Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (r Si -HLP). r Si -HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1β and TNF-α) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of r Si -HLP was heat-unstable. In subsequent studies, r Si -HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that r Si -HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si -HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.     

Faunal turnover at the end of the Cretaceous in the North Pacific region: Implications from combined magnetostratigraphy and biostratigraphy of the Maastrichtian Senpohshi Formation in the eastern Hokkaido Island,northern Japan

A combined magnetostratigraphic and biostratigraphic study has been performed on the Maastrichtian Senpohshi Formation in eastern Hokkaido Island, northern Japan, which is an approximately 1300 m thick section mainly composed of hemipelagic mudstone. The identification of magnetic polarity was possible at 51 horizons, whereby four magnetozones were recognized. These magnetozones were correlatable to geomagnetic polarity chrons C31r to C30n, suggesting that the age of the Senpohshi Formation is spanning from middle to upper part of the Maastrichtian (ca. 69–67 Ma).The magnetostratigraphy of the Senpohshi Formation established in this study enables a direct age correlation to the Maastrichtian successions in other regions. Thus, this detailed chronology of the formation contributes to paleontological studies of the Maastrichtian in the North Pacific region. For instance, this magnetostratigraphic age assessment implies the following: (1) the stratigraphic range of the ammonite Pachydiscus flexuosus contains polarity chrons from the lower part of C31r to the lower part of C31n, (2) the first occurrence (FO) of the calcareous nannofossil Nephrolithus frequens in the North Pacific region is correlatable to polarity chron C30n or below, and (3) the FO of the bivalve “Inoceramus” awajiensis is located within polarity chrons from C31r to the upper part of C31n. This suggests that the inoceramid extinction event in the North Pacific region might have occurred during polarity chrons from C31r to the upper part of C31n (ca. 70.5–67.8 Ma), which is 2.3–5.0 Myr prior to the Cretaceous/Paleogene boundary. The trend of the Maastrichtian faunal turnover in the North Pacific is well consistent with those of other regions, brings a new evidence for understanding the global faunal turnover in the Maastrichtian, just before Cretaceous/Paleogene mass extinction.     

Intracellular dynamics of topoisomerase I inhibitor,CPT-11, by slit-scanning confocal Raman microscopy

Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stability of DNA/Td3717 complexes for gene transfection,defined by the size and polydispersity of the complex

The amphiphilic α-helical peptide, Td3717, is a bi-functional synthetic peptide that acts as both a polycation for DNA binding and a ligand for targeted delivery to tumor cells. Td3717 forms a stable complex with plasmid DNA, and the complex maintained high transfection efficiency after storage at 4 °C for six months and after four freeze/thaw cycles. During the storage and freeze/thaw cycling, the particle size of the DNA/Td3717 complex remained less than 100 nm. The size of the complex is an important factor for its internalization into cells via the endocytosis pathway; therefore, the stability of the particles will strongly contribute to high transfection efficiencies after storage and repeated freezing/thawing.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

Ephrin‐A5 regulates the formation of the ascending midbrain dopaminergic pathways

Dopaminergic neurons from the substantia nigra and the ventral tegmental area of the midbrain project to the caudate/putamen and nucleus accumbens, respectively, establishing the mesostriatal and the mesolimbic pathways. However, the mechanisms underlying the development of these pathways are not well understood. In the current study, the EphA5 receptor and its corresponding ligand, ephrin‐A5, were shown to regulate dopaminergic axon outgrowth and influence the formation of the midbrain dopaminergic pathways. Using a strain of mutant mice in which the EphA5 cytoplasmic domain was replaced with β‐galactosidase, EphA5 protein expression was detected in both the ventral tegmental area and the substantia nigra of the midbrain. Ephrin‐A5 was found in both the dorsolateral and the ventromedial regions of the striatum, suggesting a role in mediating dopaminergic axon‐target interactions. In the presence of ephrin‐A5, dopaminergic neurons extended longer neurites in in vitro coculture assays. Furthermore, in mice lacking ephrin‐A5, retrograde tracing studies revealed that fewer neurons sent axons to the striatum. These observations indicate that the interactions between ephrin‐A ligands and EphA receptors promote growth and targeting of the midbrain dopaminergic axons to the striatum. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009