Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus.

The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5'-end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs.

Primary Ovarian Leiomyoma Associated with Multiple Uterine Leiomyomas.

Ovarian leiomyomas are very rare. We report the case of a primary ovarian leiomyoma accompanied by multiple uterine leiomyomas. A 50-year-old woman was referred to our department for heavy menstruation, and a hot spot in the uterine lumen was observed on positron emission tomography-computed tomography (PET-CT). Cervical and endometrial cytology and tumor marker tests were negative. Pelvic magnetic resonance imaging revealed an endometrial polyp and submucosal leiomyoma in the uterine lumen and a 5-cm right ovarian tumor. Laparoscopic total hysterectomy, right salpingo-oophorectomy, and left salpingectomy were performed for radical treatment. Histopathology showed that ovarian tumors contained interlacing bundles of fusiform cells encircled by normal ovarian tissue. Immunohistochemical staining showed strong and diffuse positive staining for α-smooth muscle actin. We diagnosed the tumor as a primary ovarian leiomyoma because the leiomyoma was localized in the ovary and was larger than the size of uterine leiomyomas. No metastatic lesion was found on PET-CT. There was no tumor recurrence at the 6-month follow-up.

Identification and structural definition of H5-specific CTL epitopes restricted by HLA-A*0201 derived from the H5N1 subtype of influenza A viruses.

The haemagglutinin (HA) glycoprotein of influenza A virus is a major antigen that initiates humoral immunity against infection; however, the cellular immune response against HA is poorly understood. Furthermore, HA-derived cytotoxic T-lymphocyte (CTL) epitopes are relatively rare in comparison to other internal gene products. Here, CTL epitopes of the HA serotype H5 protein were screened. By using in silico prediction, in vitro refolding and a T2 cell-binding assay, followed by immunization of HLA-A2.1/K(b) transgenic mice, an HLA-A*0201-restricted decameric epitope, RI-10 (H5 HA205-214, RLYQNPTTYI), was shown to elicit a robust CTL epitope-specific response. In addition, RI-10 and its variant, KI-10 (KLYQNPTTYI), were also demonstrated to be able to induce a higher CTL epitope-specific response than the influenza A virus dominant CTL epitope GL-9 (GILGFVFTL) in peripheral blood mononuclear cells of HLA-A*0201-positive patients who had recovered from H5N1 virus infection. Furthermore, the crystal structures of RI-10-HLA-A*0201 and KI-10-HLA-A*0201 complexes were determined at 2.3 and 2.2 A resolution, respectively, showing typical HLA-A*0201-restricted epitopes. The conformations of RI-10 and KI-10 in the antigen-presenting grooves in crystal structures of the two complexes show significant differences, despite their nearly identical sequences. These results provide implications for the discovery of diagnostic markers and the design of novel influenza vaccines.

Influence of polymorphism in dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related (DC-SIGNR) gene on HIV-1 trans-infection.

The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related (DC-SIGNR) molecules on the cell surface are known to enhance human immunodeficiency virus type 1 (HIV-1) infection by capturing the virions and transmitting them to CD4+ T-cell, a process termed trans-infection. The neck region and carbohydrate recognition domain of the two proteins are important for efficient binding to the HIV-1 envelope protein. DC-SIGNR is polymorphic in Exons 4 and 5 that encode the neck region and carbohydrate recognition domain, respectively; the former contains a variable number of tandem repeats, and the latter the SNP (rs2277998). Since it remains unclear whether the DC-SIGNR polymorphism is related to the risk of HIV-1 infection, we tested possible effects of the polymorphism on HIV-1 trans-infection efficiency, by constructing six kinds of cDNAs encoding DC-SIGNR variants with various numbers of repeat units and various SNP. We were able to express the variants on the surface of Raji cells, a human B cell line. Flow cytometry showed that all the tested DC-SIGNR molecules were efficiently expressed on the cell surface at various levels; the assay for HIV trans-infection efficacy showed that all the tested variants had that activity with different efficacy levels. We found a correlation between the HIV trans-infection efficiency and the mean fluorescent intensity of DC-SIGNR expression (R(2)=0.95). Thus, our results suggest that the variation of the tested DC-SIGNR genotypes affects the efficacy of trans-infection by affecting the amounts of the protein expressed on the cell surface.

Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication.

Influenza A virus matrix protein (M1) is the most abundant conservative protein that regulates the replication, assembly and budding of the viral particles upon infection. Several host cell factors have been determined to interact with M1 possibly in regulating influenza virus replication. By yeast two-hybrid screening, the isomerase cyclophilin A (CypA) was identified to interact with the M1 protein. CypA specifically interacted with M1 both in vitro and in vivo. The mutagenesis results showed CypA bound to the functional middle (M) domain of M1. The depletion of endogenous CypA by RNA interference resulted in the increase of influenza virus infectivity while overexpression of CypA caused decreasing the infectivity in affected cells. The immunofluorescence assays indicated that overexpressed CypA deduced the infectivity and inhibited the translocation of M1 protein into the nucleus while did not affect nucleoprotein entering the nucleus. Further studies indicated that overexpression of CypA significantly increased M1 self-association. Western blot with purified virions confirmed that CypA was encapsidated within the virus particle. These results together indicated that CypA interacted with the M1 protein and affected the early stage of the viral replication.

The affinity of human RANK binding to its ligand RANKL.

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re-modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombinant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was measured with surface plasmon resonance technology and K(D) value is about 1.09 x 10(-10) M.

A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells.

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon's anti-HCV action and for screening and testing new anti-HCV drugs.

F gene recombination between genotype II and VII Newcastle disease virus.

A velogenic Newcastle disease virus (NDV) strain, designated as SRZ03, was isolated from an egg layer flock with NDV vaccine immunization failure in China in 2003. Recombination was found in the F gene of SRZ03. Complete genome sequences analysis indicated that the N-terminal of SRZ03 F gene originated from a genotype II NDV strain, whereas the C-terminal of F gene and the rest of the genes originated from a prevalent velogenic genotype VII NDV strain. It provides us valuable information for understanding the recombination of nonsegmented negative-sense RNA viruses.

The polymorphisms in DC-SIGNR affect susceptibility to HIV type 1 infection.

Dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN) and its homologue DC-SIGNR (DC-SIGN related) have been thought to play an important role in establishing HIV infection by enhancing trans-infection of CD4(+)T cells in the regional lymph nodes. To identify polymorphisms associated with HIV-exposed seronegative (ESN) individuals in Thais, genomic DNA from 102 HIV-seronegative individuals of HIV-seropositive spouses, 305 HIV-seropositive individuals, and 290 HIV-seronegative blood donors was genotyped for two single nucleotide polymorphisms (SNPs) in DC-SIGN promoter (-139A/G and 336A/G), a repeat number of 69 bp in Exon 4 of DC-SIGN and DC-SIGNR, and one SNP in Exon 5 of DC-SIGNR (rs2277998A/G). We found that the proportion of individuals possessing a heterozygous 7/5 and 9/5 repeat and A allele at rs2277998 of DC-SIGNR in HIV-seronegative individuals of HIV-seropositive spouses was significantly higher than HIV-seropositive individuals [p = 0.0373, OR (95% CI) = 0.57 (0.32,1.01); p = 0.0232, OR (95% CI) = 0.38 (0.15,0.98); and p = 0.0445, OR (95% CI) = 0.61 (0.37,1.02), respectively]. Analysis after stratifying by gender showed that these associations were observed only in females but not in males. Moreover, HIV-seropositive females tend to have a homozygous 7/7 repeat more frequently than HIV-seronegative females with a marginal level of significance [p = 0.0556, OR (95% CI) = 1.79 (0.94,3.40)]. Haplotype analysis showed that the proportion of individuals possessing the 5A haplotype in HIV-seronegative females was significantly higher than HIV-seropositive females [p = 0.0133, OR = 0.50 (0.27,0.90)]. These associations suggest that DC-SIGNR may affect susceptibility to HIV infection by a mechanism that is different in females and males. Further studies are warranted to investigate the mechanisms of their function.

Medial profrontal cortex and anterior cingulate cortex in the generation of alpha activity induced by transcendental meditation: a magnetoencephalographic study.

Previous EEG studies have shown that transcendental meditation (TM) increases frontal and central alpha activity. The present study was aimed at identifying the source of this alpha activity using magnetoencephalography (MEG) and electroencephalography (EEG) simultaneously on eight TM practitioners before, during, and after TM. The magnetic field potentials corresponding to TM-induced alpha activities on EEG recordings were extracted, and we attempted to localize the dipole sources using the multiple signal classification (MUSIC) algorithm, equivalent current dipole source analysis, and the multiple spatio-temporal dipole model. Since the dipoles were mapped to both the medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC), it is suggested that the mPFC and ACC play an important role in brain activity induced by TM.

Chromatin remodeling factor encoded by ini1 induces G1 arrest and apoptosis in ini1-deficient cells.

Ini1/hsnf5 gene encodes INI1 protein, a chromatin remodeling factor associated with the SWI/SNF complex. In yeast, this complex modifies chromatin condensation to coactivate various transcriptional factors. However, in human, little is known about the SWI/SNF complex and INI1. To elucidate cellular functions of ini1, we constructed a recombinant adenovirus (AdexHA-INI1) capable of overexpressing INI1 in ini1-deficient cells. AdexHA-INI1 produced intranuclear INI1 in three ini1-deficient cell lines, changed their morphology, and decreased the proportion of viable cells. Flow cytometry and a BrdU incorporation assay showed that after the infection, growth of these cells was partially arrested at G1. In two of the three ini1-deficient cell lines, apoptosis was found to occur after the infection, as detected by the presence of cleaved poly (ADP-ribose) polymerase. To determine functional domains of INI1, we constructed plasmids expressing INI1 and its deletion mutants, which were used for a colony formation assay. Repeats 1 and 2 of INI1 were found to be required to suppress the growth of the three ini1-deficient cell lines. The results support the hypothesis that ini1 is a tumor suppressor gene and suggest a novel link between human SWI/SNF chromatin remodeling complex and apoptosis.

A novel small molecular weight compound with a carbazole structure that demonstrates potent human immunodeficiency virus type-1 integrase inhibitory activity.

The integration of reverse transcribed proviral DNA into a host genome is an essential event in the human immunodeficiency virus type 1 (HIV-1) replication life cycle. Therefore, the viral enzyme integrase (IN), which plays a crucial role in the integration event, has been an attractive target of anti-retroviral drugs. Several IN inhibitory compounds have been reported previously, yet none has been successful in clinical use. To find a new, more successful IN inhibitor, we screened a diverse library of 12 000 small molecular weight compounds randomly by in vitro strand-transfer assay. We identified a series of substituted carbazoles that exhibit strand-transfer inhibitory activity at low micromolar concentrations. Of these, the most potent compound exhibited an IC50 of 5.00+/-3.31 microM (CA-0). To analyse the structural determinants of strand-transfer inhibitory activity of the carbazole derivatives, we selected 23 such derivatives from our compound library and performed further analyses. Of these 23 compounds, six showed strong strand-transfer inhibition. The inhibition kinetics analyses and ethidium bromide displacement assays indicated that the carbazole derivatives are competitive inhibitors and not intercalators. An HeLa4.5/LTR-nEGFP cell line was employed to evaluate in vitro virus replication inhibition of the carbazole derivatives, and IC50 levels ranged from 0.48-1.52 microM. Thus, it is possible that carbazole derivatives, which possess structures different from previously-reported IN inhibitors, may become novel lead compounds in the development of IN inhibitors.

Fungal phenalenones inhibit HIV-1 integrase.

A phenalenone compound, atrovenetinone methyl acetal, was isolated from a culture broth of Penicillium sp. FKI-1463 as an HIV-1 integrase inhibitor, and it showed anti-HIV activity in vitro. HIV-1 integrase inhibition and anti-HIV activity of two other natural phenalenones were also studied. Among the tested compounds, funalenone inhibited HIV-1 integrase with an IC50 value of 10 microM and showed the best selectivity (anti-HIV, IC50=1.7 microM; cytotoxicity, IC50=87 microM).

A survey on characteristics of Japanese academic job market and evaluation.

BACKGROUND: During the Meiji era, at the end of the 19th century, Japan introduced western systems into many fields, economically developing later than other industrially developed countries. Japan introduced a higher education system modeled on the German system, focusing not on education but on research. The historical background has shaped contemporary Japanese academia differently from that of the United States and the European Union. In addition, because of geographical and linguistic barriers in Asia, intercommunication with researchers in other developed countries has been much less than that between the United States and the European Union, leaving Japanese academia relatively isolated. METHOD: We survey the characteristics of the Japanese academic system in higher education, using the latest published data. RESULT: This article indicates a concentration of research at former imperial universities and a rigidity of movement among universities both internationally and domestically. Furthermore, small differences in salary levels have provided little incentive to perform research. However, while most universities in Japan have not introduced evaluation systems for promotion and salary that are heavily dependent on journal rankings, as in the European Union and United States, Japanese academic performance has not declined. CONCLUSION: This article suggests that in Japan, salary incentives, the impact factor, and so on have had little influence on academic performance. Even though cultural and historical differences between countries affect academic behaviors, we hope that this article might trigger consideration of other possible evaluation schemes for the future.

Influence of single-nucleotide polymorphisms in the multidrug resistance-1 gene on the cellular export of nelfinavir and its clinical implication for highly active antiretroviral therapy.

Protease inhibitors (PIs) such as nelfinavir (NFV) suppress HIV replication. PIs are substrates of P-glycoprotein (P-gp), the product of the multidrug-resistance-1 (MDR1) gene. Three single-nucleotide polymorphisms (SNPs) are present in exons of the MDR1 gene: MDR1 1236, MDR1 2677 and MDR1 3435. We speculated that these genetic polymorphisms affected PI concentration in the cell. To verify this hypothesis, we first genotyped these SNPs in 79 Japanese patients by the SNaPshot method and found incomplete linkage disequilibrium between the SNPs. Because the SNP at MDR1 3435 has been reported to be associated with P-gp expression, we evaluated the effect of that SNP on the export of NFV from HIV-positive patients' lymphoblastoid cell lines by measuring time-dependent decrease in the amount of intracellular NFV by high-performance liquid chromatography. We found the intracellular concentration of NFV in lymphoblastoid cell lines (LCLs) with the homozygous T/T genotype at MDR1 3435 were higher than that with C/C genotype with statistical significance. This suggests that the activity of P-gp in patients' LCL cells with the MDR1 3435 T/T genotype was lower. In a retrospective study we evaluated the effect of the SNPs on CD4 cell count recovery in response to antiretroviral treatment with PIs, and obtained statistically significant evidence that suggested marginal association of the SNP at MDR1 1236 but not at MDR1 2677 or MDR1 3435. As in vitro results were not consistent with the clinical evaluation, clinical importance of MDR1 genotyping for antiretroviral therapy remains to be investigated in a larger, case-controlled study.

Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f. sp. hominis in Japan.

This study examined polymorphisms in the dihydrofolate reductase (DHFR) gene of Pneumocystis carinii isolates from 27 patients with P. carinii pneumonia (PCP) in Japan. Four substitution sites with two synonymous and two non-synonymous changes were found. Two synonymous substitutions at nucleotide positions 540 and 312 were identified in one and 13 patients, respectively. Two amino acid substitutions (Ala67Val, Cysl66Tyr) were found in two different patients. No linkage of amino acid substitutions in DHFR to those in dihydropteroate synthase was observed. The two patients whose isolates showed non-synonymous DHFR mutations were not exposed to DHFR inhibitors before they developed PCP and were treated successfully with co-trimoxazole.

Ictal MEG in two children with partial seizures.

UNLABELLED: We report on the successful identification of epileptic foci in two children with partial epilepsy using ictal magnetoencephalography (MEG). Case 1 is a 12-year-old male suffering with simple partial seizures with leftwards nystagmus. Ictal SPECT revealed a hyperperfusion area in the right lateral occipital area, and MRI revealed cortical dysplasia in the same area. Interictal EEG dipoles were concentrated in the right mesial occipital lobe. Both interictal and ictal MEG dipoles were concentrated in the right mesial occipital lobe, which corresponded well with neuroimaging data and his clinical features. Case 2 is a 5-year-old female suffering with simple partial seizures with left-side facial twitching. Interictal EEG dipoles were located in her left motor area, the pre-sylvian fissure, close to the location of the interictal MEG-estimated dipoles. Ictal EEGs showed no remarkable changes associated with her clinical manifestations. However, ictal MEG showed high-voltage slow waves over her left hemisphere, and ictal MEG iso-contour maps revealed a clear dipolar pattern, which suggested that the MEG dipole was located in the area of the sylvian fissure. Ictal SPECT revealed hyperperfusion areas around the left sylvian fissure. CONCLUSION: Ictal MEG is useful for determining the precise location of epileptic focus in patients with motionless seizures, including children.

Visualization of cortical projection neurons with retrograde TET-off lentiviral vector.

We are interested in identifying and characterizing various projection neurons that constitute the neocortical circuit. For this purpose, we developed a novel lentiviral vector that carries the tetracycline transactivator (tTA) and the transgene under the TET Responsive Element promoter (TRE) on a single backbone. By pseudotyping such a vector with modified rabies G-protein, we were able to express palmitoylated-GFP (palGFP) or turboFP635 (RFP) in corticothalamic, corticocortical, and corticopontine neurons of mice. The high-level expression of the transgene achieved by the TET-Off system enabled us to observe characteristic elaboration of neuronal processes for each cell type. At higher magnification, we were able to observe fine structures such as boutons and spines as well. We also injected our retrograde TET-Off vector to the marmoset cortex and proved that it can be used to label the long-distance cortical connectivity of millimeter scale. In conclusion, our novel retrograde tracer provides an attractive option to investigate the morphologies of identified cortical projection neurons of various species.

Subcellular localization and topology of porcine reproductive and respiratory syndrome virus E protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 70 or 73 amino acids (aa), termed E protein. The function and biochemical information of the E protein are currently not clear. In the present investigation, it was shown that the E protein was mainly located in the endoplasmic reticulum (ER) and Golgi complex in MARC-145 cells. Deletion studies identified the N-terminal 15 residues as an ER localization sequence of the E protein, besides two other localization sequences within positions 23-50 and 50-73, and the N-myristoylation site significantly affected the subcellular localization of the N-terminal 15 residues. The membrane association assay demonstrated that the E protein was an integral membrane protein embedded in the phospholipid bilayer. However, neither the N-myristoylation site nor the hydrophilic C-terminal domain was essential to the membrane association of the E protein. The topology analysis revealed that this protein had N-terminus oriented toward the cytoplasm and C-terminus toward the ER lumen. Finally, immunofluorescence assay indicated that the E protein colocalized with GP2, GP3, GP4 and M protein in cotransfected cells, but not N protein.

Development of a simple system for screening anti-hepatitis C virus drugs utilizing mutants capable of vigorous replication.

Replication of infectious hepatitis C virus in Huh7 cells, a human hepatocyte cell line, has become possible due to the unique properties of the JFH1 isolate. Developing reporter virus systems for a simple titration has been attempted by integrating heterologous reporter genes into the JFH1 genome, resulting in a big infectivity reduction that limits the usefulness of such reporter systems. To overcome this problem, JFH1-infected Huh7 cells were cultured continuously for 2 years to obtain Huh7-adapted JFH1 variants capable of yielding up to 1000-fold higher titers. Sequence analysis of variant genome RNA suggested that this adapted population consisted mainly of two variants. By joining the 5'-half of the obtained representative viral complementary DNA (cDNA) fragments of the variants with the 3'-half of the wild-type's, two prototype clones, A/WT and B/WT, were constructed. Replication of A/WT and B/WT viruses in Huh7 cells showed up to 100-1000-fold higher titers than the wild-type. A Renilla luciferase cDNA was inserted into the Nonstructural Protein 5A region of the A/WT and B/WT cDNA to generate A/WT-Rluc and B/WT-Rluc, respectively. Transfection of Huh7 cells with in vitro-transcribed A/WT-Rluc and B/WT-Rluc RNA resulted in production of infectious viruses with approximately 15- and 25-fold higher titers, respectively, than the wild-type RNA. The replication of A/WT-Rluc and B/WT-Rluc viruses was more vigorous than the wild-type even with insertion of the luciferase cDNA showing a good correlation of luciferase activities with infectious titers. Furthermore, interferon-alpha inhibited the replication of A/WT-Rluc and B/WT-Rluc viruses in a dose-dependent manner as determined by a luciferase assay. These results imply that our system is potentially a tool useful for screening anti-hepatitis C virus drugs in a simple and time/cost-saving manner.