Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect

Rice dwarf virus (RDV) replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi) induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.     

顺铂诱导非洲绿猴肾细胞凋亡模型的建立

目的:建立常见凋亡诱导剂顺铂(Cisplatin)诱导非洲绿猴肾细胞(Vero)凋亡的模型,为进一步研究抗凋亡基因在细胞凋亡中的分子机理打下基础。方法:分别以不同浓度的顺铂处理Vero细胞48h,用噻唑蓝(MTT)比色法、Gimesa染色、流式细胞术检测,观察处理后细胞的生长活力和凋亡情况。结果:以未处理的细胞作为对照组,1、2、3、4、5μg/mL的顺铂处理的Vero细胞生存率分别为(79.02±6.10)%、(68.84±4.42)%、(56.66±4.07)%、(46.83±3.76)%、(29.04±5.93)%(P<0.01);经顺铂诱导后细胞形态学发生明显改变,出现膜小泡和凋亡小体形成等凋亡细胞特征;流式细胞仪检测,0、1、2、3、4、5μg/mL的顺铂处理的Vero细胞后凋亡率分别为1.66%±0.19%、16.65%±1.26%、24.82%±1.03%、36.22%±1.04%、48.49%±1.24%、43.34%±1.17%(P<0.01)。结论:本实验成功建立顺铂诱导非洲绿猴肾细胞凋亡模型,将有助于进一步探讨目的基因在Vero细胞凋亡作用的的分子机制。     

顺铂诱导非洲绿猴肾细胞凋亡模型的建立

目的：建立常见凋亡诱导剂顺铂（Cisplatin）诱导非洲绿猴肾细胞（Vero）凋亡的模型,为进一步研究抗凋亡基因在细胞凋亡中的分子机理打下基础。方法：分别以不同浓度的顺铂处理Veto细胞48h,用噻唑蓝（MTT）比色法、Gimesa染色、流式细胞术检测,观察处理后细胞的生长活力和凋亡情况。结果：以未处理的细胞作为对照组,1、2、3、4,5μg／mL的顺铂处理的Vero细胞生存率分别为（79．02±6．10）％、（68．84±4．42）％、（56．66±4．07）％、（46．83±3．76）％、（29．04±5．93）％（P〈0．01）;经顺铂诱导后细胞形态学发生明显改变,出现膜小泡和凋亡小体形成等凋亡细胞特征;流式细胞仪检测,0、1、2、3、4、5μg／mL的顺铂处理的Vero细胞后凋亡率分别为1．66％±0．19％、16．65％±1．26％、24．82％±1．03％、36．22％±1．04％、48．49％±1．24％、43．34％±1．17％（P〈0．01）。结论：本实验成功建立顺铂诱导非洲绿猴肾细胞凋亡模型,将有助于进一步探讨目的基因在Vero细胞凋亡作用的的分子机制。     

植物病毒昆虫介体传播的研究进展

本文综述了目前非循回型植物病素和循回型植物病毒昆虫介体传播机理的研究现状 。     

Expression of Rat CTP:Phosphocholine Cytidylyltransferase in Insect Cells Using a Baculovirus Vector

CTP:phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in phosphatidylcholine biosynthesis. We constructed a recombinant baculovirus (bCT) containing rat CT cDNA under the control of the polyhedrin promoter. Crude cell extracts of Spodoptera frugiperda (Sf9) cells infected with bCT possessed 250-fold higher specific activities for CT compared to rat liver cytosol, and CT protein constituted 3-6% of the total cellular protein. The 42-kDa form of CT predicted from the cDNA sequence was the first immunoreactive CT protein detected at Day 2 after infection and this form continued to accumulate until Day 5. On Day 3 following infection, a 37-kDa protein immunologically related to CT began to accumulate, indicating that CT was being degraded. The active, 42-kDa form of CT was purified to homogeneity in a single step using hydroxyapatite chromatography. Antibodies raised against recombinant CT were employed to quantitatively extract and assay CT activity in mammalian cell lines. The baculovirus expression system is suitable for the preparation of large amounts of protein for investigating the structure, function, and regulation of CT.     

A Moloney Murine Leukemia Virus-Based Retroviral Vector Pseudotyped by the Insect Retroviral gypsy Envelope Can Infect Drosophila Cells

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. Previous data suggest that gypsy ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp70lucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy ENV-like protein. We have shown by immunostaining that the gypsy envelope protein is produced in 293GP/LNhsp70lucL.3 cells and that vector particles collected from these cells can infect Drosophila cells. Our results provide direct evidence that the infectious property of gypsy is due to its ORF3 gene product.     

杆状病毒反式激活蛋白IE-1诱导昆虫细胞凋亡及几种抑制剂对AcNPV诱导细胞凋亡的影响

为了探讨杆状病毒诱导细胞凋亡的机制,用含AcNPV-ie-1基因的重组质粒pGAM-ie-1转染斜纹夜蛾细胞SL－1和粉纹夜蛾细胞Tn-5B1。转染后24h通过光镜观察、DAPI荧光染料染色、DNA琼脂糖凝胶电泳等发现,SL－1发生了典型的凋亡,而同样的现象并没有在Tn-5B1细胞中出现。利用放线菌酮（cycloheximide,CHX)、莫能菌素（Monensin)及蚜栖菌素（aphidicolin)处理AcNPV感染的SL－1细胞,发现细胞凋亡被莫能菌素及蚜栖菌素抑制,被放线菌酮推迟。此结果为进一步研究杆状病毒诱导昆虫细胞凋亡的机制等奠定了基础。     

Mouse Model Reveals the Role of RERE in Cerebellar Foliation and the Migration and Maturation of Purkinje Cells

Nuclear receptors and their coregulators play a critical role in brain development by regulating the spatiotemporal expression of their target genes. The arginine-glutamic acid dipeptide repeats gene (Rere) encodes a nuclear receptor coregulator previously known as Atrophin 2. In the developing cerebellum, RERE is expressed in the molecular layer, the Purkinje cell layer and the granule cell layer but not in granule cell precursors. To study RERE''s role in cerebellar development, we used RERE-deficient embryos bearing a null allele (om) and a hypomorphic allele (eyes3) of Rere (Rere ^om/eyes3). In contrast to wild-type embryos, formation of the principal fissures in these RERE-deficient embryos was delayed and the proliferative activity of granule cell precursors (GCPs) was reduced at E18.5. This reduction in proliferation was accompanied by a decrease in the expression of sonic hedgehog (SHH), which is secreted from Purkinje cells and is required for normal GCP proliferation. The maturation and migration of Purkinje cells in Rere ^om/eyes3 embryos was also delayed with decreased numbers of post-migratory Purkinje cells in the cerebellum. During the postnatal period, RERE depletion caused incomplete division of lobules I/II and III due to truncated development of the precentral fissure in the cerebellar vermis, abnormal development of lobule crus I and lobule crus II in the cerebellar hemispheres due to attenuation of the intercrural fissure, and decreased levels of Purkinje cell dendritic branching. We conclude that RERE-deficiency leads to delayed development of the principal fissures and delayed maturation and migration of Purkinje cells during prenatal cerebellar development and abnormal cerebellar foliation and Purkinje cell maturation during postnatal cerebellar development.     

度他雄胺对大鼠附睾精子成熟和生育的影响

目的通过度他雄胺对大鼠附睾精子和生育的影响,探索调节雄性生育的睾丸后作用靶点。方法使用度他雄胺20和40 mg/(kg.d)大鼠灌胃给药,连续2周。给药结束后雄雌鼠按1∶2合笼,计算生殖指数;采用计算机辅助精子分析系统分析精子活力和形态;采用SYBR-14和PI双重荧光染色计算精子存活率;采用Elisa法测定大鼠睾酮(T)和双氢睾酮(DHT)血清浓度;采用HE染色法对各组睾丸、附睾进行组织学分析。结果度他雄胺低、高剂量组双氢睾酮浓度均显著下降,分别为0.54和0.28 nmol/L(P<0.01),精子活力明显降低,分别为39.0%和28.7%(P<0.01),畸形率分别增加为10.3%和15.6%(P<0.05),最后受孕率分别降为62.5%和38.4%。而睾酮水平和交配指数均无明显变化(P>0.05),睾丸和附睾亦无明显病理学改变。结论度他雄胺通过抑制DHT生成,影响附睾精子成熟而导致大鼠不育,为今后男性避孕和不育药物研发提供了新思路。     

Physicochemical Characterization of Recombinant Human Nerve Growth Factor Produced in Insect Cells with a Baculovirus Vector

Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion-exchange and reversed-phase chromatography to near homogeneity. The N-terminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (less than 0.08 mol of N-acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF from insect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimer's disease.     

The P2 Protein of Rice Dwarf Phytoreovirus Is Required for Adsorption of the Virus to Cells of the Insect Vector

Intact particles of rice dwarf phytoreovirus adsorbed to and entered monolayer-cultured cells of the insect vector Nephotettix cincticeps and multiplied within the cells. Particles that lacked the P2 protein neither attached to nor infected such cells. Furthermore, P2-free particles obtained from a transmission-competent isolate of the virus were unable to infect insect vectors that had been allowed to feed on these virus particles through a membrane. However, when such virus particles were injected into insects via a glass capillary tube they successfully infected the insects, which became able to transmit the virus. These results support the hypothesis that, while P2-free particles can neither interact with nor infect cells in the intestinal tract of the insect vector, they do retain the ability to infect such cells when physically introduced into the hemolymph by injection.     

Association of Rice Gall Dwarf Virus with Microtubules Is Necessary for Viral Release from Cultured Insect Vector Cells

Vector insect cells infected with Rice gall dwarf virus, a member of the family Reoviridae, contained the virus-associated microtubules adjacent to the viroplasms, as revealed by transmission electron, electron tomographic, and confocal microscopy. The viroplasms, putative sites of viral replication, contained the nonstructural viral proteins Pns7 and Pns12, as well as core protein P5, of the virus. Microtubule-depolymerizing drugs suppressed the association of viral particles with microtubules and prevented the release of viruses from cells without significantly affecting viral multiplication. Thus, microtubules appear to mediate viral transport within and release of viruses from infected vector cells.Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV), and Wound tumor virus, members of the genus Phytoreovirus in the family Reoviridae, multiply both in plants and in invertebrate insect vectors. Each virus exists as icosahedral particles of approximately 65 to 70 nm in diameter, with two concentric layers (shells) of proteins that enclose a core (1, 13). The viral genome of RGDV consists of 12 segmented double-stranded RNAs that encode six structural (P1, P2, P3, P5, P6, and P8) and six nonstructural (Pns4, Pns7, Pns9, Pns10, Pns11, and Pns12) proteins (reference 21 and references therein). The core capsid is composed of P3, the major protein, which encloses P1, P5, and P6 (12). The outer layer consists of two proteins, namely, P2 and P8 (10, 12).Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of viruses in the family Reoviridae. After infecting insect vector cell monolayers (VCMs) in culture with RDV, Wei et al. (19) examined the generation of RDV particles in and at the periphery of such viroplasms. VCMs are also useful for studies of RGDV, allowing detailed analysis of the synchronous replication and multiplication of this virus (14). In order to identify the viroplasms in RGDV-infected VCMs, we examined the subcellular localization of Pns7, Pns12, P5, and RGDV particles by confocal immunofluorescence microscopy. Pns7 and Pns12 of RGDV correspond to Pns6 and Pns11, respectively, which are components of the viroplasm of RDV (12, 19). RGDV P5 is a counterpart of RDV P5, a core protein that locates inside the viroplasm in RDV-infected cells. We inoculated VCMs with RGDV, purified by the method reported in reference 15, at a multiplicity of infection (MOI) of 1; fixed them 48 h postinfection (p.i.); probed the cells with Pns7-, Pns12-, P5-, and viral-antigen-specific antibodies (11, 12) that had been conjugated to fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO) or rhodamine (Sigma); and examined them by confocal microscopy, as described previously (19). In RGDV-infected cells, Pns7, Pns12, and P5 were detected as punctate inclusions (Fig. ​(Fig.1).1). Immunostained viral antigens formed ringlike structures around the punctate inclusions. When the images were merged, Pns7, Pns12, and P5 were colocalized in the punctate inclusions, indicating that these proteins were constituents of the viral inclusions (Fig. ​(Fig.1).1). Our observations revealed the similar respective localizations of the corresponding nonstructural proteins, core proteins, and viral particles of two phytoreoviruses, RGDV and RDV, in infected cells. Thus, Pns7 and Pns12 of RGDV had attributes common to their functional counterparts—Pns6 and Pns11, respectively—of RDV (19). The core protein P5 was located inside the viroplasms, and the viral antigens were distributed at the periphery of the viroplasms. The results, together, suggest that RGDV and RDV exploit similar replication strategies. Specific fluorescence was not detected in noninfected cells after incubation with Pns7-, Pns12-, P5-, and viral-antigen-specific antibodies (data not shown).Open in a separate windowFIG. 1.Subcellular localization of Pns7, Pns12, and P5 of RGDV and viral antigens in RGDV-infected VCMs 48 h p.i. Arrowheads show ringlike profiles of viral antigens that surround viral inclusions, which have been immunostained with the Pns12-specific antibodies. Arrows show the fibrillar profiles of immunostained viral antigens. Bars, 5 μm.In addition to the viral location at the periphery of the viral inclusions visualized as immunostained Pns12 (Fig. ​(Fig.1),1), the antigens were distributed as bundles of fibrillar structures, a form not observed in RDV-infected cells. To analyze the entity of the bundles of fibrillar structures, VCMs on coverslips were inoculated with RGDV at an MOI of 1, fixed at 48 h p.i., and examined by electron microscopy (EM), as described previously (19). We observed viral particles of approximately 70 nm in diameter in close association with the free ends, as well as along the edges, of tubules of approximately 25 nm in diameter (Fig. 2A to D). The abundant bundles of tubules with closely associated viral particles were clearly in contact with the periphery of granular, electron-dense inclusions of 800 to 1,200 nm in diameter (Fig. ​(Fig.2B),2B), namely, viroplasms. The dimensions and appearance of the tubular structures resembled those of microtubules (Fig. ​(Fig.2C)2C) (17). Transverse sections of tubules revealed arrays of closed circles of approximately 25 nm in diameter, with viral particles attached directly or via a filament to the circumference (Fig. ​(Fig.2D2D).Open in a separate windowFIG. 2.Association of RGDV particles with microtubules. (A) Electron micrograph showing RGDV particles associated with microtubules in virus-infected VCMs 48 h p.i. Bar, 300 nm. VP, electron-dense inclusion. (B) Virus-associated microtubules in contact with the periphery of the electron-dense inclusion indicated by a white rectangle in panel A. Bar, 300 nm. (C) Viral particles along the edges of tubules of approximately 25 nm in diameter. Bar, 150 nm. (D) Transverse sections of arrays of closed circles of approximately 25 nm in diameter with viral particles attached to their circumference directly (arrow) or via a filament (arrowhead). Bar, 150 nm. (E) Confocal micrograph showing the association of viral particles with microtubules in virus-infected VCMs 48 h p.i. Microtubules were stained with α-tubulin-specific antibodies conjugated to FITC; viral particles were stained with viral-antigen-specific antibodies conjugated to rhodamine. Arrowheads indicate the ringlike organization of viral antigens. Arrows show the colocalization of fibrillar profiles of viral antigens with microtubules. The insets show ringlike and fibrillar profiles of immunostained viral antigens. The circular areas inside the ringlike structures are viroplasms. Bar, 5 μm.Our observations suggested that RGDV particles might attach to microtubules in infected cells. To examine this possibility, we inoculated VCMs with RGDV at an MOI of 1, fixed the cells 48 h p.i., immunostained them with α-tubulin-specific antibodies conjugated to FITC and with viral-antigen-specific antibodies conjugated to rhodamine, and analyzed them by confocal microscopy, as described previously (19). Viral antigens were visualized as ringlike and fibrillar structures (Fig. ​(Fig.2E).2E). Double immunostaining of the infected cells revealed that a network of microtubule-based filaments colocalized with most of the fibrillar structures that represented viral antigens, confirming the association of viral particles with the microtubule-like inclusions visualized by EM (Fig. ​(Fig.2A).2A). Nonspecific reactions were not detected with either of the stainings (data not shown). Our results suggested that RGDV particles, which assembled at the periphery of viroplasms, might be transported along microtubules. Due to the lack of RGDV infectious clones fused with green fluorescent protein and the effective gene transfection system for VCMs, we could not observe the trafficking of RGDV particles along microtubules in living cells.We then used three-dimensional (3-D) electron tomographic microscopy (ET) to reveal a new level of morphological detail about the association of RGDV with microtubules. To produce 3-D reconstructions of RGDV-infected cells, we fixed, embedded, and sectioned infected leafhopper cells as described previously (5). We chose a representative region that showed numerous RGDV particles close to bundles of microtubules for this novel tomographic analysis. A single-axis tilt series was collected manually from −60° to 60° with 2° increments using an H9500SD EM (Hitachi, Tokyo) operated at 200 kV. These tomographic data were recorded at a defocus of 3.6 μm on the TVIPS 2k × 2k charge-coupled-device camera (TVIPS, Gauting, Germany). Microscopic magnification of ×15,000, providing 1.28 nm/pixel, was enough to view the microtubules and virus particles following tomographic reconstruction of the tilt series using IMOD (7). As shown in the 3-D tomogram in Fig. ​Fig.3,3, most of the RGDV particles were bound to the edges of bundles of microtubules. The RGDV particles along the edges of microtubules were arrayed in an orderly but uncrowded manner (Fig. ​(Fig.3).3). Our ET analysis also revealed that some viral particles were linked to filaments of approximately 10 nm in diameter (Fig. ​(Fig.3B).3B). Morphologically, these filaments resembled vimentin intermediate filaments (4). In many lines of cultured cells, vimentin intermediate filaments partially overlap the microtubules, and there is evidence that the two filament systems interact (3, 9, 20). Unfortunately, vimentin-specific monoclonal antibodies from mouse and rabbit did not react specifically with our leafhopper cells (data not shown), but the nature of the intermediate filaments was apparent from their dimensions, intracellular location, and organization. Thus, our ET analysis indicated that RGDV particles were able to associate directly and/or via intermediate filaments with microtubules.Open in a separate windowFIG. 3.ET analysis showing the association of RGDV particles with microtubules either directly or via intermediate filaments. (A) Translucent representation of the reconstructed viruses lining up with microtubules. (B) Slice of the reconstructed volumes from the inset of A to show the association of RGDV particles with intermediate filaments (arrows). Bars, 150 nm.To examine the role of the microtubules for RGDV activity, we added a microtubule-disrupting agent, either nocodazole (Sigma) or colchicine (Sigma), 2 hours after inoculation of VCMs with RGDV at an MOI of 1 and then continued the incubation for a further 46 h. Cells were fixed 48 h p.i. and stained with α-tubulin-specific antibodies conjugated to FITC (Sigma) and viral-particle-specific antibodies conjugated to rhodamine, with subsequent confocal fluorescence microscopy, as described previously (19). We tested a range of drug concentrations in preliminary experiments (data not shown) and determined optimal concentrations. Treatment of infected cells with 10 μM nocodazole or 5 μg/ml colchicine resulted in the complete disassembly of microtubules, with the accumulation of ringlike structures exclusively and no fibrillar structures representative of viral antigens in the cytoplasm (Fig. ​(Fig.4A).4A). These ringlike aggregates of viral antigens were confirmed to surround viroplasms when the latter were immunostained for Pns12, as described above and shown in Fig. ​Fig.1.1. Nonspecific reactions were not detected with either staining (data not shown). These results suggest that RGDV particles multiply around the viroplasm but are unable to distribute along the microtubules in the presence of the chemicals.Open in a separate windowFIG. 4.(A) Effects of microtubule-disrupting agents on the formation of microtubules and fibrillar profiles of immunostained viral antigens. Bars, 5 μm. The insets show the ringlike profiles of immunostained viral antigens after treatment with inhibitors, suggesting that viral replication occurs in the presence of each inhibitor. (B) Effects of drugs on the production of cell-associated (gray bars) and extracellular (black bars) viruses in VCMs infected with RGDV. The error bars indicate standard deviations.During the process of infection, microtubules play important roles in viral entry, intracellular trafficking, and extracellular release (2, 8, 16). We next investigated the effects of the microtubule-disrupting agents on the production in and release of viruses from virus-infected cells by the method described previously (18). Nocodazole or colchicine was added 2 h after inoculation of VCMs with RGDV at an MOI of 1, and incubation was continued for a further 46 h. The extracellular medium and the cells were collected separately. The medium was centrifuged for 30 min at 15,000 × g, and the supernatant was collected. The cells were subjected to three cycles of freezing and thawing to release viral particles. The viral titer of each sample was determined, in duplicate, by the fluorescent focus assay as described previously (6), with VCMs and a magnification of ×10. As shown in Fig. ​Fig.4B,4B, nocodazole (20 μM) and colchicine (10 μg/ml) caused a fivefold reduction in the number of released viruses, compared to that from untreated control infected cells. In contrast, each inhibitor at the selected dose failed to significantly reduce the titer of cell-associated viruses (less then 5% compared to that from untreated control). These results suggest that the inhibitors impeded the release of viruses into the medium without affecting viral production in infected cells. We do not yet understand why the viral titer was not elevated in drug-treated cells from which viral release was inhibited. However, our data show clearly that disruption of microtubules directly inhibited the release of mature viral particles from infected cells.In conclusion, EM, ET, immunofluorescence staining, and experiments with two inhibitors support the hypothesis that the transport of RGDV from viroplasms to the plasma membrane and into the medium is dependent on microtubules. In the case of RDV, vesicular compartment-containing viral particles that locate adjacent to the viroplasms were considered to play an important role in the transport and release of the virus from the viroplasm to the culture medium in infected VCMs (18). On the other hand, a fibrillar structure (Fig. ​(Fig.11 and ​and2),2), not observed in RDV-infected cells, was considered to function in the trafficking of RGDV from viroplasm into the culture medium (Fig. ​(Fig.4)4) in the present study. RGDV and RDV, both members of the Phytoreovirus genus, have some common biological and biochemical properties but are distinct from each other (13). For example, viruses are restricted to phloem-related cells in RGDV-infected plants but distributed in many types of cells in RDV-infected plants, and a P2 protein with a function to adsorb to and/or penetrate into insect vector cells is present in RGDV and absent in RDV in particles purified using carbon tetrachloride. The present molecular cytopathological study revealed one more difference between the viruses: they have different means for transporting and releasing infectious particles to the cell exterior. The presence of such a molecular mechanism may accelerate the secondary infections by the viruses in infected vector insects, and the high propagation speed would allow the viruses to complete infection cycles through insects and plants.     

The Role of Cytochrome c on Apoptosis Induced by Anagrapha falcifera Multiple Nuclear Polyhedrosis Virus in Insect Spodoptera litura Cells

There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell- free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.     

转基因小鼠模型的载体构建和在人肝癌HepG2细胞中的表达

目的:构建转基因小鼠模型的载体并检测在人肝癌HepG2中的表达效果.方法:将n-3多不饱和脂肪酸脱氢酶基因fat-1插入到真核表达载体(pcDNA3.1(+)myc-HisA)中,构建重组表达载体pcDNA3.1(+)myc-His A-fat-1,用脂质体介导的方法转染到人肝癌HepG2细胞中,RT-PCR检测fat-l基因的表达,MTT法分析fat-l基因对HepG2细胞增殖的影响,气相色谱分析检测fat-l基因对HepG2细胞n-6/n-3多聚不饱和脂肪酸(PUFAs)比例的影响.结果:成功地构建了真核表达裁体peDNA3.1(+)myc-HisA-fat-1,并能在HepG2细胞内有效异源表达.48h后可检测到fat-l mRMA的条带.与对照细胞相比,fat-l基因有效地抑制了人肝癌细胞HepG2细胞的增殖(70%,p＜0.01),降低了n-6/n-3 PUFAs比例.结论:pcDNA3.1(+)myc-His A-fat-l重组载体构建成功并能在肝癌细胞中有效的表达,可以作为下一步转基因小鼠的合适载体.     

Maturation of Autophagic Vacuoles in Mammalian Cells

The autophagic process was first described in mammalian cells several decades ago. After their formation as double-membraned vacuoles containing cytoplasmic material, autophagic vacuoles or autophagosomes undergo a stepwise maturation including fusion with both endosomal and lysosomal vesicles. However, the molecular mechanisms regulating these fusion steps have begun to emerge only recently. The list of newly discovered molecules that regulate the maturation of autophagosomes to degradative autolysosomes includes the AAA ATPase SKD1, the small GTP binding protein Rab7, and possibly also the Alzheimer-linked presenilin 1. This review combines previous data on the endo/lysosomal fusion steps during autophagic vacuole maturation with recent findings on the molecules regulating these fusion steps. Interestingly, autophagic vacuole maturation appears to be blocked in certain human diseases including neuronal ceroid lipofuscinosis and Danon disease. This suggests that autophagy has important housekeeping or protective functions, because a block in autophagic maturation causes a disease.     

诱导多能干细胞来源的肝细胞在HCV感染模型中的应用*

诱导多能干细胞(induced pluripotent stem cells,iPSCs)与胚胎干细胞(embryonic stem cells,ESCs)类似,是一类具有自我更新和无限增殖潜能的细胞, 并且能诱导分化为机体各胚层所有类型的细胞。因iPSCs来源于机体本身,规避了ESCs的免疫排斥和医学伦理等问题,具有极大的研究前景及应用潜能。大量研究表明,诱导多能干细胞分化的肝样细胞(iPS-derived hepatocyte-like cells,iHLCs)已广泛运用于HCV体内外感染模型的建立,并用于研究HCV的发病机制、宿主基因在HCV致病机制和筛选新型抗HCV药物及疫苗的研发。主要对iPSCs的来源、从不同策略诱导iPSCs成为功能性肝细胞的研究方法及其在HCV感染模型中的应用进行归纳总结。     

诱导多能干细胞来源的肝细胞在HCV感染模型中的应用*

诱导多能干细胞(induced pluripotent stem cells,iPSCs)与胚胎干细胞(embryonic stem cells,ESCs)类似,是一类具有自我更新和无限增殖潜能的细胞, 并且能诱导分化为机体各胚层所有类型的细胞。因iPSCs来源于机体本身,规避了ESCs的免疫排斥和医学伦理等问题,具有极大的研究前景及应用潜能。大量研究表明,诱导多能干细胞分化的肝样细胞(iPS-derived hepatocyte-like cells,iHLCs)已广泛运用于HCV体内外感染模型的建立,并用于研究HCV的发病机制、宿主基因在HCV致病机制和筛选新型抗HCV药物及疫苗的研发。主要对iPSCs的来源、从不同策略诱导iPSCs成为功能性肝细胞的研究方法及其在HCV感染模型中的应用进行归纳总结。