Fluorescence contributions of the individual Trp residues in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.     

The role of mitochondria in direct cell-to-cell connection dependent rescue of postischemic cardiomyoblasts

In this in vitro study we induced ischemic injury on H9c2 rat cardiomyoblasts using the oxygen-glucose deprivation model (OGD). We monitored if the addition of healthy or mitochondria-depleted cells can save OGD treated cells from post-ischemic injury. We were able to significantly improve the surviving cell number of oxidatively damaged H9c2 cells by the addition of healthy cells to the culture. On the contrary, cells with disturbed mitochondria did not increase the number of surviving cells. High-resolution confocal time-lapse imaging also proved that mitochondria are drifting from cell-to-cell through tunneling membrane bridges, however, they do not get into the cytoplasm of the other cell. We conclude that addition of healthy cells to severly injured post-ischemic cardiomyoblasts can rescue them from death during the first 24h after reoxigenation. Grafted cells must maintain their mitochondria in an actively respiring state, and although cell contact is required for the mechanism, neither cell fusion nor organelle transfer occurs. This novel mechanism opens a new possiblity for cell-based cardiac repair in ischemic heart disease.     

Women with and without metabolic disorder differ in their gut microbiota composition

The aim of this study was to investigate whether overweight/obese women in metabolic disorder group (MDG, n = 27) differ in their gut microbiota composition from overweight/obese women in non-metabolic disorder group (NMDG, n = 47) and normal weight women group (NWG, n = 11). Gut microbiota was profiled from fecal samples by 16S rRNA fluorescence in situ hybridization and flow cytometry in 85 premenopausal women. Body composition was measured by bioimpedance, and dietary intakes were collected via food diaries. Standard procedures were used to assess plasma glucose, serum insulin, lipids, and inflammatory status. We found that the proportion of bacteria belonging to Eubacterium rectale-Clostridium coccoides group, indicating efficient energy harvest from nutrients in gut, was higher in MDG compared to NMDG and NWG, while no difference was found between NMDG and NWG. The proportion of Eubacterium rectale-Clostridium coccoides group correlated positively with weight, BMI, total fat, fat mass percentage (FM%), visceral fat area, and serum triglycerides, and negatively with high-density lipoprotein (HDL). Our results indicate that certain members of Eubacterium rectale-Clostridium coccoides group are associated with obesity-related MDs not obesity per se.     

Telomere-mediated truncation of barley chromosomes

Engineered minichromosomes offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in barley (Hordeum vulgare) by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of Arabidopsis-like telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into immature embryos of diploid and tetraploid barley, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by fluorescent in situ hybridisation, primer extension telomere repeat amplification and DNA gel blot analysis in regenerated plants. Telomere seeding connected to chromosome truncation was found in tetraploid plants only, indicating that genetic redundancy facilitates recovery of shortened chromosomes. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.     

Stereoselective synthesis of spiro and condensed pyrazolines of steroidal α,β-unsaturated ketones and nitrilimines by 1,3-dipolar cycloaddition

Effective syntheses of endo- and exocyclic α,β-unsaturated ketones as CC dipolarophiles were carried out in the 13α-estrone series. The 1,3-dipolar cycloadditions of 15,16α,β-unsaturated ketones of 13α-estrone 3-methyl and 3-benzyl ether with nitrilimines stereoselectively furnished two regioisomers of new condensed pyrazolines in a ratio of 2:1. The main product was the isomer obtained by the attack of the N-terminus of the 1,3-dipole on the carbon atom β to the carbonyl group of the dipolarophile. The nitrilimine cycloadditions to the 16-methylene-17-ketones of 13α-estrone 3-methyl and 3-benzyl ether stereo- and regioselectively furnished spiropyrazolines. The attack of the N-terminus of the dipole occurred on the α-carbon of the α,β-unsaturated ketones. The reactions were performed under both homogeneous and heterogeneous conditions. Silver acetate as a base proved more effective than its triethylamine counterpart. Changes in regio- and stereoselectivities were not observed on variation of the conditions of the cycloaddition reactions. The structures of the new products were determined by NMR (one- and two-dimensional) and MALDI TOF MS techniques, with C₇₀ fullerenes as matrix in the latter case.     

The effect of parental quality and malaria infection on nestling performance in the Collared Flycatcher (Ficedula albicollis)

Plumage ornamentation often signals the quality of males and, therefore, female birds may choose elaborately ornamented mates to increase their fitness. Such mate choice may confer both direct and indirect benefits to the offspring. Males with elaborate ornaments may provide good genes, which can result in better nestling growth, survival or resistance against parasitic infections. However, these males may also provision their offspring with more food or food of better quality, resulting in nestlings growing at a higher rate or fledging in better condition. In this study, we examined if there was an association between male ornamentation and malaria infection in Collared Flycatchers (Ficedula albicollis). We also investigated offspring performance in relation to malaria infection in the parents and the quality of the genetic and rearing fathers (assessed by the size of two secondary sexual characters) under simulated good and bad conditions (using brood size manipulation). We found that secondary sexual characters did not signal the ability of males to avoid parasitic infections, and malaria infection in the genetic and the rearing parents had no effect on nestling growth and fledging size. Our results do show, however, that it may be beneficial for the females to mate with males with a large forehead patch because wing feathers of nestlings reared by large-patched males grew at a higher rate. Fast feather growth can result in earlier fledging which, in turn, could improve nestling survival in highly variable environments or under strong nest predation.     

Activation of poly(ADP-ribose) polymerase by myocardial ischemia and coronary reperfusion in human circulating leukocytes

Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.     

Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections

Background  

Bone marrow derived mesenchymal stem cells (MSCs) are promising candidates for cell based therapies in myocardial infarction. However, the exact underlying cellular mechanisms are still not fully understood. Our aim was to explore the possible role of direct cell-to-cell interaction between ischemic H9c2 cardiomyoblasts and normal MSCs. Using an in vitro ischemia model of 150 minutes of oxygen glucose deprivation we investigated cell viability and cell interactions with confocal microscopy and flow cytometry.     

Pre-clustered TCR complexes

The T-cell antigen receptor (TCR) is a multisubunit transmembrane complex that mediates the antigen-specific activation of T cells. Using a variety of techniques, several research groups have shown that TCRs are at least partially pre-clustered before antigen binding. These new findings are contradictory to the “classical” view, according to which TCRs are randomly distributed on the cell surface and only associate upon antigen binding. In this review we try to answer the following questions: What are the experimental evidences for the existence of pre-clustered TCRs? How can the TCR pre-clusters be activated upon antigen binding? Which functional consequences for T-cell activation arise from the pre-clustering of TCRs.     

Organogold complexes probe a large beta-barrel cavity for human serum alpha1-acid glycoprotein

Human alpha(1)-acid glycoprotein (AAG) is an acute phase component of the plasma, binding numerous drugs and natural compounds with high-affinity. Using circular dichroism (CD) spectroscopy, strong AAG binding of organogold complexes was found, the molecular size and chemical structure of which differ from known AAG binding agents. The 16-membered Au(2)P(4)C(8)O(2) macrocycles interconvert rapidly between two helical forms and produce enantiomeric conformations which are in dynamic equilibrium in solution. AAG binds preferentially one of the chiral conformers as indicated by strong Cotton effects generated by intramolecular exciton coupling between the pairs of hetercyclic chromophores. Lipophilic nature of the guest molecules suggests the dominant contribution of hydrophobic interactions in the AAG binding. Comparison of the main genetic variants of AAG revealed that both the 'F1/S' and 'A' variants bind with high-affinity the gold(I) macrocycles (K(a) approximately 10(6) M(-1)). CD/fluorescence displacement, and fluorescence quenching experiments indicated inclusion of the compounds into the central beta-barrel cavity of AAG of which exact tertiary structure is yet unknown. Molecular dimensions of the gold(I) macrocycles (13 x 14 x 14 A) indicate that the principal ligand binding cavity of both the 'F1/S' and 'A' variants must be larger compared to the models published to date. Based on these findings, a novel homology model of AAG 'F1' variant was constructed using the human neutrophil gelatinase-associated lipocalin as a template. The organogold complexes were successfully docked into the central cavity of this model.