The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis.

Bicarbonate (HCO3-) ions maintain pH homeostasis in eukaryotic cells and serve as a carbonyl donor to support cellular metabolism. However, whether the abundance of HCO3- is regulated or harnessed to promote cell growth is unknown. The mechanistic target of rapamycin complex 1 (mTORC1) adjusts cellular metabolism to support biomass production and cell growth. We find that mTORC1 stimulates the intracellular transport of HCO3- to promote nucleotide synthesis through the selective translational regulation of the sodium bicarbonate cotransporter SLC4A7. Downstream of mTORC1, SLC4A7 mRNA translation required the S6K-dependent phosphorylation of the translation factor eIF4B. In mTORC1-driven cells, loss of SLC4A7 resulted in reduced cell and tumor growth and decreased flux through de novo purine and pyrimidine synthesis in human cells and tumors without altering the intracellular pH. Thus, mTORC1 signaling, through the control of SLC4A7 expression, harnesses environmental bicarbonate to promote anabolic metabolism, cell biomass, and growth.

Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis.

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

Improvement of in vitro thrombolysis employing magnetically-guided microspheres.

Significant shortcomings in clinical thrombolysis efficiencies and arterial recanalization rates still exist to date necessitating the development of additional thrombolysis-enhancing technologies. For example, to improve tPA-induced systemic clot lysis several supplementary treatment methods have been proposed, among them ultrasound-enhanced tissue plasminogen activator (tPA) thrombolysis which has already found some clinical applicability. The rationale of this study was to investigate whether biodegradable, magnetic spheres can be a useful adjuvant to currently existing tPA-induced thrombolysis and further enhance clot lysis results. Based on an envisioned, novel thrombolysis technology--magnetically-guided, tPA-loaded nanocarriers with triggered release of the shielded drug at an intravascular target site--we evaluated the lysis efficiencies of magnetically-guided, non-medicated magnetic spheres in various combinations with tPA and ultrasound. When tPA was used in conjunction with magnetic spheres and a magnetic field, the lysis efficiency under static, no-flow conditions improved by 1.7 and 2.7 fold for red and white clots, respectively. In dynamic lysis studies, the addition of ultrasound and magnetically-guided spheres to lytic tPA dosages resulted in both maximum clot lysis efficiency and shortest reperfusion time corresponding to a 2-fold increase in lysis and 7-fold reduction in recanalization time, respectively. Serial microscopic evaluations on histochemical sections reconfirmed that tPA penetration into and fragmentation of the clot increased with escalating exposure time to tPA and magnetic spheres/field. These results delineate the effectiveness of magnetic spheres as an adjuvant to tPA therapy accelerating in vitro lysis efficiencies beyond values found for tPA with and without ultrasound. We demonstrated that the supplementary use of magnetically-guided, non-medicated magnetic spheres significantly enhances in vitro static and dynamic lysis of red and white blood clots.

A novel human detoxification system based on nanoscale bioengineering and magnetic separation techniques.

We describe the conceptual approach, theoretical background and preliminary experimental data of a proposed platform technology for specific and rapid decorporation of blood-borne toxins from humans. The technology is designed for future emergent in-field or in-hospital detoxification of large numbers of biohazard-exposed victims; for example, after radiological attacks. The proposed systems is based on nanoscale technology employing biocompatible, superparamagnetic nanospheres, which are functionalized with target-specific antitoxin receptors, and freely circulate within the human blood stream after simple intravenous injection. Sequestration of the blood-borne toxins onto the nanosphere receptors generates circulating nanosphere-toxin complexes within a short time interval; mathematical modeling indicates prevailing of unbound nanosphere receptors over target toxin concentrations at most therapeutic injection dosages. After a toxin-specific time interval nanosphere-toxin complexes are generated within the blood stream and, after simple arterial or venous access, the blood is subsequently circulated via a small catheter through a portable high gradient magnetic separator device. In this device, the magnetic toxin complexes are retained by a high gradient magnetic field and the detoxified blood is then returned back to the blood circulation (extracorporeal circulation). Our preliminary in vitro experiments demonstrate >95% first pass capture efficiency of magnetic spheres within a prototype high gradient magnetic separation device. Further, based on the synthesis of novel hydrophobic magnetite nanophases with high magnetization ( approximately 55 emu/g), the first biodegradable magnetic nanospheres at a size range of approximately 280 nm and functionalized with PEG-maleimide surface groups for specific antibody attachment are described here. In future applications, we envision this technology to be suitable for emergent, in-field usage for acutely biohazard exposed victims as both the injectable toxin-binding magnetic spheres and the separator device are made to be portable, light-weight, zero-power, and self- or helper-employed. Details of the technology are presented and the state-of-knowledge and research is discussed.

Research imaging in an academic medical center.

RATIONALE AND OBJECTIVES: Managing and supervising the complex imaging examinations performed for clinical research in an academic medical center can be a daunting task. Coordinating with both radiology and research staff to ensure that the necessary imaging is performed, analyzed, and delivered in accordance with the research protocol is nontrivial. The purpose of this communication is to report on the establishment of a new Human Imaging Research Office (HIRO) at our institution that provides a dedicated infrastructure to assist with these issues and improve collaborations between radiology and research staff. MATERIALS AND METHODS: The HIRO was created with three primary responsibilities: 1) coordinate the acquisition of images for clinical research per the study protocol, 2) facilitate reliable and consistent assessment of disease response for clinical research, and 3) manage and distribute clinical research images in a compliant manner. RESULTS: The HIRO currently provides assistance for 191 clinical research studies from 14 sections and departments within our medical center and performs quality assessment of image-based measurements for six clinical research studies. The HIRO has fulfilled 1806 requests for medical images, delivering 81,712 imaging examinations (more than 44.1 million images) and related reports to investigators for research purposes. CONCLUSIONS: The ultimate goal of the HIRO is to increase the level of satisfaction and interaction among investigators, research subjects, radiologists, and other imaging professionals. Clinical research studies that use the HIRO benefit from a more efficient and accurate imaging process. The HIRO model could be adopted by other academic medical centers to support their clinical research activities; the details of implementation may differ among institutions, but the need to support imaging in clinical research through a dedicated, centralized initiative should apply to most academic medical centers.

Human biliverdin reductase: a member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity.

We describe here the tyrosine kinase activity of human biliverdin reductase (BVR) and its potential role in the insulin-signaling pathway. BVR is both a substrate for insulin receptor (IR) tyrosine kinase (IRK) activity and a kinase for serine phosphorylation of IR substrate 1 (IRS-1). Our previous studies have revealed serine/threonine kinase activity of BVR. Y198, in the YMKM motif found in the C-terminal domain of BVR, is shown to be a substrate for insulin-activated IRK. This motif in IRS proteins provides a docking site for proteins that contain a Src homology 2 domain. Additionally, Y228 in the YLSF sequence and Y291 are IRK substrates; the former sequence provides optimum recognition motif in the tyrosine phosphatase, SHP-1, and for SHC (Src homology 2 domain containing transfroming protein 1). BVR autophosphorylates N-terminal tyrosines Y72 and Y83. Serine residues in IRS-1 are targets for BVR phosphorylation, and point mutation of serine residues in the kinase domain of the reductase inhibits phosphotransferase activity. Because tyrosine phosphorylation of IRS-1 activates the insulin signaling pathway and serine phosphorylation of IRS-1 blocks insulin action, our findings that insulin increases BVR tyrosine phosphorylation and that there is an increase in glucose uptake in response to insulin when expression of BVR is "knocked down" by small interfering RNA suggest a potential role for BVR in the insulin signaling pathway.

Small interference RNA-mediated gene silencing of human biliverdin reductase, but not that of heme oxygenase-1, attenuates arsenite-mediated induction of the oxygenase and increases apoptosis in 293A kidney cells.

BVR reduces biliverdin, the HO-1 and HO-2 product, to bilirubin. Human biliverdin (BVR) is a serine/threonine kinase activated by free radicals. It is a leucine zipper (bZip) DNA-binding protein and a regulatory factor for 8/7-bp AP-1-regulated genes, including HO-1 and ATF-2/CREB. Presently, small interference (si) RNA constructs were used to investigate the role of human BVR in sodium arsenite (As)-mediated induction of HO-1 and in cytoprotection against apoptosis. Activation of BVR involved increased serine/threonine phosphorylation but not its protein or transcript levels. The peak activity at 1 h (4-5-fold) after treatment of 293A cells with 5 mum As preceded induction of HO-1 expression by 3 h. The following suggests BVR involvement in regulating oxidative stress response of HO-1: siBVR attenuated As-mediated increase in HO-1 expression; siBVR, but not siHO-1, inhibited As-dependent increased c-jun promoter activity; treatment of cells with As increased AP-1 binding of nuclear proteins; BVR was identified in the DNA-protein complex; and AP-1 binding of the in vitro translated BVR was phosphorylation-dependent and was attenuated by biliverdin. Most unexpectedly, cells transfected with siBVR, but not siHO-1, displayed a 4-fold increase in apoptotic cells when treated with 10 mum As as detected by flow cytometry. The presence of BVR small interference RNA augmented the effect of As on levels of cytochrome c, TRAIL, and DR-5 mRNA and cleavage of poly(ADP-ribose) polymerase. The findings describe the function of BVR in HO-1 oxidative response and, demonstrate, for the first time, not only that BVR advances the role of HO-1 in cytoprotection but also affords cytoprotection independent of heme degradation.

Biliverdin reductase, a novel regulator for induction of activating transcription factor-2 and heme oxygenase-1.

Biliverdin IXalpha reductase (BVR) catalyzes reduction of the HO activity product, biliverdin, to bilirubin. hBVR is a serine/threonine kinase that contains a bZip domain. Presently, regulation of gene expression by hBVR was examined. 293A cells were infected with adenovirus-doxycycline (Ad-Dox)-inducible hBVR cDNA. High level expression of hBVR was determined at mRNA, protein, and activity levels 8 h after induction. Cell signal transduction microarray analysis of cells infected with expression or with the control Ad-inverted (INV)-hBVR vector identified ATF-2 among several up-regulated genes. ATF-2 is a bZip transcription factor for activation of cAMP response element (CRE) and a dimeric partner to c-jun in MAPK pathway that regulates the stress protein, HO-1, expression. Northern and Western blot analyses showed increases of approximately 10-fold in ATF-2 mRNA and protein at 16 and 24 h after Dox addition. Ad-INV-hBVR did not effect ATF-2 expression. In hBVR-infected cells, levels of HO-1 mRNA and protein were increased. In vitro translated hBVR and nuclear extract containing hBVR in gel mobility-shift assay bound to AP-1 sites in the ATF-2 promoter region and to an oligonucleotide containing the CRE site. Both bindings could be competed out by excess unlabeled probe; in the presence of hBVR antibody, they displayed shifted bands. Co-transfection of hBVR with ATF-2 or c-jun promoters caused a severalfold increase in luciferase activity. hBVR modulation of ATF-2 and HO-1 expression suggests it has a potential role in regulation of AP-1 and cAMP-regulated genes and a role in cell signaling. We propose that increased expression of the protein can be used to alter the gene expression profile in the cell.