Abnormal Organization of White Matter Network in Patients with No Dementia after Ischemic Stroke

Structural changes after ischemic stroke could affect information communication extensively in the brain network. It is likely that the defects in the white matter (WM) network play a key role in information interchange. In this study, we used graph theoretical analysis to examine potential organization alteration in the WM network architecture derived from diffusion tensor images from subjects with no dementia and experienced stroke in the past 5.4–14.8 months (N = 47, Mini-Mental Screening Examination, MMSE range 18–30), compared with a normal control group with 44 age and gender-matched healthy volunteers (MMSE range 26–30). Region-wise connectivity was derived from fiber connection density of 90 different cortical and subcortical parcellations across the whole brain. Both normal controls and patients with chronic stroke exhibited efficient small-world properties in their WM structural networks. Compared with normal controls, topological efficiency was basically unaltered in the patients with chronic stroke, as reflected by unchanged local and global clustering coefficient, characteristic path length, and regional efficiency. No significant difference in hub distribution was found between normal control and patient groups. Patients with chronic stroke, however, were found to have reduced betweenness centrality and predominantly located in the orbitofrontal cortex, whereas increased betweenness centrality and vulnerability were observed in parietal-occipital cortex. The National Institutes of Health Stroke Scale (NIHSS) score of patient is correlated with the betweenness centrality of right pallidum and local clustering coefficient of left superior occipital gyrus. Our findings suggest that patients with chronic stroke still exhibit efficient small-world organization and unaltered topological efficiency, with altered topology at orbitofrontal cortex and parietal-occipital cortex in the overall structural network. Findings from this study could help in understanding the mechanism of cognitive impairment and functional compensation occurred in patients with chronic stroke.     

Auto-FACE: An NMR Based Binding Site Mapping Program for Fast Chemical Exchange Protein-Ligand Systems

Background

Nuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, N Heteronuclear Single Quantum Correlation (HSQC) experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately.

Methodology/Principal Findings

We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues. Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant () calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event.

Conclusion/Significance

Detail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called “Auto-FACE”, which also allowed quantitative information of each interaction site and elucidation of binding mechanism.     

NADPH oxidase and cyclooxygenase mediate the ultraviolet B-induced generation of reactive oxygen species and activation of nuclear factor-kappaB in HaCaT human keratinocytes

The detrimental effects of ultraviolet B (UVB) irradiation have been connected with the enhanced generation of reactive oxygen species (ROS) by UVB. However, the exact source of ROS produced by UVB has not been clearly revealed yet. In this study, we determined the source of ROS production and its role in the UVB-induced activation of nuclear factor (NF)-kappaB in HaCaT human keratinocytes. UVB irradiation generated ROS in a dose-dependent manner, and this was significantly inhibited by diphenylene iodonium (DPI), apocynin (Apo) and neopterine (Neo), inhibitors of the NADPH oxidase, and indomethacin (Indo), a cyclooxygenase (COX) inhibitor, but not by the mitochondrial electron transport inhibitors and other cytosolic enzyme inhibitors. In addition, these inhibitors of the NADPH oxidase and COX significantly blocked the UVB irradiation-induced nuclear translocation of NF-kappaB. These results suggest that the NADPH oxidase and COX may be major sources for the UVB-induced ROS generation, and play an essential role in the activation of NF-kappaB which is involved in the expression of a variety of genes induced by UVB in HaCaT cells. These results further suggest that these enzymes may be good targets for the preventive strategy of UVB-induced skin injury.     

Proteome Analysis of Light-induced Proteins in Synechocystis sp. PCC 6803: Identification of Proteins Separated by 2D-PAGE Using N-terminal Sequencing and MALDI-TOF MS

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.     

Direct effects of varying doses of oestradiol on early embryonic development in in vitro culture of rat's two-cell embryos

Hyperstimulation in the rat using pregnant mares' serum gonadotrophin (PMSG) has been known to cause death in pre-implantation embryos, as well as enhancement of oestradiol production. This study examines the effect of oestradiol, in levels that are found in hyperstimulated pregnant rats, on pre-implantation embryonic development. Using a simplified in vitro system, 2-cell embryos retrieved from rats on the 2nd day of pregnancy were cultured in rat two-cell embryo culture medium (R2ECM) containing pharmacological doses of oestradiol for 96 h and scored daily in the morning. Three ngxmL(-1) oestradiol reduced the incidence of >8-cell embryos to morulae on the 5th day and blastocysts on the 6th day of development. Most embryos were retarded at the lower cell stages on the 5th day and degenerated by the 6th day. None of the blastocysts expanded on the last day of culture. Fifteen ngxmL(-1) oestradiol accelerated embryo development on the 3rd day but retarded development on the 4th day, and increased the incidence of degenerated embryos by the 5th and 6th day of development. These results suggest that the elevated oestradiol may constitute a mechanism by which PMSG induces death in pre-implantation rat embryos, possibly via a direct action on the embryos.     

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.