Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRβ, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding-defective TRβ leads to disruption of the hypothalamic-pituitary-thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.

The occludin-like proteins belong to a family of tetraspan transmembrane proteins carrying a marvel domain. The intrinsic function of the occludin family is not yet clear. Occludin is a unique marker of any tight junction and is found in polarized endothelial and epithelial tissue barriers, at least in the adult vertebrate organism. Occludin is able to oligomerize and to form tight junction strands by homologous and heterologous interactions, but has no direct tightening function. Its oligomerization is affected by pro- and antioxidative agents or processes. Phosphorylation of occludin has been described at multiple sites and is proposed to play a regulatory role in tight junction assembly and maintenance and, hence, to influence tissue barrier characteristics. Redox-dependent signal transduction mechanisms are among the pathways modulating occludin phosphorylation and function. This review discusses the novel concept that occludin plays a key role in the redox regulation of tight junctions, which has a major impact in pathologies related to oxidative stress and corresponding pharmacologic interventions.

In memory of Rainer RudolphBreast cancer is still one of the most frequently occurring tumors in women. Severe and often therapy-limiting side effects are a major obstacle in chemotherapy. New delivery concepts that reduce systemic side effects are needed to optimize anticancer therapies, and selective targeting concepts are required for early and selective tumor diagnosis. Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is a C-terminal amidated peptide hormone consisting of 36 amino acid residues.[1, 2] NPY-mediated functions are transmitted by so-called Y receptors, named Y 1 , Y 2 , and Y 5 receptors, which bind NPY with nanomolar affinity. All Y receptors are members of the class A of heptahelix receptors, that signal through heterotrimeric G proteins. [3,4] Reubi et al. have recently described Y-receptor expression in human breast cancer. They have shown that over 90 % of all breast tumors and 100 % of the examined metastases express Y 1 receptors.[5] Interestingly, a shift of the receptor subtype from Y 2 receptors in healthy tissue to Y 1 receptors during neoplasm was found, which is potentially related to reduced differentiation. Based on NPY and the known structure-activity relationships for Y 1 -receptor binding, [6] we designed, synthesized, and characterized two analogues for tumor labeling that vary in the position of the chelator to conjugate 99m Tc. Peptides 1 a and 2 a were synthesized with a N a -histidinyl acetyl (N a His-ac) chelator [7] at the N terminus, whereas peptides 1 b and 2 b were modified at the N e side chain of Lys 4 . The tridentate ligand N a His-ac is able to form stable and biologically active complexes. [8,9] Modification of the resin-bound peptide was performed by an efficient strategy (Scheme 1). In the first step, bromoacetic acid was activated by diisopropylcarbodiimide to form the corresponding anhydride. His(Trt)-OtBu was then added and the NHÀ CH bond was formed by HBr elimination. Cleavage of the peptide yielded His-acetyl peptides either at the N terminus or at the N e side chain of Lys 4 . Rhenium was used as a cold surrogate for 99m Tc and introduced for in vitro studies

ContextAnaplastic thyroid carcinoma (ATC) represents one of the most aggressive carcinomas with no consistent survival benefit when treated with conventional radiochemotherapy. Approaches targeting “oncogene addiction” of ATC are increasingly explored and first promising results have been reported in single case studies.ObjectiveTo determine the prevalence of mutations in known thyroid oncogenes and signalling pathways amendable to targeted therapy in a large cohort of ATC.ResultsIn 118 ATC (57 male/ 61 female) a total of 165 mutations were found. Genes involved in the MAPK/ERK and PI3K pathway (BRAF 11.0%, HRAS 4.2%, KRAS 7.6%, NRAS 7.6%, PI3KCA 11.8%) were altered in 33%. Targetable receptor tyrosine kinases were mutated in 11%. The most frequently altered genes were TERT in 86/118 (73%) and p53 in 65/118 (55%) cases. No mutations were found analysing ALK, KIT, MET and mTOR.Materials and MethodsNext generation sequencing (NGS) was performed in FFPE samples from 118 ATC using MiSeq (Illumina) and CLC Cancer Research Workbench (CLCbio; Qiagen) for mutation analysis in: ALK, BRAF, CDKN2A, EGFR, ERBB2, HRAS, KIT, KRAS, MET, mTOR, NRAS, PDGFRA, PI3KCA, p53, RB1, RET and TSC2. Sanger sequencing was used to detect TERT promotor mutations.ConclusionsTo our knowledge this is the largest study analysing mutations for targeted therapy of ATC. We found that 33% of ATC harbour mutations in pathways amendable to targeted therapy. Molecular screening in ATC is suggested for targeted therapies since current conventional treatment for ATC proved mainly futile.

Selective NPY analogues are potent tools for tumour targeting. Their Y(1)-receptors are significantly over-expressed in human breast tumours, whereas normal breast tissue only expresses Y(2)-receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y(1)-receptor agonists enhance the tumour to background ratio compared to Y(1)-receptor antagonists, we were interested in the development of Y(1)-receptor selective agonists. We designed 19 peptides containing the C-terminus of NPY (28-36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y(1)-receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N-terminally 5(6)-carboxyfluorescein (CF) labelled peptides resulted in half-lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y(1)-receptor internalization. For the first time, here we show the design and characterization of a small Y(1)-receptor selective agonist. This agonist might be a useful novel ligand for NPY-mediated tumour diagnostics and therapeutics.