Increased affinity for copper mediated by cysteine 111 in forms of mutant superoxide dismutase 1 linked to amyotrophic lateral sclerosis

Mutations in Cu,Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (ALS). It has been proposed that neuronal cell death might occur due to inappropriately increased Cu interaction with mutant SOD1. Using Cu immobilized metal-affinity chromatography (IMAC), we showed that mutant SOD1 (A4V, G85R, and G93A) expressed in transfected COS7 cells, transgenic mouse spinal cord tissue, and transformed yeast possessed higher affinity for Cu than wild-type SOD1. Serine substitution for cysteine at the Cys111 residue in mutant SOD1 abolished the Cu interaction on IMAC. C111S substitution reversed the accelerated degradation of mutant SOD1 in transfected cells, suggesting that the Cys111 residue is critical for the stability of mutant SOD1. Aberrant Cu binding at the Cys111 residue may be a significant factor in altering mutant SOD1 behavior and may explain the benefit of controlling Cu access to mutant SOD1 in models of familial ALS.     

A Novel System for Simultaneous or Sequential Integration of Multiple Gene-Loading Vectors into a Defined Site of a Human Artificial Chromosome

Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.     

The 3′–5′ DNA Exonuclease TREX1 Directly Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) during the DNA Damage Response

The main function of the 3′–5′ DNA exonuclease TREX1 is to digest cytosolic single-stranded DNA to prevent activation of cell-intrinsic responses to immunostimulatory DNA. TREX1 translocates to the nucleus following DNA damage with its nuclear activities being less well defined. Although mutations in human TREX1 have been linked to autoimmune/inflammatory diseases, the mechanisms contributing to the pathogenesis of these diseases remain incompletely understood. Here, using mass spectrometry and co-immunoprecipitation assays and in vivo overexpression models, we show that TREX1 interacts with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear enzyme involved in the DNA damage response. Two zinc finger domains at the amino terminus of PARP1 were required for the interaction with TREX1 that occurs after nuclear translocation of TREX1 in response to DNA damage. Functional studies suggested that TREX1 may contribute to stabilization of PARP1 levels in the DNA damage response and its activity. These results provide new insights into the mechanisms of single-stranded DNA repair following DNA damage and alterations induced by gene mutations.     

Harmful or Not: Trichostatin A treatment of embryos generated by ICSI or ROSI

Trichostatin A (TSA), a histone deacetylase inhibitor, is a known teratogen causing malformations such as vertebral fusions when applied during the postimplantation period; TSA also causes developmental arrest when applied during the preimplantation period. Regardless of these hindrances, we have succeeded in the establishment of an efficient somatic cloning method for the mouse where reconstructed embryos are treated with TSA. To elucidate this apparent discrepancy, we treated fertilized mouse embryos generated either by intracytoplasmic sperm injection (ICSI) or round spermatid injection (ROSI) with 50 nM TSA for 20 h after fertilization as well as parthenogenetic embryos and found that TSA treatment inhibited the preimplantation development of ICSI embryos but not ROSI or parthenogenetic embryos. And, although we often observed hypomorphism following TSA treatment in embryos grown to full term produced by both ICSI (av. of body weight: 1.7 g vs. 1.5 g) and ROSI (1.6 g vs. 1.2 g), TSA treatment reduced the offspring production rate for ICSI from 57% to 34% but not for ROSI from 30% to 36%. Thus, these data indicate that the effects, harmful or not, of TSA treatment on embryonic development depend on their nuclear derivations. Also, the resulting hypomorphism after TSA treatment is a caveat for this procedure in current Assisted Reproductive Technologies.     

Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids

Although round spermatid injection can be used to create progeny for males who do not produce mature sperm, the rate of successful embryogenesis after such procedures is significantly lower than that for similar procedures using mature spermatozoa. The mechanisms underlying this difference are unknown. In this study, we demonstrate that, unlike the normal paternal genome, the paternal zygotic genome derived from a round spermatid is highly remethylated before first mitosis after demethylation. Genomes from elongated spermatids exhibited an intermediate level of DNA methylation, between those of round spermatids and mature spermatozoa, suggesting that the male germ cell acquires the ability to maintain its undermethylated state in the paternal zygotic genome during this phase of spermiogenesis. In addition, treatment of zygotes with trichostatin A led to a significant reduction in DNA methylation, specifically in the spermatid-derived paternal genome, except for the pericentromeric regions enriched by trimethylation of Lys9 of histone H3. These data provide insight into epigenetic errors that may be associated with the poor development of embryos generated from immature spermatozoa.     

Overexpression of ganglioside GM1 results in the dispersion of platelet-derived growth factor receptor from glycolipid-enriched microdomains and in the suppression of cell growth signals.

To investigate the molecular mechanisms of gangliosides for the regulation of cell proliferation, Swiss 3T3 cells were transfected with GM2/GD2 synthase and GM1 synthase cDNAs, resulting in the establishment of GM1-expressing (GM1(+)) lines. Compared with the vector control (GM1(-)) cell lines, GM1(+) cells exhibited reduced cell proliferation by stimulation with platelet-derived growth factor (PDGF). In accordance with the reduced cell growth, GM1(+) cells showed earlier decreases in the phosphorylation levels of PDGF receptor and less activation of MAP kinases than GM1(-) cells. To analyze the effects of GM1 expression on the PDGF/PDGF receptor (PDGFR) signals, the glycolipid-enriched microdomain (GEM) was isolated and the following results were obtained. (i) PDGFR predominantly distributed in the non-GEM fraction in GM1(+) cells, while it was present in both GEM and non-GEM fractions in GM1(-) cells. (ii) Activation of PDGFR as detected by anti-phosphotyrosine antibody occurred almost in parallel with existing amounts of PDGFR in each fraction. (iii) GM1 binds with PDGFR in GEM fractions. These findings suggested that GM1 regulates signals via PDGF/PDGFR by controlling the distribution of PDGFR in- and outside of GEM, and also interacting with PDGFR in the GEM fraction as a functional constituent of the microdomain.     

Genotypic variability in phosphorus solubilizing activity of root exudates by pigeonpea grown in low-nutrient environments

A pot experiment confirmed that pigeonpea could efficiently utilize various sources of phosphorus (P) (aluminium phosphate, iron phosphate and apatite), irrespective of genotype. A qualitative assay method for iron (Fe)-P solubilizing activity showed that root exudates collected from P-deficient pigeonpea contained Fe-P solubilizing substances and that they were released mainly from root tips. Citric, malic, malonic, succinic and piscidic acids were identified in root exudates. Citric and piscidic acids release from roots was increased by low-P treatment in all the genotypes tested. The release rates of citric and piscidic acids were affected by the P concentration of shoots rather than that of roots. The pigeonpea roots released approximately 5–100 times more piscidic acid than citric acid depending on P stress status, plant age and genotype. When organic acids were added to Alfisols, citric acid was most capable of mobilizing P from the soil, followed by piscidic acid and malic acid. No correlation was found between genotypic variability in the release rates of citric and piscidic acids from the roots under low-P treatment at hydroponic culture and in the growth and P uptake of plants on Alfisols. Although citric and piscidic acids released from pigeonpea roots may play a partial role in solubilizing unavailable insoluble P in soils, the releases were thought to be an unsatisfactory strategy for explaining genotypic variation in low P availability of pigeonpea.