Far-red fluorescence: a direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching

Time-resolved fluorescence on oligomers of the main light-harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter-trimer chlorophyll-chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far-red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400ps fluorescence component appearing in intact leaves of Arabidopsis when non-photochemical quenching (NPQ) is induced. The fluorescence components with high far-red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far-red emitting state is shown to be an emissive Chl-Chl charge transfer state which plays a crucial part in the quenching.     

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).     

High-throughput, nontargeted metabolite fingerprinting using nominal mass flow injection electrospray mass spectrometry

Flow injection electrospray-mass spectrometry (FIE-MS) is finding utility as a first-pass metabolite fingerprinting tool in many research fields. We provide a protocol that has proved reliable in large-scale research projects involving diverse sample matrices originating from plants, microbes and mammalian biofluids. Using Brachypodium leaf material as an example matrix all steps are summarized from sample extraction to data quality assessment. Alternative procedures for dealing with other common matrices such as bloods and urine are included. With little sample pretreatment, no chromatography and instrument cycle times of <5 min it is feasible to analyze >1,000 samples per week. Analysis of a typical batch of 240 samples (including first-pass data analysis) can be accomplished within 48 h.     

Les blasons de Lascaux

Between 1952 and 1963, the Abbé Glory conducted excavation in the Lascaux Cave. He discovered many artefacts useful for the Prehistoric painter. Today this material is kept in the “Institut de Paléontologie Humaine” in Paris. Some of this material, as well as the painting matter of the “Blazons” of the Black Cow Panel of the Nef, were examined and analysed by electron microscopy: SEM and TEM. Yellow, ocher and red pigments are iron oxides and black pigments are manganese oxides. The results indicate that the pigments were by no means heated, nor associated with any extender. Nevertheless the variety of the raw matter indicates a real strategy to exploit nature in order to match the desired hue and the texture of the painted panel.     

Centrosome overduplication and mitotic instability in PKD2 transgenic lines

Polycystin-2 (PC-2), a protein encoded by PKD2 and involved in autosomal dominant polycystic kidney disease (ADPKD), is a non-selective cationic channel recently implicated in the function of primary cilia. We recently constructed a new animal model in the form of a transgenic mouse with a BAC-containing human PKD2 inserted in its genome. Two transgenic mouse lines overexpressing human PKD2 showed mitotic instability. Fibroblasts from these transgenic mouse lines have abnormal chromosomal numbers. These lines also have supernumerary centrosomes. PC-2 overexpression is associated with mitotic instability and centrosome overduplication. PC-2 therefore seems to play a role in centrosome duplication, and this hypothesis is being evaluated in other models.     

Meiotic weakness: the first division

Cell division is probably the most dramatic event in the life of a cell : the entire genetic material has to be equally distributed into the two daughter cells. Segregation errors have severe consequences and lead to either cell death or the generation of aneuploid cells and may cause the formation of tumors or tumor promoting mutations in somatic cells. In meiosis, they provoke the generation of aneuploid embryos and/or spontaneous abortions. Trisomies in humans, such as trisomy 21, are due to the missegregation of one chromosome in the first meiotic division in the oocyte. This review deals with the molecular mechanisms regulating the two meiotic divisions required for the generation of female haploid germ cells. Here we focus mainly on spindle assembly, and cell cycle regulation especially during the first meiotic division in mouse oocytes (excellent reviews have been written on the peculiar aspects of cell cycle regulation in meiosis II, such as the CSF arrest).     

A conserved cysteine motif essential for ceramide kinase function

Ceramide kinase (CerK) is a sphingolipid metabolizing enzyme very sensitive to oxidation; however, the determinants are unknown. We show here that the thiol-modifying agent N-ethyl-maleimide abrogates CerK activity in vitro and in a cell based assay, implying that important cysteine residues are accessible in purified as well as endogenous CerK. We replaced every 22 residues in human CerK, by an alanine, and measured activity in the resulting mutant proteins. This led to identification of a cluster of cysteines, C(347)XXXC(351)XXC(354), essential for CerK function. These findings are discussed based on homology modeling of the catalytic domain of CerK.