Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response.     

Does the direct effect of atmospheric CO2 concentration on leaf respiration vary with temperature? Responses in two species of Plantago that differ in relative growth rate

The objective of this study was to investigate the direct effect of elevated atmospheric CO2 concentrations on leaf respiration in darkness (R) over a broad range of measurement temperatures. Our aim was to further elucidate the underlying mechanism(s) of the often-reported inhibition of leaf R by a doubling of the atmospheric CO2 concentration. Experiments were conducted using two species of Plantago that differed in maximum relative growth rate (fast-growing Plantago lanceolata L. and the slow-growing P. euryphylla Briggs, Carolin & Pulley). Rates of leaf respiration (R) were measured at atmospheric CO2 concentrations ranging from 75 to 2000 &mgr;mol mol-1 at temperatures from 12 to 42 degrees C. R was measured as CO2 release with a portable gas exchange system with infrared gas analysers. Our hypothesis was that the changes in temperature alter the flux coefficient (i.e. the extent to which changes in potential enzyme activity has an effect on the rate of a reaction) of enzymes potentially affected by CO2. Initial analysis of our results suggested that R was inhibited by elevated CO2 in both species, with the apparent degree of inhibition being greatest at low temperature. Moreover, the apparent degree of inhibition following a doubling of atmospheric CO2 concentration from 350 to 700 &mgr;mol mol-1 was similar to that reported by several previous studies (approximately 14% and 26% for P. lanceolata and P. euryphylla, respectively) at a temperature equal to the mean of the previous studies. However, subsequent correction for diffusion leaks of CO2 across the gas exchange's cuvette gaskets revealed that no significant inhibition had occurred in either species, at any temperature. The inhibitory effect of elevated CO2 on leaf respiratory CO2 release reported by previous studies may therefore have been overestimated.     

The synthesis and SAR of 2-arylsulfanyl-phenyl piperazinyl acetic acids as glyT-1 inhibitors

Elevation of glycine levels and activation of the NMDA receptor by inhibition of the glycine transporter 1 (GlyT-1) is a potential strategy for the treatment of schizophrenia. A novel series of GlyT-1 inhibitors have been identified containing the 2-arylsulfanyl-phenylpiperazine motif. The most prominent member of this series, (R)-4-[5-chloro-2-(4-methoxy-phenylsulfanyl)-phenyl]-2-methyl-piperazin-1-yl-acetic acid (31) is a potent glycine transporter-1 inhibitor (IC(50)=150 nM), which elevated glycine levels in rat ventral hippocampus as measured by microdialysis in vivo at doses of 1.2-4.6 mg/kg s.c.     

Identification of five hemoglobins in B6C3F1 mice by mass spectrometry and sequence analysis

The aim of the work is to identify and characterize the hemoglobins found in B6C3F1 mice using mass spectrometry. The primary structures are compared to those reported for BALB/c mice. Individual hemoglobin chains were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the globins were determined using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The purified globin chains were enzymatically cleaved and the resulting peptides were separated by RP-HPLC. The chains were identified by N-terminal sequencing and mass spectrometry (MALDI). Selected peptides were analysed by Edman degradation. ESI analysis indicates that B6C3F1 mice have two -globin chains (-1 and -2) and at least three β-globin chains, β-1, β-2 and β-3. This is one additional - and one additional β-globin chain than reported in the literature for BALB/c mice. Mass and sequence analysis of enzymatically generated peptides showed variations in the amino acid sequence in the -1, -2, β-2 and β-3 chains compared to the BALB/c mouse hemoglobins (, β^minor and β^major). The study showed that mass spectrometry in combination with traditional protein chemistry is able to identify and locate minor protein sequence variations.     

A putative plant aminophospholipid flippase, the Arabidopsis P4 ATPase ALA1, localizes to the plasma membrane following association with a β-subunit

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein β-subunit.     

Effects of elevated CO₂, warming and drought episodes on plant carbon uptake in a temperate heath ecosystem are controlled by soil water status

The impact of elevated CO₂, periodic drought and warming on photosynthesis and leaf characteristics of the evergreen dwarf shrub Calluna vulgaris in a temperate heath ecosystem was investigated. Photosynthesis was reduced by drought in midsummer and increased by elevated CO₂ throughout the growing season, whereas warming only stimulated photosynthesis early in the year. At the beginning and end of the growing season, a T × CO₂ interaction synergistically stimulated plant carbon uptake in the combination of warming and elevated CO₂. At peak drought, the D × CO₂ interaction antagonistically down‐regulated photosynthesis, suggesting a limited ability of elevated CO₂ to counteract the negative effect of drought. The response of photosynthesis in the full factorial combination (TDCO₂) could be explained by the main effect of experimental treatments (T, D, CO₂) and the two‐factor interactions (D × CO₂, T × CO₂). The interactive responses in the experimental treatments including elevated CO₂ seemed to be linked to the realized range of treatment variability, for example with negative effects following experimental drought or positive effects following the relatively higher impact of night‐time warming during cold periods early and late in the year. Longer‐term experiments are needed to evaluate whether photosynthetic down‐regulation will dampen the stimulation of photosynthesis under prolonged exposure to elevated CO₂.     

Abatacept Treatment Does Not Preserve Renal Function in the Streptozocin-Induced Model of Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the most severe complications of diabetes and remains the largest cause of end-stage renal disease in the Western world. Treatment options are limited and novel therapies that effectively slow disease progression are warranted. Previous work suggested that treatment with CTLA4-Ig (abatacept), a molecule that binds and blocks B7-1 and is licensed for the treatment of rheumatoid arthritis, could ameliorate DN. This study was designed to assess whether B7-1 signalling constitutes a promising therapeutic pathway for DN. Mice injected with streptozotocin (STZ) were treated with abatacept and glycemia and renal function were assessed. No differences were found in diabetes progression, albumin excretion rates or albumin/creatine ratios, while mesangial expansion was unaltered at endpoint. Except for increased renal CCL5, treatment did not affect a panel of gene expression endpoints reflecting early fibrotic changes, inflammation and kidney injury. Finally, abatacept treatment effectively reduced the accumulation of activated CD4⁺ T cells in the kidney, suggesting that renal T cell inflammation is not a driving factor in the pathology of the STZ model. In conjunction with the recent data discounting the expression of B7-1 on podocytes, our present data do not support a role for abatacept in DN treatment.