Functional importance of inositol-1,4,5-triphosphate-induced intracellular Ca2+ mobilization in galanin-induced microglial migration

Galanin (GAL) is a neuropeptide which is up-regulated following neuronal axotomy or inflammation. One subtype of GAL receptor (GalR2) is reported to be expressed in the brain's immune cell population, microglia. In the present study, we investigated the effect of GAL on microglial migration and compared the mechanism with that of bradykinin (BK). GAL significantly increased the migration of rat cultured microglia at 0.1 pM. The GAL-induced signal cascade was partly similar to that induced by BK. It was not dependent on G(i/o) protein but involved activation of protein kinase C, phosphoinositide 3-kinase and Ca(2+)-dependent K(+) channels. However, reverse-mode activation of the Na(+) /Ca(2+) -exchanger 1 was not involved in GAL-induced microglial migration, unlike BK-induced migration. Likewise, nominally-free extracellular Ca(2+) inhibited BK-induced migration but not GAL-induced migration. An inositol-1,4,5-triphosphate receptor antagonist significantly inhibited GAL-induced migration. GAL-induced Ca(2+) signaling did not induce nitric oxide synthase expression, but up-regulated class II major histocompatibility complex expression. These results indicate that activation of inositol-1,4,5-triphosphate receptor and increase in intracellular Ca(2+) are important for GAL-induced migration and immunoreactivity in microglia. The differences in down-stream signal transduction induced by GAL and BK suggest that GAL and BK may control distinct microglial functions under pathological conditions.     

Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7

CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.     

Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.     

Participation of hup gene product in ori2-dependent replication of fertility plasmid F

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.     

DGCR8‐mediated disruption of miRNA biogenesis induces cellular senescence in primary fibroblasts

The regulation of gene expression by microRNAs (miRNAs) is critical for normal development and physiology. Conversely, miRNA function is frequently impaired in cancer, and other pathologies, either by aberrant expression of individual miRNAs or dysregulation of miRNA synthesis. Here, we have investigated the impact of global disruption of miRNA biogenesis in primary fibroblasts of human or murine origin, through the knockdown of DGCR8, an essential mediator of the synthesis of canonical miRNAs. We find that the inactivation of DGCR8 in these cells results in a dramatic antiproliferative response, with the acquisition of a senescent phenotype. Senescence triggered by DGCR8 loss is accompanied by the upregulation of the cell‐cycle inhibitor p21CIP1. We further show that a subset of senescence‐associated miRNAs with the potential to target p21CIP1 is downregulated during DGCR8‐mediated senescence. Interestingly, the antiproliferative response to miRNA biogenesis disruption is retained in human tumor cells, irrespective of p53 status. In summary, our results show that defective synthesis of canonical microRNAs results in cell‐cycle arrest and cellular senescence in primary fibroblasts mediated by specific miRNAs, and thus identify global miRNA disruption as a novel senescence trigger.     

Conformational changes of α-lactalbumin induced by the stepwise reduction of its disulfide bridges: The effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution

Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.     

Interaction analysis between tmRNA and SmpB from Thermus thermophilus

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.     

Brain 5-HT synthesis in the Flinders Sensitive Line rat model of depression: an autoradiographic study

Alterations of serotonin (5-HT) levels and serotonergic transmission have been associated with depression. 5-HT synthesis is an important factor of serotonergic neurotransmission that may also be altered in depression. Many studies of the relationships between brain serotonergic functions and affective disorders have been performed in different animal models. In this study, brain regional 5-HT synthesis was examined using the alpha-[(14)C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method in a genetic rat model of depression, Flinders Sensitive Line (FSL) rats, and was compared to both the Flinders Resistant Line (FRL) rats and the control Sprague-Dawley (SD) rats. The plasma concentration of free tryptophan in the FSL rats was not significantly different (p > 0.05; ANOVA and post-hoc Bonferroni correction) when compared to that of the FRL and SD rats. The FSL rats had significantly lower 5-HT synthesis (one sample two-tailed t-test on the ratio) than both the FRL and SD rats (the mean ratios were 0.78 +/- 0.12 and 0.73 +/- 0.15, respectively). Overall, the 5-HT synthesis in the FRL rats was not significantly different (p > 0.05) from that in the SD rats (one sample two-tailed t-test on the ratio and the mean ratio was 0.93 +/- 0.13). Studies of individual brain structures, such as the raphe nuclei and their many terminal areas, including the nucleus accumbens, cingulate and frontal cortex, hippocampus, amygdala, and thalamus revealed significant reductions (typically 25-50%) in 5-HT synthesis in the FSL rats compared to the non-depressive FRL and SD rats. These results suggest that significantly reduced 5-HT synthesis in the raphe nuclei and limbic areas in FSL rats may contribute to their depressive features.