Functional CD8<Superscript>+</Superscript> T cells infiltrate into nonsmall cell lung carcinoma

Infiltration of CD3(+)CD8(+) cytotoxic T cells was analyzed by multiparameter confocal laser microscopy in a panel of 16 randomly selected stage I nonsmall cell lung carcinomas. T-cell infiltration was observed in the stroma (range 57-2,093 T cells/mm(2)) but also in the tumor epithelium (range 21-892 T cells/mm(2)) and showed wide variation between individual tumors. Interestingly, a significantly higher percentage of CD3(+)CD8(+) T cells was detected in the tumor epithelium compared to the stroma illustrating that cytotoxic T cells may preferentially migrate into tumor epithelium. Aberrant HLA class I antigen expression was observed in 69% of the nonsmall-cell lung carcinoma (NSCLC) tumors. One tumor of a squamous cell lung carcinoma patient with the highest number of tumor infiltrating CD3(+) and CD3(+)CD8(+) cells was studied in detail and the majority (90%) of these cells were shown to be functionally activated granzyme B-positive cytotoxic T cells. DNA oligotyping of a lung carcinoma cell line established from this tumor revealed loss of one HLA haplotype corresponding with a translocation involving chromosome 6, as observed by COBRA-FISH. HLA class I-restricted tumor specific T cells could be isolated from PBMC. One further characterized cytotoxic CD8(+) T cell clone, that released TNF-alpha, IFN-gamma, and granzyme B upon co-incubation with the autologous tumor cells, was shown to be restricted by the remaining HLA-A11 allele, which was also shown to be expressed in the tumor tissue. Our data indicate that, despite HLA-haplotype loss a vigorous antitumor immune response mediated by CD8(+ )T-cells can be present in NSCLC offering possibilities for specific immunotherapy.     

Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale

The coevolution of ticks and the pathogens that they transmit has ensured their mutual survival. In these studies, we used a functional genomics approach to characterize tick genes regulated in response to Anaplasma marginale infection. Differentially regulated genes/proteins were identified by suppression-subtractive hybridization and differential in-gel electrophoresis analyses of cultured IDE8 tick cells infected with A. marginale. Nine of 17 of these genes were confirmed by real-time RT-PCR to be differentially regulated in ticks and/or IDE8 tick cells in response to A. marginale infection. RNA interference was used for functional studies. Six genes, which encode putative selenoprotein W2a, hematopoietic stem/progenitor cells protein-like, proteasome 26S subunit, ferritin, GST, and subolesin control, were found to affect A. marginale infection in IDE8 tick cells. Four genes, which encode putative GST, salivary selenoprotein M, vATPase, and ubiquitin, affected A. marginale infection in different sites of development in ticks. The results of these studies demonstrated that a molecular mechanism occurs by which tick cell gene expression mediates the A. marginale developmental cycle and trafficking through ticks.     

Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes

The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C_16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C_18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C_16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes.     

Optimized co-solute paramagnetic relaxation enhancement for the rapid NMR analysis of a highly fibrillogenic peptide

Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T ₁ relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T ₁ values across the entire sequence of the intrinsically disordered protein α-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in ‘proton-less’ NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from α₁-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.     

Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux

ATP can be produced in the cytosol by glycolytic conversion of glucose (GLC) into pyruvate. The latter can be metabolized into lactate, which is released by the cell, or taken up by mitochondria to fuel ATP production by the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) system. Altering the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival during mitoenergetic dysfunction, which is observed in a large variety of human disorders including cancer. To gain insight into the kinetic properties of this adaptive mechanism we determined here how acute (30 min) inhibition of OXPHOS affected cytosolic GLC homeostasis. GLC dynamics were analyzed in single living C2C12 myoblasts expressing the fluorescent biosensor FLII¹²Pglu-700μδ6 (FLII). Following in situ FLII calibration, the kinetic properties of GLC uptake (V₁) and GLC consumption (V₂) were determined independently and used to construct a minimal mathematical model of cytosolic GLC dynamics. After validating the model, it was applied to quantitatively predict V₁ and V₂ at steady-state (i.e., when V₁ = V₂ = V_steady-state) in the absence and presence of OXPHOS inhibitors. Integrating model predictions with experimental data on lactate production, cell volume, and O₂ consumption revealed that glycolysis and mitochondria equally contribute to cellular ATP production in control myoblasts. Inhibition of OXPHOS induced a twofold increase in V_steady-state and glycolytic ATP production flux. Both in the absence and presence of OXPHOS inhibitors, GLC was consumed at near maximal rates, meaning that GLC consumption is rate-limiting under steady-state conditions. Taken together, we demonstrate here that OXPHOS inhibition increases steady-state GLC uptake and consumption in C2C12 myoblasts. This activation fully compensates for the reduction in mitochondrial ATP production, thereby maintaining the balance between cellular ATP supply and demand.     

Amazonian Dark Earth Shapes the Understory Plant Community in a Bolivian Forest

Amazonian Dark Earths (ADE) are the result of human modification of the Amazonian landscape since pre‐Columbian times. ADE are characterized by increased soil fertility compared to natural soils. In the Amazonian forest, soil fertility influences understory herb and fern species composition. However, little research has been done to evaluate the effect of ADE on the composition of the understory community. We evaluated the effects of ADE and soil in 36 plots (150 m × 4 m) established in a Bolivian moist forest (La Chonta). For each plot, we determined soil nutrients, and the composition, richness, and abundance of terrestrial fern, angiosperm herb, and understory palm species. We found that the presence of ADE created a gradient in soil nutrients and pH that affected the understory species composition especially of ferns and palms. Additionally, the higher nutrient concentration and more neutral soil pH on ADE soils caused a decrease of ferns species richness. We therefore conclude that the current composition of the understory community in this particular Bolivian forest is a reflection of past human modifications of the soil.     

Tick anti-hemostatics: targets for future vaccines and therapeutics

For ticks, a significant obstacle in obtaining a blood meal is counteracting the hemostatic system of the host. To this end, ticks have developed a broad array of anti-hemostatics, which is reflected in the presence of structurally related tick proteins with different functions. Disruption of blood flow which blocks successful tick feeding makes anti-hemostatics attractive targets for anti-tick vaccines. Moreover, the limited number of drugs currently available for a range of important cardio-vascular diseases makes ticks a potential source of novel therapeutics. This review aims to summarize the key features of tick anti-hemostatics, their structures, mode of action and possible future application as vaccines and novel therapeutic agents.     

Advances in the genomics of ticks and tick-borne pathogens

Ticks and the diseases for which they are vectors engage in complex interactions with their mammalian hosts. These interactions involve the developmental processes of tick and pathogen, and interplay between the defensive responses and counter responses of host, tick and pathogen. Understanding these interactions has long been an intractable problem, but progress is now being made thanks to the flood of genomic information on host, tick and pathogen, and the attendant, novel experimental tools that have been generated. Each advance reveals new levels of complexity, but there are encouraging signs that genomics is leading to novel means of parasite control.     

Smoothelin and Intermediate Filament Proteins in Humanaortocoronary Saphenous Vein By-pass Grafts

The aim of this immunohistochemical investigation was to study the distribution of the novel cytoskeletal protein smoothelin and the intermediate filament proteins vimentin and desmin in normal human great saphenous vein and in human aortocoronary by-pass vein grafts. Smoothelin was present in most smooth muscle cells in the media of the native vein. In the neointima of the vein grafts that had been in situ for three months or more, smoothelin was, in general, present only in few smooth muscle cells. Desmin was distributed in the same pattern as smoothelin in the native great saphenous vein. When desmin and smoothelin were present in the neointima, smoothelin was detected in more cells than desmin. Vimentin was present in most cells in all wall layers of both the native saphenous vein and the vein grafts. Vascular smooth muscle cells containing vimentin but not desmin or smoothelin are the principal cells in the neointima of human aortocoronary vein grafts. In some grafts, however, all three cytoskeletal proteins were detected in the neointima. The distribution of smoothelin and desmin in aortocoronary vein grafts support the postulate that these proteins are expressed mainly in the contractile smooth muscle cell phenotype.