Recent advances in reverse genetics of hepatitis C virus (HCV) made it possible to determine the properties and biochemical compositions of HCV virions. Sedimentation analysis and characterization of HCV RNAcontaining particles produced in the cultured cells revealed that HCV virions cover a large range of heterogeneous densities in sucrose gradient. The fractions of low densities are infectious, while the higher-density fractions containing the majority of HCV virion RNA are not. HCV core protein and apolipoprotein B and apolipoprotein E (apoE) were detected in the infectious HCV virions. The level of apoE correlates very well with HCV infectivity. Both apoE-and HCV E2-specific monoclonal antibodies precipitated HCV, demonstrating that HCV virions contain apoE and E2 proteins. apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity in a dose-dependent manner, resulting in a reduction of infectious HCV by nearly 4 orders of magnitude. The knockdown of apoE expression by specific small interfering RNAs (siRNAs) remarkably reduced the levels of intracellular as well as secreted HCV virions. The apoE siRNA suppressed HCV production by more than 100-fold at 50 nM. These findings demonstrate that apoE is required for HCV virion infectivity and production, suggesting that HCV virions are assembled as apoE-enriched lipoprotein particles. Our findings also identified apoE as a novel target for discovery and development of antiviral drugs and monoclonal antibodies to suppress HCV virion formation and infection.Hepatitis C virus (HCV) is a major cause of liver diseases, affecting approximately 170 million people worldwide (59). Most (ϳ85%) acutely HCV-infected individuals become chronic carriers that can develop cirrhosis and hepatocellular carcinoma (50). HCV is an enveloped RNA virus with a singlestrand and positive-sense RNA genome and is classified as Hepacivirus in the Flaviviridae family (47). The genomic RNA consists of a long open reading frame and relatively short untranslated regions (UTR) at the 5Ј and 3Ј ends (11,32,36,46,53). The 5Ј and 3Ј UTR contain cis-acting RNA elements important for HCV polyprotein translation and RNA replication (16-18, 38, 39, 61, 62). The translation of HCV polyprotein is mediated by the internal ribosomal entry site within the 5Ј UTR (46, 58). Upon translation, the HCV polyprotein is cleaved by cellular peptidases and viral proteases into different viral proteins in the order of C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (36, 38). A number of studies demonstrated that the NS3 to NS5B proteins are sufficient for HCV RNA replication (4, 6, 37), which occurs in the membranebound replication complex consisting of HCV RNA and proteins as well as cellular proteins (13,14,42,57). The core and NS5B coding regions also contain cis-acting RNA elements important for HCV RNA replication and regulation (40,63). Last, the newly synthesized HCV proteins and genomic RNA are packaged to form progeny virus particles. However, the molecular aspects underlying HCV virion assembly, matur...

In the developing brain, transcription factors (TFs) direct the formation of a diverse array of neurons and glia. We identifed 1445 putative TFs in the mouse genome. We used in situ hybridization to map the expression of over 1000 of these TFs and TF-coregulator genes in the brains of developing mice. We found that 349 of these genes showed restricted expression patterns that were adequate to describe the anatomical organization of the brain. We provide a comprehensive inventory of murine TFs and their expression patterns in a searchable brain atlas database.

Identification of common mechanisms underlying organ development and primary tumor formation should yield new insights into tumor biology and facilitate the generation of relevant cancer models. We have developed a novel method to project the gene expression profiles of medulloblastomas (MBs)-human cerebellar tumors-onto a mouse cerebellar development sequence: postnatal days 1-60 (P1-P60). Genomically, human medulloblastomas were closest to mouse P1-P10 cerebella, and normal human cerebella were closest to mouse P30-P60 cerebella. Furthermore, metastatic MBs were highly associated with mouse P5 cerebella, suggesting that a clinically distinct subset of tumors is identifiable by molecular similarity to a precise developmental stage. Genewise, down-and up-regulated MB genes segregate to late and early stages of development, respectively. Comparable results for human lung cancer vis-a-vis the developing mouse lung suggest the generalizability of this multiscalar developmental perspective on tumor biology. Our findings indicate both a recapitulation of tissue-specific developmental programs in diverse solid tumors and the utility of tumor characterization on the developmental time axis for identifying novel aspects of clinical and biological behavior.[Keywords: cerebellar development; medulloblastoma; comparative genomics; multiscale models; metastasis; principle component analysis] Supplemental material is available at http://www.genesdev.org. Received December 30, 2003; revised version accepted February 25, 2004. In the 19th century, Lobstein and Cohnheim were among the first to theorize similarities between human embryogenesis and the biology of cancer cells (Rather 1978). The brain tumor classification system of Bailey and Cushing (1926), from which modern taxonomies derive, emphasizes the histologic resemblance to cells of the developing central nervous system (CNS; Bailey and Cushing 1926). Nevertheless, the putative relationship between underlying mechanisms in normal development and tumorigenesis remains controversial for most types of cancer, particularly the solid tumors such as medulloblastomas (MBs) and carcinomas.Here, we focused on the relationship between genes regulated during oncogenesis in the human cerebellar tumor, MB, and the developing wild-type mouse cerebellum during postnatal days 1-60 (P1-P60). The cerebellum is the brain structure largely responsible for coordinating motor activities. Granule neurons, the most abundant cell type in the cerebellum during development, are derived from precursors of the embryonic hindbrain (Hallonet et al. 1990). In mice, the major phase of granule cell proliferation commences at birth and peaks by P8-P10 (Altman and Bayer 1987). Differentiation is complete by P60 in mice, and at ∼18 mo of age in humans. Granule neuron progenitors are thought to be the predominant dysregulated cell type from which the majority of MB cases arise (Kadin et al. 1970;Reddy and Packer 1999). MBs are the most common pediatric CNS malignancy and comprise two primary histological...

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive-and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10 8 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.Discovered in 1989 by molecular cloning (10), hepatitis C virus (HCV) has been recognized as a major cause of viral hepatitis in humans. HCV infection is characterized by the establishment of chronic infection in the majority (up to 85%) of individuals exposed to HCV. It is estimated that approximately 4 million people in the United States and 170 million people worldwide are persistently infected (9, 38). The chronic HCV infection carries an increased risk of developing fatal liver diseases such as cirrhosis, liver failure, and hepatocellular carcinoma. HCV is a single-stranded positive-sense RNA virus belonging to the Hepacivirus genus of the family Flaviviridae (30). The 9.6-kb RNA genome encodes a single polyprotein that is cleaved by cellular and viral proteases into at least 10 structural (C, E1, E2, and probably p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins that play important roles in virus entry, replication, assembly, and pathogenesis (24, 29). The sequence and structures of the untranslated regions (UTR) at both the 5Ј and 3Ј ends of the HCV RNA genome, which contain cis-acting RNA elements required for HCV RNA translation and replication, are highly conserved (7,13,20,24,25,35,40,41).A great deal of progress has been made with respect to the HCV genome organization, properties and roles of viral proteins and conserved RNA sequence/structures, virus-host interactions, and mechanisms of HCV replication since the dis...

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development, and it can be regulated by multiple environmental cues and endogenous signals. The mitogen-activated protein kinase (MAPK) cascades play diverse roles in intracellular and extracellular signaling in plants. Roles of the MAPK signaling module in leaf senescence are unknown. Here, a MAPK cascade involving MKK9-MPK6 is shown to play an important role in regulating leaf senescence in Arabidopsis (Arabidopsis thaliana). Both MKK9 and MPK6 possess kinase activities, with MPK6 an immediate target of MKK9, as revealed by in vitro, in vivo, and in planta assays. The constitutive and inducible overexpression of MKK9 causes premature senescence in leaves and in whole Arabidopsis plants. The premature senescence phenotype is suppressed when MKK9 is overexpressed in the mpk6 null background. When either MKK9 or MPK6 is knocked out, leaf senescence is delayed.

The guts of metazoans are in permanent contact with the microbial realm that includes beneficial symbionts, nonsymbionts, food-borne microbes and life-threatening pathogens. However, little is known concerning how host immunity affects gut bacterial community. Here, we analyze the role of a dual oxidase gene (BdDuox) in regulating the intestinal bacterial community homeostasis of the oriental fruit fly Bactrocera dorsalis. The results showed that knockdown of BdDuox led to an increased bacterial load, and to a decrease in the relative abundance of Enterobacteriaceae and Leuconostocaceae bacterial symbionts in the gut. The resulting dysbiosis, in turn, stimulates an immune response by activating BdDuox and promoting reactive oxygen species (ROS) production that regulates the composition and structure of the gut bacterial community to normal status by repressing the overgrowth of minor pathobionts. Our results suggest that BdDuox plays a pivotal role in regulating the homeostasis of the gut bacterial community in B. dorsalis.

We previously found that 82% of glial fibrillary acidic protein (GFAP)‐positive hippocampal astrocytes acutely isolated from P1–10 rats responded to glutamate (Glu) with transient intracellular calcium increases via activation of a Group I metabotropic glutamate receptor (mGluR). Fewer cells responded to ATP and none to serotonin (5‐HT). In this study we asked the question whether hippocampal astrocytes in older animals retain this relative pattern of expression. We have found that 77% of GFAP (+) cells from P11–20 rats responded to 50 μM Glu, 43% to ATP, and none to 5‐HT. Thirty‐three percent of GFAP (+) cells from P25–35 rats responded to Glu, 12% to ATP and 3% to 5‐HT. In the case of the responses to Glu, pharmacological characterization and single‐cell RT‐PCR data confirmed that these responses were mediated by the mGluR5 subtype of group I mGluRs. Also, fewer (36%) GFAP mRNA (+) cells from P25–35 rats expressed detectable mGluR5 mRNA than those from P11–20 rats (77%). This number essentially corresponds to the number of GFAP(+) showing a Ca2+ response to Glu. Both mGluR5a and b were expressed with equal frequency in cells from P11–20 rats, but the b form predominated in cells from older animals. Overall, our studies show that expression of mGluR5 in hippocampal astrocytes decreases with increasing age and the “a” splice variant declines to a greater extent than the “b” splice variant, corresponding to the developmental changes shown in total tissue for mGluR5. GLIA 29:70–80, 2000. © 2000 Wiley‐Liss, Inc.

Development of full-length hepatitis C virus (HCV) RNAs replicating efficiently and producing infectious cell-cultured virions, HCVcc, in hepatoma cells provides an opportunity to characterize immunogenic domains on viral envelope proteins involved in entry into target cells. A panel of immunoglobulin G1 human monoclonal antibodies (HMAbs) to three immunogenic conformational domains (designated A, B, and C) on HCV E2glycoprotein showed that epitopes within two domains, B and C, mediated HCVcc neutralization, whereas HMAbs to domain A were all nonneutralizing. For the neutralizing antibodies to domain B (with some to conserved epitopes among different HCV genotypes), the inhibitory antibody concentration reducing HCVcc infection by 90%, IC 90 , ranged from 0.1 to 4 g/ml. For some neutralizing HMAbs, HCVcc neutralization displayed a linear correlation with an antibody concentration between the IC 50 and the IC 90 while others showed a nonlinear correlation. The differences between IC 50 /IC 90 ratios and earlier findings that neutralizing HMAbs block E2 interaction with CD81 suggest that these antibodies block different facets of virus-receptor interaction. Collectively, these findings support an immunogenic model of HCV E2 having three immunogenic domains with distinct structures and functions and provide added support for the idea that CD81 is required for virus entry.Hepatitis C virus (HCV) is a positive-stranded RNA virus containing at least three structural proteins-core and two envelope glycoproteins, E1 and E2-and six nonstructural proteins (1). Studies with infectious retroviral particles pseudotyped with HCV E1E2 (HCVpp) showed that virus entry requires both E1 and E2 glycoproteins associated as a heterodimer, involves interactions with CD81, is low pH dependent, and is blocked by antibodies to HCV E2 and sera from HCV-infected individuals (3,8,13). Detailed information on the immunogenic and functional organization of HCV envelope glycoproteins and in particular E2 is needed to facilitate immunotherapeutics and vaccine development. Cross-competition studies with a panel of HCV E2 human monoclonal antibodies (HMAbs) showed that the HCVpp E2 glycoprotein contains three immunogenic conformational domains, designated A, B, and C, that are accessible on the surface of HCVpp (10). Each domain carries epitopes that are either highly conserved among diverse HCV genotypes or more restrictedly conserved. Epitopes within two domains, B and C, are targets of HCVpp-neutralizing antibodies, and the other domain, A, contains nonneutralizing epitopes that participate in structural changes as part of a pH-dependent virus envelope fusion process (9). Recently, three groups developed full-length HCV RNA genomes replicating efficiently when transfected into human hepatoma cells (Huh7) and producing infectious virions (11,15,17). The availability of these and other cell-cultured infectious HCV virions, HCVcc, should greatly accelerate studies of HCV biology (4,11,(14)(15)(16)(17). This report focuses on the immunogenic and f...