Metabolic profile and quantification of deoxyribose synthesis pathways in HepG2 cells

Traditional tracer studies of cell proliferation fail to distinguish between label enrichment due to increased DNA repair versus DNA replication. We used the emerging stable (non-radiating) isotope-based dynamic metabolic profiling technique on HepG2 cells to determine synthesis pathways of nucleic acids from glucose and rates of proliferation using CG-MS assay of RNA and DNA enrichment. Comparing the isotopic enrichment curve in DNA with the theoretical curve based on cell growth, we observed that the measured tracer enrichment was significantly higher, indicating that surplus label was acquired during DNA repair. In particular, after the first duplication (3 days), 80.13% of the total enrichment observed corresponds to duplication and 19.87% corresponds to DNA repair as calculated from the [1, 2-¹³C₂]-glucose incorporation curve. Our data indicate contemporary measurements of cell proliferation rates relying on tracer incorporation may be overestimated. ¹³C label was distributed between m1 (m1/Σm = 80) and m2 (m2/Σm = 14) of deoxyribose, indicating that most of the glucose carbon was acquired via direct glucose oxidation in the pentose cycle. The stable isotope technique distinguishes rates of DNA synthesis and repair via the oxidative and non-oxidative pentose cycle, separately, in one test, without inhibition of either process. The contribution of DNA repair in malignant cells to isotope accumulation in deoxyribose remains to be investigated.     

Fungal transformation of natural and synthetic cobalt-bearing manganese oxides and implications for cobalt biogeochemistry

Manganese oxide minerals can become enriched in a variety of metals through adsorption and redox processes, and this forms the basis for a close geochemical relationship between Mn oxide phases and Co. Since oxalate-producing fungi can effect geochemical transformation of Mn oxides, an understanding of the fate of Co during such processes could provide new insights on the geochemical behaviour of Co. In this work, the transformation of Mn oxides by Aspergillus niger was investigated using a Co-bearing manganiferous laterite, and a synthetic Co-doped birnessite. A. niger could transform laterite in both fragmented and powder forms, resulting in formation of biomineral crusts that were composed of Mn oxalates hosting Co, Ni and, in transformed laterite fragments, Mg. Total transformation of Co-doped birnessite resulted in precipitation of Co-bearing Mn oxalate. Fungal transformation of the Mn oxide phases included Mn(III,IV) reduction by oxalate, and may also have involved reduction of Co(III) to Co(II). These findings demonstrate that oxalate-producing fungi can influence Co speciation in Mn oxides, with implications for other hosted metals including Al and Fe. This work also provides further understanding of the roles of fungi as geoactive agents which can inform potential applications in metal bioremediation, recycling and biorecovery.     

The role of the calcium-sensing receptor in epidermal differentiation

Calcium regulates the proliferation and differentiation of keratinocytes both in vivo and in vitro. Elevated extracellular Ca2+ concentration ([Ca2+]o) raises the intracellular free calcium ([Ca2+]i) and activates differentiation-related genes. Cells lacking the calcium-sensing receptor (CaR) fail to respond to [Ca2+]o and to differentiate, indicating a role for CaR in keratinocyte differentiation. These concepts derived from in vitro experiments have been tested and confirmed in two mouse models.     

Recognition of lumenal prion protein aggregates by post-ER quality control mechanisms is mediated by the preoctarepeat region of PrP

Prion diseases are fatal transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We have shown previously that the chemical compound suramin induced aggregation of fully matured PrP(c) in post-ER compartments, thereby, activating a post-ER quality control mechanism and preventing cell surface localization of PrP by intracellular re-routing of aggregated PrP from the Golgi/TGN directly to lysosomes. Of note, drug-induced PrP aggregates were not toxic and could easily be degraded by neuronal cells. Here, we focused on determining the PrP domains mediating these effects. Using PrP deletion mutants we show that intracellular re-routing but not aggregation depends on the N-terminal PrP (aa 23-90) and, more precisely, on the preoctarepeat domain (aa 23-50). Fusion of the PrP N-terminus to the GPI-anchored protein Thy-1 did not cause aggregation or re-routing of the chimeric protein, indicating that the N-terminus is only active in re-routing when prion protein aggregation occurs. Insertion of a region with a comparable primary structure contained in the PrP paralogue prnd/doppel (aa 27-50) into N-terminally deleted PrP re-established the re-routing phenotype. Our data reveal an important role for the conserved preoctarepeat region of PrP, namely controlling the intracellular trafficking of misfolded PrP.     

Hürthle cell carcinoma: diagnostic and therapeutic implications

Background  

Hürthle cell carcinoma is a variant of follicular cell carcinoma of thyroid. It may present as a low-grade tumour or as a more aggressive type. Prognosis depends upon the age of the patient, tumour size, extent of invasion and initial nodal or distant metastasis.     

The Production of Reactive Oxygen Species in Intact Isolated Nerve Terminals Is Independent of the Mitochondrial Membrane Potential

Dependence on mitochondrial membrane potential (m) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H₂O₂ generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250nM) or DNP (50mM) and then the rate of H₂O₂ production was followed. Neither of the uncouplers had a significant effect on the rate of H₂O₂ production induced by rotenone or antimycin. Inhibition of the F₀F₁-ATPase by oligomycin, which also eliminates m in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H₂O₂ production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of m. In addition, we detected a significant antimycin-induced H₂O₂ production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.     

Hepatitis C virus core and nonstructural protein 3 proteins induce pro- and anti-inflammatory cytokines and inhibit dendritic cell differentiation

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-alpha production in monocytes. Degradation of I-kappa B alpha suggested involvement of NF-kappa B activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-alpha and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients' DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.     

Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood-brain barrier

Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood-brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent.