Involvement of the linker histone H1Foo in the regulation of oogenesis.

In brief: In oocytes, chromatin structure is loosened during their growth, which seems to be essential for the establishment of competence to accomplish the maturation and further development after fertilization. This paper shows that a linker histone variant, H1foo, is involved in the formation of loosened chromatin structure in growing oocytes. Abstract: During oogenesis, oocytes show a unique mode of division and gene expression patterns. Chromatin structure is thought to be involved in the regulation of these processes. In this study, we investigated the functions of linker histones, which modulate higher-order chromatin structure during oogenesis. Because H1foo is highly expressed in oocytes, we knocked down H1foo using siRNA and observed oocyte growth, maturation, and fertilization. However, H1foo knockdown had no effect on any of these processes. Overexpression of H1b or H1d, which has a high ability to condense chromatin and is expressed at a low level in oocytes, resulting in tightened chromatin and a decreased success rate of oocyte maturation. By contrast, overexpression of H1a, which is expressed at a high level in oocytes and has a low ability to compact chromatin, did not affect growth or maturation. Therefore, H1a, but not other variants, might compensate for the function of H1foo in H1foo-knockdown oocytes. These results implicate H1foo in the formation of loose chromatin structure, which is necessary for oocyte maturation. In addition, the low expression of somatic linker histone variants, for example, H1b and H1d, is important for loosened chromatin and meiotic progression.

Minor zygotic gene activation is essential for mouse preimplantation development.

In mice, transcription initiates at the mid-one-cell stage and transcriptional activity dramatically increases during the two-cell stage, a process called zygotic gene activation (ZGA). Associated with ZGA is a marked change in the pattern of gene expression that occurs after the second round of DNA replication. To distinguish ZGA before and after the second-round DNA replication, the former and latter are called minor and major ZGA, respectively. Although major ZGA are required for development beyond the two-cell stage, the function of minor ZGA is not well understood. Transiently inhibiting minor ZGA with 5, 6-dichloro-1-ß-d-ribofuranosyl-benzimidazole (DRB) resulted in the majority of embryos arresting at the two-cell stage and retention of the H3K4me3 mark that normally decreases. After release from DRB, at which time major ZGA normally occurred, transcription initiated with characteristics of minor ZGA but not major ZGA, although degradation of maternal mRNA normally occurred. Thus, ZGA occurs sequentially starting with minor ZGA that is critical for the maternal-to-zygotic transition.

The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3' processing.

Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1-cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA-Seq, we have found that transcription in 1-cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core-promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1-cell embryos because of inefficient splicing and 3' processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1-cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1-cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1-cell embryos.

Characterization of gene expression in mouse embryos at the 1-cell stage.

In mice, transcription from the zygotic genome is initiated at the mid-1-cell stage after fertilization. Although a recent high-throughput sequencing (HTS) analysis revealed that this transcription occurs promiscuously throughout almost the entire genome in 1-cell stage embryos, a detailed investigation of this process has yet to be conducted using protein-coding genes. Thus, the present study utilized previous RNA sequencing (RNAseq) data to determine the characteristics and regulatory regions of genes transcribed at the 1-cell stage. While the expression patterns of protein-coding genes of mouse embryos were very different at the 1-cell stage than at other stages and in various tissues, an analysis for the upstream and downstream regions of actively expressed genes did not reveal any elements that were specific to 1-cell stage embryos. Therefore, the unique gene expression pattern observed at the 1-cell stage in mouse embryos appears to be governed by mechanisms independent of a specific promoter element.

Automated selection of regions of interest for intensity-based FRET analysis of transferrin endocytic trafficking in normal vs. cancer cells.

The overexpression of certain membrane-bound receptors is a hallmark of cancer progression and it has been suggested to affect the organization, activation, recycling and down-regulation of receptor-ligand complexes in human cancer cells. Thus, comparing receptor trafficking pathways in normal vs. cancer cells requires the ability to image cells expressing dramatically different receptor expression levels. Here, we have presented a significant technical advance to the analysis and processing of images collected using intensity based Förster resonance energy transfer (FRET) confocal microscopy. An automated Image J macro was developed to select region of interests (ROI) based on intensity and statistical-based thresholds within cellular images with reduced FRET signal. Furthermore, SSMD (strictly standardized mean differences), a statistical signal-to-noise ratio (SNR) evaluation parameter, was used to validate the quality of FRET analysis, in particular of ROI database selection. The Image J ROI selection macro together with SSMD as an evaluation parameter of SNR levels, were used to investigate the endocytic recycling of Tfn-TFR complexes at nanometer range resolution in human normal vs. breast cancer cells expressing significantly different levels of endogenous TFR. Here, the FRET-based assay demonstrates that Tfn-TFR complexes in normal epithelial vs. breast cancer cells show a significantly different E% behavior during their endocytic recycling pathway. Since E% is a relative measure of distance, we propose that these changes in E% levels represent conformational changes in Tfn-TFR complexes during endocytic pathway. Thus, our results indicate that Tfn-TFR complexes undergo different conformational changes in normal vs. cancer cells, indicating that the organization of Tfn-TFR complexes at the nanometer range is significantly altered during the endocytic recycling pathway in cancer cells. In summary, improvements in the automated selection of FRET ROI datasets allowed us to detect significant changes in E% with potential biological significance in human normal vs. cancer cells.

Platelet neuropeptide Y is critical for ischemic revascularization in mice.

We previously reported that the sympathetic neurotransmitter neuropeptide Y (NPY) is potently angiogenic, primarily through its Y2 receptor, and that endogenous NPY is crucial for capillary angiogenesis in rodent hindlimb ischemia. Here we sought to identify the source of NPY responsible for revascularization and its mechanisms of action. At d 3, NPY(-/-) mice demonstrated delayed recovery of blood flow and limb function, consistent with impaired collateral conductance, while ischemic capillary angiogenesis was reduced (~70%) at d 14. This biphasic temporal response was confirmed by 2 peaks of NPY activation in rats: a transient early increase in neuronally derived plasma NPY and increase in platelet NPY during late-phase recovery. Compared to NPY-null platelets, collagen-activated NPY-rich platelets were more mitogenic (~2-fold vs. ~1.6-fold increase) for human microvascular endothelial cells, and Y2/Y5 receptor antagonists ablated this difference in proliferation. In NPY(+/+) mice, ischemic angiogenesis was prevented by platelet depletion and then restored by transfusion of platelets from NPY(+/+) mice, but not NPY(-/-) mice. In thrombocytopenic NPY(-/-) mice, transfusion of wild-type platelets fully restored ischemia-induced angiogenesis. These findings suggest that neuronally derived NPY accelerates the early response to femoral artery ligation by promoting collateral conductance, while platelet-derived NPY is critical for sustained capillary angiogenesis.

Active wide-field illumination for high-throughput fluorescence lifetime imaging.

Wide-field fluorescence lifetime imaging allows for fast imaging of large sample areas at the cost of low sensitivity to weak fluorescence signals. To overcome this challenge, we developed an active wide-field illumination (AWFI) strategy to optimize the impinging spatial intensity for acquiring optimal fluorescence signals over the whole sample. We demonstrated the ability of AWFI to accurately estimate lifetimes from a multiwell plate sample with concentrations ranging over two orders of magnitude. We further reported its successful application to a quantitative Förster resonance energy transfer lifetime cell-based assay. Overall, this method allows for enhanced accuracy in lifetime-based imaging at high acquisition speed over samples with large fluorescence intensity distributions.

Role of TRP channels in the regulation of the endosomal pathway.

Some members of the transient receptor potential (TRP) channel superfamily have proved to be essential in maintaining adequate ion homeostasis, signaling, and membrane trafficking in the endosomal pathway. The unique properties of the TRP channels confer cells the ability to integrate cytosolic and intraluminal stimuli and allow maintained and regulated release of Ca(2+) from endosomes and lysosomes.

Effect of body posture on stability and balance of occlusal contacts.

OBJECTIVE: To evaluate the effect of body posture on occlusal contact. METHODS: A total of 30 healthy subjects were evaluated. T-Scan™ III was used to analyze the center of occlusal force (COF) and occlusal force distribution while subjects remained supine (SP), upright sitting with the head fixed (UP-HFI), upright sitting with the head free (UP-HFR), and natural standing (NS). RESULTS: The total trajectory length of COF was significantly longer in NS than in SP, UP-HFI, and UP-HFR. The COF area was significantly larger in UP-HFR than in SP and UP-HFI and also significantly larger in NS than in SP, UP-HFI, and UP-HFR. The anteroposterior occlusal force distribution (AOD) in NS shifted significantly forward, compared to SP, UP-HFI, and UP-HFR. AOD in UP-HFI and UP-HFR shifted significantly forward, compared to the SP position. CONCLUSION: Changes in body posture affect the stability and anteroposterior balance of occlusal contacts.

Anti-ulcer agent teprenone inhibits hepatitis C virus replication: potential treatment for hepatitis C.

BACKGROUND: Previously we reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, inhibited hepatitis C virus (HCV) RNA replication. Furthermore, recent reports revealed that the statins are associated with a reduced risk of hepatocellular carcinoma and lower portal pressure in patients with cirrhosis. The statins exhibited anti-HCV activity by inhibiting geranylgeranylation of host proteins essential for HCV RNA replication. Geranylgeranyl pyrophosphate (GGPP) is a substrate for geranylgeranyltransferase. Therefore, we examined the potential of geranyl compounds with chemical structures similar to those of GGPP to inhibit HCV RNA replication. METHODS: We tested geranyl compounds [geranylgeraniol, geranylgeranoic acid, vitamin K(2) and teprenone (Selbex)] for their effects on HCV RNA replication using genome-length HCV RNA-replicating cells (the OR6 assay system) and a JFH-1 infection cell culture system. Teprenone is the major component of the anti-ulcer agent, Selbex. We also examined the anti-HCV activities of the geranyl compounds in combination with interferon (IFN)-α or statins. RESULTS: Among the geranyl compounds tested, only teprenone exhibited anti-HCV activity at a clinically achievable concentration. However, other anti-ulcer agents tested had no inhibitory effect on HCV RNA replication. The combination of teprenone and IFN-α exhibited a strong inhibitory effect on HCV RNA replication. Although teprenone alone did not inhibit geranylgeranylation, surprisingly, statins' inhibitory action against geranylgeranylation was enhanced by cotreatment with teprenone. CONCLUSIONS: The anti-ulcer agent teprenone inhibited HCV RNA replication and enhanced statins' inhibitory action against geranylgeranylation. This newly discovered function of teprenone may improve the treatment of HCV-associated liver diseases as an adjuvant to statins.

General anesthetics activate a nociceptive ion channel to enhance pain and inflammation.

General anesthetics (GAs) have transformed surgery through their actions to depress the central nervous system and blunt the perception of surgical insults. Counterintuitively, many of these agents activate peripheral nociceptive neurons. However, the underlying mechanisms and significance of these effects have not been explored. Here, we show that clinical concentrations of noxious i.v. and inhalation GAs excite sensory neurons by selectively activating TRPA1, a key ion channel in the pain pathway. Further, these GAs induce pain-related responses in mice that are abolished in TRPA1-null animals. Significantly, TRPA1-dependent neurogenic inflammation is greater in mice anesthetized with pungent compared with nonpungent anesthetics. Thus, our results show that TRPA1 is essential for sensing noxious GAs. The pronociceptive effects of GAs combined with surgical tissue damage could lead to a paradoxical increase in postoperative pain and inflammation.

Oxidative stress induces anti-hepatitis C virus status via the activation of extracellular signal-regulated kinase.

UNLABELLED: Recently, we reported that beta-carotene, vitamin D(2), and linoleic acid inhibited hepatitis C virus (HCV) RNA replication in hepatoma cells. Interestingly, in the course of the study, we found that the antioxidant vitamin E negated the anti-HCV activities of these nutrients. These results suggest that the oxidative stress caused by the three nutrients is involved in their anti-HCV activities. However, the molecular mechanism by which oxidative stress induces anti-HCV status remains unknown. Oxidative stress is also known to activate extracellular signal-regulated kinase (ERK). Therefore, we hypothesized that oxidative stress induces anti-HCV status via the mitogen activated protein kinase (MAPK)/ERK kinase (MEK)-ERK1/2 signaling pathway. In this study, we found that the MEK1/2-specific inhibitor U0126 abolished the anti-HCV activities of the three nutrients in a dose-dependent manner. Moreover, U0126 significantly attenuated the anti-HCV activities of polyunsaturated fatty acids, interferon-gamma, and cyclosporine A, but not statins. We further demonstrated that, with the exception of the statins, all of these anti-HCV nutrients and reagents actually induced activation of the MEK-ERK1/2 signaling pathway, which was inhibited or reduced by treatment not only with U0126 but also with vitamin E. We also demonstrated that phosphorylation of ERK1/2 by cyclosporine A was attenuated with N-acetylcysteine treatment and led to the negation of inhibition of HCV RNA replication. We propose that a cellular process that follows ERK1/2 phosphorylation and is specific to oxidative stimulation might lead to down-regulation of HCV RNA replication. CONCLUSION: Our results demonstrate the involvement of the MEK-ERK1/2 signaling pathway in the anti-HCV status induced by oxidative stress in a broad range of anti-HCV reagents. This intracellular modulation is expected to be a therapeutic target for the suppression of HCV RNA replication.

Excitatory and inhibitory synapses in neuropeptide Y-expressing striatal interneurons.

Although rare, interneurons are pivotal in governing striatal output by extensive axonal arborizations synapsing on medium spiny neurons. Using a genetically modified mouse strain in which a green fluorescent protein (GFP) is driven to be expressed under control of the neuropeptide Y (NPY) promoter, we identified NPY interneurons and compared them with striatal principal neurons. We found that the bacteria artificial chromosome (BAC)-npy mouse expresses GFP with high fidelity in the striatum to the endogenous expression of NPY. Patch-clamp analysis from NPY neurons showed a heterogeneous population of striatal interneurons. In the majority of cells, we observed spontaneous firing of action potentials in extracellular recordings. On membrane rupture, most NPY interneurons could be classified as low-threshold spiking interneurons and had high-input resistance. Voltage-clamp recordings showed that both GABA and glutamate gated ion channels mediate synaptic inputs onto these striatal interneurons. AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) were small in amplitude and infrequent in NPY neurons. Evoked EPSCs did not show short-term plasticity but some rectification. Evoked N-methyl-d-aspartate (NMDA) EPSCs had fast decay kinetics and were poorly sensitive to an NR2B subunit containing NMDA receptor blocker. Spontaneous inhibitory postsynaptic currents (sIPSCs) were mediated by GABA(A) receptors and were quite similar among all striatal neurons studied. On the contrary, evoked IPSCs decayed faster in NPY neurons than in other striatal neurons. These data report for the first time specific properties of synaptic transmission to NPY striatal interneurons.

The DNA damage sensors ataxia-telangiectasia mutated kinase and checkpoint kinase 2 are required for hepatitis C virus RNA replication.

Cellular responses to DNA damage are crucial for maintaining genome integrity, virus infection, and preventing the development of cancer. Hepatitis C virus (HCV) infection and the expression of the HCV nonstructural protein NS3 and core protein have been proposed as factors involved in the induction of double-stranded DNA breaks and enhancement of the mutation frequency of cellular genes. Since DNA damage sensors, such as the ataxia-telangiectasia mutated kinase (ATM), ATM- and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2), play central roles in the response to genotoxic stress, we hypothesized that these sensors might affect HCV replication. To test this hypothesis, we examined the level of HCV RNA in HuH-7-derived cells stably expressing short hairpin RNA targeted to ATM, ATR, PARP-1, or Chk2. Consequently, we found that replication of both genome-length HCV RNA (HCV-O, genotype 1b) and the subgenomic replicon RNA were notably suppressed in ATM- or Chk2-knockdown cells. In addition, the RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were suppressed in these knockdown cells after inoculation of the cell culture-generated HCV. Consistent with these observations, ATM kinase inhibitor could suppress the HCV RNA replication. Furthermore, we observed that HCV NS3-NS4A interacted with ATM and that HCV NS5B interacted with both ATM and Chk2. Taken together, these results suggest that the ATM signaling pathway is critical for HCV RNA replication and may represent a novel target for the clinical treatment of patients with chronic hepatitis C.

HCV genotype 1b chimeric replicon with NS5B of JFH-1 exhibited resistance to cyclosporine A.

Cyclosporine A (CsA) is a well-characterized anti-HCV reagent. Recently it was reported that the genotype 2a JFH-1 strain was more resistant than genotype 1 HCV strains to CsA in a cell culture system. However, the JFH-1 responsible region for the resistance to CsA remains unclear. It was also demonstrated that in genotype 1b HCVs, NS5B interacts with cyclophilin (CyP). To clarify whether or not NS5B of JFH-1 is significant for CsA resistance, we developed a chimeric replicon with NS5B of JFH-1 in the genotype 1b backbone. The chimeric replicon was more resistant to CsA than the parental genotype 1b replicon. Furthermore, reduction of CyPA had a greater effect on HCV RNA replication and sensitivity to CsA than reduction of CyPB. Here, we demonstrated that NS5B of JFH-1 contributed to this strain's CsA-resistant phenotype. NS5B and CyPA are significant for determining HCV's sensitivity to CsA.

Development of a hepatitis C virus relapse model using genome-length hepatitis C virus ribonucleic acid-harboring cells possessing the interferon-alpha-resistance phenotype.

AIM: The cure rate of current interferon (IFN) therapy is limited to approximately 50% and most of the relapses after therapy are caused by genotype-1. To develop a relapse model in cell culture, we attempted to obtain genome-length hepatitis C virus ribonucleic acid (HCV RNA) harboring cells possessing the IFN-alpha-resistance phenotype from previously established OR6 cells, which enabled the luciferase reporter assay for monitoring of HCV RNA replication. METHODS: The IFN-alpha-resistant HCV RNA-harboring cells and control cells were obtained by the treatment of OR6 cells with and without IFN-alpha, respectively. Then, we examined the relapse of HCV in IFN-alpha-resistant HCV RNA-harboring cells. RESULTS: Only type I IFN (alpha and beta) showed significantly different anti-HCV activity between IFN-alpha-resistant HCV RNA-harboring cells and control cells. There was no significant difference in the anti-HCV activity of IFN-gamma, fluvastatin, or cyclosporine A between the two types of cells. Furthermore, we showed that fluvastatin or cyclosporine A in combination with IFN-alpha could prevent the relapse after therapy in the IFN-alpha-resistant HCV RNA-harboring cells. CONCLUSION: We developed a HCV relapse model in cell culture using IFN-alpha-resistant HCV RNA-harboring cells. Thus anti-HCV reagents, which have a mechanism different from IFN-alpha, were shown to be useful for preventing a relapse of IFN-alpha-resistant HCV.

New efficient replication system with hepatitis C virus genome derived from a patient with acute hepatitis C.

We report for the first time a new RNA replication system with a hepatitis C virus (HCV) strain (AH1) derived from a patient with acute hepatitis C. Using an HCV replicon RNA library constructed with the AH1 strain (genotype 1b), we first established a cloned cell line, sAH1, harboring the HCV replicon. Cured cells obtained with interferon treatment of sAH1 cells were used for transfection with genome-length HCV RNA possessing four mutations found in sAH1 replicon. Consequently, one cloned cell line, AH1, supporting efficient replication of genome-length HCV RNA was obtained. By the comparison of AH1 cells with the O cells supporting genome-length HCV RNA (HCV-O strain) replication, we found different anti-HCV profiles of interferon-gamma and cyclosporine A between AH1 and O cells. Reporter assay analysis suggests that the diverse effects of interferon-gamma are due to the difference in HCV strains, but not the cellular environment.

DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication.

DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.

A new living cell-based assay system for monitoring genome-length hepatitis C virus RNA replication.

We previously developed a cell-based luciferase reporter assay system for monitoring genome-length hepatitis C virus (HCV) RNA replication (OR6 assay system). Here, we aimed to develop a new living cell-based reporter assay system using enhanced green fluorescent protein (EGFP). Genome-length HCV RNAs encoding EGFP were introduced into a subline of HuH-7 cells and G418 selection was performed. One cloned cell line, OGF7, was successfully selected from among the several G418-resistant cell lines obtained, and the robust expression of HCV RNA and proteins in OGF7 cells was confirmed. The fluorescent intensity of OGF7 cells was decreased by interferon-alpha treatment in a dose-dependent manner, and it correlated well with the HCV RNA concentration. We demonstrated that the interferon-alpha sensitivity in the OGF7 assay system measuring the fluorescent intensity was equivalent to that of the OR6 assay system, and that the OGF7 assay system was useful for quantitative evaluation of anti-HCV reagents. The OGF7 assay system is expected to be the most time-saving and inexpensive assay system for high-throughput screening of anti-HCV reagents.

Cell culture-adaptive NS3 mutations required for the robust replication of genome-length hepatitis C virus RNA.

We recently established a genome-length HCV RNA-replicating cell line (O strain of genotype 1b; here called O cells) using cured cells derived from sO cells, in which HCV subgenomic replicon RNA with an adaptive NS5A mutation (S2200R) is replicated. Characterization of the O cells revealed a second adaptive NS3 mutation (K1609E) required for genome-length HCV RNA replication. To clarify the role of adaptive mutation in genome-length HCV RNA replication, we newly established one and three kinds of genome-length HCV RNA-replicating cell lines possessing the cell background of sO and O cells, respectively, and found additional adaptive NS3 mutations (Q1112R, P1115L, and E1202G) required for the robust replication of genome-length HCV RNA. We further found that specific combinations of adaptive NS3 mutations drastically enhanced HCV RNA replication, regardless of the cell lines examined. These findings suggest that specific viral factors may affect the replication level of genome-length HCV RNA.