Assessment of the Novel Anti-Seizure Potential of Validamycin A Using Zebrafish Epilepsy Model.

Epilepsy is a prevalent neurological disorder characterized by recurrent seizures. Validamycin A (VA) is an antibiotic fungicide that inhibits trehalase activity and is widely used for crop protection in agriculture. In this study, we identified a novel function of VA as a potential anti-seizure medication in a zebrafish epilepsy model. Electroencephalogram (EEG) analysis demonstrated that VA reduced pentylenetetrazol (PTZ)-induced seizures in the brains of larval and adult zebrafish. Moreover, VA reduced PTZ-induced irregular movement in a behavioral assessment of adult zebrafish. The developmental toxicity test showed no observable anatomical alteration when the zebrafish larvae were treated with VA up to 10 µM within the effective range. The median lethal dose of VA in adult zebrafish was > 14,000 mg/kg. These results imply that VA does not demonstrate observable toxicity in zebrafish at concentrations effective for generating anti-seizure activity in the EEG and alleviating abnormal behavior in the PTZ-induced epileptic model. Furthermore, the effectiveness of VA was comparable to that of valproic acid. These results indicate that VA may have a potentially safer anti-seizure profile than valproic acid, thus offering promising prospects for its application in agriculture and medicine.

Ipconazole Disrupts Mitochondrial Homeostasis and Alters GABAergic Neuronal Development in Zebrafish.

Ipconazole, a demethylation inhibitor of fungal ergosterol biosynthesis, is widely used in modern agriculture for foliar and seed treatment, and is authorized for use in livestock feed. Waste from ipconazole treatment enters rivers and groundwater through disposal and rain, posing potential toxicity to humans and other organisms. Its metabolites remain stable under standard hydrolysis conditions; however, their neurodevelopmental toxicity is unknown. We investigated the potential neurodevelopmental toxicity of ipconazole pesticides in zebrafish (Danio rerio). Our behavioral monitoring demonstrated that the locomotive activity of ipconazole-exposed zebrafish larvae was reduced during early development, even when morphological abnormalities were undetected. Molecular profiling demonstrated that the mitochondrial-specific antioxidants, superoxide dismutases 1 and 2, and the genes essential for mitochondrial genome maintenance and functions were specifically reduced in ipconazole-treated (0.02 µg/mL) embryos, suggesting underlying ipconazole-driven oxidative stress. Consistently, ipconazole treatment substantially reduced hsp70 expression and increased ERK1/2 phosphorylation in a dose-dependent manner. Interrupted gad1b expression confirmed that GABAergic inhibitory neurons were dysregulated at 0.02 µg/mL ipconazole, whereas glutamatergic excitatory and dopaminergic neurons remained unaffected, resulting in an uncoordinated neural network. Additionally, ipconazole-treated (2 µg/mL) embryos exhibited caspase-independent cell death. This suggests that ipconazole has the potential to alter neurodevelopment by dysregulating mitochondrial homeostasis.

Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation.

While transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNALeu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNALeu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNALeu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNALeu-binding protein. We suspected that the overexpression of free tRNALeu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNALeu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNALeu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.

Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNA(Met), leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Fbw7γ-mediated degradation of KLF13 prevents RANTES expression in resting human but not murine T lymphocytes.

RANTES (CCL5) is a chemokine implicated in many human diseases. We previously showed that the transcription factor Kruppel-like factor 13 (KLF13) controls the late (3-5 days after activation) expression of RANTES in T lymphocytes and that KLF13 itself is translationally regulated through the 5'-untranslated region of its mRNA. Here, we show that KLF13 levels are further regulated by ubiquitination and degradation. KLF13 protein is undetectable in resting human T lymphocytes, but treatment with either proteosomal or lysosomal inhibitors increases KLF13 protein levels. Glycogen synthase kinase 3ß (GSK3ß)-mediated phosphorylation of KLF13 triggers the ubiquitination of KLF13 by the E3 ligase Fbw7γ, resulting in KLF13 protein degradation. Knockdown of either Fbw7γ or GSK3ß by small interfering RNA increases KLF13 expression in resting human T lymphocytes. In contrast, in murine T lymphocytes, KLF13 protein is abundant because of the absence of Fbw7γ. Treatment of unactivated human lymphocytes with lysosomal inhibitors stabilizes KLF13 protein, resulting in an increase of RANTES mRNA and protein. Taken together, these studies found that tightly regulated control of both synthesis and degradation allows rapid changes in the level of KLF13 in human T lymphocytes.

Antagonism of VEGF-A-induced increase in vascular permeability by an integrin α3ß1-Shp-1-cAMP/PKA pathway.

In cancer, VEGF-induced increase in vascular permeability results in increased interstitial pressure, reducing perfusion and increasing hypoxia, which reduce delivery of chemotherapeutic agents and increase resistance to ionizing radiation. Here, we show that both TIMP-2 and Ala + TIMP-2, a TIMP-2 mutant without matrix metalloproteinase inhibitory activity, antagonize the VEGF-A-induced increase in vascular permeability, both in vitro and in vivo. Like other agents known to preserve endothelial barrier function, TIMP-2 elevates cytosolic levels of cAMP and increases cytoskeletal-associated vascular endothelial cadherin in human microvascular endothelial cells. All of these effects are completely ablated by selective knockdown of integrin α3ß1 expression, expression of a dominant negative protein tyrosine phosphatase Shp-1 mutant, administration of the protein tyrosine phosphatase inhibitor orthovanadate, or the adenylate cyclase inhibitor SQ22536. This TIMP-2-mediated inhibition of vascular permeability involves an integrin α3ß1-Shp-1-cAMP/protein kinase A-dependent vascular endothelial cadherin cytoskeletal association, as evidenced by using siRNAs to integrin α3ß1 and Shp-1, or treatment with Shp-1 inhibitor NSC87877 and protein kinase A inhibitor H89. Our results demonstrate the potential utility for TIMP-2 in cancer therapy through "normalization" of vascular permeability in addition to previously described antiangiogenic effects.

Differential Neuroprotective Effects of N-Acetylcysteine against Dithianon Toxicity in Glutamatergic, Dopaminergic, and GABAergic Neurons: Assessment Using Zebrafish.

Despite the widespread agricultural use of dithianon as an antifungal agent, its neurotoxic implications for humans and wildlife have not been comprehensively explored. Using zebrafish embryonic development as our model, we found that dithianon treatment induced behavioral alterations in zebrafish larvae that appeared normal. Detailed quantitative analyses showed that dithianon at ≥0.0001 µgmL-1 induced cytoplasmic and mitochondrial antioxidant responses sequentially, followed by the disruption of mitochondrial and cellular homeostasis. Additionally, dithianon at 0.01 and 0.1 µgmL-1 downregulated the expressions of glutamatergic (slc17a6b), GABAergic (gad1b), and dopaminergic (th) neuronal markers. Contrarily, dithianon upregulated the expression of the oligodendrocyte marker (olig2) at concentrations of 0.001 and 0.01 µgmL-1, concurrently suppressing the gene expression of the glucose transporter slc2a1a/glut1. Particularly, dithianon-induced increase in reactive oxygen species (ROS) production was reduced by both N-acetylcysteine (NAC) and betaine; however, only NAC prevented dithianon-induced mortality of zebrafish embryos. Moreover, NAC specifically prevented dithianon-induced alterations in glutamatergic and dopaminergic neurons while leaving GABAergic neurons unaffected, demonstrating that the major neurotransmission systems in the central nervous system differentially respond to the protective effects. Our findings contribute to a better understanding of the neurotoxic potential of dithianon and to developing preventive strategies.

TNNT1 myopathy with novel compound heterozygous mutations.

Nemaline myopathies are clinically and genetically heterogeneous disorders caused by several different genes. One of them is TNNT1, which was initially described in Amish families and has not been reported in Asian populations. Although most TNNT1 myopathies are caused by loss-of-function mutations, several recent studies have shown that missense mutations can also be pathogenic. A 16-year-old Korean boy with progressive muscle weakness visited the Seoul National University Hospital. He showed generalized myopathy, which was predominant in the paraspinal and neck muscles. Moreover, nemaline rods were observed in a muscle biopsy. Whole-exome sequencing of DNA samples of the patient and his younger brother, who had a similar phenotype, revealed novel compound heterozygous mutations in TNNT1 (c.724G>C (p.Ala242Pro) and c.611+1G>A). Sanger sequencing of cDNA extracted from muscle samples of the patient confirmed partial or total skipping of exon 11 in the splicing variant. The impact of the missense variant on muscle integrity and locomotor activity was verified using a zebrafish loss-of-function model. Here, we reported novel familial cases of TNNT1 myopathy with intermediate clinical presentations caused by compound heterozygous mutations and demonstrated their functional defects using an animal model.

Genome annotation of Caenorhabditis briggsae by TEC-RED identifies new exons, paralogs, and conserved and novel operons.

The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin Caenorhabditis elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the trans-spliced exon coupled RNA end determination technique. In addition to identifying the 5' ends of expressed genes, we have discovered operons and paralogs. In summary, our analysis yielded 10,243 unique 5' end sequence tags with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. These genes included 14 that are exclusively trans-spliced in C. briggsae when compared with C. elegans orthologs. A major contribution of this study is the identification of 492 high confidence operons, of which two-thirds are fully supported by tags. In addition, 2 SL1-type operons were discovered. Interestingly, comparisons with C. elegans showed that only 40% of operons are conserved. Of the remaining operons, 73 are novel, including 12 that entirely lack orthologs in C. elegans. Further analysis revealed that 4 of the 12 novel operons are conserved in Caenorhabditis nigoni. Altogether, the work described here has significantly advanced our understanding of the C. briggsae system and serves as a rich resource to aid biological studies involving this species.

SynMuv genes redundantly inhibit lin-3/EGF expression to prevent inappropriate vulval induction in C. elegans.

Activation of EGFR-Ras-MAPK signaling in vulval precursor cells (VPCs) by LIN-3/EGF from the gonad induces vulval development in C. elegans. The prevailing view is that LIN-3 overcomes an "inhibitory signal" from the adjacent hyp7 hypodermal syncytium. This view originated from observations indicating that inactivation of functionally redundant Synthetic Multivulva (SynMuv) genes in hyp7 can activate EGFR-Ras-MAPK signaling in the VPCs. Many SynMuv genes encode transcription and chromatin-associated factors, including the Rb ortholog. Here, we show that the SynMuv A and SynMuv B gene classes are functionally redundant for transcriptional repression of the key target gene, lin-3/EGF, in the hypodermis. These observations necessitate a revision of the concept of "inhibitory signaling." They also underscore the importance of preventing inappropriate cell signaling during development and suggest that derepression of growth factors may be the mechanism by which tumor suppressor genes such as Rb can have cell nonautonomous effects.

Histopathological assessment of laterality defects in zebrafish development.

Laterality defects during embryonic development underlie the aetiology of various clinical symptoms of neuropathological and cardiovascular disorders; however, experimental approaches to understand the underlying mechanisms are limited due to the complex organ systems of vertebrate models. Zebrafish have the ability to survive even when the heart stops beating for a while during early embryonic development and those adults with cardiac abnormalities. Therefore, we induced laterality defects and investigated the occurrence of situs solitus, situs inversus, and situs ambiguus in zebrafish development. Histopathological analysis revealed heterotaxy in both embryos and juvenile fish. Additionally, randomization of left-right asymmetry of the brain and heart in individual zebrafish embryos under artificial experimental pressure further demonstrated the advantage of transparent zebrafish embryos as an experimental tool to select or reduce the embryos with laterality defects during early embryonic development for long-term studies, including behavioural and cognitive neuroscience investigations.

Promoter cross-talk affects the inducible expression of intronic shRNAs from the tetracycline response element.

BACKGROUND: RNA interference (RNAi), defined as double-stranded, RNA-mediated gene silencing, is a useful tool for functional genomic studies. Along with increasing information about genomic sequences due to the innovative development of genome-sequencing technologies, functional genomic technologies are needed to annotate the genome and determine the processes by which each gene is regulated. Lentiviral vectors have been used to efficiently deliver reagents, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), into cells and tissues for functional genomic analyses. OBJECTIVE: We developed a lentiviral vector that efficiently expresses intronic shRNA from the tetracycline regulatory element (TRE) promoter in a doxycycline-dependent manner. METHODS: We developed a lentiviral vector system that contains reverse tetracycline-controlled transactivator 3 (rtTA3) and the TRE promoter, which are necessary for the doxycycline-inducible expression of shRNAs that are expressed as intronic miR-30a precursors. We then measured the cross-talk between the cytomegalovirus (CMV) and TRE promoters in the vector. RESULTS: We found that nearby promoters influence each other and that the TRE promoter should be located far from other promoters, such as the CMV promoter, in a vector. The orientation of a promoter with respect to other promoters also influences its transcriptional activity. A head-to-head orientation of the CMV and TRE promoters maintains the lowest level of transcription from TRE in the absence of doxycycline, compared to the tail-to-tail and head-to-tail orientations. CONCLUSION: Based on these findings, we were able to construct a lentiviral vector that faithfully expresses intronic miR-30a shRNA precursors in a doxycycline-inducible manner.

Enhancement of gene knockdown efficiency by CNNC motifs in the intronic shRNA precursor.

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Of several methods to express shRNA, a method of transcribing shRNAs embedded in microRNA precursors has been more widely used than the one that directly expresses shRNA from RNA polymerase III promoters because the microRNA precursor form of shRNA is known to cause lower levels of cytotoxicity and off-target effects than the overexpressed shRNAs from the RNA polymerase III promoters. OBJECTIVE: We study the primary sequence features of microRNA precursors, which enhance their processing into mature form, helps design more potent shRNA precursors embedded in microRNA precursors. METHODS: We measure the enhancement of gene knockdown efficiency by adding CNNC motifs in the 3' flanking region of shRNA precursor embedded in the human miR-30a microRNA precursor. RESULTS: By systemically adding three CNNC motifs in the 3' flanking region of shRNA precursor, we found that addition of two CNNC motifs saturates their enhanced knockdown ability of shRNA and that the CNNC motif in the + 17 to + 20 from the drosha cleavage site is most important for the shRNA-mediated target gene knock down. We also did see little knockdown of target gene expression by the shRNA precursor lacking CNNC motif. CONCLUSION: Since genetic studies generally require techniques that could reduce gene expression at different degrees, the findings in this study will allow us to use RNAi for genetic studies of reducing gene expression at different degrees.

Enzymatic construction of shRNA library from oligonucleotide library.

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. OBJECTIVE: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. METHODS: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. RESULTS: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. CONCLUSION: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.

3D light-sheet assay assessing novel valproate-associated cardiotoxicity and folic acid relief in zebrafish embryogenesis.

Precise in vivo toxicological assays to determine the cardiotoxicity of pharmaceuticals and their waste products are essential in order to evaluate their risks to humans and the environment following industrial release. In the present study, we aimed to develop the sensitive imaging-based cardiotoxicity assay and combined 3D light-sheet microscopy with a zebrafish model to identify hidden cardiovascular anomalies induced by valproic acid (VPA) exposure. The zebrafish model is advantageous for this assessment because its embryos remain transparent. The 3D spatial localization of fluorescence-labeled cardiac cells in and around the heart using light-sheet technology revealed dislocalization of the heart from the outflow tract in two-day-old zebrafish embryos treated with 50 µM and 100 µM VPA (P < 0.01) and those embryos exposed to 20 µM VPA presented hypoplastic distal ventricles (P < 0.01). These two observed phenotypes are second heart field-derived cardiac defects. Quantitative analysis of the light-sheet imaging demonstrated that folic acid (FA) supplementation significantly increased the numbers of endocardial and myocardial cells (P < 0.05) and the accretion of second heart field-derived cardiomyocytes to the arterial pole of the outflow tract. The heart rate increased in response to the cellular changes occurring in embryonic heart development (P < 0.05). The present study disclosed the cellular mechanism underlying the role of FA in spontaneous cellular changes in cardiogenesis and in VPA-associated cardiotoxicity. The 3D light-sheet assay may be the next-generation test to evaluate the risks of previously undetected pharmaceutical and environmental cardiotoxicities in both humans and animals.

Improved dynamic monitoring of transcriptional activity during longitudinal analysis in the mouse brain.

Bioluminescence imaging has proven to be a highly sensitive technique for assessing in vitro transcriptional activity toward understanding gene regulation patterns; however, application of this technique is limited for brain research. In particular, the poor spatiotemporal resolution is a major hurdle for monitoring the dynamic changes of transcriptional activity in specific regions of the brain during longitudinal analysis of living animals. To overcome this limitation, in this study, we modified a lentivirus-based luciferase glucocorticoid receptor (GR) reporter by inserting destabilizing sequence genes, and then the reporter was stereotaxically injected in the mouse infralimbic prefrontal cortex (IL-PFC). Using this strategy, we could successfully pin-point and monitor the dynamic changes in GR activity in IL-PFC during normal stress adaptation. The modified reporter showed a 1.5-fold increase in temporal resolution for monitoring GR activity compared to the control, with respect to the intra-individual coefficients of variation. This novel in vivo method has broad applications, as it is readily adaptable to different types of transcription factor arrays as well spanning wide target regions of the brain to other organs and tissues.

Biochemical analysis on the parallel pathways of methionine biosynthesis in Corynebacterium glutamicum.

Two alternative pathways for methionine biosynthesis are known in Corynebacterium glutamicum: one involving transsulfuration (mediated by metB and metC) and the other involving direct sulthydrylation (mediated by metY). In this study, MetB (cystathionine gamma-synthase) and MetY (O-acetylhomoserine sulfhydrylase) from C. glutamicum were purified to homogeneity and the biochemical parameters were compared to assess the functional and evolutionary importance of each pathway. The molecular masses of the native MetB and MetY proteins were measured to be approximately 170 and 280 kDa, respectively, showing that MetB was a homotetramer of 40-kDa subunits and MetY was a homohexamer of 45-kDa subunits. The Km values for the O-acetylhomoserine catalysis effected by MetB and MetY were 3.9 and 6.4 mM, and the maximum catalysis rates were 7.4 (kcat = 21 s(-1)) and 6.0 (kcat=28 s(-1)) micromol mg(-1) min(-1), respectively. This suggests that both MetB and MetY can be comparably active in vivo. Nevertheless, the Km value for sulfide ions by MetY was 8.6 mM, which was too high, considering the physiological condition. Moreover, MetB was active at a broad range of temperatures (30 and 65 degrees C) and pH (6.5 and 10.0), as compared with MetY, which was active in a range from 30 to 45 degrees C and at pH values from 7.0 to 8.5. In addition, MetY was inhibited by methionine, but MetB was not. These biochemical data may provide insight on the role of the parallel pathways of methionine biosynthesis in C. glutamicum with regard to cell physiology and evolution.

N-acylhomoserine lactonase producing Rhodococcus spp. with different AHL-degrading activities.

N-acylhomoserine lactones (AHLs) are conserved signal molecules that control diverse biological activities in quorum sensing system of Gram-negative bacteria. Recently, several soil bacteria were found to degrade AHLs, thereby interfering with the quorum sensing system. Previously, Rhodococcus erythropolis W2 was reported to degrade AHLs by both oxido-reductase and AHL-acylase. In the present study, two AHL-utilizing bacteria, strains LS31 and PI33, were isolated and identified as the genus Rhodococcus. They exhibited different AHL-utilization abilities: Rhodococcus sp. strain LS31 rapidly degraded a wide range of AHLs, including N-3-oxo-hexanoyl-l-homoserine lactone (OHHL), whereas Rhodococcus sp. strain PI33 showed relatively less activity towards 3-oxo substituents. Coculture of strain LS31 with Erwinia carotovora effectively reduced the amount of OHHL and pectate lyase activity, compared with coculture of strain PI33 with E. carotovora. A mass spectrometry analysis indicated that both strains hydrolyzed the lactone ring of AHL to generate acylhomoserine, suggesting that AHL-lactonases (AHLases) from the two Rhodococcus strains are involved in the degradation of AHL, in contrast to R. erythropolis W2. To the best of our knowledge, this is the first report on AHLases of Rhodococcus spp.

Electrophoretic mobility shift assays to study protein binding to damaged DNA.

The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA-PKcs, which are involved in double-strand break repair. This chapter describes EMSA protocols for detecting proteins that bind to UV-damaged DNA, cisplatin-damaged DNA, and DNA ends. The chapter also describes variations of the EMSA that can be used to obtain additional information about these important proteins. The variations include the reverse EMSA, which can detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay, which can define the composition of protein-DNA complexes.