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At least 703 records · Page 39Linked to original sources
PubMed1999

A new picorna-like virus, PnPV, isolated from ficus transparent wing moth, Perina nuda (Fabricius).

Two viruses, Perina nuda nucleopolyhedrovirus and a new picorna-like virus, were previously isolated from P. nuda larvae with flacherie. In this study the new picorna-like virus was characterized using physical and biochemical methods. This small virus appears to belong to the family Picornaviridae and we propose the name PnPV. PnPV can be propagated in its homogenous cell line, NTU-PN-HH. PnPV purified from the cell line resembles PnPV isolated from insects: under electron microscopy, it exhibits icosahedral symmetry, measures 30 nm in diameter, and has no envelope and no distinct surface structure in negatively stained preparations. In addition, we show here that PnPV has a buoyant density of 1.381 g/ml in cesium chloride, the viral genome was composed of one single-stranded RNA molecule with a length of 10 kb, and poly(A) tract and polyacrylamide gel electrophoresis of purified viral particles revealed three major (31.5, 29.7, and 28.4 kDa) and three minor (27. 0, 24.5, and 4.0 kDa) structural proteins.

Animals↗
PubMed1997

Production and purification of novel secreted human proteins.

Our objective during the last year was to produce and purify 50-80 novel, secreted human proteins identified via high throughput cDNA sequencing and computer analysis. We chose the baculovirus expression vector system in order to obtain secreted, correctly folded, bioactive proteins. Recombinant (re-)baculoviruses (BV) were plaque purified, and pulse-labeling was used to verify the synthesis and secretion of the re-proteins. N-terminal microsequencing was performed to simultaneously confirm the identity of the protein(s) as well as the signal peptide (SP) cleavage site(s). Following sequence confirmation, the proteins were purified to homogeneity and functional assays carried out to determine potential therapeutic applications. We identified proteins with antiviral activity, several novel growth factors, proteins influencing the differentiation of specific cell types, novel proteases and protease inhibitors among others. Certain proteins were expressed both in insect cells and in CHO stable cell lines. In the cases analyzed, we found that the same SP cleavage site was utilized in the two expression systems. Significant differences were observed in the carbohydrate moieties attached to the proteins, though no effects on the biological activity due to these differences have been demonstrated. The BV system has served as a viable alternative for the high throughput, high fidelity expression of many novel secreted human genes. To date, more than 75 new genes have been expressed, and the re-proteins purified. This expression system combines many favorable traits including relative speed, moderate cost but perhaps most importantly, the production of biologically active proteins.

Baculoviridae↗
PubMed1999

Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti.

Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5'-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes.

Aedes↗
PubMed2000

Phosphine-induced oxidative damage in rats: attenuation by melatonin.

Phosphine (PH(3)), from hydrolysis of aluminum, magnesium and zinc phosphide, is an insecticide and rodenticide. Earlier observations on PH(3)-poisoned insects, mammals and a mammalian cell line led to the proposed involvement of oxidative damage in the toxic mechanism. This investigation focused on PH(3)-induced oxidative damage in rats and antioxidants as candidate protective agents. Male Wistar rats were treated ip with PH(3) at 2 mg/kg. Thirty min later the brain, liver, and lung were analyzed for glutathione (GSH) levels and lipid peroxidation (as malondialdehyde and 4-hydroxyalkenals) and brain and lung for 8-hydroxydeoxyguanosine (8-OH-dGuo) in DNA. PH(3) caused a significant decrease in GSH concentration and elevation in lipid peroxidation in brain (36-42%), lung (32-38%) and liver (19-25%) and significant increase of 8-OH-dGuo in DNA of brain (70%) and liver (39%). Antioxidants administered ip 30 min before PH(3) were melatonin, vitamin C, and beta-carotene at 10, 30, and 6 mg/kg, respectively. The PH(3)-induced changes were significantly or completely blocked by melatonin while vitamin C and beta-carotene were less effective or inactive. These findings establish that PH(3) induces and melatonin protects against oxidative damage in the brain, lung and liver of rats and suggest the involvement of reactive oxygen species in the genotoxicity of PH(3).

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Phage display selection on whole cells yields a peptide specific for melanocortin receptor 1.

A phage display system for the selection of peptides binding to heterologously expressed human melanocortin receptor 1 on the surface of insect cells has been established. It could be shown that phage particles displaying the natural ligand alpha-melanocyte-stimulating hormone bind selectively to cells expressing this receptor and that these phages exhibit biological activity on mouse B16F1 melanoma cells. Insect cells were superior to other cell lines tested and have been used to select binders from a small library, in which critical determinants (Phe7-Arg8-Trp9) were kept, whereas the flanking regions where allowed to variate freely. One peptide displaying little similarity with native hormone was found that binds to the receptor also in its free form with an affinity of 7 nM. It showed a remarkable selectivity for this receptor, because it binds to the other melanocortin receptor subtypes with a maximum affinity of 21 microM. This is the first time phage display has been used successfully with G-protein-coupled receptors lacking an extracellular binding domain.

Amino Acid Sequence↗
PubMed2002

Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction.

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (P(hCMV)) or the human elongation factor 1alpha (P(hEF1)(alpha)), (iii) P(hCMV-) or P(PGK-) (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5'U3, R-U5-psi+-SD, cPPT-RRE-SA and 3'LTR(DeltaU3) modules or placed within the 5'U3 (-78 bp) and 3'LTR(DeltaU3) (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5'LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3'LTR(DeltaU3) element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.

Animals↗
PubMed1999

Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins.

Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. Here, we show that the expression of human cyclin A1 and cyclin A1-associated kinase activities was regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein were present at very low levels in cells at the G0 phase. They increased during the progression of the cell cycle and reached the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaked at the G2/M phase. We report that cyclin A1 could bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 was greatly enhanced when cyclin A1 was complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 were observed in vivo in several cell lines. When cyclin A1 was coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex had kinase activities for histone H1, E2F-1, and the Rb family of proteins. Our results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells.

Animals↗
PubMed2005

Minos as a genetic and genomic tool in Drosophila melanogaster.

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5'-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient "jump-starting" scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and approximately 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.

Animals↗
PubMed1999

An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins.

A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expressed in locust oocytes, fat body, brain, and midgut. This receptor appeared to be a homolog of the mammalian very low density lipoprotein receptor as it contains eight cysteine-rich repeats in its putative ligand-binding domain. When transiently expressed in COS-7 or stably expressed in LDL receptor-deficient CHO cells, the receptor mediates endocytic uptake of high density lipophorin (HDLp), an abundant lipoprotein in the circulatory compartment of insects. Moreover, in the latter cell line, we demonstrated that an excess of unlabeled HDLp competed with fluorescent labeled HDLp for uptake whereas an excess of human LDL did not affect uptake. Expression of the receptor mRNA in fat body cells is down-regulated during adult development, which is consistent with the previously reported down-regulation of receptor-mediated endocytosis of lipophorins in fat body tissue (Dantuma, N. P., M.A.P. Pijnenburg, J. H. B. Diederen, and D. J. Van der Horst. 1997. J. Lipid Res. 38: 254-265). The expression of this receptor in various tissues that internalize circulating lipophorins and its capability to mediate endocytosis of HDLp indicate that this novel member of the LDL receptor family may function as an endocytic lipophorin receptor in vivo.

Adipocytes↗
PubMed2004

Biosynthesis of human-type N-glycans in heterologous systems.

Insects, yeasts and plants generate widely different N-glycans, the structures of which differ significantly from those produced by mammals. The processing of the initial Glc2Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 in the endoplasmic reticulum shows significant similarities among these species and with mammals, whereas very different processing events occur in the Golgi compartments. For example, yeasts can add 50 or even more Man residues to Man(8-9)GlcNAc2, whereas insect cells typically remove most or all Man residues to generate paucimannosidic Man(3-1)GlcNAc2N-glycans. Plant cells also remove Man residues to yield Man(4-5)GlcNAc2, with occasional complex GlcNAc or Gal modifications, but often add potentially allergenic beta(1,2)-linked Xyl and, together with insect cells, core alpha(1,3)-linked Fuc residues. However, genomic efforts, such as expression of exogenous glycosyltransferases, have revealed more complex processing capabilities in these hosts that are not usually observed in native cell lines. In addition, metabolic engineering efforts undertaken to modify insect, yeast and plant N-glycan processing pathways have yielded sialylated complex-type N-glycans in insect cells, and galactosylated N-glycans in yeasts and plants, indicating that cell lines can be engineered to produce mammalian-like glycoproteins of potential therapeutic value.

Animals↗
PubMed2001

Dynamic nuclear localization of the baculovirus proteins IE2 and PE38 during the infection cycle: the promyelocytic leukemia protein colocalizes with IE2.

The early gene products IE2 and PE38 of Autographa californica multicapsid nuclear polyhedrosis virus localize to distinct nuclear domains after transient expression. Here, the nuclear localization pattern and the putative association with cellular proteins have been determined during virus infection to shed light on the functional significance of the nuclear domains. IE2 was always localized to distinct nuclear structures while PE38 was partly present in nuclear dots. Confocal imaging indicated colocalization of PE38 and IE2 to common domains, prominently at 2 h p.i. The nuclear dot localization of PE38 in infected cells was different from that in transfected cells. Hence, we have performed cotransfection experiments that suggested that a viral factor influences the nuclear distribution. Since the promyelocytic leukemia protein (PML) that localizes to distinct nuclear multiprotein complexes termed ND10/PODs in mammalian cells functions as a target for some immediate early viral proteins, we have investigated whether baculovirus proteins act similarly. Transiently expressed IE2 and PE38 were found to be associated with endogenous PML in the mammalian cell line BHK21. Infection with a recombinant virus that expresses the human pml gene in insect cells reveals IE2 and PML to be colocalized during the early phase of infection followed by a redistribution of both proteins. Taken together our results provide first evidence that the early baculovirus protein IE2 associates at least with one component of mammalian PODs during virus infection, suggesting that POD-like structures can be formed in insect cells.

Animals↗
PubMed2000

Insect cells as HLA-restricted antigen-presenting cells for the IFN-gamma elispot assay.

Measurement of specific cellular immune responses in patients undergoing immunotherapy is difficult. Established approaches, including cytotoxicity (e.g., 51Cr release) and cytokine release assays, require in vitro culturing for several weeks or more of patients' peripheral blood mononuclear cells (PBMC) and the addition of exogenous cytokines. Therefore, the immunological response does not reflect in vivo conditions. To address these disadvantages, we have used an interferon-gamma (IFN-gamma) Elispot assay for detecting peptide-specific CD8(+) lymphocytes in PBMC. A limitation of this assay is the lack of a reproducible source of antigen-presenting cells (APCs). Currently available APCs often lead to significant background levels. It has been shown that transfected insect cells can express empty MHC class I molecules on their surface. We have transfected Drosophila melanogaster S2 cells and the Lepidopteran line Sf9 with the gene encoding human HLA-A2.1. We demonstrate that insect cells expressing a human HLA molecule effectively function as APCs in the IFN-gamma Elispot assay. Initially the feasibility of the assay was assessed using CD8(+) T cells from HLA-A2.1(+) donors with known reactivity against an HLA-A2.1-binding epitope of the influenza matrix protein. Use of insect cells as APCs abrogated background spots, increasing sensitivity. We further observed that a short-term prestimulation of PBMC with peptide-pulsed insect cells markedly enhanced the frequency of peptide-specific T cells that could be measured in the Elispot assay without increasing the background. This approach was then used to measure CD8(+) T cell reactivity to a peptide from tyrosinase, an antigen that is processed and presented by melanoma cells. Insect cells expressing human HLA molecules provide a standard APC for monitoring CD8(+) T cell responses to tumor and viral peptides during immunotherapy.

Animals↗
PubMed1980

Chemically-defined media for production of insect cells and viruses in vitro.

Two chemically-defined media are described. They support the growth of a) an established cell line of Spodoptera frugiperda cells and b) two established mosquito cell lines from Aedes aegypti and Anopheles gambiae. The replication of Autographa californica Nuclear Polyhedrosis Virus (ACNPV) in S. frugiperda cells grown in a defined medium is reported.

Aedes↗
PubMed1990

Expression of the gene encoding firefly luciferase in insect cells using a baculovirus vector.

A cDNA encoding the firefly luciferase [Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was cloned downstream from the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda clone-9 cells. Synthesis of luciferase (Luc) was accurately measured in insect cells growing in a 96-well plate, by a simple, rapid, nonradioactive, inexpensive and sensitive method based on fogging of x-ray film. Luc was produced in a coordinate fashion during virus infection. The Luc synthesized in insect cells was not secreted into the medium but was contained within the cell. Our findings suggest that Luc can be used as a superior reporter enzyme for molecular genetic analyses of baculovirus regulatory signals involved in high level expression of foreign genes, protein processing, targeting and stability in insect cells.

Animals↗
PubMedDate not supplied

Autoinduction of nuclear hormone receptors during metamorphosis and its significance.

Metamorphosis is a most dramatic example of hormonally regulated genetic reprogramming during postembryonic development. The initiation and sustenance of the process are under the control of ecdysteroids in invertebrates and thyroid hormone, 3,3', 5-triiodothyronine, in oviparous vertebrates. Their actions are inhibited or potentiated by other endogenous or exogenous hormones - juvenile hormone in invertebrates and prolactin and glucocorticoids in vertebrates. The nuclear receptors for ecdysteroids and thyroid hormone are the most closely related members of the steroid/retinoid/thyroid hormone receptor supergene family. In many pre-metamorphic amphibia and insects, the onset of natural metamorphosis and the administration of the exogenous hormones to the early larvae are characterized by a substantial and rapid autoinduction of the respective nuclear receptors. This review will largely deal with the phenomenon of receptor autoinduction during amphibian metamorphosis, although many of its features resemble those in insect metamorphosis. In the frog Xenopus, thyroid hormone receptor autoinduction has been shown to be brought about by the direct interaction between the receptor protein and the thyroid-responsive elements in the promoter of its own gene. Three lines of evidence point towards the involvement of receptor autoinduction in the process of initiation of amphibian metamorphosis: (1) a close association between the extent of inhibition or potentiation by prolactin and glucocorticoid, respectively, and metamorphic response in whole tadpoles and in organ and cell cultures; (2) thyroid hormone fails to upregulate the expression of its own receptor in obligatorily neotenic amphibia but does so in facultatively neotenic amphibia; and (3) dominant-negative receptors known to block hormonal response prevent the autoinduction of wild-type Xenopus receptors in vivo and in cell lines. Autoinduction is not restricted to insect and amphibian metamorphic hormones but is also a characteristic of other nuclear receptors (e.g., retinoid, sex steroids, vitamin D(3) receptors) where the ligand is involved in a postembryonic developmental function. A wider significance of such receptor autoregulation is that the process may also be important for mammalian postembryonic development.

Amphibians↗
PubMed1990

Immunochemical analysis of a monoclonal antibody recognizing a terminal glucuronic acid-containing epitope of insect acidic glycolipids.

A cloned, hybridoma cell-line was established that secreted the monoclonal antibody CAF-I following stimulation of the donor Balb/c mouse spleen cells by the total acidic fraction glycolipids of the third-instar larvae of Calliphora vicina (Insecta:Diptera). The monoclonal antibody isotype was IgG3 By qualitative (TLC-immunostaining) and semi-quantitative (enzyme-linked immunosorbent assay) methods, and comparison with the cross-reactivity of known monoclonal antibodies, the epitope was specifically located on the terminal, non-reducing end of the oligosaccharide chain of most of the insect acidic glycolipids. Following isolation of the two main acidic glycolipids of C. vicina larvae (A5c and Az5c), exoglycosidase treatment characterized the terminal disaccharide CAF-I epitope as glucuronic acid bound to subterminal galactose, both in the beta-anomeric configuration: G1cA beta-Ga1 beta-. The immunohistological distribution of this epitope in the dipteran, Drosophila melanogaster, showed its main expression to be in the imaginal discs and brain of the third-instar larva, and the retinula cells of the ommatidial elements of the compound eye retina of the adult female.

Animals↗
PubMed2003

Providence virus: a new member of the Tetraviridae that infects cultured insect cells.

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia beta virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.

Animals↗