In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera)

Summary The in vitro host range of a newly isolated baculovirus from the diamondback moth Plutella xylostella was tested against six lepidopteran cell lines. Two baculoviruses with host ranges from the alfalfa looper Autographa californica (A. californica multiple nucleopolyhedrovirus, AcMNPV) and the celery looper Anagrapha falcifera (AfMNPV) were also included in this study for comparative purposes. PxMNPV replicated in all six cell lines and produced occlusion bodies, with HV-AMI and TN-CLI cells producing the highest viral titers and greatest number of occlusion bodies. There was no significant replication of AcMNPV and AfMNPV in the HZ-FB33 cell line and thus no production of occlusion bodies. The restriction endonuclease profiles of the three baculoviruses showed similarities but could be readily distinguished from each other. Either HV-AM1 or TN-CL1 would be suitable cell lines for the in vitro production of PxMNPV.     

Asp 187 and Phe 190 residues in lethal factor are required for the expression of anthrax lethal toxin activity

Anthrax toxin consists of three proteins, protective antigen, lethal factor, and edema factor. Protective antigen translocates lethal factor and edema factor to the cytosol of mammalian cells. The amino-termini of lethal factor and edema factor have several homologous stretches. These regions are presumably involved in binding to protective antigen. In the present study we have determined the role of one such homologous stretch in lethal factor. Residues 187AspLeuLeuPhe190 were replaced by alanine. Asp187Ala and Phe190Ala were found to be non-toxic in combination with protective antigen. Their protective antigen-binding ability was drastically reduced. We propose that Asp187 and Phe190 are crucial for the expression of anthrax lethal toxin activity.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

Characterization of invertebrate cell lines

Summary Cell extracts of five mosquito cell lines and a tick cell line were examined for four cellular isozymes using a cellulose-acetate electrophoretic technique. This method distinguished the cell lines that were derived from the different species. Intraspecies distinctions were not made using the cell lines tested; the significance of this finding is discussed. The usefulness of this technique in identifying a potentially mislabeled cell line was demonstrated. This research was supported by contracts, DADA 17-72C-2170 of the U.S. Army and N00014-78C-0104 of the U.S. Office of Naval Research and grants from the World Health Organization and the Rockefeller Foundation.     

Replication ofAutographa californica nuclear polyhedrosis virus in a thymidine kinse deficientSpodoptera exigua cell line (SE-UCR-1A)

Summary Cultivation of aSpodoptera exigua cloned cell line (SE-UCR-1A) for 8 to 9 mo. in a medium containing increasing amounts of bromodeoxyuridine (BUdr) resulted in the selection of a BUdr-resistant subline unable to grow in TNMFH medium supplemented with HAT (hypoxanthine, 10⁻⁴ M; aminopterin, 10⁻⁷ M; thymidine, 10⁻³ M). Subsequent assay of this subline revealed an absence of thymidine kinase (TK) activity. The specific activity of the wild-type (wt) cells was 878±192 counts per min (cpm)/μg supernatant protein compared to 9 cpm/μg for the BUdr-resistant, HAT-sensitive subline. In addition the wt activity was inhibited >90% by addition of BUdr to the assay, indicating that the activity is predominantly due to TK and not to a nonspecific nucleoside phosphotransferase. The morphology of the TK-deficient (−) cells was indistinguishable from that of wt cells. The doubling time for wt cells in TNMFH was 16 h; however, in TNMFH-HAT it was 36 h. In comparison the TK(−) cells in TNMFH had a doubling time of 61 h. Cultivation of TK(−) cells in nonselective TNMFH for 14 mo. to date has not changed the TK(−) characteristic of the subline. The host-cell TK was not required for development of progeny virus fromAutographa californica NPV inoculum. Although similar numbers of cells were infected (80 to 90%) in the wt and TK(−) lines and extracellular virus was generated at similar rates to similar titers in both media, the initial appearance of virus in the medium of TK(−) cell was delayed 10 to 20 h compared to wt cells. In addition, polyhedra appearance was similarly delayed in TK(−) cells and only 20 to 25% of the cells contained >10 polyhedra per cell compared to 75 to 90% for wt cells. Also, during infection of wt cells, specific activity of TK increased twofold peaking at 20 to 30 h postinfection, yet there was no stimulation of TK activity in infected TK(−) cells.     

炭疽毒素受体结构和功能研究进展

肿瘤血管内皮标志物8(TEMS)和毛细血管形态发生蛋白2(CMG2)是已知的两个炭疽毒素受体,它们的主要功能是当炭疽杆菌侵染细胞时介导炭疽毒素进入宿主细胞.这两个受体都是涉及到细胞外基质动态平衡的I型跨膜蛋白,并且都因为它们在血管生成或血管内皮细胞中表达增强而被发现.有研究发现TEM8能够调节内皮细胞迁移和血管形成,而CMG2则在内皮细胞增殖过程中起重要作用.进一步的研究显示它们与整联蛋白同源性较高,但它们确切的生理功能和作用机制尚不明确.本文中,主要讨论这两种蛋白的结构和它们作为炭疽毒素受体介导炭疽毒素进入细胞的分子机制,然后我们简单探讨一下TEM8在靶向肿瘤血管内皮细胞的抗血管生成和抗肿瘤疗法方面的研究进展,最后我们展望了下一步炭疽毒素受体研究的热点-它们的配体及生理功能研究.     

RNAi介导的棉铃虫氨肽酶N基因Haapn1和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N（aminopeptidase N, APN）和钙粘蛋白（cadherin）是存在于鳞翅目昆虫中肠刷状缘膜囊（brush border membrane vesicles, BBMV）上Bt毒素Cry1A的受体。本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapn1和钙粘蛋白基因Ha_BtR双链RNA（dsRNA）注入棉铃虫4龄幼虫体内, 以研究这两种受体基因沉默后对Cry1Ac毒力的影响。结果表明: 注射dsRNA（1 μg/头）进行基因沉默后, Haapn1 mRNA表达量比注射缓冲液（elution solution, ES）的对照下降了30%~49%, Ha_BtR mRNA表达量下降了30%~37%。注射Haapn1 dsRNA的幼虫在40和70 μg/cm² Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫, 而在 100 和 170 μg/cm² Cry1Ac原毒素处理下两者死亡率无显著差异; Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异。当同时注射Haapn1及Ha_BtR dsRNA后, 干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降。本研究进一步证明了棉铃虫Haapn1和Ha_BtR均是Bt毒素Cry1Ac的功能受体, 这两种受体蛋白共同参与Cry1Ac的毒杀作用过程。该结果也提示, Haapn1或Ha_BtR基因产生突变都可能导致棉铃虫对Cry1Ac产生抗性。     

Characterization of invertebrate cell lines

Summary The usefulness of four serologic techniques for distinguishing five selected lepidopteran cell lines was evaluated; the techniques included complement fixation, hemagglutination. immunodiffusion, and immunoelectrophoresis. The five selected lepidopteran cell lines represent three taxonomic families of Lepidoptera with one family, Noctuidae, containing two cell lines derived from insects within the same genus. The five cell lines were crossreactive in complement-fixation tests, but the lines were distinguishable at a familic level when two units of antigens were used in the test. Agglutination of goose erythrocytes was not observed with the antigens over a pH range of 5.8 to 7.2 at 4°C or ambient temperature. Immunodiffusion tests demonstrated a common cross-reactive antigen(s), but spurs of partial identity and the presence of extra precipitin bands were indicative that differentiation at a familic level was possible. Immunoelectrophoresis of the cellular antigens also revealed common cross-reactive precipitin arcs, but the number and clarity of arcs in homologous systems was increased such that four of the five cell lines were distinguishable. A basic protein was consistently seen in the homologous system, but it was absent in the heterologous systems. Although these data suggest that immunoelectrophoresis was the best serologic technique for distinguishing the five lepidopteran cell lines, the shortcomings of this approach are also discussed. This research was supported in part by the World Health Organization, The Rockefeller Foundation, and U.S. Public Health Service Grant AI-13727.     

Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin

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Opioid Receptors in Magnesium-Digitonin-Solubilized Rat Brain Membranes Are Tightly Coupled to a Pertussis Toxin-Sensitive Guanine Nucleotide-Binding Protein

Opioid receptors solubilized in Mg2+-digitonin (2%, wt/vol) from Mg2+-pretreated rat brain membranes maintain, in addition to high-affinity opioid agonist binding, the modulation by guanine nucleotides. One of the modes of expression of the latter property is an attenuation of agonist binding by guanine nucleotides in the presence of Na+. To investigate the molecular basis of this modulation and to identify the G protein(s) involved, the soluble receptors were [32P]ADP-ribosylated by means of Bordetella pertussis toxin and subjected to molecular size exclusion chromatography. In addition, soluble extracts were chromatographed on lectin and hydrophobic affinity columns. The binding of 35S- and 3H-labelled analogues of GTP was also monitored in the species separated. The oligomeric G protein-coupled opioid receptors and the guanine nucleotide/pertussis toxin-sensitive species showed similar chromatographic properties in all three systems. This indicates that the biochemically functional G protein-opioid receptor complex formed in Mg2+-pretreated membranes in the absence of an agonist is stable in digitonin solution and to chromatographic separation. Further analysis showed that the guanine nucleotide modulation of opioid receptors is via the pertussis toxin substrates with Mr of 41,000 and 39,000, which are identified as Gi and Go alpha subunits, respectively.     

Multiple Coupling of Human D5 Dopamine Receptors to Guanine Nucleotide Binding Proteins GS and Gz

Abstract: We have demonstrated previously that D1 dopamine receptors are coupled to both G_sα and G_oα. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH₄C₁ cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both G_sα and cholera toxin-insensitive G proteins. The interaction between D5 receptors and G_sα was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to G_oα or G_iα. D5 receptors were also not coupled to G_qα and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF⁻₄-sensitive, N -ethylmaleimide-resistant G protein. Anti-G_zα caused immunoprecipitation of 24.2 ± 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and G_zα. The coupling to G_zα was specific for D5 receptors, because similar associations were not detected between D1 receptors and G_zα.     

The electrical and mechanical properties of the proleg retractor muscle of the Chinese oak silkmoth larva

ABSTRACT. The main proleg retractor muscle (y) of Antheraea pernyi Guer. (Lepidoptera, Saturniidae) larvae consists of three layers of fibres. The innermost layer of fibres is dually innervated. Cobalt backfills of the two motor neurones, in nerve 2d, showed the somata to be situated ventrally and anteriorly in the same segmental ganglion, ipsilateral to the filled nerve. Intramuscular microelectrode recordings showed excitatory junction potentials (EJPs) of two distinct amplitudes, both of which were relatively slow. However, 26% of the larger amplitude EJPs had an active membrane response. The EJPs and mechanical responses both summated at low stimulation frequencies. Large EJPs resulted in a much greater development of tension than small ones. Extracellular stimulation of nerve lbiii modulated peak tension and peak rate of relaxation.     

Internalization and Proteolytic Action of Botulinum Toxins in CNS Neurons and Astrocytes

Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red-conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins.     

Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua

Hemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14 +/- 0.45 microm and 2.84 +/- 0.14 microm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10 +/- 0.21 microm x 2.00 +/- 0.11 microm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.