Objective: Primary hyperparathyroidism (PHPT) has been associated with low-grade inflammation and increased risk of cardiovascular disease (CVD). The aim of the study was to investigate systemic levels of pro-inflammatory proteins that previously have not been examined in patients with PHPT. The selection of the pro-inflammatory biomarkers included in this study, MMP9, S100A4, S100A8/A9 and the receptors sCD14 and RAGE, was based on a previous microarray screen of mRNAs in adipose tissue from PHPT patients.Design: A prospective study was conducted on a total of 57 patients with PHPT and a control group of 20 healthy blood donors.Methods: PHPT patients with normalisation of serum calcium levels after parathyroidectomy were followed for 6 months. Forty-two patients participated in the longitudinal study, in which blood samples were taken at inclusion, and 1, 3 and 6 months after surgery.Results: We observed increased serum levels of MMP9 (P=0.029), S100A4 (P<0.001) and sCD14 (P=0.002) in the 57 patients with PHPT compared to the control-group. During 6 months of follow up, S100A4 (P=0.022) and sCD14 (0.002) decreased significantly, while serum levels of MMP9 increased (P=0.025).Conclusions: The results demonstrate an increased inflammatory response in PHPT patients shown by elevated MMP9, S100A4 and sCD14 at inclusion. During the 6 months of follow-up, MMP9 increased further, possibly due to the tissue repair process after parathyroidectomy. S100A4 and sCD14 decreased after surgery demonstrating a partial reversal of the systemic inflammation.

Objectives Primary hyperparathyroidism (PHPT) has been associated with low-grade inflammation and elevated risk of cardiovascular disease (CVD). In inflammatory conditions, interferon-gamma (IFN-gamma) activity is enhanced and a decreased circulating concentration of vitamin B6 is often observed. Such changes in IFN-gamma activity or vitamin B6 levels have been associated with increased incidence of CVD. The aim of the study was to investigate systemic markers of IFN-gamma-mediated immune activation, such as neopterin, the kynurenine-to-tryptophan ratio (KTR) and kynurenine pathway metabolites, as well as B6 vitamers in patients with PHPT. Design/subjects A total of 57 patients with PHPT and a control group of 20 healthy blood donors were included in this study. PHPT patients who responded positively to parathyroidectomy were followed for 6 months. Forty-three patients participated in the longitudinal study in which blood samples were taken at inclusion and 1, 3 and 6 months after surgery. Results Plasma concentrations of the B6 vitamers pyridoxal 5'-phosphate (PLP) (P = 0.007) and pyridoxal (P = 0.013) were significantly lower in the patient group compared to healthy control subjects. An increase in the KTR indicated that the kynurenine pathway of tryptophan metabolism was altered in PHPT patients (P = 0.015). During the initial 6 months after surgery, levels of PLP (P < 0.001) and anthranilic acid (P < 0.001) increased significantly, whereas neopterin decreased (P = 0.018). Conclusions The results of this study demonstrate altered levels of vitamin B6 and the KTR in PHPT patients, both of which may reflect cellular immune activation. These abnormalities should be considered in relation to the increased risk of CVD previously observed in patients with PHPT.

BACKGROUND:Clinical and histopathological distinction between benign and malignant adrenocortical tumors can be a challenge.Report on 2 patients with cortisol producing apparently benign adrenal adenomas ≥ 5 cm in diameter with local malignant recurrence and peritoneal carcinomatosis after endoscopic surgery. RESULTS:Case 1: The 59-year-old male presented with adrenal hypercortisolism due to a 5.0 cm large adrenal tumor on the left side. A retroperitoneoscopic total adrenalectomy was performed. Histologically, a benign adrenal adenoma (Weiss score 1, Ki-67 < 2%) was found. 6 months later, the patient developed clinically and biochemically recurrent disease with recurrent tumor in the left adrenal region and peritoneal carcinomatosis. The patient died 5 months after second surgery. Case 2: The 32-year-old female was pregnant in 27th week when presenting with adrenal hypercortisolism due to a 5.5 cm large adrenal tumor on the left side. She was operated on using a laparoscopic approach and a total adrenalectomy was carried out. Histological examination revealed a benign adrenocortical adenoma (Weiss score 1, Ki-67 < 5%). 4 years later, the patient came back with clinically and biochemically recurrent disease. Imaging showed a 10 cm large tumor in the left retroperitoneum and a diffuse peritoneal carcinomatosis. The patient died 2 months after diagnosis. CONCLUSION:Cortisol producing adrenal tumors ≥ 5 cm in diameter are at risk to be misdiagnosed as apparently benign. Regular surveillance should be considered in patients presenting with large cortisol producing tumors.

BACKGROUNDWe have previously described the essential role of the retinoid-inducible nuclear factor (RINF) during differentiation of hematopoietic cells and suggested its putative involvement in myeloid leukemia and preleukemia. Here, we have investigated whether this gene could have a deregulated expression in malignant tissues compared with their normal tissues of origin and if this potential deregulation could be associated with important clinicopathological parameters.PATIENTS AND METHODSRINF messenger RNA expression was examined in biopsies from locally advanced breast tumors, metastatic malignant melanomas, and papillary thyroid carcinomas and compared with their paired or nonpaired normal reference samples. Further, the prognostic role of RINF expression was evaluated in locally advanced breast cancer.RESULTSRINF expression was significantly higher in all tumor forms (primary breast, and thyroid cancers and metastatic melanomas) as compared with normal control tissues (P < 0.001 for each comparison). Importantly, high levels of RINF expression correlated to a poor overall survival in breast cancer (P = 0.013). This finding was confirmed in three independent public microarray datasets (P = 0.043, n = 234; P = 0.016, n = 69; P = 0.001, n = 196) and was independent of tamoxifen therapy. Notably, high levels of RINF was strongly associated with TP53 wild-type status (P = 0.002) possibly indicating that high levels of RINF could substitute for TP53 mutations as an oncogenic mechanism during the malignant development of some cases of breast cancer.CONCLUSIONSOur data indicate that (i) RINF overexpression is associated with the malignant phenotype in solid tumors and (ii) RINF overexpression represents an independent molecular marker for poor prognosis in breast tumors.

Pharmacogenetics has arrived in clinical psychiatric practice with the FDA approval of the AmpliChip CYP450 Test that genotypes for two cytochrome P450 2D6 (CYP2D6) and 2C19 (CYP2C19) genes. Other pharmacogenetic tests, including those focused on pharmacodynamic genes, are far from ready for clinical application. CYP2D6 is important for the metabolism of many antidepressants and antipsychotics, and CY2C19 is important for some antidepressant metabolism. Poor metabolizers (PMs), lacking the enzyme, account for up to 7% of Caucasians for CYP2D6 and up to 25% of East Asians for CYP2C19. Patients having three or more active CYP2D6 alleles (up to 29% in North Africa and the Middle East), are called CYP2D6 ultrarapid metabolizers (UMs). CYP2D6 phenotypes (particularly PMs) are probably important in patients taking tricyclic antidepressants (TCAs), venlafaxine, typical antipsychotics, and risperidone. The CYP2C19 PM phenotype is probably important in patients taking TCAs and perhaps citalopram, escitalopram, and sertraline. On the basis of the literature and the authors’ clinical experience, the authors provide provisional recommendations for identifying and treating CYP2D6 PMs, CYP2C19 PMs, and CYP2D6 UMs. The next few years will determine whether CYP2D6 genotyping is beneficial for patients taking the new drugs aripiprazole, duloxetine, and atomoxetine. Practical recommendations for dealing with laboratories offering CYP2D6 and CYP2C29 genotyping are provided.

CYP2D6 and SULT1A1 genotypes may partly explain the wide inter-individual variations in the serum levels of tamoxifen and its metabolites. We propose that therapeutic drug monitoring should be included in studies linking CYP2D6 and SULT1A1 genotypes to clinical outcome.

We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PTC) specimens. Seven clinically aggressive carcinomas were included, comprising poorly differentiated PTC and tumors with extensive local invasion or synchronous distant metastases. Ten differentiated (classic) papillary thyroid carcinomas (PTC) and non-neoplastic thyroid tissues were also investigated. TaqMan quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry verified the differential gene expression. The B-Raf gene was mutated with a T-->A transversion at nucleotide 1799 (V600E) in 8 of 10 differentiated PTC, and in 4 of 7 aggressive carcinomas. Among genes markedly and equally over-expressed in carcinomas of both the aggressive and classic PtC groups, compared to normal thyroid tissue, were CBP/p300 transactivator (CItED1), fibronectin, growth/differentiation factor 15, potassium inwardly rectifying channel KCNJ2, glutaminyl peptide cyclotransferase, WNT7A, and dipeptidyl peptidase IV. A marked upregulation in carcinomas of P-cadherin mRNA and protein concomitant with E-cadherin downregulation, indicates a possible P-E cadherin "switch" in PTC. The growth factor homologue Nel-like 2, dual specificity phosphatase 5, the serine protease kallikrein 10, and also the tight junction genes claudin 1 and claudin 16, were upregulated in classic PTC but not in aggressive tumors, which may be consistent with altered cell polarity in the dedifferentiated PtC. The aggressive, poorly differentiated PtC group was specifically characterized by marked upregulation of several genes related to cell proliferation such as cell division cycle 2 (CDC2), CDC7, kinesin-like 5, ubiquitin conjugating enzyme E2C, and topoisomerase IIalpha, and by upregulation of genes encoding extracellular matrix proteins such as seprase, extracellular matrix protein 1, and several collagens. These aggressive tumors were also characterized by overexpression of the integrin ligand periostin, and in some biopsies also of osteopontin and of the upstream Rac-regulator dedicator of cytokinesis 10 (DOCK10). These data are interpreted to be consistent with altered cell motility, extracellular matrix remodeling and increased cell proliferation, as important processes in PTC tumor progression.

Protein N-ɛ-acetylation is recognized as an important modification influencing many biological processes, and protein deacetylase inhibitors leading to N-ɛ-hyperacetylation of histones are being clinically tested for their potential as anticancer drugs. In contrast to N-ɛ-acetyltransferases, the N-α-acetyltransferases transferring acetyl groups to the α-amino groups of protein N-termini have only been briefly described in mammalians. Human arrest defective 1 (hARD1), the only described human enzyme in this class, complexes with N-acetyltransferase human (NATH) and cotranslationally transfers acetyl groups to the N-termini of nascent polypeptides. Here, we demonstrate that knockdown of NATH and/or hARD1 triggers apoptosis in human cell lines. Knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis, potentially pointing at the NATH–hARD1 acetyltransferase complex as a novel target for chemotherapy. Our results argue for an essential role of the NATH–hARD1 complex in cell survival and underscore the importance of protein N-α-acetylation in mammalian cells.

We investigated post-translational modification and subcellular localisation of endogenous platelet-derived growth factor-C (PDGF-C) in human thyroid papillary carcinomas (PTC), non-neoplastic thyroid tissues, and a selection of cultured cell lines. PDGF-C expressed nuclear localisation in 95% of all tested cell types in culture and in 10% of the thyrocytes from both PTC and non-neoplastic tissue. The cell lines expressed two forms of full-length PDGF-C, approximately 39 and approximately 55 kDa, in cell membrane and cytosol, while the approximately 55 kDa form dominated in the nucleus where it was partly chromatin-associated. The approximately 55 kDa form was post-translationally modified by SUMO-1. The putative PDGF-C SUMOylation site is the surface exposed (314)lysine part of a positively charged loop ((312)RPKTGVRGLHK(322)) with characteristics of a nuclear localisation signal. The tissue thyrocytes expressed a non-SUMOylated approximately 43 kDa and the 55 kDa PDGF-C. The SUMO-1 modified approximately 55 kDa PDGF-C expression was low in PTC where the approximately 43 kDa PDGF-C dominated. This is in contrast to non-neoplastic tissue and cultured cells where the SUMOylated approximately 55 kDa PDGF-C was strongly expressed. Our data provide novel evidence for nuclear localisation of PDGF-C, post-translational modification by SUMOylation and the expression of a novel form of PDGF-C in human papillary thyroid carcinomas.

The human hARD1–NATH complex, cotranslationally acetylating the α-amino groups of proteins, was recently described. In S. cerevisiae and D. melanogaster this NatA complex contains a third subunit, Nat5p or San, respectively. Based on phylogenetic analyses and database searches, we here describe the human homologue, hNAT5, of these proteins. RT-PCR experiments demonstrated that hNat5 mRNA was expressed in several human cell lines. The candidacy of hNAT5 as a third subunit of the hARD1–NATH complex was investigated using anti-NATH or anti-hARD1 in co-immunoprecipitation experiments followed by Mass Spectrometry analysis of tryptic peptides. Oligopeptides specific for hNAT5 were identified. This verified the expression of endogenous hNAT5 protein in human cells and also identified hNAT5 as a NATH and hARD1 interacting partner. hNAT5 localized to the cytoplasm in accordance with hNAT5–hARD1–NATH complexes playing a role in cotranslational N-α-acetylation. Sequence alignment revealed a high degree of similarity of the NAT5 protein between species supporting its conserved role as a part of the complex. The predicted acetyltransferase domain within hNAT5 suggested that this protein, like hARD1, is an enzymatically active component. In summary, we present the first description of the human homologue of Nat5p/San, hNAT5, the third component of the human NatA N-α-acetyltransferase complex.

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1 alpha and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1 alpha and thereby enhances HIF-1 alpha ubiquitination and degradation. Furthermore, ARD1 was proposed to be down-regulated in hypoxia thus facilitating the stabilization of HIF-1 alpha. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1 alpha. However, we find that hARD1 specifically binds HIF-1 alpha, suggesting a putative, still unclear, connection between these proteins.

Protein acetylation is an important posttranslational modification regulating oncogenesis, apoptosis and cell cycle. NATH (N-acetyl transferase human) is overexpressed at the mRNA level in papillary thyroid carcinomas relative to non-neoplastic thyroid tissue. The NATH protein has recently been demonstrated to be the partner of hARD1 (human Arrest defective 1) and this complex acetylates the N-termini of proteins. ARD1 has also been implicated in the destabilization of the transcription factor HIF-1alpha (hypoxia inducible factor-1alpha). Using human thyroid papillary carcinoma biopsies and NATH- and hARD1-specific antibodies, we examined the levels of endogenous NATH and hARD1 proteins in 27 patients. We demonstrate that NATH protein level is upregulated in neoplastic versus non-neoplastic tissue in good accordance with our previous mRNA findings. In all tumors in which NATH was downregulated compared to non-neoplastic tissue, the hARD1 protein level was concomitantly reduced. SiRNA-mediated knockdown of NATH resulted in decreased levels of hARD1 protein. Taken together, these results suggest that NATH positively affects the level of hARD1 protein both in vivo and in cell cultures.

Protein acetyltransferases and deacetylases have been implicated in oncogenesis, apoptosis and cell cycle regulation. Most of the protein acetyltransferases described acetylate ε-amino groups of lysine residues within proteins. Mouse ARD1 (homologue of yeast Ard1p, where Ard1p stands for arrest defective 1 protein) is the only known protein acetyltransferase catalysing acetylation of proteins at both α- (N-terminus) and ε-amino groups. Yeast Ard1p interacts with Nat1p (N-acetyltransferase 1 protein) to form a functional NAT (N-acetyltransferase). We now describe the human homologue of Nat1p, NATH (NAT human), as the partner of the hARD1 (human ARD1) protein. Included in the characterization of the NATH and hARD1 proteins is the following: (i) endogenous NATH and hARD1 proteins are expressed in human epithelial, glioma and promyelocytic cell lines; (ii) NATH and hARD1 form a stable complex, as investigated by reciprocal immunoprecipitations followed by MS analysis; (iii) NATH–hARD1 complex expresses N-terminal acetylation activity; (iv) NATH and hARD1 interact with ribosomal subunits, indicating a co-translational acetyltransferase function; (v) NATH is localized in the cytoplasm, whereas hARD1 localizes both to the cytoplasm and nucleus; (vi) hARD1 partially co-localizes in nuclear spots with the transcription factor HIF-1α (hypoxia-inducible factor 1α), a known ε-amino substrate of ARD1; (vii) NATH and hARD1 are cleaved during apoptosis, resulting in a decreased NAT activity. This study identifies the human homologues of the yeast Ard1p and Nat1p proteins and presents new aspects of the NATH and hARD1 proteins relative to their yeast homologues.

OBJECTIVE:To study the clinical effect of thymectomy in a well-defined early-onset MG subgroup and to correlate it to MG severity, the presence of circulating muscle autoantibodies, and the need for pharmacological treatment in a long-term setting.METHODS:Fifty-two consecutive AChR antibody-positive early-onset MG patients (34 thymectomized and 18 nonthymectomized) were included. Severity was assessed and the pharmacological treatment monitored on a yearly basis, starting from the year of MG onset, for 5, 10, 15, and 20 consecutive years; AChR, titin, and RyR antibodies were assayed.RESULTS:In the four follow-up groups, MG severity was significantly higher in nonthymectomized compared to thymectomized MG patients. The postthymectomy MG improvement was significant and persistent. There were 21/34 remissions in thymectomized patients and only 4/18 in the nonthymectomized group. Patients with initially high or low AChR antibody concentration had a similar thymectomy outcome. Only 6 patients had titin antibodies, and none had RyR antibodies.CONCLUSION:The present study indicates a benefit of thymectomy in early-onset MG. The muscle autoantibody concentration does not influence the outcome of thymectomy in early-onset MG.

Platelet-derived growth factor (PDGF)-C is a novel member of the PDGF family that binds to PDGF alphaalpha and alphabeta receptors. The growth factor domain of PDGF-C (GFD-PDGF-C) was expressed in high yields in Escherichia coli and was purified and refolded from inclusion bodies obtaining a biologically active growth factor with dimeric structure. The GFD-PDGF-C contains 12 cysteine residues, and Ellman assay analysis indicates that it contains three intramonomeric disulfide bonds, which is in accordance with GFD-PDGF-C being a member of the cystine knot superfamily of growth factors. The recombinant GFD-PDGF-C was characterized by CD, fluorescence, NMR, and infrared spectroscopy. Together, our data indicate that GFD-PDGF-C is a highly thermostable protein that contains mostly beta-sheet secondary structure and some (6%) alpha-helix structure. The structural model of PDGF-C, obtained by homology-based molecular modeling using the structural representatives of this family of growth factors, shows that GFD-PDGF-C has a higher structural homology to the vascular endothelial growth factor than to PDGF-B. The modeled structure can give further insights into the function and specificity of this molecule.