PCR-based identification of Staphylococcus epidermidis targeting gseA encoding the glutamic-acid-specific protease

The frequency of the gseA gene encoding a glutamic acid-specific serine protease, GluSE, of Staphylococcus epidermidis was investigated. DNA hybridization analysis demonstrated that gseA existed exclusively in S. epidermidis but not in other bacteria examined. A single step PCR assay with a set of designed primers yielded amplification of gseA from all 69 clinical isolates of S. epidermidis taken from patients and healthy adults, whereas production of GluSE was observed in 74% (51/69) of the isolates. Furthermore, none of the 46 clinical isolates of other species of coagulase-negative staphylococci and 45 clinical isolates of Staphylococcus aureus showed amplification, except a Staphylococcus capitis strain. However, this strain was positive for a S. epidermidis-specific DNA region and the DNA sequence of the 16S rRNA gene showed 99% identity with that of S. epidermidis. Therefore, these results indicated that the present PCR assay for gseA was ubiquitous and highly specific for detection of S. epidermidis.     

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.     

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.     

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks

Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.     

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.

     

Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells

Hepatic stellate cells (HSCs) play a central role in the development of hepatic fibrosis. Recent evidence has revealed that HSCs also play a role in its resolution, where HSC apoptosis was determined. Moreover, induction of HSC apoptosis caused a reduction of experimental hepatic fibrosis in rats. Thus knowing the mechanism of HSC apoptosis might be important to clarify the pathophysiology and establish the therapeutic strategy for hepatic fibrosis. In HSCs, Rho and Rho kinase are known to regulate contraction, migration, and proliferation with modulation of cell morphology. Controversy exists as to the participation of Rho and Rho kinase on cell survival, and little is known regarding this matter in HSCs. In this study, we directed our focus on the role of the Rho pathway in the regulation of HSC survival. C3, an inhibitor of Rho, increased histone-associated DNA fragmentation and caspase 3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs. Moreover, Y-27632, an inhibitor of Rho kinase, had the same effects, suggesting that inhibition of the Rho/Rho kinase pathway causes HSC apoptosis. On the other hand, lysophosphatidic acid, which stimulates the Rho/Rho kinase pathway, decreased histone-associated DNA fragmentation in HSCs. Inhibition of the Rho/Rho kinase pathway did not affect p53, Bcl-2, or Bax levels in HSCs. Thus we concluded that the Rho/Rho kinase pathway may play a role in the regulation of HSC survival.     

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

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.