatment in the next future.

The increasing incidence of thyroid cancer is associated with a higher number of advanced disease characterized by the loss of cancer differentiation and metastatic spread. The knowledge of the molecular pathways involved in the pathogenesis of thyroid cancer has made possible the development of new therapeutic drugs able to blockade the oncogenic kinases (BRAF V600E, RET/PTC) or signaling kinases [vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptors (PDGFR)] involved in cellular growth and proliferation. Some clinical trials have been conducted showing the ability of targeted therapies (sorafenib, sunitinib, axitinib, imanitib, vandetanib, pazopanib, gefitinib) in stabilizing the course of the disease. Until now, however, no consensus guidelines have been established for patient selection and more data on toxicities and side effects are needed to be collected.

Background: In a number of human malignancies, the presence of lymphocytic infiltration in or around tumor tissue is commonly considered to be part of the host tumor immune response. An association between thyroid carcinoma and chronic inflammation has been described. This relationship is not fully understood, so we performed a systematic study on a follicular variant of papillary thyroid carcinoma (FVPTC), to evaluate the type and distribution of certain immunological cells and their relationship with prognostic factors., Methods: We selected 91 consecutive cases of FVPTC, in which we evaluated the presence of three different immunological cells: dendritic cells (DC), immature CD1a+ and mature DC-Lamp+; mast cells (MC), tryptase+; and macrophages (M), CD68+, in the intratumoral, peritumoral, and extratumoral areas. As a control we analyzed 44 cases of thyroid adenomas (A)., Results: In the intratumoral and peritumoral areas, the expression of CD1a, tryptase, and CD68 was significantly higher in FVPTC than in adenomas. Expression of CD1a and tryptase was comparable in the extratumoral compartment, whereas CD68 expression in the extratumoral area was significantly higher in FVPTC than in adenoma (p=0.0015). DC-Lamp expression was not significantly different among the intra-tumor, peri-tumor, and extra-tumor areas of FVPTC or adenoma. It was also very interesting that nonencapsulated FVPTC were more positive to tryptase., Conclusion: We highlight a higher presence of immunological cells in carcinomas than in adenomas. On this basis, it is possible to speculate that these inflammatory elements could be involved in tumor progression and invasion, as appears to be the case for MC and M.

Aim: We have studied the antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3 and CLM29) in primary papillary dedifferentiated thyroid cancer (DePTC) cells., Methods: The antiproliferative effect was tested in DePTC cells obtained at reoperation from patients with recurrence of the tumor. The concentrations of CLM3 and CLM29 used in the in vitro experiments were 1, 10, 30, and 50 μm., Results: Proliferation assays in DePTC cells showed a significant reduction of proliferation by CLM3 and CLM29, which was by 12% with CLM3 (the most potent compound) 10 μm, 43% with CLM3 30 μm, and 60% with CLM3 50 μm. CLM3 and CLM29 increased the percentage of apoptotic cells in DePTC cells dose dependently (P < 0.001) and inhibited migration (P < 0.001). A DePTC cell line (AL) was injected sc in CD nu/nu mice, and tumor masses became detectable 10 d after xenotransplantation. CLM3 (40 mg/kg · die) significantly inhibited tumor growth and weight, and the therapeutic effect was significant starting on the 19th day after cell implantation (4 d after the beginning of treatment). The CLM3-treated group of animals did not show any appreciable toxicity. CLM3 and CLM29 increased thrombospondin-1 expression in the AL cell line. A significant reduction of microvessels and in the percentage of antivascular endothelial growth factor antibody immunoreactivity was observed in the CLM3 treated tumors, with a simultaneous increase of the percentage of necrosis., Conclusion: The antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3, CLM29) in vitro and CLM3 in vivo in DePTC has been shown, opening the way to a future clinical evaluation.

Different cultivars of Vitis vinifera vary in their potential to form embryogenic tissues. The WUSCHEL (WUS)-related homeobox (WOX) genes have been shown to play an important role in coordinating the gene transcription involved in the early phases of embryogenesis. The expression dynamics of 12 VvWOX genes present in the V. vinifera genome in embryogenic and other tissues of 'Chardonnay' were analysed. In order to understand the influence of WOX genes on the somatic embryogenic process, their expression profiles were compared in two cultivars of V. vinifera ('Chardonnay' and 'Cabernet Sauvignon') that show different aptitudes for embryogenesis. The expression of all VvWOX genes was influenced by culture conditions. VvWOX2 and VvWOX9 were the principal WOX genes expressed during the somatic embryogenesis process, and the low aptitude for embryogenesis of 'Cabernet Sauvignon' was generally correlated with the low expression levels of these VvWOX genes. VvWOX3 and VvWOX11 were strongly activated in correspondence to torpedo and cotyledonary stages of somatic embryos, with low expression in the earlier developmental stages (pre-embryogenic masses and globular embryos) and during embryo germination. VvWOX genes appeared to be key regulators of somatic embryogenesis in grapevine, and the regulation of these genes during early phases of somatic embryogenesis differed between the two cultivars of the same species.

Introduction: Prenatal diagnosis of pheocromocytoma, although rare, is important as it allows a reduction in both maternal mortality and foetal loss. Pheocromocytoma operated on in the first trimester of pregnancy with survival of both patient and foetus is rare in literature. Our case was operated on with success after a correct and early diagnosis was obtained despite a chronic hypertension which existed long before pregnancy. Our case study well illustrates that a correct multidisciplinary approach involving endocrinologists, anesthesiologists, surgeons and gynecologists is fundamental for a positive outcome., Case Report: The case of a white caucasian pregnant woman at 13th weeks of gestation with pheocromocytoma and severe and unstable hypertension that could not be pharmacologically controlled is described. Morphological diagnosis was safely obtained by Magnetic Resonance Imaging (MRI) without intravenous medium contrast agent. Pre-operative treatment consisted of therapy with alpha-blockers and rehydration. Adrenalectomy was performed through a laparotomy. Postoperative treatment consisted of rehydration and ephedrine continued until the fourth post-operative day. The post-operative period was uneventful and a new ultrasound (US) scan confirmed foetal vitality. The patient was discharged seven days after surgery. A live newborn was physiologically delivered after a nine-month pregnancy., Conclusion: A correct diagnosis in all pregnant women with severe hypertension particularly those not screened for secondary hypertension and a multidisciplinary management are mandatory to obtain optimal results and avoid deleterious effects at delivery.

In papillary thyroid carcinomas (PTCs), oncogenes activate a transcriptional program including the upregulation of CXCL10 chemokine, which stimulates proliferation and invasion. Furthermore, peroxisome proliferator-activated receptor-gamma (PPARgamma) activators thiazolidinediones (TZDs) modulate CXCL10 secretion in normal thyroid follicular cells (TFC), and inhibit PTC growth. Until now, no study has evaluated the effect of cytokines on CXCL10 secretion in PTCs, nor the effect of PPARgamma activation. The combined effects of interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) stimulation on CXCL10 secretion in primary cells from PTCs and TFC were tested. Furthermore, the effect of PPARgamma activation by TZDs, on CXCL10 secretion and proliferation in these cell types was studied. In primary cultures of TFC and PTCs CXCL10 production was absent under basal conditions; a similar dose-dependent secretion of CXCL10 was induced by IFNgamma in both cell types. TNFalpha alone induced a slight but significant CXCL10 secretion only in PTCs. The stimulation with IFNgamma+TNFalpha induced a synergistic CXCL10 release in both cell types; however, a secretion more than ten times higher was induced in PTCs. Treatment of TFC with TZDs dose-dependently suppressed IFNgamma+TNFalpha-induced CXCL10 release, while TZDs stimulated CXCL10 secretion in PTCs. A significant antiproliferative effect by TZDs was observed only in PTCs. In conclusion, a dysregulation of CXCL10 secretion has been shown in PTCs. In fact, a CXCL10 secretion more than ten times higher has been induced by IFNgamma+TNFalpha in PTCs with respect to TFC. Moreover, TZDs inhibited CXCL10 secretion in TFC and stimulated it in PTCs. The effect of TZDs on CXCL10 was unrelated to the significant antiproliferative effect in PTCs.

During the 1990s, with the general tendency to develop minimally invasive operations, an endoscopic approach has been applied to neck surgery for both parathyroidectomy and thyroidectomy. The most widely spread minimally invasive technique for thyroidectomy is minimally invasive video assisted thyroidectomy (MIVAT), described and developed for the first time at our institution in 1998. Ideal candidates for MIVAT are patients with a thyroid volume lower than 25ml with nodules smaller than 35 mm. Consequently, MIVAT will present restricted indications, being suitable only for the treatment of about 10-15% of the whole standard surgical case load. Thus, together with small follicular lesions, "low risk" papillary carcinoma will result the main indication for MIVAT, being this small cancer usually harboured in normal glands of young females. On the other hand, in case of locally invasive carcinomas and/or lymph node metastasis the procedure must be immediately converted to the conventional technique. MIVAT also is not indicated for the treatment of medullary and anaplastic carcinomas. Recent prospective randomized studies clearly demonstrate that MIVAT allows achieving same clearance at the thyroid bed level and same outcome as conventional technique, when dealing with "low risk" papillary carcinoma. At the same time, patients can benefit from the main advantages of this minimally invasive technique: lower postoperative pain, faster postoperative recovery and excellent cosmetic outcome.

Objective: No study has evaluated the antiproliferative effects of thiazolidinediones and antiblastics in 'primary cultured human anaplastic thyroid cancer cells'., Design: Primary anaplastic cells proliferation was evaluated after incubation with increasing concentrations of rosiglitazone or pioglitazone or antiblastics (bleomycin, cisplatin, gemcitabine) by a proliferation assay (WST-1-tetrazolium reaction) and cell counting., Measurements and Results: A reduction of proliferation by thiazolidinediones at 1 h (from the start of tetrazolium reaction) [of 11% and 25%, with rosiglitazone, 10 or 20 (P = 0.0001) microM, respectively; of 7% and 17%, with pioglitazone, 10 or 20 (P = 0.0125) microM, respectively], and at 2 h [of 14% and 24%, with rosiglitazone, 10 (P = 0.0043) or 20 (P < 0.0001) microM, respectively; of 9% and 21%, with pioglitazone, 10 (P = 0.0397) or 20 (P = 0.0001) microM, respectively] was shown. No significant thiazolidinediones effect was observed in normal thyroid follicular cells. Bleomycin, cisplatin and gemcitabine significantly (P < 0.0001) inhibited (> 50%) anaplastic cells proliferation. Cell counting confirmed the above mentioned results. Inhibition of proliferation was similar in tumours with or without (V600E)BRAF mutation, both for thiazolidinediones and antiblastics., Conclusions: Thiazolidinediones exert an antiproliferative effect in primary cultured human anaplastic carcinoma cells in vitro, such as antiblastics.

Human papillary dedifferentiated thyroid cancer (HPDTC) represents a therapeutic dilemma. Targeted therapy (RET proto-oncogene or BRAF-targeting drugs) are promising treatments for HPDTC. Also PPARg agonists are another exciting field for redifferentiating therapy of HPDTC. However, even if many new approaches for the therapy of HPDTC are emerging, until now a significant clinical impact on survival by the use of these drugs is still lacking. In the future, the identification of patients who are likely to benefit from each therapeutic option will be important. In this view particular importance should be given to development of primary cells from the single patient by fine needle aspiration samples, as recently observed in anaplastic thyroid cancer. In fact, chemosensitivity tests in primary tumoral cells may help in detecting responsive patients and in preventing the administration of inactive drugs to those unresponsive.

Context: Because very few studies have examined the correlation between BRAF mutations and clinicopathological features of papillary thyroid carcinoma (PTC), we analyzed here a large and homogeneous cohort of patients with PTC for the presence of the BRAF mutation., Objective: We examined BRAF mutations in a consecutive series of 500 PTC patients who underwent surgery in the Department of Surgery of the University of Pisa, and we correlated the presence of the mutation with clinicopathological parameters of the patients: age, gender, tumor size, presence of tumor capsule, extrathyroidal invasion, multicentricity, presence of node metastases, and tumor class., Design: BRAF (exon 15) mutation was examined by PCR-single strand conformational polymorphism followed by DNA sequencing in laser-capture microdissected tissue samples., Results: In this study, BRAF mutation was found in 219 of 500 cases (43.8%). In particular, we found the most common BRAF V600E mutation in 214 cases (42.8%), BRAF K601E mutation in three cases (0.6%), BRAF VK600-1E (0.2%) in one case, whereas in one case we found a new 14-bp deletion with concomitant 2-bp insertion, VKSR600-3del and T599I, respectively. BRAF V600E was associated with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), class III vs. classes I and II (P < 0.00000006), and absence of tumor capsule (P < 0.0001), in particular in follicular- and micro-PTC variants. By multivariate analysis, the absence of tumor capsule remained the only parameter associated (P = 0.0005) with BRAF V600E mutation., Conclusions: Our data suggest that BRAF V600E mutation is associated with high-risk PTC and in particular in follicular variant with invasive tumor growth.

The histologic diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) may be troublesome, especially in its encapsulated form. We evaluated the expression of galectin-3 (gal-3) and Hector Battifora mesothelial cell (HBME-1) in 200 formalin-fixed thyroid tissues with diagnosis of classical variant of papillary thyroid carcinoma or FVPTC, encapsulated or with infiltrative growth, with or without lymph node metastasis. All cases of classical variant of papillary thyroid carcinoma were consistently positive for gal-3; similar results have been obtained by using HBME-1. Interestingly, the invasive type of FVPTC, with or without metastasis, was strongly positive for gal-3 (78.2% and 96%, respectively), whereas only 46.8% of encapsulated FVPTCs without metastasis showed immunostaining for this marker. In the latter group, the HBME-1 expression achieved a significantly higher percentage of positivity (87%). Surprisingly, gal-3 immunodetection showed negative results in 4 encapsulated FVPTCs, despite the strong immunoreactivity in corresponding metastasis. Our data suggest that gal-3 immunodetection alone is not able to support the diagnosis of encapsulated FVPTCs.

The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H(2)O(2) and O(2)(.-) in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.

Background: In the last decade, development of videolaparoscopic surgery allowed several operations to be performed with minimally invasive techniques, making them less invasive and painful. Neck surgery was also involved in this effort, in spite of the skepticism shown by some authors., Study Design: Minimally invasive video-assisted thyroidectomy was developed in 1998, and since then, about 600 operations have been performed. Access was the same as was previously described for parathyroidectomy; it was based on a small central incision (1.5 cm) and on external retraction without neck insufflation., Results: From July 1998 to October 2003, 579 patients were selected from 5,450 for minimally invasive video-assisted thyroidectomy. The operation consisted of a total thyroidectomy in 312 patients and lobectomy in 267 patients. Mean operative time was 41 +/- 19.5 minutes (range 15 to 120 minutes) for lobectomy and 51.6 +/- 18.8 minutes (range 30 to 140 minutes) for total thyroidectomy. Postoperative hospital stay was 24 hours (overnight discharge) for all patients. Complications were postoperative bleeding (0.1%), recurrent nerve palsy (1.3%), and definitive hypoparathyroidism (0.2%)., Conclusions: After 5 years of experience using this approach for various indications, we achieved a good esthetic result with an operative time comparable to that of conventional open surgery. Minimally invasive video-assisted thyroidectomy was found to be a safe operation, with advantages over traditional procedures represented by better cosmetic outcomes and postoperative course, as demonstrated by visual analogue scales and statistically analyzed numeric scales.

Introduction: This study reviews four years of Minimally Invasive Video Assisted Thyroidectomy (MIVAT) technique and compares the results to those of traditional thyroid surgery., Materials and Methods: Between 1999 and 2002, a series of 427 patients were submitted to MIVAT at our Department. Selection criteria were: thyroid nodule maximum diameter of 3.5 cm, total thyroid volume under 25 cc, no signs associated thyroiditis, diagnosis of benign thyroid disease or "low risk" thyroid tumor, no evidence of nodal disease of the neck., Results: We operated on 362 females and 65 males and the mean age of the population was 39.6 years (range 10-77). A total thyroidectomy was performed in 208 cases, and 219 patients underwent a single-side procedure. Mean operative time was 30.4 minutes for lobectomy (range 20-140 minutes) and 50.2 for total thyroidectomy (range 35-140). Complications were represented by definitive recurrent nerve palsy in 3 patients (0.7%) and one case of definitive hypoparathyroidism (0.4%). A wound infection is reported in 3 cases and we had no major bleeding that required surgical revision. A conversion to open procedure was performed in 5 cases (1.2%); mean hospitalisation was 1.28 days (range: 1-4)., Conclusions: This series demonstrates that MIVAT is not different to conventional open surgery in terms of complications, radicality of the procedure and operative time. Moreover, even if not statistically proved, MIVAT appears to offer some advantages in terms of cosmetic results and postoperative pain. In conclusion, we believe that MIVAT is a perfectly reproducible and safe technique for both benign and low-risk malignant thyroid disease, when correct indications are strictly followed.

Introduction: In 1997 a Minimally Invasive Video Assisted Technique for Parathyroidectomy (MIVAP) was developed in the University of Pisa. In this review we examine the last three years of MIVAP (240 cases) in order to identify the advantages and the limits of the procedure after the first-period development of the technique., Results: In our experience, 65% of patients affected by primary hyperparathyroidism (PHPT) turned out to be eligible for MIVAP. During the first years several selection criteria were strictly followed; more recently, some initially absolute contraindications to the operation have been interpreted more flexibly. Mean operative time is 35 minutes. 18 conversions (7.5%) to traditional open cervicotomy were needed and in 4 cases (1.6%) no affected parathyroid tissue was removed., Conclusions: At present, we consider absolutely necessary for MIVAP: preoperative localization of an adenoma with at least one imaging study (US or MIBI scintiscan) and the availability of QPTH intraoperative assay. No absolute contraindications other than the size of the lesions and the suspect of parthyroid carcinoma are identified for patients with PHPT. Moreover, MIVAP has proved to have further advantages when compared to other mini invasive procedures such as the demonstrated possibility to perform a traditional bilateral exploration, when indicated. Nevertheless, a great degree of experience is requested for this procedure. In conclusion, MIVAP permits to significantly reduce postoperative pain, size of the incision, days of hospitalisation and, finally, the cost of the entire procedure without affecting in any way the success rate of the traditional operation and without an increase of the complications.

Purpose: To evaluate the usefulness of intraoperative radiofrequency thermoablation of liver tumours in association or not with hepatic resection., Materials and Methods: 21 patients were treated between January 1998 and December 2001, there were 4 hepatocellular carcinoma and 17 metastasis. In 13 cases radiofrequency was associated to hepatectomy, in 3 cases to resection of extraepatic disease and in 5 cases were performed alone. 23 lesions were treated by radiofrequency (range 1-3); the mean dimension was 26 millimetres (range 8-70). A clamping of the liver pedicle was always done., Results: There were no operative deaths, 3 (14.3%) patients developed complications related to radiofrequency (2 biliary leakages, 1 hepatic abscess). 14 (66.7%) patients were alive after a mean follow up of 14.5 months, 2 of all (9.5%) had a recurrence in the site previously treated with thermoablation. Association between hepatectomy and radiofrequency increased the number of curative liver resections from 10.1% to 16.3% (in case of colorectal metastasis)., Discussion: Intraoperative radiofrequency is useful to increase the number of curative hepatectomies, to treat liver masses which demonstrate unresectable or found by ultrasonography at the operating time and even to reach tumours difficult to manage by percutaneous approach. In any case the aim is to obtain the absence of macroscopic neoplastic disease (RO status). It is a safe and effective therapeutic strategy, anyway all procedures and indications are still not completely cleared., Conclusions: Intraoperative thermoablation of liver tumour is safe and effective and increases therapeutic the number of curative hepatectomies. Further progresses may improve the efficacy and extend the indications of this strategy.

Parathyroid cysts are rare, representing 1% of all neck swellings. A correct preoperative diagnosis is rarely formulated, especially because of the non-specific clinical and ultrasonographic findings; for this reason, patients are often submitted to surgery for thyroid nodules. In the present report we describe a case where the preoperative diagnosis was correctly formulated following the aspiration and PTH assay of clear, colorless, watery fluid from the cyst. While cyst aspiration is considered the elective treatment for these lesions, recurrences being uncommon, surgery was indicated in the present case because of two recurrences after aspiration and the onset of compressive symptoms. Cystic masses of the neck should be accurately diagnosed to recognize their true nature and to allow their correct and non-invasive treatment: surgery is indicated only when recurrences and symptoms are present. Indications for aspiration of all solitary cysts of the neck should be considered to correctly identify their nature.

Background: The aim of this study was to ascertain the utility of echo-Doppler in the analysis of the low resistance thyroid vascularization in diffuse toxic goiter (DTG), and the effectiveness of Lugol's solution (iodine-iodide solution) in patients undergoing thyroidectomy., Study Design: Twenty-five patients with diffuse toxic goiter were evaluated and compared with 19 normal subjects. Patients were treated with increasing doses of Lugol's solution 2% for 7 days until a total dose of 75 mg of iodine was given. Echo-Doppler was performed on the last day of treatment, 12 hours before operation., Results: Mean basal Doppler Resistance Index (RI) of intrathyroid arterial flow was significantly lower in patients with DTG compared with normal controls (0.4718 +/- 0.0625 versus 0.55 +/- 0.05, range: 0.472 to 0.643; p = 0.008). Moreover, the RI was significantly increased in patients with DTG after Lugol's solution (+16.46 +/- 10.22%, range: -2.59 to +39.97; p< 0.0005)., Conclusions: Echo-Doppler RI allowed documenting lower arterial resistances within the thyroid gland in patients with DTG. The use of preoperative Lugol's solution therapy induces normalization of those changes for safer thyroidectomy.

Background: The utility of intraoperative parathyroid hormone (PTH) monitoring is unclear in the surgical management of renal hyperparathyroidism. Our goal was to define the normal pattern of decay during operation for renal hyperparathyroidism by using the rapid intact (1-84) parathyroid hormone (PTH) assay., Methods: Eighty consecutive patients underwent neck exploration for renal hyperparathyroidism. Intact PTH levels were monitored with a rapid immunochemiluminometric assay. Samples were assayed at the induction of anesthesia, after dissection before resection, and 20 and 40 minutes after resection. Follow-up ranged from 3 to 24 months., Results: Twenty minutes after resection, PTH levels remained many-fold supranormal. Seventy-seven patients (96%) were cured. Of these, 75 patients (94%) had PTH decay of more than 50% from the preoperative level; 74 (99%) were cured. Only 1 of 3 patients (33%) in whom the PTH level decreased less than 40% from the preoperative level was cured. Two patients had intermediate values and both were cured., Conclusions: The intraoperative decay of PTH during operation for renal hyperparathyroidism is slower than for patients with normal renal function. However, 20 minutes after resection, a decline to less than 50% of the preoperative level predicts cure, while a level greater than 60% predicts failure.

A study is presented on the coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H(+) release per heme a, Cu(A) oxidized (H(+)/heme a, Cu(A) ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme a, Cu(A), and added cytochrome c oxidized and the H(+) released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H(+)/heme a, Cu(A) ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. The pH dependence of the H(+)/heme a, Cu(A) ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu(A) and heme a exhibited superimposed E'(m) values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A model in which oxido-reduction of heme a and Cu(A) are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the pH dependence of the H(+)/heme a, Cu(A) ratios, provided a satisfactory fit for the pH dependence of the E'(m) of heme a and Cu(A). The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and Cu(A) in the proton-pumping activity of cytochrome c oxidase.

Background: It is known that factors other than thyroid-stimulating hormone, such as insulin-like growth factor-I (IGF-I) and epidermal growth factor, have a goitrogenic effect. It has been established that insulin-like growth factor-binding proteins (IGFBPs) may play a role as autocrine/paracrine factors in regulating the local actions of IGFs. Both an inhibitory and a stimulatory effect for different IGFBPs have been shown in several cell systems. The aim of this study was to assess the involvement of IGFBPs in the goitrogenic process in patients with goiter recurrence., Methods: The IGFBP-1 and -3 content in normal and nodular tissues obtained at the time of thyroidectomy from 10 patients with recurrent goiters, unresponsive to thyroid-stimulating hormone suppressive therapy, was studied. In all patients, a fragment of normal tissue was also obtained. The IGF-I, IGFBP-1, and -3 content was evaluated by specific immunoassays and/or immunoblotting with anti-IGFBP specific antiserum., Results: The IGF-I content was significantly higher (P < .05) in nodular tissues (8.0 +/- 1.6 ng/g of tissue) than what was found in normal tissue (4.8 +/- 0.9 ng/g). Radioimmunoassay IGFBP-3 concentration in nodular tissue was 111.5 +/- 18.2 ng/g significantly higher (P < .001) than values found in normal tissue (77.5 +/- 18.6 ng/g). By immunoblot, IGFBP-1 appeared higher in all but 1 nodular tissue., Conclusions: These data raise the possibility that IGFBPs are important in the proliferative activities entailed in the goitrogenic process. Three mechanisms are potentially involved: (1) reduction of the potency of locally produced IGF peptide to downregulate type I receptors (potentiating effect on the autocrine/paracrine mitogenic action of IGFs); (2) increase of the IGF-I tissue concentration restraining its passage to circulation; and (3) protection of IGF-I from degradation. Further studies are needed to define a more precise link between these factors and the recurrence of goiter.

The effect of different anions on the steady-state proton translocation in bovine bc1 complex reconstituted in liposomes was studied. The H+/e- ratio for vectorial proton translocation is at the steady state definitely lower than that measured at level flow, (0.3 vs. 1.0). The presence of azide or arachidonate at micro- and submicromolar concentrations, respectively, gave a substantial reactivation of the proton pumping activity at the steady state, without any appreciable effect on respiration-dependent transmembrane pH difference. Addition of azide to turning-over bc1 vesicles also caused a transition of b cytochromes toward oxidation. The results are discussed in terms of possible involvement of an acidic residue in the protonation of the semiquinone/quinol couple at the N side of the membrane.

Peptide segments of the inhibitor protein (IF1) of the F0F1 ATP synthase complex from bovine-heart mitochondria have been constructed by chemical synthesis. The IF1-(42-58)-peptide was equally effective as IF1 in inhibiting the ATPase activity of both the F0F1 complex in the mitochondrial membrane deprived of IF1 (SMP) and soluble F1. The IF1-(22-46)-peptide inhibited the ATPase activity in the soluble F1 but had no effect on either the ATPase activity or H+ conduction in SMP. Substitution of the His or Lys residues with Ala in the IF1-(42-58)-peptide decreased the inhibition of ATP hydrolysis. The inhibition exerted by the IF1-(42-58)-peptide on ATP hydrolysis in SMP exhibited a pH dependence, similar to that observed with IF1, which was lost upon replacement of His or Lys with Ala. In soluble F1, inhibition of ATP hydrolysis by IF1, the IF1-(42-58)-peptide and the IF1-(22-46)-peptide was pH dependent when F1 was first incubated with ATP. The IF1-(42-58)-peptide also caused inhibition of passive H+ conduction in SMP. This activity of the synthetic peptide was weaker, as compared to that of IF1, and practically unaffected by substitution of His or Lys with Ala. An antibody against the IF1-(42-58)-synthetic peptide stimulated ATP hydrolysis in the membrane-bound F0F1 complex with associated IF1 but was without effect on H+ conduction. An antibody against IF1 stimulated both processes.

The effect of pH and transmembrane delta pH on the efficiency of the proton pump of the mitochondrial bc1 complex both in situ and in the reconstituted state was studied. In both cases the H+/e- ratio for vectorial proton translocation by the bc1 complex respiring at the steady state, under conditions in which the transmembrane pH difference (delta pH) represents the only component of the proton motive force (delta p), was significantly lower than that measured under level flow conditions. The latter amounts, at neutral pH, to 1 (2 including the scalar H+ release). In the reconstituted system steady-state delta pH was modulated by changing the intravesicular buffer as well as the intra/extra-liposomal pH. Under these conditions the H+/e- ratio varied inversely with the delta pH. The data presented show that delta pH exerts a critical control on the proton pump of the bc1 complex. Increasing the external pH above neutrality caused a decrease of the level flow H+/e- ratio. This effect is explained in terms of proton/electron linkage in b cytochromes.

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.

A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine-heart cytochrome-bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome-c reductase activity of either the cholate-solubilized or liposome-reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre-steady-state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome-b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin-promoted oxidant-induced extrareduction of b cytochromes. Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe-S protein. This is interpreted as an interaction of Zn2+ with the 2Fe-2S-cluster region of the Fe-S protein, thus giving rise to inhibition of the reductase activity and of the antimycin-insensitive reduction route of b cytochromes. A Scatchard-plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 x 10(-6) M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.