Differential inhibitory effects of mu-opioids on substance P- and capsaicin-induced nociceptive behavior in mice

The antinociceptive mechanisms of the selective mu-opioid receptor agonists [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA) or H-Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA-NH2) against substance P (SP)- or capsaicin-elicited nociceptive behaviors was investigated in mice. DAMGO, TAPA or TAPA-NH2 given intrathecally inhibited the nociceptive behaviors elicited by intrathecally administered SP or capsaicin, and these antinociceptive effects were completely eliminated by intrathecal co-administration with D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist. Pretreatment subcutaneously with naloxonazine, a selective mu1-opioid receptor antagonist, partially attenuated the antinociceptive effect of TAPA-NH2, but not DAMGO and TAPA, against SP. However, the antinociception induced by TAPA, but not DAMGO and TAPA-NH2, against capsaicin was significantly inhibited by naloxonazine. On the other hand, co-administration intrathecally with Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a selective mu2-opioid receptor antagonist, significantly attenuated the antinociceptive effects of DAMGO, but not TAPA and TAPA-NH2, against capsaicin, while the antinociceptions induced by three opioid peptides against SP were significantly inhibited by D-Pro2-Tyr-W-MIF-1. These results suggest that differential inhibitory mechanisms on pre- and postsynaptic sites in the spinal cord contribute to the antinociceptive effects of the three mu-opioid peptides.     

ATM-Chk2-p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency

BRCA1 is a checkpoint and DNA damage repair gene that secures genome integrity. We have previously shown that mice lacking full-length Brca1 (Brca1(delta11/delta11)) die during embryonic development. Haploid loss of p53 completely rescues embryonic lethality, and adult Brca1(delta11/delta11)p53+/- mice display cancer susceptibility and premature aging. Here, we show that reduced expression and/or the absence of Chk2 allow Brca1(delta11/delta11) mice to escape from embryonic lethality. Compared to Brca1(delta11/delta11)p53+/- mice, lifespan of Brca1(delta11/delta11)Chk2-/- mice was remarkably extended. Analysis of Brca1(delta11/delta11)Chk2-/- mice revealed that p53-dependent apoptosis and growth defect caused by Brca1 deficiency are significantly attenuated in rapidly proliferating organs. However, in later life, Brca1(delta11/delta11)Chk2-/- female mice developed multiple tumors. Furthermore, haploid loss of ATM also rescued Brca1 deficiency-associated embryonic lethality and premature aging. Thus, in response to Brca1 deficiency, the activation of the ATM-Chk2-p53 signaling pathway contributes to the suppression of neoplastic transformation, while leading to compromised organismal homeostasis. Our data highlight how accurate maintenance of genomic integrity is critical for the suppression of both aging and malignancy, and provide a further link between aging and cancer.     

Binding of AlF-C, an Orc1-binding transcriptional regulator, enhances replicator activity of the rat aldolase B origin

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.     

Glycogen synthase kinase 3 and h-prune regulate cell migration by modulating focal adhesions

h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the activation of Rac were suppressed in GSK-3 or h-prune knocked-down cells. GSK-3 inhibitors suppressed the disassembly of paxillin and the activation of FAK and Rac. Furthermore, h-prune was highly expressed in colorectal and pancreatic cancers, and the positivity of the h-prune expression was correlated with tumor invasion. These results suggest that GSK-3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration and that h-prune is useful as a marker for tumor aggressiveness.     

Evidence of cross-fertilization in a gonochoric population of the tadpole shrimp Triops numidicus (Crustacea: Branchiopoda: Notostraca)

Cross-fertilization was evident in a gonochoric population of the notostracan Triops numidicus, in which a thick, hard eggshell had been suspected of preventing the sperm from reaching the egg. Most of the eggs from copulated females hatched into larvae, but the eggs from virgin females did not develop. In the larvae, paternal DNA fragments were detected by AFLP. In histological sections, several spermatozoa were found in the shell of newly oviposited eggs in the brood pouches of copulated females, suggesting that the shell was still soft enough to be penetrated by spermatozoa. These results showed that copulation and subsequent fertilization achieved by penetration of sperm through the newly deposited eggshell were indispensable for the normal development of the eggs.     

Myxococcus xanthus twin-arginine translocation system is important for growth and development

The twin-arginine translocation (Tat) system serves to export fully folded proteins across the cytoplasmic membrane. In many bacteria, three major components, TatA, TatB and TatC, are the functionally essential constituents of the Tat system. A Myxococcus xanthus tatB–tatC deletion mutant could aggregate and form mounds, but was unable to form fruiting bodies under nutritionally limiting conditions. When tatB–tatC mutant vegetative cells were cultured with 0.5 M glycerol, the cell morphology changed to spore-like spherical cells, but the spores were not resistant to heat and sonication treatments. In contrast to the wild-type strain, the tatB–tatC mutant also showed a decreased cell growth rate and a lower maximum cell concentration. These results suggest possibility that the Tat system may contribute to export of various important proteins for development and growth for M. xanthus.     

DNA repair defect in AT cells and their hypersensitivity to low-dose-rate radiation

Ataxia telangiectasia (AT) and normal cells immortalized with the human telomerase gene were irradiated in non-proliferative conditions with high- (2 Gy/min) or low-dose-rate (0.3 mGy/min) radiation. While normal cells showed a higher resistance after irradiation at a low dose rate than a high dose rate, AT cells showed virtually the same survival after low- and high-dose-rate irradiation. Although the frequency of micronuclei induced by low-dose-rate radiation was greatly reduced in normal cells, it was not reduced significantly in AT cells. The number of gamma-H2AX foci increased in proportion to the dose in both AT and normal cells after high-dose-rate irradiation. Although few gamma-H2AX foci were observed after low-dose-rate irradiation in normal cells, significant and dose-dependent numbers of gamma-H2AX foci were observed in AT cells even after low-dose-rate irradiation, indicating that DNA damage was not completely repaired during low-dose-rate irradiation. Significant phosphorylation of ATM proteins was detected in normal cells after low-dose-rate irradiation, suggesting that the activation of ATM plays an important role in the repair of DNA damage during low-dose-rate irradiation. In conclusion, AT cells may not be able to repair some fraction of DNA damage and are severely affected by low-dose-rate radiation.     

Molecular epidemiology of echoviruses 11 and 13, based on an environmental surveillance conducted in Toyama Prefecture, 2002-2003

Nineteen echovirus 11 (E11) and 12 E13 isolates were isolated from three rivers in Toyama Prefecture, Japan, during an environmental surveillance conducted from April 2002 to March 2003. The nucleotide sequences of E13 isolates were closely related to those from patients with aseptic meningitis, with less than 1.3% divergence in the VP1 region of the viral capsid gene, and belonged to the same clade responsible for a worldwide outbreak that started in 2000. In contrast, E11 isolates were clustered into three genomic groups and were not closely related to echovirus strains isolated from patients. These results suggest that the combination of both virus isolation from environmental sources and phylogenetic analysis could be complementary assessment approaches to trace prevalent and minor circulating enteroviruses in the human population.     

Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.