Radicals in Berkeley?

In a previous autobiographical sketch for DNA Repair (Linn, S. (2012) Life in the serendipitous lane: excitement and gratification in studying DNA repair. DNA Repair 11, 595–605), I wrote about my involvement in research on mechanisms of DNA repair. In this Reflections, I look back at how I became interested in free radical chemistry and biology and outline some of our bizarre (at the time) observations. Of course, these studies could never have succeeded without the exceptional aid of my mentors: my teachers; the undergraduate and graduate students, postdoctoral fellows, and senior lab visitors in my laboratory; and my faculty and staff colleagues here at Berkeley. I am so indebted to each and every one of these individuals for their efforts to overcome my ignorance and set me on the straight and narrow path to success in research. I regret that I cannot mention and thank each of these mentors individually.     

Eavesdropping on plant volatiles by a specialist moth: significance of ratio and concentration

We investigated the role that the ratio and concentration of ubiquitous plant volatiles play in providing host specificity for the diet specialist grape berry moth Paralobesia viteana (Clemens) in the process of locating its primary host plant Vitis sp. In the first flight tunnel experiment, using a previously identified attractive blend with seven common but essential components ("optimized blend"), we found that doubling the amount of six compounds singly [(E)- & (Z)-linalool oxides, nonanal, decanal, β-caryophyllene, or germacrene-D], while keeping the concentration of other compounds constant, significantly reduced female attraction (average 76% full and 59% partial upwind flight reduction) to the synthetic blends. However, doubling (E)-4,8-dimethyl 1,3,7-nonatriene had no effect on female response. In the second experiment, we manipulated the volatile profile more naturally by exposing clonal grapevines to Japanese beetle feeding. In the flight tunnel, foliar damage significantly reduced female landing on grape shoots by 72% and full upwind flight by 24%. The reduction was associated with two changes: (1) more than a two-fold increase in total amount of the seven essential volatile compounds, and (2) changes in their relative ratios. Compared to the optimized blend, synthetic blends mimicking the volatile ratio emitted by damaged grapevines resulted in an average of 87% and 32% reduction in full and partial upwind orientation, respectively, and the level of reduction was similar at both high and low doses. Taken together, these results demonstrate that the specificity of a ubiquitous volatile blend is determined, in part, by the ratio of key volatile compounds for this diet specialist. However, P. viteana was also able to accommodate significant variation in the ratio of some compounds as well as the concentration of the overall mixture. Such plasticity may be critical for phytophagous insects to successfully eavesdrop on variable host plant volatile signals.     

Nerve agent hydrolysis activity designed into a human drug metabolism enzyme

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.     

ConDeTri--a content dependent read trimmer for Illumina data

During the last few years, DNA and RNA sequencing have started to play an increasingly important role in biological and medical applications, especially due to the greater amount of sequencing data yielded from the new sequencing machines and the enormous decrease in sequencing costs. Particularly, Illumina/Solexa sequencing has had an increasing impact on gathering data from model and non-model organisms. However, accurate and easy to use tools for quality filtering have not yet been established. We present ConDeTri, a method for content dependent read trimming for next generation sequencing data using quality scores of each individual base. The main focus of the method is to remove sequencing errors from reads so that sequencing reads can be standardized. Another aspect of the method is to incorporate read trimming in next-generation sequencing data processing and analysis pipelines. It can process single-end and paired-end sequence data of arbitrary length and it is independent from sequencing coverage and user interaction. ConDeTri is able to trim and remove reads with low quality scores to save computational time and memory usage during de novo assemblies. Low coverage or large genome sequencing projects will especially gain from trimming reads. The method can easily be incorporated into preprocessing and analysis pipelines for Illumina data. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://code.google.com/p/condetri.     

The Reproductive Behavior of Acanthurus nigrofuscus (Forskal) and other Surgeonfishes (Fam. Acanthuridae) off Eilat,Israel (Gulf of Aqaba,Red Sea)

The reproductive behavior of Acanthurus nigrofuscus, a common surgeonfish of the Red Sea, is described for two overlapping populations inhabiting the coral reefs off Eilat, Israel (Gulf of Aqaba). The spectacular daily spawning activities, often involving aggregations of thousands of individuals, as well as the often long distance migrations to and from the spawning grounds by groups in highly organized formations are characterized in detail. Evidence is provided that the precise timing of these various phenomena is controlled by ambient light levels during the late afternoon. Less detailed information is also provided on comparable activities of two other surgeonfishes from the same reefs: Ctenochaetus striatus and Zebrasoma xanthurum. Finally, comparisons are made of reproduction and its associated environmental controls among populations of the same and related species of surgeonfishes at widely distributed locations in the Indo-Pacific.     

Preferential binding and structural distortion by Fe2+ at RGGG-containing DNA sequences correlates with enhanced oxidative cleavage at such sequences

Certain DNA sequences are known to be unusually sensitive to nicking via the Fe2+-mediated Fenton reaction. Most notable are a purine nucleotide followed by three or more G residues, RGGG, and purine nucleotides flanking a TG combination, RTGR. Our laboratory previously demonstrated that nicking in the RGGG sequences occurs preferentially 5' to a G residue with the nicking probability decreasing from the 5' to 3'end of these sequences. Using 1H NMR to characterize Fe2+ binding within the duplex CGAGTTAGGGTAGC/GCTACCCTAACTCG and 7-deazaguanine-containing (Z) variants of it, we show that Fe2+ binds preferentially at the GGG sequence, most strongly towards its 5' end. Substitutions of individual guanines with Z indicate that the high affinity Fe2+ binding at AGGG involves two adjacent guanine N7 moieties. Binding is accompanied by large changes in specific imino, aromatic and methyl proton chemical shifts, indicating that a locally distorted structure forms at the binding site that affects the conformation of the two base pairs 3' to the GGG sequence. The binding of Fe2+ to RGGG contrasts with that previously observed for the RTGR sequence, which binds Fe2+ with negligible structural rearrangements.     

Structural organization and Zn2+-dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro52

Ro52 is one of the major autoantigens targeted in the autoimmune disease Sj?gren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn2+-binding Ring-B-box region. Primarily associated with Sj?gren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn2+-binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn2+ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities.     

Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling

Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the bound ligand. Furthermore, it documents the complexity and uniqueness of the ligand-receptor structure, and highlights the fact that structural differences exist between the receptor bound with ligands of different pharmacological character that, nevertheless, produce similar crystal structures.     

Effect of a single in ovo injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin on protein expression in liver and ovary of the one-day-old chick analyzed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry

The polyhalogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an ubiquitously distributed environmental pollutant which can induce a broad spectrum of toxic responses in animals, including birds. In this study, we investigated the impact of 0 or 20 ng TCDD injections into the yolk of chicken eggs before start of development, on liver and ovarian protein expression in hatchlings using fluorescent two-dimensional difference gel electrophoresis (2-D-DIGE) under a pH range of 4-7, combined with MS. Despite considerable interindividual variability, exposure to TCDD prior to the start of embryonic development resulted in significant changes in expression of a small set of proteins. Expression of fibrinogen gamma chain precursor in the liver and 60 kDa heat shock protein in the ovary were significantly higher as a result of the very early exposure to TCDD. NADH ubiquinone oxidoreductase (42 kDa subunit) and regucalcin expression was decreased by early TCDD treatment in the liver and ovary, respectively. These proteins could not be directly linked with drug metabolism per se but are involved in blood clotting, oxidative stress, electron transport, and calcium regulation. It remains to be elucidated how these changes in the hatchling might be linked to the observed long-term consequences during posthatch life of the chicken.