Detection of Free Radicals by Microdialysis/Spin Trapping Epr Following Focal Cerebral Ischemia-Reperfusion and a Cautionary Note on the Stability of 5,5-Dimethyl-1-Pyrroline N-Oxide (DMPO)

We have examined free radical production in a rat model of focal cerebral ischemia using microdialysis coupled with EPR analysis. A microdialysis probe was inserted 2 mm into the cerebral cortex, supplied by the right middle cerebral artery (MCA), and after a 2-hour washout period with artificial cerebral spinal fluid (ACSF), the perfusate solution was changed to ACSF containing the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). No free radicals were detected by DMPO during the pre-ischemia period. Both common carotid arteries and the right MCA were then ligated for 90 minutes. Microdialysate collected every 15 min during the ischemic period demonstrated predominantly superoxide or peroxyl radical production. After release of the occlusive sutures, hydroxyl radical became apparent initially, then thiyl and carbon centered radicals appeared later in samples collected every 15 min for two hours following cortical reperfusion. Careful studies on the purification and stability of DMPO solution were performed to circumvent artifacts and spurious signals.     

Experimental retracement of the origins of a protocell

Although Oparin used coacervate droplets from two or more types of polymer to model the first cell, he hypothesized homacervation from protein, consistent with Pasteur and Darwin. Herrera made two amino acids and numerous cell-like structures (sulfobes) in the laboratory, which probably arose from intermediate polymers. Our experiments have conformed with a homoacervation of thermal proteinoid, in which amino acid sequences are determined by the reacting amino acids themselves. All proteinoids that have been tested assemble themselves alone in water to protocells. The protocells have characteristics of life defined by Webster's Dictionary: metabolism, growth, reproduction and response to stimuli in the environment. The protocells are able also to evolve to more modern cells including the initiation of a nucleic acid coding system.Principal spinoffs from the results are revised evolutionary theory, models for protoneurons and networks thereof, and numerous industrial applications of thermal polyamino acids. Life itself has thus been reaffirmed to be rooted in protein, not in DNA nor RNA, which are however crucial to inheritance in modern life as instruction manual (Kornberg).Recognition of the advances have been considerably delayed by the deeply held assumption that life began by chance from random polymerization of amino acids, in contrast to the experimental findings. The concepts of DNA/RNA-first and protein-first are reconciled by a rise-and-fall progression as often seen in biochemical and biological evolution.The fact that amino acids order themselves explains in turn that thermal copolyamino acids are finding numerous applications. The entire sequence of processes in the proteinoid origins theory is now seen to be highly deterministic, in close accord with Einstein.     

Polymerase chain reaction engineering

A mathematical model for polymerase chain reaction (PCR) is developed, taking into account the three steps in this process: melting of DNA; primer annealing; and DNA synthesis (polymerization). Activity and deactivation of the polymerase enzyme as a function of temperature is incorporated in the kinetic model to get a better understanding of the amplification of DNA. Computer simulation of the model is carried out to determine the effects of various parameters, such as the cycle number, initial DNA concentration (copynumber), initial enzyme concentration, extension time, temperature ramp, and enzyme deactivation on the DNA generation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 359-366, 1997.     

Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.     

Genetics of Coronary Artery Disease in Taiwan: A Cardiometabochip Study by the Taichi Consortium

By means of a combination of genome-wide and follow-up studies, recent large-scale association studies of populations of European descent have now identified over 46 loci associated with coronary artery disease (CAD). As part of the TAICHI Consortium, we have collected and genotyped 8556 subjects from Taiwan, comprising 5423 controls and 3133 cases with coronary artery disease, for 9087 CAD SNPs using the CardioMetaboChip. We applied penalized logistic regression to ascertain the top SNPs that contribute together to CAD susceptibility in Taiwan. We observed that the 9p21 locus contributes to CAD at the level of genome-wide significance (rs1537372, with the presence of C, the major allele, the effect estimate is -0.216, standard error 0.033, p value 5.8x10^-10). In contrast to a previous report, we propose that the 9p21 locus is a single genetic contribution to CAD in Taiwan because: 1) the penalized logistic regression and the follow-up conditional analysis suggested that rs1537372 accounts for all of the CAD association in 9p21, and 2) the high linkage disequilibrium observed for all associated SNPs in 9p21. We also observed evidence for the following loci at a false discovery rate >5%: SH2B3, ADAMTS7, PHACTR1, GGCX, HTRA1, COL4A1, and LARP6-LRRC49. We also took advantage of the fact that penalized methods are an efficient approach to search for gene-by-gene interactions, and observed that two-way interactions between the PHACTR1 and ADAMTS7 loci and between the SH2B3 and COL4A1 loci contribute to CAD risk. Both the similarities and differences between the significance of these loci when compared with significance of loci in studies of populations of European descent underscore the fact that further genetic association of studies in additional populations will provide clues to identify the genetic architecture of CAD across all populations worldwide.     

Assessment of Median Nerve Mobility by Ultrasound Dynamic Imaging for Diagnosing Carpal Tunnel Syndrome

Carpal tunnel syndrome (CTS) is the most common peripheral neuropathy and is characterized by median nerve entrapment at the wrist and the resulting median nerve dysfunction. CTS is diagnosed clinically as the gold standard and confirmed with nerve conduction studies (NCS). Complementing NCS, ultrasound imaging could provide additional anatomical information on pathological and motion changes of the median nerve. The purpose of this study was to estimate the transverse sliding patterns of the median nerve during finger movements by analyzing ultrasound dynamic images to distinguish between normal subjects and CTS patients. Transverse ultrasound images were acquired, and a speckle-tracking algorithm was used to determine the lateral displacements of the median nerve in radial-ulnar plane in B-mode images utilizing the multilevel block-sum pyramid algorithm and averaging. All of the averaged lateral displacements at separate acquisition times within a single flexion–extension cycle were accumulated to obtain the cumulative lateral displacements, which were curve-fitted with a second-order polynomial function. The fitted curve was regarded as the transverse sliding pattern of the median nerve. The R² value, curvature, and amplitude of the fitted curves were computed to evaluate the goodness, variation and maximum value of the fit, respectively. Box plots, the receiver operating characteristic (ROC) curve, and a fuzzy c-means clustering algorithm were utilized for statistical analysis. The transverse sliding of the median nerve during finger movements was greater and had a steeper fitted curve in the normal subjects than in the patients with mild or severe CTS. The temporal changes in transverse sliding of the median nerve within the carpal tunnel were found to be correlated with the presence of CTS and its severity. The representative transverse sliding patterns of the median nerve during finger movements were demonstrated to be useful for quantitatively estimating median nerve dysfunction in CTS patients.