In Vivo Formation and Binding of SeHg Complexes to the Erythrocyte Surface

The in vivo dynamics of selenium (Se) and mercury (Hg) interaction was studied in mouse tissues using direct visualization of individual Se, Hg, and SeHg particles on the surface of circulating erythrocytes. This high-resolution detection of Se and Hg was obtained by scanning electron microscopy coupled to X-ray microanalysis. BALB/c mice were injected in the peritoneal cavity with Se and Hg salts, and the animals were sacrificed 3 min after the Hg injection. Only a minority (9%) of the metal dots seen on mouse liver erythrocytes were SeHg complexes when Se and Hg salts were mixed together before injection. In contrast, the majority (73%) of metal dots on liver erythrocytes were SeHg complexes if Se was injected at least 5 min before Hg injection. All metal dots on liver erythrocytes were of SeHg complexes if Se was injected 9 or 12 min before the Hg injection. We conclude that the formation of stable in vivo SeHg complexes requires preliminary interaction of Se with a putative serum factor before complexes between Se and Hg are formed and are bound to the erythrocyte cell surface.     

Mutations in methylenetetrahydrofolate reductase and in cysthationine beta synthase: is there a link to homocysteine levels in peripheral arterial disease?

Peripheral arterial disease (PAD) is an atherosclerotic disturbance characterized by a progressive obstruction of lower limb arteries. Many risk factors associated with PAD development have being reported in the literature. The present study aimed to investigate whether mutations in the methylenetetrahydrofolate reductase (MTHFR) or in the cystathionine beta synthase (CBS) genes are associated with higher levels of homocysteine and the risk of PAD in patients from Brazil. This study analyzed 39 patients with PAD and 32 without PAD in whom risk factors and C677T mutations in the MTHFR gene and both 844ins68 and T833C mutations in the CBS gene were investigated. Although higher levels of homocysteine could be observed in patients with PAD compared to controls, no association between the increase of homocysteine and the frequency of C677T, 844ins68, and T833C mutations could be observed. The results suggest that these mutations do not appear to be related to either homocysteine levels or the development of the disease. However, hyperhomocysteinemia and smoking are important factors in PAD development.     

Ammonium Transformation in a Nitrogen-Rich Tidal Freshwater Marsh

The fate and transport of watershed-derived ammonium in a tidal freshwater marsh fringing the nutrient rich Scheldt River, Belgium, was quantified in a whole ecosystem ¹⁵N labeling experiment. In late summer (September) we added ¹⁵N-NH₄⁺ to the flood water entering a 3477 m² tidal freshwater marsh area, and traced the ammonium processing and retention in four subsequent tide cycles. In this paper we present the results for the water-phase components of the marsh system and compare them to a similar experiment conducted in spring/early summer (May). Changes in concentration and isotopic enrichment of NO₃⁻ + NO₂⁻, N₂O, N₂, NH₄⁺ and suspended particulate nitrogen (SPN) were measured in concert with a mass balance study. All analyzed N-pools were labeled, and 49% of the added ¹⁵NH₄⁺ was retained or transformed. The most important pool for ¹⁵N was nitrate, accounting for 17% of ¹⁵N-transformation. N₂, N₂O and SPN accounted for 2.4, 0.02 and 1.4%, respectively. The temporal and spatial patterns of ¹⁵N transformation in the water phase component of the system were remarkably similar to those observed in May, indicating good reproducibility of the whole ecosystem labeling approach, but the absolute ammonium transformation rate was 3 times higher in May. While the marsh surface area was crucial for nitrification in May this was less pronounced in September. Denitrification, on the other hand, appeared more important in September compared to May.     

Antitumor effect of miR-197 targeting in p53 wild-type lung cancer

Lung cancer is the leading cause of tumor-related death. The lack of effective treatments urges the development of new therapeutic approaches able to selectively kill cancer cells. The connection between aberrant microRNA (miRNA – miR) expression and tumor progression suggests a new strategy to fight cancer by interfering with miRNA function. In this regard, LNAs (locked nucleic acids) have proven to be very promising candidates for miRNA neutralization. Here, we employed an LNA-based anti-miR library in a functional screening to identify putative oncogenic miRNAs in non-small-cell lung cancer (NSCLC). By screening NIH-H460 and A549 cells, miR-197 was identified as a new functional oncomiR, whose downregulation induces p53-dependent lung cancer cell apoptosis and impairs the capacity to establish tumor xenografts in immunodeficient mice. We further identified the two BH3-only proteins NOXA and BMF as new miR-197 targets responsible for induction of apoptosis in p53 wild-type cells, delineating miR-197 as a key survival factor in NSCLC. Thus, we propose the inhibition of miR-197 as a novel therapeutic approach against lung cancer.     

Synthesis,characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.     

Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain

Abstract: Elevated extracellular potassium concentration ([K⁺]_e) has been shown to induce reversal of glial Na⁺-dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K⁺]_e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K⁺]_e (by equimolar substitution of K⁺ for Na⁺), and glutamate accumulation was measured by HPLC. With [K⁺]_e elevations to 60 m M , medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d -aspartate, and parallel results were obtained; no increase in medium d -aspartate content resulted from [K⁺]_e elevation up to 90 m M , whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K⁺]_e attainable in brain.     

CD36 or alphavbeta3 and alphavbeta5 integrins are not essential for MHC class I cross-presentation of cell-associated antigen by CD8 alpha+ murine dendritic cells

Cross-presentation of cell-associated Ag is thought to involve receptor-mediated uptake of apoptotic cells by dendritic cells (DC), and studies with human DC strongly implicate the endocytic receptor CD36 and the integrins alpha(v)beta(3) and/or alpha(v)beta(5) in this process. In the mouse, cross-presentation was recently shown to be a function of CD8alpha(+) DC. Here we report that CD36 is expressed on CD8alpha(+), but not on CD8alpha(-), DC. To address the role of CD36 in cross-presentation we compared CD36(-/-) and CD36(+/+) H-2(b) DC for their ability to stimulate naive OT-1 T cells specific for OVA plus H-2K(b) in the presence of OVA-loaded MHC-mismatched splenocytes as a source of cell-associated Ag for cross-presentation. Surprisingly, no difference was seen between CD36(-/-) and CD36(+/+) CD8alpha(+) DC in their ability to cross-present cell-associated OVA or to capture OVA-bearing cells. Furthermore, the proliferation of CFSE-labeled OT-1 cells in response to OVA cross-presentation in vivo was normal in CD36(-/-) bone marrow chimeras, also arguing against a necessary role for CD36 in cross-presentation by DC or other APC. DC doubly deficient for beta(3) and beta(5) integrins were similarly unimpaired in their ability to cross-present OVA-bearing cells in vitro. These data demonstrate that in the mouse, receptors other than CD36 or beta(3) and beta(5) integrins can support the specialized cross-presenting function of CD8alpha(+) DC.     

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.