Transcriptional Regulation of N-Acetylglutamate Synthase

The urea cycle converts toxic ammonia to urea within the liver of mammals. At least 6 enzymes are required for ureagenesis, which correlates with dietary protein intake. The transcription of urea cycle genes is, at least in part, regulated by glucocorticoid and glucagon hormone signaling pathways. N-acetylglutamate synthase (NAGS) produces a unique cofactor, N-acetylglutamate (NAG), that is essential for the catalytic function of the first and rate-limiting enzyme of ureagenesis, carbamyl phosphate synthetase 1 (CPS1). However, despite the important role of NAGS in ammonia removal, little is known about the mechanisms of its regulation. We identified two regions of high conservation upstream of the translation start of the NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Within the promoter, we identified multiple transcription start sites that differed between liver and small intestine. Several transcription factor binding motifs were conserved within the promoter and enhancer regions while a TATA-box motif was absent. DNA-protein pull-down assays and chromatin immunoprecipitation confirmed binding of Sp1 and CREB, but not C/EBP in the promoter and HNF-1 and NF-Y, but not SMAD3 or AP-2 in the enhancer. The functional importance of these motifs was demonstrated by decreased transcription of reporter constructs following mutagenesis of each motif. The presented data strongly suggest that Sp1, CREB, HNF-1, and NF-Y, that are known to be responsive to hormones and diet, regulate NAGS transcription. This provides molecular mechanism of regulation of ureagenesis in response to hormonal and dietary changes

Second generation tyrosine kinase inhibitors prevent disease progression in high-risk (high CIP2A) chronic myeloid leukaemia patients.

High cancerous inhibitor of PP2A (CIP2A) protein levels at diagnosis of chronic myeloid leukaemia (CML) are predictive of disease progression in imatinib-treated patients. It is not known whether this is true in patients treated with second generation tyrosine kinase inhibitors (2G TKI) from diagnosis, and whether 2G TKIs modulate the CIP2A pathway. Here, we show that patients with high diagnostic CIP2A levels who receive a 2G TKI do not progress, unlike those treated with imatinib (P=<0.0001). 2G TKIs induce more potent suppression of CIP2A and c-Myc than imatinib. The transcription factor E2F1 is elevated in high CIP2A patients and following 1 month of in vivo treatment 2G TKIs suppress E2F1 and reduce CIP2A; these effects are not seen with imatinib. Silencing of CIP2A, c-Myc or E2F1 in K562 cells or CML CD34+ cells reactivates PP2A leading to BCR-ABL suppression. CIP2A increases proliferation and this is only reduced by 2G TKIs. Patients with high CIP2A levels should be offered 2G TKI treatment in preference to imatinib. 2G TKIs disrupt the CIP2A/c-Myc/E2F1 positive feedback loop, leading to lower disease progression risk. The data supports the view that CIP2A inhibits PP2Ac, stabilising E2F1, creating a CIP2A/c-Myc/E2F1 positive feedback loop, which imatinib cannot overcome

SUDOSCAN: A Simple, Rapid, and Objective Method with Potential for Screening for Diabetic Peripheral Neuropathy.

Clinical methods of detecting diabetic peripheral neuropathy (DPN) are not objective and reproducible. We therefore evaluated if SUDOSCAN, a new method developed to provide a quick, non-invasive and quantitative assessment of sudomotor function can reliably screen for DPN. 70 subjects (45 with type 1 diabetes and 25 healthy volunteers [HV]) underwent detailed assessments including clinical, neurophysiological and 5 standard cardiovascular reflex tests (CARTs). Using the American Academy of Neurology criteria subjects were classified into DPN and No-DPN groups. Based on CARTs subjects were also divided into CAN, subclinical-CAN and no-CAN. Sudomotor function was assessed with measurement of hand and foot Electrochemical Skin Conductance (ESC) and calculation of the CAN risk score. Foot ESC (μS) was significantly lower in subjects with DPN [n = 24; 53.5(25.1)] compared to the No-DPN [77.0(7.9)] and HV [77.1(14.3)] groups (ANCOVA p<0.001). Sensitivity and specificity of foot ESC for classifying DPN were 87.5% and 76.2%, respectively. The area under the ROC curve (AUC) was 0.85. Subjects with CAN had significantly lower foot [55.0(28.2)] and hand [53.5(19.6)] ESC compared to No-CAN [foot ESC, 72.1(12.2); hand ESC 64.9(14.4)] and HV groups (ANCOVA p<0.001 and 0.001, respectively). ROC analysis of CAN risk score to correctly classify CAN revealed a sensitivity of 65.0% and specificity of 80.0%. AUC was 0.75. Both foot and hand ESC demonstrated strong correlation with individual parameters and composite scores of nerve conduction and CAN. SUDOSCAN, a non-invasive and quick test, could be used as an objective screening test for DPN in busy diabetic clinics, insuring adherence to current recommendation of annual assessments for all diabetic patients that remains unfulfilled