Huntingtin-interacting protein 14, a palmitoyl transferase required for exocytosis and targeting of CSP to synaptic vesicles

Posttranslational modification through palmitoylation regulates protein localization and function. In this study, we identify a role for the Drosophila melanogaster palmitoyl transferase Huntingtin-interacting protein 14 (HIP14) in neurotransmitter release. hip14 mutants show exocytic defects at low frequency stimulation and a nearly complete loss of synaptic transmission at higher temperature. Interestingly, two exocytic components known to be palmitoylated, cysteine string protein (CSP) and SNAP25, are severely mislocalized at hip14 mutant synapses. Complementary DNA rescue and localization experiments indicate that HIP14 is required solely in the nervous system and is essential for presynaptic function. Biochemical studies indicate that HIP14 palmitoylates CSP and that CSP is not palmitoylated in hip14 mutants. Furthermore, the hip14 exocytic defects can be suppressed by targeting CSP to synaptic vesicles using a chimeric protein approach. Our data indicate that HIP14 controls neurotransmitter release by regulating the trafficking of CSP to synapses.     

Auxin response, but not its polar transport, plays a role in hydrotropism of Arabidopsis roots

Plants are sessile in nature, and need to detect and respond to many environmental cues in order to regulate their growth and orientation. Indeed, plants sense numerous environmental cues and respond via appropriate tropisms, and it is widely accepted that auxin plays an important role in these responses. Recent analyses using Arabidopsis have emphasized the importance of polar auxin transport and differential auxin responses to gravitropism. Even so, the involvement of auxin in hydrotropism remains unclear. To clarify whether or not auxin is involved in the hydrotropic response, Arabidopsis seedlings were treated with inhibitors of auxin influx (3-chloro-4-hydroxyphenylacetic acid), efflux (1-naphthylphthalemic acid and 2,3,5-triiodobenzoic acid), and response (p-chlorophenoxyisobutylacetic acid), and their effects were examined on both hydrotropic and gravitropic responses. In agreement with previous reports, gravitropism was inhibited by all the chemicals tested. By contrast, only an inhibitor of the auxin response (p-chlorophenoxyisobutylacetic acid) reduced hydrotropism, whereas inhibitors for influx or efflux of auxin had no effect. These results suggest that auxin response, apart from its polar transport, plays a definite role in hydrotropic response, and will evoke a new concept for the auxin-mediated regulation of tropisms.     

Possible function of caudal nuclear pocket: degradation of nucleoproteins by ubiquitin-proteasome system in rat spermatids and human sperm.

Many temporarily functioning proteins are generated during the replacement of nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus. This study was designed to clarify the involvement of the ubiquitin-proteasome degradation system in the nucleus of rat developing spermatids. Thus, we studied the nuclear distribution of polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm using postembedding immunoelectron microscopy. We divided the nuclear area of late spermatids into two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocket in the neck region. The latter was located in the caudal nuclear region and was surrounded by redundant nuclear envelope. We demonstrated the presence of pUP in the dense area and nuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of rat spermatids. Using quantitative analysis of immunogold labeling, we found that fluctuation of pUP and proteasome levels in late spermatogenesis was mostly synchronized with disappearance of histones and transitional proteins reported previously. In the nuclei of human sperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuoles and clear spots. These results strongly suggest that pUP occur in the dense nuclear area of developing spermatids and that the ubiquitin-proteasome system is more actively operational in the nuclear pocket than dense area. Thus, the nuclear pocket might be the degradation site for temporarily functioning proteins generating during condensation of chromatin in late spermatids.     

Fiber-modified adenovirus vectors decrease liver toxicity through reduced IL-6 production

Adenovirus (Ad) vectors are one of the most commonly used viral vectors in gene therapy clinical trials. However, they elicit a robust innate immune response and inflammatory responses. Improvement of the therapeutic index of Ad vector gene therapy requires elucidation of the mechanism of Ad vector-induced inflammation and cytokine/chemokine production as well as development of the safer vector. In the present study, we found that the fiber-modified Ad vector containing poly-lysine peptides in the fiber knob showed much lower serum IL-6 and aspartate aminotransferase levels (as a maker of liver toxicity) than the conventional Ad vector after i.v. administration, although the modified Ad vector showed higher transgene production in the liver than the conventional Ad vector. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine, chemokine, and IFN expression. Splenic CD11c(+) cells were found to secret cytokines. The tissue distribution of Ad vector DNA showed that spleen distribution was much reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. Liver toxicity by the conventional Ad vector was reduced by anti-IL-6R Ab, suggesting that IL-6 signaling is involved in liver toxicity and that decreased liver toxicity of the modified Ad vector was due in part to the reduced IL-6 production. This study contributes to an understanding of the biological mechanism in innate immune host responses and liver toxicity toward systemically administered Ad vectors and will help in designing safer gene therapy methods that can reduce robust innate immunity and inflammatory responses.     

Fibroblast growth factor receptor 3 kinase domain mutation increases cortical progenitor proliferation via mitogen-activated protein kinase activation

We have previously shown that mice carrying the K644E kinase domain mutation in fibroblast growth factor receptor 3 (Fgfr3) (EIIa;Fgfr3(+/K644E)) have enlarged brains with increased proliferation and decreased apoptosis of the cortical progenitors. Despite its unique rostral-low caudal-high gradient expression in the cortex, how Fgfr3 temporally and spatially influences progenitor proliferation is unknown. In vivo BrdU labelling now showed that progenitor proliferation was 10-46% higher in the EIIa;Fgfr3(+/K644E) cortex compared with wild type during embryonic day 11.5 (E11.5)-E13.5. The difference in proliferation between the EIIa;Fgfr3(+/K644E) and wild-type cortices was the greatest in the caudal cortex at E12.5 and E13.5. Inhibition of mitogen-activated or extracellular signal-regulated protein kinase (MEK) in vitro at E11.5 reduced the proliferation rate of the EIIa;Fgfr3(+/K644E) cortical progenitors to similar levels observed in the wild type, indicating that the majority of the increase in cell proliferation caused by the Fgfr3 mutation is mitogen-activated protein kinase (MAPK) pathway-dependent at this stage. In addition, elevated levels of Sprouty were observed in the EIIa;Fgfr3(+/K644E) telencephalon at E14.5, indicating the presence of negative feedback that may have suppressed further MAPK activation. We suggest that temporal activation of MAPK is largely responsible for cell proliferation caused by the Fgfr3 mutation during early stages of cortical development.     

CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection

Huh7 cells constitute a permissive cell line for cell culture of hepatitis C virus (HCV) particles. However, our Huh7 line shows limited permissiveness for HCV. Thus, in this study we set out to determine which host factors are important for conferring permissiveness. To analyze the limited permissiveness of our Huh7 cells, 70 clones were obtained after single-cell cloning of parental Huh7 cells. The cloned Huh7 cells exhibited various levels of HCV pseudoparticles and JFH-1 virus infection efficiency, and some clones were not permissive. A subgenomic replicon was then transfected into the cloned Huh7 cells. While the replication efficiencies differed among the cloned Huh7 cells, these efficiencies did not correlate with infectious permissibility. Flow cytometry showed that CD81, scavenger receptor class B type I, and low-density-lipoprotein receptor expression on the cell surfaces of the Huh7 clones differed among the clones. Interestingly, we found that all of the permissive cell clones expressed CD81 while the nonpermissive cell clones did not. To confirm the importance of CD81 expression for HCV permissiveness, CD81 was then transiently and stably expressed on a nonpermissive Huh7 cell clone, which was consequently restored to HCV infection permissiveness. Furthermore, permissiveness was down-regulated upon transfection of CD81 silencing RNA into a CD81-positive cell clone. In conclusion, CD81 expression is an important determinant of HCV permissiveness of Huh7 cell clones harboring different characteristics.     

Potassium bromate treatment predominantly causes large deletions, but not GC>TA transversion in human cells

Potassium bromate (KBrO(3)) is strongly carcinogenic in rodents and mutagenic in bacteria and mammalian cells in vitro. The proposed genotoxic mechanism for KBrO(3) is oxidative DNA damage. KBrO(3) can generate high yields of 8-hydroxydeoxyguanosine (8OHdG) DNA adducts, which cause GC>TA transversions in cell-free systems. In this study, we investigated the in vitro genotoxicity of KBrO(3) in human lymphoblastoid TK6 cells using the comet (COM) assay, the micronucleus (MN) test, and the thymidine kinase (TK) gene mutation assay. After a 4h treatment, the alkaline and neutral COM assay demonstrated that KBrO(3) directly yielded DNA damages including DNA double strand breaks (DSBs). KBrO(3) also induced MN and TK mutations concentration-dependently. At the highest concentration (5mM), KBrO(3) induced MN and TK mutation frequencies that were over 30 times the background level. Molecular analysis revealed that 90% of the induced mutations were large deletions that involved loss of heterozygosity (LOH) at the TK locus. Ionizing-irradiation exhibited similar mutational spectrum in our system. These results indicate that the major genotoxicity of KBrO(3) may be due to DSBs that lead to large deletions rather than to 8OHdG adducts that lead to GC>TA transversions, as is commonly believed. To better understand the genotoxic mechanism of KBrO(3), we analyzed gene expression profiles of TK6 cells using Affymetrix Genechip. Some genes involved in stress, apoptosis, and DNA repair were up-regulated by the treatment of KBrO(3). However, we could not observe the similarity of gene expression profile in the treatment of KBrO(3) to ionizing-irradiation as well as oxidative damage inducers.     

The real factor for polypeptide elongation in Dictyostelium cells is EF-2B, not EF-2A

Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method. Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells.     

Membrane microdomain formation is crucial in epiboly during gastrulation of medaka

Membrane microdomain (microdomain) was isolated from early gastrula embryos. The isolated microdomain was characterized by enrichment of cholesterol and sphingomyelin, and by the presence of huge glycoproteins containing Lewis X structure. Importance of the microdomain in the progress of epiboly was assessed using methyl beta-cyclodextrin (MBCD) and C2-ceramide that disrupt microdomains through different mechanisms. Both reagents efficiently disrupted the microdomain structure and concomitantly impaired epiboly. Interestingly, when embryos pretreated with MBCD, a cholesterol-binding molecule, were exogenously supplemented with cholesterol, the embryos underwent not only reconstitution of the microdomain, but also complete restoration to the normal epiboly. Thus, normal or impaired development is reversibly controlled by the cholesterol-dependent formation or disruption of microdomains. The most typical phenotype of the microdomain-disrupted embryos is detachment of cells from the blastoderm, suggesting that a major contribution of microdomains to epiboly is cell adhesion of blastodermal cells.     

Two distinct human POM121 genes: requirement for the formation of nuclear pore complexes

Pom121 is one of the integral membrane components of the nuclear pore complex (NPC) in vertebrate cells. Unlike rodent cells carrying a single POM121 gene, human cells possess multiple POM121 gene loci on chromosome 7q11.23, as a consequence of complex segmental-duplications in this region during human evolution. In HeLa cells, two "full-length" Pom121 are transcribed and translated by two distinct genetic loci. RNAi experiments showed that efficient depletion of both Pom121 proteins significantly reduces assembled NPCs on nuclear envelope. Pom121-depletion also induced clustering of NPCs, indicating its role on maintenance of NPC structure/organization.