Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first ~600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl

Virtually Coupled Train Sets - A Comprehensive Analysis

In the X2Rail-3 project of the Shift2Rail Joint Undertaking, a detailed virtual coupling concept is developed and analysed in an extensive theoretical framework. Based on the paradigm shift from absolute to relative braking distance, various operational scenarios and potential hazards are identified. According to these scenarios and the general functional architecture, the project provides a first definition of the system requirements, detailing both the virtual coupling components as well as the interfaces and interactions with external systems. The subsequent analysis shows that virtual coupling can enable multiple performance benefits for railway operation. Beside the significant increase in capacity through shorter headways and reduced coupling times, additional flexibility and robustness are gained with situationally appropriate, dynamic coupling manoeuvres with a free choice of vehicles. Moreover, virtual coupling can contribute to reducing costs and delays by replacing the mechanical coupler. Aiming for technology readiness level 3, the results of the project illustrate that the concept is feasible from a technological and operational point of view, highlighting the most critical implementation parts in sensors, control and communication. A two-stage implementation approach is proposed to facilitate the introduction of virtual coupling. This is based on a minimum-complexity first stage to introduce the core functionalities and a second stage with incremental implementation of additional virtual coupling functionalities to further improve the operation. This stepwise approach is also corroborated by the results of the impact analysis, which shows a reduced impact for the first stage. The impact analysis illustrates that the fundamental principle of the external systems remains untouched and a full rework is not required. This also implies that virtual coupling will not interfere with the progress of the European Train Control System (ETCS) development and deployment. Therefore, the X2Rail-3 virtual coupling concepts provides a feasible, non-disruptive solution for a more efficient railway transport

Technical feasibility analysis and introduction strategy of the virtually coupled train set concept

Todays railway network capacity is limited by constraints imposed by traditional train protection systems. A way to overcome those limitations, maximize the railway network performance and also increase the operational flexibility is presented by the Virtually Coupled Train Set (VCTS) concept. This paper evaluates the technical feasibility of this approach, that was developed and is further evaluated in the framework of the Shift2Rail (S2R) project X2Rail-3. The main functionality of virtually coupled train sets is achieved by replacing the mechanical coupler between two railway vehicles by an electronic (virtual) coupling link. This operational change requires a permanent vehicle-to-vehicle communication and precise distance measurement, while enabling much faster coupling and decoupling procedures, increased interoperability and the operation of trains with a headway below absolute braking distance. To evaluate the technical feasibility of the VCTS concept, a series of technical and operational subsystem have been identified and analyzed. Interviews with experts from a variety of VCTS linked topics have been conducted, to evaluate the state of the art and new developments for those subsystems. Subsequently, the capabilities of the subsystems have been compared with the requirements of the VCTS system. In addition, different mitigations to overcome possible obstacles have been identified and evaluated. As the result, the most critical technical aspects for the implementation and success of VCTS have been identified as the requirement of controllable, fast and accurate responding braking systems, the availability of suitable communication technologies and frequency bands, the need for highly-accurate measurement of distance, speed and acceleration and the fast detection and monitoring of train integrity. Considering those results, a qualitative roadmap for the future VCTS development and introduction strategy is derived

Glucagon effect on rat liver protein synthesis in vivo

The in vivo effect of glucagon administration on hepatic polyribosomal profiles has been studied. Glucagon did not change significantly total, free or bound polyribosomal fractions 30-45 minutes after its administration. The combined administration of glucagon plus antiinsulin serum failed to show any significant effect of glucagon over the antiinsulin serum treated control. Glucagon increased valine production in the perfused isolated liver. These results suggest that the well known amino acid catabolic action of glucagon may be preferentially mediated through an increased proteolysis. Since it is known that glucagon increases considerably in vivo the liver cyclic AMP levels then its lack of effect on polyribosomal profiles might indicate that the postulated role for the cyclic nucleotide on liver protein synthesis must be taken cautiously.This work has been supported by a Grant-in-aid from Lilly Indiana S.A. and a research grant (612/g) from Fondo Nacional para el Desarrollo de la Investigación.Peer reviewe

Cellular metabolite distribution and the control of gluconeogenesis in the perfused isolated rat liver

Glucose production was measured in isolated rat livers perfused with 100 ml of blood-free recirculating medium. The gluconeogenic rate using l-alanine as substrate was only 55% of that obtained with l-lactate. The steady-state concentration of gluconeogenic and tricarboxylic acid cycle intermediates were measured in freeze clamped biopsies. Livers perfused with l-lactate displayed higher concentrations of malate, α-glycerophosphate and β-hydroxybutyrate probably as a result of a higher state of reduction of the nicotinamide system. Hexose-phosphate intermediates were also increased when l-lactate was the substrate. Phosphoenolpyruvate and 3-phosphoglycerate were considerably elevated when l-alanine was the glucose precursor. Livers perfused with l-lactate displayed higher cytosolic concentration of all the tricarboxylic acid cycle intermediates except oxaloacetate while glutamate was slightly and aspartare considerably higher when alanine was the substrate. In the mitochondrial compartment the pattern of distribution tended to be the opposite; that is, livers perfused with l-lactate showed lower concentrations of all the intermediates except α-ketoglutarate. The mitochondrial: cytosolic metabolite gradients of all the intermediates whose distribution was studied were higher in livers perfused with l-alanine. The relevance of these findings to the observed differences in the gluconeogenic fluxes are discussed.This work has been supported in part by a Grant-in-aid from Lilly Indiana and a research grant (612/9) from the Spanish Advisory Commission for the Development of Research.Peer reviewe

Effect of vasopressin on the regulation of protein synthesis initiation in liver cells.

Vasopressin was found to be an effective inhibitor of protein labelling in isolated liver cells. Its effect shows the following distinct characteristics: (1) in contrast with alpha-adrenergic agonists, its effect is observable under a wide range of cellular Ca2+-loading conditions; (2) it is not influenced by the nutritional state of the animal. The lack of vasopressin effect on valine production, and its ability to decrease protein labelling from near-saturation concentrations of [3H]valine, indicate that the observed variations in protein labelling reflect actual changes in the rate of protein synthesis. The action of vasopressin is primarily exerted on the initiation step of protein synthesis and this effect is accompanied by a decreased activity of eukaryotic initiation factor 2. Activators of protein kinase C showed similar but not additive effects on protein synthesis, as did vasopressin. It seems plausible to conclude that protein kinase C activation may play an important regulatory role in hepatic protein synthesis as a transducer of hormonal and perhaps other type of signals

Cellular redistribution of metabolites during glucagon and insulin control of gluconeogenesis in the isolated perfused rat liver

Livers isolated from fasted rats were perfused in a blood-free recirculating system using alanine (10 mm) as the carbon source. Glucagon at a concentration of 2.1 × 10−9m enhanced gluconeogenesis, ureogenesis, and ketogenesis. The proportion of alanine utilized to glucose formed remained rather constant in all the situations studied, suggesting that the contribution of glycogen breakdown to the total glucose output was negligible. The glucagon stimulation of gluconeogenesis was accompanied by a decrease in the [ATP]/[ADP]ratio and a rise in the reduction state of the cytosolic and mitochondrial NAD systems. The calculation of the intracellular distribution of metabolites indicates that glucagon increases the intramitochondrial oxaloacetate concentration. This finding seems to support the hypothesis of pyruvate carboxylation, the first nonequilibrium enzymic step in the gluconeogenic sequence, as one of the main sites of glucagon action. The rise in the mitochondrial:cytosolic concentration gradient of malate suggests that glucagon may also act by facilitating the transfer of three-carbon units from the mitochondria to the cytosol. The fact that insulin reversed virtually all the glucagon-induced changes strongly suggests that both hormones act on common steps. It is remarkable that these insulin effects occur at glucagon/insulin ratios similar to those normally found in the portal vein of the intact animal.This work has been supported by grants from Lilly Indiana S.A., Fundación Rodríguez Pascual and Comisión Asesora para el Desarrollo de la InvestigaciónPeer reviewe