Temperature dependences of carbon assimilation processes in four dominant species from mountain grassland ecosystem

Temperature responses of carbon assimilation processes were studied in four dominant species from mountain grassland ecosystem, i.e. Holcus mollis (L.), Hypericum maculatum (Cr.), Festuca rubra (L.), and Nardus stricta (L.), using the gas exchange technique. Leaf temperature (T _L) of all species was adjusted within the range 13–30 °C using the Peltier thermoelectric cooler. The temperature responses of metabolic processes were subsequently modelled using the Arrhenius exponential function involving the temperature coefficient Q ₁₀. The expected increase of global temperature led to a significant increase of dark respiration rate (R _D; Q ₁₀ = 2.0±0.5), maximum carboxylation rate (V _Cmax; Q ₁₀ = 2.2±0.6), and maximum electron transport rate (J _max; Q ₁₀ = 1.6±0.4) in dominant species of mountain grassland ecosystems. Contrariwise, the ratio between J _max and V _Cmax linearly decreased with T _L [y = −0.884 T _L + 5.24; r ² = 0.78]. Hence temperature did not control the ratio between intercellular and ambient CO₂ concentration, apparent quantum efficiency, and photon-saturated CO₂ assimilation rate (P _max). P _max primarily correlated with maximum stomatal conductance irrespective of T _L. Water use efficiency tended to decrease with T _L [y = −0.21 T _L + 8.1; r ² = 0.87].     

Multiplexed immuno-precipitation with 1725 commercially available antibodies to cellular proteins

Antibody array analysis of complex samples requires capture reagents with exceptional specificity. The frequency of antibodies with label-based detection may be as low as 5%. Here, however, we show that as many as 25% of commercially available antibodies are useful when biotinylated cellular proteins are fractionated by size exclusion chromatography (SEC) first. A microsphere multiplex with 1725 antibodies to cellular proteins was added to 24 SEC fractions, labelled with streptavidin and analyzed by flow cytometry (microsphere-based affinity proteomics, MAP) The SEC-MAP approach resolved different targets captured by each antibody as reactivity peaks across the separation range of the SEC column (10-670kDa). Complex reactivity profiles demonstrated that most antibodies bound more than one target. However, specific binding was readily detected as reactivity peaks common for different antibodies to the same protein. We optimized sample preparation and found that amine-reactive biotin rarely inhibited antibody binding when the biotin to lysine ratio was kept below 1:1 during labelling. Moreover, several epitopes that were inaccessible to antibodies in native proteins were unmasked after heat denaturation with 0.1% of SDS. The SEC-MAP format should allow researchers to build multiplexed assays with antibodies purchased for use in e.g. Western blotting.     

Age-related increase of kynurenic acid in human cerebrospinal fluid - IgG and beta2-microglobulin changes

Kynurenic acid (KYNA) is an endogenous metabolite in the kynurenine pathway of tryptophan degradation and is an antagonist at the glycine site of the N-methyl-D-aspartate as well as at the alpha 7 nicotinic cholinergic receptors. In the brain tissue KYNA is synthesised from L-kynurenine by kynurenine aminotransferases (KAT) I and II. A host of immune mediators influence tryptophan degradation. In the present study, the levels of KYNA in cerebrospinal fluid (CSF) and serum in a group of human subjects aged between 25 and 74 years were determined by using a high performance liquid chromatography method. In CSF and serum KAT I and II activities were investigated by radioenzymatic assay, and the levels of beta(2)-microglobulin, a marker for cellular immune activation, were determined by ELISA. The correlations between neurochemical and biological parameters were evaluated. Two subject groups with significantly different ages, i.e. <50 years and >50 years, p < 0.001, showed statistically significantly different CSF KYNA levels, i.e. 2.84 +/- 0.16 fmol/microl vs. 4.09 +/- 0.14 fmol/microl, p < 0.001, respectively; but this difference was not seen in serum samples. Interestingly, KYNA is synthesised in CSF principally by KAT I and not KAT II, however no relationship was found between enzyme activity and ageing. A positive relationship between CSF KYNA levels and age of subjects indicates a 95% probability of elevated CSF KYNA with ageing (R = 0.6639, p = 0.0001). KYNA levels significantly correlated with IgG and beta(2)-microglobulin levels (R = 0.5244, p = 0.0049; R = 0.4253, p = 0.043, respectively). No correlation was found between other biological parameters in CSF or serum. In summary, a positive relationship between the CSF KYNA level and ageing was found, and the data would suggest age-dependent increase of kynurenine metabolism in the CNS. An enhancement of CSF IgG and beta(2)-microglobulin levels would suggest an activation of the immune system during ageing. Increased KYNA metabolism may be involved in the hypofunction of the glutamatergic and/or nicotinic cholinergic neurotransmission in the ageing CNS.     

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical-physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TMwt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TMKK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TMKK elutes much earlier than TMwt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.     

Forster resonance energy transfer in liposomes: measurements of transmembrane helix dimerization in the native bilayer environment

The lipid bilayer vesicle is a model of the cellular membrane. Even in this simple system, however, measuring the thermodynamics of membrane protein association is a challenge. Here we discuss Forster resonance energy transfer (FRET) in liposomes as a method to probe the dimerization of transmembrane helices in a membrane environment. Although the measurements are labor intensive, FRET in liposomes can be measured accurately provided that attention is paid to sample homogeneity and sample equilibration. One must also take into account statistical expectations and the FRET that results from random colocalization of donors and acceptors in the bilayer. Without careful attention to these details, misleading results are easy to obtain in membrane FRET experiments. The results that we obtain in model systems are reproducible and depend solely on the concentration of the protein in the bilayer (i.e., on the protein-to-lipid ratio), thereby yielding thermodynamic parameters that are directly relevant to processes in biological membranes.     

Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein

Human respiratory syncytial virus (HRSV) is the most common etiological agent of acute lower respiratory tract disease in infants and can cause repeated infections throughout life. In this study, we have analyzed nucleotide sequences encompassing 629 bp at the carboxy terminus of the G glycoprotein gene for HRSV subgroup A strains isolated over 47 years, including 112 Belgian strains isolated over 19 consecutive years (1984 to 2002). By using a maximum likelihood method, we have tested the presence of diversifying selection and identified 13 positively selected sites with a posterior probability above 0.5. The sites under positive selection correspond to sites of O glycosylation or to amino acids that were previously described as monoclonal antibody-induced in vitro escape mutants. Our findings suggest that the evolution of subgroup A HRSV G glycoprotein is driven by immune pressure operating in certain codon positions located mainly in the second hypervariable region of the ectodomain. Phylogenetic analysis revealed the prolonged cocirculation of two subgroup A lineages among the Belgian population and the possible extinction of three other lineages. The evolutionary rate of HRSV subgroup A isolates was estimated to be 1.83 x 10(-3) nucleotide substitutions/site/year, projecting the most recent common ancestor back to the early 1940s.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.