Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

Niche Separation of Methanotrophic Archaea (ANME-1 and -2) in Methane-Seep Sediments of the Eastern Japan Sea Offshore Joetsu

In this study, we investigated the diversity and spatial distribution of anaerobic methanotrophic archaea (ANMEs) in sediments of a gas hydrate field off Joetsu in the Japan Sea. Distribution of ANMEs in sediments was identified by targeting the gene for methyl coenzyme M reductase alpha subunit (mcrA), a phylogenetically conserved gene that occurs uniquely in methanotrophic and methanogenic archaea, in addition to 16S rRNA genes. Quantitative PCR analyses of mcrA genes in 14 piston core samples suggested that members of ANME-1 group would dominate AOM communities in sulfate-depleted sediments, even below the sulfate-methane interface, while ANME-2 archaea would prefer to populate in shallower sediments containing comparatively higher sulfate concentrations. These results suggest that, although the potential electron acceptors in sulfate-depleted habitats remain elusive, the niche separation of ANME-1 and -2 may be controlled by in situ concentration of sulfate and the availability in sediments.     

A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 X g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P-450.     

PI3K/Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-α

Gap junctional communication, which is mediated by the connexin protein family, is essential for the maintenance of normal tissue function and homeostasis. Loss of intercellular communication results in a failure to coordinately regulate cellular functions, and it can facilitate tumorigenesis. Expression of oncogenes and stimulation with cytokines has been shown to suppress intercellular communication; however, the exact mechanism by which intercellular communication is disrupted by these factors remains uncertain. In this report, we show that Akt is essential for the disruption of gap junctional communication in v-Src-transformed cells. In addition, inhibition of Akt restores gap junctional communication after it is suppressed by TNF-α signaling. Furthermore, we demonstrate that the expression of a constitutively active form of Akt1, but not of Akt2 or Akt3, is sufficient to suppress gap junctional communication. Our results clearly define Akt1 as one of the critical regulators of gap junctional communication.     

EML4 promotes the loading of NUDC to the spindle for mitotic progression

Echinoderm microtubule-associated protein (EMAP)-like (EML) family proteins are microtubule-associated proteins that have a conserved hydrophobic EMAP-like protein (HELP) domain and multiple WD40 domains. In this study, we examined the role of EML4, which is a member of the EML family, in cell division. Time-lapse microscopy analysis demonstrated that EML4 depletion induced chromosome misalignment during metaphase and delayed anaphase initiation. Further analysis by immunofluorescence showed that EML4 was required for the organization of the mitotic spindle and for the proper attachment of kinetochores to microtubules. We searched for EML4-associating proteins by mass spectrometry analysis and found that the nuclear distribution gene C (NUDC) protein, which is a critical factor for the progression of mitosis, was associated with EML4. This interaction was mediated by the WD40 repeat of EML4 and by the C-terminus of NUDC. In the absence of EML4, NUDC was no longer able to localize to the mitotic spindle, whereas NUDC was dispensable for EML4 localization. Our results show that EML4 is critical for the loading of NUDC onto the mitotic spindle for mitotic progression.     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility

Rho GTPases are molecular switches that transmit biochemical signals in response to extracellular stimuli to elicit changes in the actin cytoskeleton. Rho GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. These states are regulated by two distinct families of proteins-guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We studied the role of a previously uncharacterized GAP, ARHGAP18 (MacGAP). Overexpression of ARHGAP18 suppressed the activity of RhoA and disrupted stress fiber formation. Conversely, silencing of ARHGAP18 by small interfering RNA transfection-enhanced stress fiber formation and induced rounding of cells. We examined the role of ARHGAP18 in cell spreading and migration. Immunofluorescence analysis revealed that ARHGAP18 was localized to the leading edge during cell spreading and migration. ARHGAP18-knockdown cells showed impaired spreading, premature formation of stress fibers, and sustained activation of RhoA upon cell attachment. In addition, knockdown and overexpression of ARHGAP18 resulted in the inhibition and promotion of cell migration, respectively. Furthermore, ARHGAP18 was required for the polarization of cells for migration. Our results define ARHGAP18 as one of the crucial factors for the regulation of RhoA for the control of cell shape, spreading, and migration.     

Misshapen-like kinase 1 (MINK1) is a novel component of striatin-interacting phosphatase and kinase (STRIPAK) and is required for the completion of cytokinesis

Cytokinesis is initiated by constriction of the cleavage furrow and terminated by abscission of the intercellular bridge that connects two separating daughter cells. The complicated processes of cytokinesis are coordinated by phosphorylation and dephosphorylation mediated by protein kinases and phosphatases. Mammalian Misshapen-like kinase 1 (MINK1) is a member of the germinal center kinases and is known to regulate cytoskeletal organization and oncogene-induced cell senescence. To search for novel regulators of cytokinesis, we performed a screen using a library of siRNAs and found that MINK1 was essential for cytokinesis. Time-lapse analysis revealed that MINK1-depleted cells were able to initiate furrowing but that abscission was disrupted. STRN4 (Zinedin) is a regulatory subunit of protein phosphatase 2A (PP2A) and was recently shown to be a component of a novel protein complex called striatin-interacting phosphatase and kinase (STRIPAK). Mass spectrometry analysis showed that MINK1 was a component of STRIPAK and that MINK1 directly interacted with STRN4. Similar to MINK1 depletion, STRN4-knockdown induced multinucleated cells and inhibited the completion of abscission. In addition, STRN4 reduced MINK1 activity in the presence of catalytic and structural subunits of PP2A. Our study identifies a novel regulatory network of protein kinases and phosphatases that regulate the completion of abscission.