Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice

The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interest in these drugs as potential therapeutic agents in genetic diseases. However, the toxicity of aminoglycoside antibiotics may result in severe side effects during long-term treatment. In this paper, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of the mdx mouse, an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in the dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrate that negamycin binds to a partial sequence of the eukaryotic rRNA-decoding A-site. We conclude that negamycin is a promising new therapeutic candidate for DMD and other genetic diseases caused by nonsense mutations.     

Novel types of COMP mutations and genotype-phenotype association in pseudoachondroplasia and multiple epiphyseal dysplasia

Mutations in the gene encoding cartilage oligomeric matrix protein ( COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). More than 40 mutations have been identified; however, genotype-phenotype relationships are not well delineated. Further, mutations other than in-frame insertion/deletions and substitutions have not been found, and currently known mutations are clustered within relatively small regions. Here we report the identification of nine novel and three recurrent COMP mutations in PSACH and MED patients. These include two novel types of mutations; the first, a gross deletion spanning an exon-intron junction, causes an exon deletion. The second, a frameshift mutation that results in a truncation of the C-terminal domain, is the first known truncating mutation in the COMP gene. The remaining mutations, other than a novel exon 18 mutation, affected highly conserved aspartate or cysteine residues in the calmodulin-like repeat (CLR) region. Genotype-phenotype analysis revealed a correlation between the position and type of mutations and the severity of short stature. Mutations in the seventh CLR produced more severe short stature compared with mutations elsewhere in the CLRs ( P=0.0003) and elsewhere in the COMP gene ( P=0.0007). Patients carrying mutations within the five-aspartates repeat (aa 469-473) in the seventh CLR were extremely short (below -6 SD). Patients with deletion mutations were significantly shorter than those with substitution mutations ( P=0.0024). These findings expand the mutation spectrum of the COMP gene and highlight genotype-phenotype relationships, facilitating improved genetic diagnosis and analysis of COMP function in humans.     

Oxidative stress-induced cell death of human oral neutrophils

Polymorphonuclear leukocytes (PMN) playcrucial roles in protecting hosts against invading microbes and in thepathogenesis of inflammatory tissue injury. Although PMN migrate intomucosal layers of digestive and respiratory tracts, only limitedinformation is available of their fate and function in situ. Wepreviously reported that, unlike circulating PMN (CPMN), PMN in theoral cavity spontaneously generate superoxide radical and nitric oxide (NO) in the absence of any stimuli. When cultured for 12 h under physiological conditions, oral PMN (OPMN) showed morphological changesthat are characteristic of those of apoptosis. Upon agarose gelelectrophoresis, nuclear DNA samples isolated from OPMN revealed ladder-like profiles characteristic of nucleosomal fragmentation. L-cysteine, reduced glutathione (GSH), and herbimycin A, aprotein tyrosine kinase inhibitor, suppressed the activation ofcaspase-3 and apoptosis of OPMN. Neither thiourea, superoxidedismutase (SOD), nor catalase inhibited the activation of caspase-3 and apoptosis. Moreover,N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibitorfor caspase-3, inhibited the fragmentation of DNA. These resultssuggested that oxidative stress and/or tyrosine-kinase-dependent pathway(s) activated caspase-3 in OPMN, thereby inducing their apoptosis.

     

Characterization of Sleeping Beauty transposition and its application to genetic screening in mice

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.     

Circadian dynamics of cytosolic and nuclear Ca2+ in single suprachiasmatic nucleus neurons

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.     

Immunological detection of proteolytically activated epidermal-type transglutaminase (TGase 3) using cleavage-site-specific antibody

Transglutaminase 3 (TGase 3), involved in the cross-linking of structural proteins in the epidermis, is activated by limited proteolysis of zymogen into two fragments during keratinocyte differentiation. Using recombinant TGase 3, the N-terminus sequence of the proteolyzed fragment was analyzed. Antibody against the synthetic peptide corresponding to the cleavage site specifically detected the fragment in the mouse forestomach extract.     

Estrogen-responsive RING finger protein controls breast cancer growth

Most of the breast cancers initially respond to endocrine therapy that reduces the levels of estrogens or competes with estrogen for binding to its receptor. Most of the patients, however, acquire resistance to endocrine therapy with tamoxifen and aromatase inhibitors later. We assumed that identification of estrogen-responsive genes those regulate the growth of breast cancer is indispensable to develop new strategies targeting the genes and overcome the resistance to current endocrine therapy. Estrogen-responsive finger protein (Efp) is one of the estrogen receptor (ER)-target genes we have cloned using genomic binding site cloning. Efp features a structure of the RING-finger B-box coiled-coil (RBCC) motif. We postulated that Efp is a critical factor in proliferation of breast tumors. In a model system using MCF7 cells grown in xenografts, we showed that inhibition of Efp expression by antisense oligonucleotide reduced the tumor growth. MCF7 cells overexpressing Efp formed tumors in xenografts even in estrogen deprivation environment. By yeast two-hybrid screen, we identified that Efp interacts with 14-3-3σ, which is known as a cell cycle brake that causes G2 arrest and expressed in normal mammary glands. In vitro studies have revealed that Efp functions as a ubiquitin-protein ligase (E3) that targets 14-3-3σ. These data suggest that Efp controls breast cancer growth through ubiquitin-dependent proteolysis of 14-3-3σ. Future studies may provide a new therapy to block breast tumor proliferation by targeting Efp.     

Oxysterol regulation of estrogen receptor alpha-mediated gene expression in a transcriptional activation assay system using HeLa cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor alpha-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17beta-estradiol > genistein > beta-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = beta-epoxysitosterol = 7beta-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.     

Expressions of adrenomedullin mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Adrenomedullin (AM) has diuretic, natriuretic, and hypotensive effects. To study the possible effects of HHE on the AM synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of a 5,500-m altitude level for 21 days. After 14 days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased (compared with control rats). The plasma AM protein level was significantly increased on day 21 of exposure to HHE. In the right ventricle (RV), right atrium, and left atrium of the heart, the expressions of AM mRNA and protein were increased in the middle to late phase (5-21 days) of HHE, whereas in the brain and lung they were increased much earlier (0.5-5 days). In situ hybridization and immunohistochemistry showed AM mRNA and protein staining to be more intense in the RV in animals in the middle to late phase of HHE exposure than in the controls. During HHE, these changes in AM synthesis, which occurred strongly in the RV, occurred alongside the increase in PAP. Conceivably, AM may play a role in modulating pulmonary hypertension in HHE.     

Effect of Rabbit Anti-Asialo-GM1 (GA1) Polyclonal Antibodies on Neuromuscular Transmission and Acetylcholine-Induced Action Potentials: Neurophysiological and Immunohistochemical Studies

We produced anti-asialo-GM1 (GA1) polyclonal antibodies by sensitizing New Zealand rabbits with GA1 and investigated the epitopes and pathogenic role of anti-GA1 antibodies that appeared in serum. The serum blocked neuromuscular transmission, but not acetylcholine (ACh)-induced potentials, in muscle-spinal cord cocultured cells. The effect was complement independent. The antibodies inhibited voltage-gated Ca2+ channel (VGCC). The epitopes recognized by the antibodies were located in the outer membrane of Schwann cells and motor axons of Wistar rat ventral roots and on motor axons extended from spinal cord to muscle cells in muscle-spinal cocultured cells. The ACh-induced potential was not reduced by the addition of sera, suggesting the blockade is presynaptic. Thus, anti-GA1 antibodies may block neuromuscular transmission by suppressing VGCC on axonal terminals of motor nerves.