Interleukin-1ß induces tumor necrosis factor-α secretion from rat hepatocytes.

AIM: Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine involved in various inflammatory diseases. The only production of TNF-α in the liver is thought to be from hepatic macrophages known as Kupffer cells, predominantly in response to bacterial lipopolysaccharide (LPS). METHODS: Primary cultured rat hepatocytes were used to analyze TNF-α expression in response to the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). Livers of rats subjected to LPS-induced endotoxemia were analyzed. RESULTS: Immunocytochemistry and enzyme-linked immunosorbent assays demonstrated that IL-1ß-treated rat hepatocytes secreted TNF-α, and RNA analyses indicated that TNF-α mRNA was induced specifically by IL-1ß. Northern blot analysis showed that not only mRNA, but also a natural antisense transcript (asRNA), was transcribed from the rat Tnf gene in IL-1ß-treated hepatocytes. TNF-α was detected in the hepatocytes of LPS-treated rats. Both TNF-α mRNA and asRNA were expressed in the hepatocytes of LPS-treated rats, human hepatocellular carcinoma and human monocyte/macrophage cells. To disrupt the interaction between TNF-α asRNA and TNF-α mRNA, sense oligonucleotides corresponding to TNF-α mRNA were introduced into rat hepatocytes resulting in significantly increased levels of TNF-α mRNA. One of these sense oligonucleotides increased a half-life of TNF-α mRNA, suggesting that the TNF-α asRNA may reduce the stability of TNF-α mRNA. CONCLUSION: IL-1ß-stimulated rat hepatocytes are a newly identified source of TNF-α in the liver. TNF-α mRNA and asRNA are expressed in rats and humans, and the TNF-α asRNA reduces the stability of the TNF-α mRNA. Hepatocytes and TNF-α asRNA may be therapeutic targets to regulate levels of TNF-α mRNA.

Rate control of cell sheet recovery by incorporating hydrophilic pattern in thermoresponsive cell culture dish.

Thready stripe-polyacrylamide (PAAm) pattern was fabricated on a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) surface, and their surface properties were characterized. A PIPAAm surface spin-coated with positive photoresist was irradiated through a 5 µm/5 µm or a 10 µm/10-µm black and white striped photomask, resulting in the radical polymerization of AAm on the photoirradiated area. After staining with Alexa488 bovine serum albumin, the stripe-patterned surface was clearly observed and the patterned surface was also observed by a phase contrast image of an atomic force microscope. NIH-3T3 (3T3) single cells were able to be cultured at 37°C on the patterned surfaces as well as on a PIPAAm surface without pattern, and the detachment of adhered cells was more rapidly from the patterned surface after reducing temperature. Furthermore, the rate of detachment of 3T3 confluent cell sheet on the patterned surface was accelerated, compared with on a conventional PIPAAm surface under the static condition. The rate control of cell sheet recovery should contribute the preservations of cell phenotype and biological functions of cell sheet for applying to clinical trials.

Stabilization of human interferon-α1 mRNA by its antisense RNA.

Antisense transcription is a widespread phenomenon in the mammalian genome and is believed to play a role in regulating gene expression. However, the exact functional significance of antisense transcription is largely unknown. Here, we show that natural antisense (AS) RNA is an important modulator of interferon-α1 (IFN-α1) mRNA levels. A ~4-kb, spliced IFN-α1 AS RNA targets a single-stranded region within a conserved secondary structure element of the IFN-α1 mRNA, an element which was previously reported to function as the nuclear export element. Following infection of human Namalwa lymphocytes with Sendai virus or infection of guinea pig 104C1 fetal fibroblasts with influenza virus A/PR/8/34, expression of IFN-α1 AS RNA becomes elevated. This elevated expression results in increased IFN-α1 mRNA stability because of the cytoplasmic (but not nuclear) interaction of the AS RNA with the mRNA at the single-stranded region. This results in increased IFN-α protein production. The silencing of IFN-α1 AS RNA by sense oligonucleotides or over-expression of antisense oligoribonucleotides, which were both designed from the target region, confirmed the critical role of the AS RNA in the post-transcriptional regulation of IFN-α1 mRNA levels. This AS RNA stabilization effect is caused by the prevention of the microRNA (miRNA)-induced destabilization of IFN-α1 mRNA due to masking of the miR-1270 binding site. This discovery not only reveals a regulatory pathway for controlling IFN-α1 gene expression during the host innate immune response against virus infection but also suggests a reason for the large number of overlapping complementary transcripts with previously unknown function.

Imaging of light and heavy atomic columns by spherical aberration corrected middle-angle bright-field STEM.

Simultaneous detection of both light and heavy atomic columns is theoretically and experimentally explored with spherical aberration (C(s))-corrected middle-angle bright-field (MABF) scanning transmission electron microscopy (STEM). Optimized MABF STEM visualizes both light O atomic columns and heavy Sr and Ti-O atomic columns for SrTiO3(001) as distinct bright spots and dark spots with characteristic bright rings, respectively, over practical ranges of the probe-forming lens defocus and sample thickness, although medium-heavy Ti-O atomic columns appear as blurred dark spots. The difference in contrast between heavy and light atomic columns is greater than that of annular BF STEM images. The formation of distinctive bright and dark spots is interpreted simply as the difference in the degrees of localization and inelastic absorption of channeling electrons in individual atomic columns by analyses of convergent wave fields inside the crystal in both real and reciprocal space. In addition, Bloch wave expansion of MABF STEM images suggests that bright rings are formed mainly by 2p-like convergent Bloch wave fields localized on heavy atomic columns.

[Psychological effects of emotional crying in adults: events that elicit crying and social reactions to crying].

This research focused on both the psychological benefits and costs of crying. We investigated the relationships of intrapersonal and interpersonal consequences of crying. Female nurses (N = 300) were requested to describe one of the most impressive negative episodes where they had cried. Then, they were asked to complete a questionnaire including a scale of their psychological changes after the crying episode and the social reactions when they cried. Factor analysis revealed five components of the psychological changes scale. Solitary crying had greater effects for both psychological benefits and costs after crying than crying in front of others. Factor analysis revealed three components of the scale of social reactions. When they cried in front of others, "catharsis", "positive attitude", and "recognition of the relationship with others" after crying were associated with "empathy and social support" from others. The factors of "recognition of negative reality" and "negative attitude" were associated with "criticism and slander" from others. These results were discussed in terms of the communicative functions and the reflective functions of adult crying.

Nuclear and cytoplasmic effects of human CRM1 on HIV-1 production in rat cells.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced gag and singly spliced env mRNAs by bridging viral RNA and the export receptor, CRM1. Recently, rat CRM1 was found to be less efficient than human CRM1 in supporting Rev function in rats. In this study, to understand the role of CRM1 in HIV propagation, the mechanism underlying the function of human and rat CRM1 in HIV-1 replication was investigated in rat cells. The production of viral particles, represented by the p24 Gag protein, was greatly enhanced by hCRM1 expression in rat cells; however, this effect was not simply because of the enhanced export of gag mRNA. The translation initiation rate of gag mRNA was not increased, nor was the Gag protein stabilized in the presence of hCRM1. However, the processing of the p55 Gag precursor and the release of viral particles were facilitated. These results indicated that hCRM1 exports gag mRNA to the cytoplasm, not only more efficiently than rCRM1 but also correctly, leading to efficient processing of Gag proteins and particle formation.

Novel cis-active structures in the coding region mediate CRM1-dependent nuclear export of IFN-α 1 mRNA.

We recently reported the chromosome region maintenance 1 (CRM1)-dependent nuclear export of intron-less human interferon-α1 (IFN-α1) mRNA, which encodes a main effecter of host innate immunity. We show that the coding region of IFN-α1 mRNA forms novel secondary structures that are responsible for the CRM1-dependent export of the transcript. Deletion-mutagenesis, in vivo export assays, and computer analyses of the folding potentials of export-competent fragments revealed the presence of a domain, termed the conserved secondary structure (CSS), comprising two adjacent putative stable stem-loop structures (nt 208-452). Internal deletion-mutagenesis and constitutive export assays of each stem-loop structure demonstrated that subregions 308-322 and 352-434 act as a core element by conferring the export function on the CSS. Leptomycin B (LMB) inhibition of the CRM1 pathway decreased the export of core element RNA, implying that the principal site of CRM1 action for exporting IFN-α1 mRNA resides within the core element. An RNPS1 (RNA-binding protein S1, serine-rich domain) cDNA was isolated by yeast three-hybrid screening, using bait containing two CSS regions. We showed that RNPS1 might recognize IFN-α1 mRNP that includes CRM1. The data demonstrate that interaction between RNA structures in the coding region and CRM1 affects the nucleocytoplasmic translocation of IFN-α1 mRNA.

Effect of convergent beam semiangle on image intensity in HAADF STEM images.

In this study, we experimentally and theoretically show that the intensities of bright spots in a spherical aberration (C(s))-uncorrected high-angle annular dark-field (HAADF) scanning transmission electron microscope (STEM) image of [011]-oriented Co(3)O(4), which has two different numbers of Co atoms in the projected atomic columns, are reversed with increasing sample thickness. However, C(s)-corrected HAADF STEM images produce intensities that correctly depend on the average number of atoms in the projected atomic columns. From an analysis based on the Bloch-wave theorem, it is found that an insufficient semiangle of the incident convergent beam yields intensities that do not depend on the average atomic number in the atomic columns.

Effect of chromatic aberration on atomic-resolved spherical aberration corrected STEM images.

The effect of the chromatic aberration (C(c)) coefficient in a spherical aberration (C(s))- corrected electromagnetic lens on high-resolution high-angle annular dark field (HAADF) scanning transmission electron microscope (STEM) images is explored in detail. A new method for precise determination of the C(c) coefficient is demonstrated, requiring measurement of an atomic-resolution one-frame through-focal HAADF STEM image. This method is robust with respect to instrumental drift, sample thickness, all lens parameters except C(c), and experimental noise. It is also demonstrated that semi-quantitative structural analysis on the nanometer scale can be achieved by comparing experimental C(s)- corrected HAADF STEM images with their corresponding simulated images when the effects of the C(c) coefficient and spatial incoherence are included.

Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase.

Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.

Many-beam dynamical simulation for multilayer structures without a superlattice cell.

A many-beam dynamical theory for plan-view high-resolution transmission electron microscopy (HRTEM) images of multilayer systems without the limitation of a superlattice cell is proposed. The accuracy of our method is examined by comparing convergent-beam electron-diffraction calculations of Si(011) and HRTEM calculations of a system of epitaxial Al(100) on GaAs(100). Furthermore, this method is applied to CdSe clusters embedded in MgO, where it is revealed that the relative shift of their crystal-lattice planes produces moiré-like fringes.

Quantitative and easy estimation of a crystal bending effect using low-order CBED patterns.

The quantitative measurement of a crystal bending effect is performed using low-order zone-axis convergent beam electron diffraction (CBED) patterns. Although the accuracy of the present method is inferior to that of the method of using split higher order Laue zone lines, this method enables us to estimate the crystal bending effect at a region very close to the interface and to easily judge whether the crystal bending effect results in a tensile bend or a compressive bend. As an application of the present method, the crystal bending effect at a region close to the SiGe/Si interface was measured. It was found that the crystal bending effect is due to a thin-foil relaxation of almost 0.3 degrees at a region that is approximately 10 nm away from the interface.

Quantitative structural analysis of twin boundary in alpha-Zn7Sb2O12 using HAADF STEM method.

The structure and composition of the 1/4{110} twin boundary in alpha-Zn7Sb2O12 have been determined by using quantitative high-angle annular dark field scanning transmission electron microscopy (HAADF STEM) analysis. The noise in the experimental HAADF STEM images is reduced by using the maximum entropy method and average processing, and the parameters used in dynamical simulations are experimentally determined. From the analysis, it has been found that octahedral sites in the twin boundary slightly shift parallel to the [110] direction, and a reduction of the Sb concentration at the octahedral sites on the plane adjacent to the twin boundary was detected. The reduction was measured from three regions in the same twin boundary, and the Sb concentrations were 4 +/- 3, 8 +/- 3 and 19 +/-2 at% from 33 at%.

Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes.

UNLABELLED: During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1beta (IL-1beta) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3'-untranslated region (3'UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5-kilobase region 3'-flanking to iNOS gene exon 27 was involved in IL-1beta induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3'UTR. These results together with the data in a yeast RNA-hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The iNOS mRNA colocalized with the AU-rich element-binding protein HuR, a human homolog of embryonic lethal-abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA-binding proteins may mutually interact. CONCLUSION: The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA-RNA interaction may be a new therapeutic target for NO-mediating inflammatory diseases.

Measurement of twofold astigmatism of probe-forming lens using low-order zone-axis ronchigram.

By using a low-order zone-axis ronchigram of a crystalline sample, a simple method for measuring twofold astigmatism of a probe-forming lens is proposed. This method allows precise measurement of the value of astigmatism from only one experimental ronchigram.

Extended dynamical HAADF STEM image simulation using the Bloch-wave method.

An extended method is proposed for the precise simulation of high-angle annular dark-field (HAADF) scanning transmission electron-microscope (STEM) images for materials containing elements with large atomic numbers and for thick specimens. The approach combines a previously reported method utilizing two kinds of optical potential [Watanabe, Yamazaki, Hashimoto & Shiojiri (2001). Phys. Rev. B, 64, 115432] with a representation of a crystal sliced into multiple layers. The validity of the method is demonstrated by simulated images for elements with the diamond structure (Si, Ge and alpha-Sn) and for the perovskite BaTiO3.

Precise measurement of local strain fields with energy-unfiltered convergent-beam electron diffraction.

A simple and robust method to precisely determine local strain fields using energy-unfiltered convergent-beam electron diffraction is presented. This method involves the subtraction of background intensity, the extraction of higher-order Laue-zone lines by tracing using a Radon transformation and a system of analytical strain determination without the need for an optimization routine such as chi2-based minimization. As an example, the measurement of residual strain in a silicon-on-insulator wafer is demonstrated. It is found from micro-Raman spectroscopy analysis that, at the nanometre scale, this measurement succeeds with an accuracy of 0.06%.

CRM1-dependent, but not ARE-mediated, nuclear export of IFN-alpha1 mRNA.

While the bulk of cellular mRNA is known to be exported by the TAP pathway, export of specific subsets of cellular mRNAs may rely on chromosome region maintenance 1 (CRM1). One line of evidence supporting this hypothesis comes from the study of mRNAs of certain early response genes (ERGs) containing the adenylate uridylate-rich element (ARE) in their 3' untranslated regions (3' UTRs). It was reported that HuR-mediated nuclear export of these mRNAs was CRM1-dependent under certain stress conditions. To further examine potential CRM1 pathways for other cellular mRNAs under stress conditions, the nuclear export of human interferon-alpha1 (IFN-alpha1) mRNA, an ERG mRNA induced upon viral infection, was studied. Overproduction of human immunodeficiency virus type 1 Rev protein reduced the expression level of the co-transfected IFN-alpha1 gene. This inhibitory effect, resulting from nuclear retention of IFN-alpha1 mRNA, was reversed when rev had a point mutation that made its nuclear export signal unable to associate with CRM1. Leptomycin B sensitivity experiments revealed that the cytoplasmic expression of IFN-alpha1 mRNA was arrested upon inhibition of CRM1. This finding was further supported by overexpression of DeltaCAN, a defective form of the nucleoporin Nup214/CAN that inhibits CRM1 in a dominant-negative manner, which resulted in the effective inhibition of IFN-alpha1 gene expression. Subsequent RNA fluorescence in situ hybridisation and immunocytochemistry demonstrated that the IFN-alpha1 mRNA was colocalised with CRM1, but not with TAP, in the nucleus. These results therefore imply that the nuclear export of IFN-alpha1 mRNA is mediated by CRM1. However, truncation of the 3' UTR did not negatively affect the nuclear export of IFN-alpha1 mRNA that lacked the ARE, unexpectedly indicating that this CRM1-dependent mRNA export may not be mediated via the ARE.