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1.
Cranio ; : 1-11, 2022 Sep 13.
Article in English | MEDLINE | ID: mdl-36101939

ABSTRACT

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.

2.
Reproduction ; 164(2): 19-29, 2022 08 01.
Article in English | MEDLINE | ID: mdl-35666814

ABSTRACT

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.


Subject(s)
Histones , Oogenesis , Chromatin/genetics , Chromatin/metabolism , Histones/metabolism , Oocytes/metabolism , Oogenesis/genetics
3.
Proc Natl Acad Sci U S A ; 115(29): E6780-E6788, 2018 07 17.
Article in English | MEDLINE | ID: mdl-29967139

ABSTRACT

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.


Subject(s)
Blastocyst/metabolism , Embryonic Development/physiology , Gene Expression Regulation, Developmental/physiology , Zygote/metabolism , Animals , Blastocyst/cytology , Dichlororibofuranosylbenzimidazole/pharmacology , Embryonic Development/drug effects , Gene Expression Regulation, Developmental/drug effects , Histones/metabolism , Mice , Zygote/cytology
4.
J Reprod Dev ; 62(1): 87-92, 2016.
Article in English | MEDLINE | ID: mdl-26599803

ABSTRACT

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.


Subject(s)
Blastocyst/cytology , Gene Expression Regulation, Developmental , Animals , Cluster Analysis , CpG Islands , Embryo, Mammalian/metabolism , Embryonic Development , Female , Gene Expression Profiling , Mice , Oocytes/cytology , Phenotype , Phylogeny , Placenta/metabolism , Pregnancy , Promoter Regions, Genetic , RNA, Messenger/metabolism , Regulatory Sequences, Nucleic Acid , Sequence Analysis, RNA , Transcription, Genetic , Transcriptome , Zygote/metabolism
5.
EMBO J ; 34(11): 1523-37, 2015 Jun 03.
Article in English | MEDLINE | ID: mdl-25896510

ABSTRACT

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.


Subject(s)
3' Untranslated Regions/physiology , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental/physiology , RNA Splicing/physiology , Transcription, Genetic/physiology , Zygote/metabolism , Animals , Chromatin/metabolism , Chromatin Assembly and Disassembly/physiology , Embryo, Mammalian/cytology , Mice , Zygote/cytology
6.
Regen Ther ; 2: 32-41, 2015 Dec.
Article in English | MEDLINE | ID: mdl-31245457

ABSTRACT

Human mesenchymal stem cells (MSCs) are expected to have utility as a cell source in regenerative medicine. Because we previously reported that suppression of the Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we synthesized twenty-three derivatives of small molecule compounds originally reported to suppress the Wnt/ß-catenin signal in human colorectal cancer cells. We then screened these compounds for their ability to induce hepatic differentiation of human UE7T-13 MSCs. After screening using WST assay, TCF reporter assay, and albumin mRNA expression, IC-2, a derivative of ICG-001, was identified as a potent inducer of hepatic differentiation of human MSCs. IC-2 potently induced the expression of albumin, complement C3, tryptophan 2,3-dioxygenase (TDO2), EpCAM, C/EBPα, glycogen storage, and urea production. Furthermore, we examined the effects of IC-2 on human bone marrow mononuclear cell fractions sorted according to CD90 and CD271 expression. Consequently, CD90+ CD271+ cells were found to induce the highest production of urea and glycogen, important hepatocyte functions, in response to IC-2 treatment. CD90+ CD271+ cells also highly expressed albumin mRNA. As the CD90+ CD271+ population has been reported to contain a rich fraction of MSCs, IC-2 apparently represents a potent inducer of hepatic differentiation of human MSCs.

7.
Biomed Opt Express ; 5(3): 944-60, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24688826

ABSTRACT

Fluorescence lifetime imaging is playing an increasing role in drug development by providing a sensitive method to monitor drug delivery and receptor-ligand interactions. However, the wide dynamic range of fluorescence intensity emitted by ex vivo and in vivo samples presents challenges in retrieving information over the whole subject accurately and quantitatively. To overcome this challenge, we developed an active wide-field illumination (AWFI) strategy based on a spatial light modulator that acquires optimal fluorescence signals by enhancing the dynamic range, signal to noise ratio, and estimation of lifetime-based parameters. We demonstrate the ability of AWFI to estimate Förster resonance energy transfer (FRET) donor fraction from dissected organs with high accuracy (standard deviation <6%) over the whole field of view, in contrast with the homogenous wide-field illumination. We further report its successful application to quantitative FRET imaging in a live mouse. AWFI allows improved detection of weak signals and enhanced quantitative accuracy in ex vivo and in vivo molecular fluorescence quantitative imaging. The technique allows for robust quantitative estimation of the bio-distribution of molecular probes and lifetime-based parameters over an extended imaging field exhibiting a large range of fluorescence intensities and at a high acquisition speed (less than 1 min).

8.
Methods ; 66(2): 139-52, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-23994873

ABSTRACT

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.


Subject(s)
Endocytosis , Image Processing, Computer-Assisted , Transferrins/metabolism , Animals , Cell Line, Tumor , Dogs , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Humans , Madin Darby Canine Kidney Cells , Microscopy, Fluorescence , Protein Transport , Software
9.
PLoS One ; 8(11): e80269, 2013.
Article in English | MEDLINE | ID: mdl-24278268

ABSTRACT

The conjugation of anti-cancer drugs to endogenous ligands has proven to be an effective strategy to enhance their pharmacological selectivity and delivery towards neoplasic tissues. Since cell proliferation has a strong requirement for iron, cancer cells express high levels of transferrin receptors (TfnR), making its ligand, transferrin (Tfn), of great interest as a delivery agent for therapeutics. However, a critical gap exists in the ability to non-invasively determine whether drugs conjugated to Tfn are internalized into target cells in vivo. Due to the enhanced permeability and retention (EPR) effect, it remains unknown whether these Tfn-conjugated drugs are specifically internalized into cancer cells or are localized non-specifically as a result of a generalized accumulation of macromolecules near tumors. By exploiting the dimeric nature of the TfnR that binds two molecules of Tfn in close proximity, we utilized a Förster Resonance Energy Transfer (FRET) based technique that can discriminate bound and internalized Tfn from free, soluble Tfn. In order to non-invasively visualize intracellular amounts of Tfn in tumors through live animal tissues, we developed a novel near infrared (NIR) fluorescence lifetime FRET imaging technique that uses an active wide-field time gated illumination platform. In summary, we report that the NIR fluorescence lifetime FRET technique is capable of non-invasively detecting bound and internalized forms of Tfn in cancer cells and tumors within a live small animal model, and that our results are quantitatively consistent when compared to well-established intensity-based FRET microscopy methods used in in vitro experiments.


Subject(s)
Breast Neoplasms/diagnosis , Fluorescence Resonance Energy Transfer/methods , Spectroscopy, Near-Infrared/methods , Transferrin/metabolism , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude
10.
Opt Lett ; 38(19): 3976-9, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-24081103

ABSTRACT

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.


Subject(s)
Light , Optical Imaging/methods , Algorithms , Time Factors
11.
FASEB J ; 27(6): 2244-55, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23457218

ABSTRACT

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.


Subject(s)
Blood Platelets/metabolism , Ischemia/blood , Neovascularization, Physiologic , Neuropeptide Y/physiology , Animals , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Endothelial Cells/pathology , Hindlimb , Humans , Ischemia/genetics , Ischemia/physiopathology , Male , Mice , Mice, 129 Strain , Mice, Knockout , Neovascularization, Physiologic/genetics , Neuropeptide Y/deficiency , Neuropeptide Y/genetics , Rats , Rats, Wistar
12.
Dis Model Mech ; 6(2): 323-31, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23324329

ABSTRACT

The primary purpose of this investigation was to determine whether ApoE(-/-) mice, when subjected to chronic stress, exhibit lesions characteristic of human vulnerable plaque and, if so, to determine the time course of such changes. We found that the lesions were remarkably similar to human vulnerable plaque, and that the time course of lesion progression raised interesting insights into the process of plaque development. Lard-fed mixed-background ApoE(-/-) mice exposed to chronic stress develop lesions with large necrotic core, thin fibrous cap and a high degree of inflammation. Neovascularization and intraplaque hemorrhage are observed in over 80% of stressed animals at 20 weeks of age. Previously described models report a prevalence of only 13% for neovascularization observed at a much later time point, between 36 and 60 weeks of age. Thus, our new stress-induced model of advanced atherosclerotic plaque provides an improvement over what is currently available. This model offers a tool to further investigate progression of plaque phenotype to a more vulnerable phenotype in humans. Our findings also suggest a possible use of this stress-induced model to determine whether therapeutic interventions have effects not only on plaque burden, but also, and importantly, on plaque vulnerability.


Subject(s)
Atherosclerosis/etiology , Atherosclerosis/pathology , Disease Models, Animal , Plaque, Atherosclerotic/etiology , Plaque, Atherosclerotic/pathology , Stress, Psychological/complications , Animals , Atherosclerosis/blood , Atherosclerosis/complications , Blood Pressure , Cholesterol/blood , Coronary Stenosis/complications , Coronary Stenosis/pathology , Corticosterone/blood , Hemorrhage/complications , Hemorrhage/pathology , Humans , Inflammation/blood , Inflammation/complications , Inflammation/pathology , Mice , Mice, Inbred C57BL , Necrosis , Neovascularization, Pathologic/complications , Neovascularization, Pathologic/pathology , Neuropeptide Y/blood , Plaque, Atherosclerotic/complications , Stress, Psychological/blood
13.
Science ; 333(6046): 1116-9, 2011 Aug 26.
Article in English | MEDLINE | ID: mdl-21868668

ABSTRACT

Meteorite studies suggest that each solar system object has a unique oxygen isotopic composition. Chondrites, the most primitive of meteorites, have been believed to be derived from asteroids, but oxygen isotopic compositions of asteroids themselves have not been established. We measured, using secondary ion mass spectrometry, oxygen isotopic compositions of rock particles from asteroid 25143 Itokawa returned by the Hayabusa spacecraft. Compositions of the particles are depleted in (16)O relative to terrestrial materials and indicate that Itokawa, an S-type asteroid, is one of the sources of the LL or L group of equilibrated ordinary chondrites. This is a direct oxygen-isotope link between chondrites and their parent asteroid.

14.
Liver Int ; 31(6): 871-80, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21645219

ABSTRACT

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.


Subject(s)
Anti-Ulcer Agents/pharmacology , Antiviral Agents/pharmacology , Diterpenes/pharmacology , Hepacivirus/drug effects , Hepatocytes/drug effects , RNA, Viral/biosynthesis , Virus Replication/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Synergism , Genes, Reporter , Hepacivirus/genetics , Hepacivirus/metabolism , Hepatocytes/metabolism , Hepatocytes/virology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Interferon-gamma/pharmacology , Prenylation , Protein Processing, Post-Translational/drug effects , Time Factors , Transfection , Viral Proteins/metabolism
15.
Physiology (Bethesda) ; 26(1): 14-22, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21357899

ABSTRACT

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.


Subject(s)
Endosomes/metabolism , Transient Receptor Potential Channels/metabolism , Amino Acid Sequence , Animals , Calcium/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Protein Transport , Signal Transduction
16.
Exp Biol Med (Maywood) ; 235(10): 1179-84, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20881322

ABSTRACT

Neuropeptide Y (NPY) is a central neuromodulator and peripheral sympathetic neurotransmitter that also has important regulatory roles in cardiovascular, neuroendocrine, immune and metabolic functions during stress. Focusing on the peripheral actions of the peptide in rodent models, we summarize recent studies from our laboratory demonstrating that stress-induced release of NPY mediates accelerated atherosclerosis/restenosis, obesity and metabolic-like syndrome, particularly when combined with a high fat, high sugar diet. In this review, we propose mechanisms of NPY's actions, its receptors and cellular substrates that increase the risk for cardiovascular and metabolic diseases when chronic stress is associated with pre-existing vascular injury and/or states of hypernutrition.


Subject(s)
Neuropeptide Y/physiology , Overnutrition/physiopathology , Stress, Physiological , Animals , Atherosclerosis/etiology , Cardiovascular Diseases/etiology , Humans , Metabolic Diseases/etiology , Models, Biological , Obesity/etiology
17.
J Reprod Dev ; 56(5): 502-7, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20562521

ABSTRACT

As mouse oocytes approach maturity, a global repression of gene transcription occurs. Here, we investigated the involvement of RPB1, the largest subunit of RNA polymerase II (RNAP II), in the regulation of this transcriptional silencing mechanism. Using BrUTP to follow transcription in an in vitro run-on assay, we observed an abrupt decrease in transcriptional activity when oocytes reached their full size (approximately 80 µm). Immunoblotting using antibodies specific for the phosphorylated and unphosphorylated forms of RPB1 revealed that RPB1 is phosphorylated at Ser-2 and Ser-5 in the small growing oocytes in which active transcription occurs. By contrast, in transcriptionally inactive, full-grown oocytes, RPB1 is predominantly unphosphorylated. When we permeabilized the nuclear membrane using Triton X-100 during fixation for immunocytochemistry, the unphosphorylated form of RPB1 diffused out of the nucleus in the full-grown oocytes but still remained there in the small growing oocytes, indicating that RPB1 is not bound to DNA in full-grown oocytes. These results suggest that the immediate cause of global transcriptional silencing is the dissociation of RNAP II from the DNA. We also observed dissociation of RPB1 from the DNA in full-grown oocytes treated with trichostatin A to decondense their chromatin, suggesting that chromatin condensation is not an essential process in gene silencing during oocyte growth.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Gene Silencing/physiology , Oocytes/physiology , RNA Polymerase II/genetics , Animals , Chromatin/genetics , Mice , Mice, Inbred Strains , NIH 3T3 Cells , Oocytes/growth & development , Phosphorylation/physiology , Protein Subunits/genetics , Protein Subunits/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic/physiology
18.
Virus Res ; 146(1-2): 41-50, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19720094

ABSTRACT

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a serious global health problem. Cell culture-based persistent HCV RNA replication systems and infectious HCV production systems are widely used in HCV research. However, persistent HCV production systems have been developed only for HuH-7 hepatoma cells. Here we found a new human hepatoma cell line, Li23, that enables persistent HCV production and anti-HCV reagent assay. Li23's cDNA expression profile differed from HuH-7's, although the two cells had similar liver-specific expression profiles. We used HCV RNA with a specific combination of adaptive mutations to develop an HCV replicon system and genome-length HCV RNA replicating systems including a reporter assay system. Finally, Li23-derived cells persistently produced infectious virus of an HCV strain. Li23-derived cells are potentially useful for understanding the HCV life cycle and for finding antiviral targets.


Subject(s)
Cell Culture Techniques/methods , Hepacivirus/growth & development , Hepatocytes/virology , Cell Line, Tumor , Hepacivirus/drug effects , Humans , Male , Middle Aged
19.
J Neurophysiol ; 102(5): 3038-45, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19759327

ABSTRACT

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.


Subject(s)
Corpus Striatum/cytology , Interneurons/physiology , Neural Inhibition/physiology , Neuropeptide Y/metabolism , Synapses/physiology , Synaptic Potentials/physiology , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Biophysical Phenomena/drug effects , Biophysical Phenomena/physiology , Biophysics , Choline O-Acetyltransferase/metabolism , Electric Stimulation , Excitatory Amino Acid Antagonists/pharmacology , GABA Antagonists/pharmacology , Green Fluorescent Proteins/genetics , Mice , Mice, Transgenic , Neural Inhibition/drug effects , Neuropeptide Y/genetics , Organophosphonates/pharmacology , Parvalbumins/metabolism , Patch-Clamp Techniques/methods , Piperazines/pharmacology , Quinoxalines/pharmacology , Sodium Channel Blockers/pharmacology , Synapses/drug effects , Synaptic Potentials/drug effects , Tetrodotoxin/pharmacology
20.
Arch Virol ; 154(10): 1671-7, 2009.
Article in English | MEDLINE | ID: mdl-19779801

ABSTRACT

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.


Subject(s)
Cyclosporine/pharmacology , Hepacivirus/drug effects , Replicon/genetics , Blotting, Western , Cell Line , Drug Resistance, Viral/genetics , Genotype , Hepacivirus/genetics , Humans , Immunoprecipitation , Phenotype , RNA, Viral/biosynthesis , Recombinant Fusion Proteins/genetics , Virus Replication/drug effects , Virus Replication/genetics
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