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H A Wood

Publications and source records attributed to H A Wood.

At least 19 recordsLinked to original sources
PubMed2000

Effects of incubation temperature on the dose-survival time relationship of Trichoplusia ni larvae infected with Autographa californica nucleopolyhedrovirus.

The interaction between virus multiplication and host development was studied by determining the survival time of Trichoplusia ni larvae inoculated with a wide range of doses of Autographa californica nucleopolyhedrovirus and incubated at five different temperatures spanning the biologically relevant range. The results support earlier findings that the course of baculovirus infection follows the mathematical description of the birth-death model. Both in vivo rate of virus increase and larval growth rate increased linearly with increasing temperature from 14 to 29 degrees C; developmental zeros for virus replication and larval growth were estimated from these data to be 10.2 and 10.4 degrees C, respectively. The data were used to generate a description of the combined effects of dose and temperature on median survival time at doses greater than the LD(50). Implications of the lag times before onset of viral replication and between cessation of replication and larval death with respect to model-based estimation of the critical population level (i.e., amount of virus in the host just prior to death) are discussed.

Animals↗
PubMed1999

Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: influence of ammonia, time of harvest, temperature, and dissolved oxygen.

Glycosylation is both cell line and protein dependent. Culture conditions can also influence the profile of glycoforms produced. To examine this possibility in the insect cell/baculovirus system, structures of N-linked oligosaccharides attached to SEAP (human secreted alkaline phosphatase), expressed under various culture conditions in BTI Tn5B1-4 cells, were characterized using FACE (fluorescence-assisted carbohydrate electrophoresis). Parameters varied were time of harvest, ammonia added during infection, dissolved oxygen, and temperature. It was found that glycosylation in the insect cell/baculovirus expression system is a robust, stable system that is less perturbed by variations in culture conditions than the level of protein expression. Addition of ammonia and low oxygen conditions affected SEAP expression, but not the oligosaccharide profile of SEAP. Time of SEAP harvest increased the amount of alpha-mannosidase resistant structures from 4.1% at 34 hours postinfection (h pi), to 5.0% at 100 h pi, and to 7.5% at 120 h pi. These structures were primarily sensitive to N-acetylhexosaminidase digest, although a small amount was insensitive to both mannosidase and N-acetyl-hexosaminidase digests. Lowering the temperature from 28 degrees C to 24 degrees C or even 20 degrees C, resulted in a twofold increase in oligosaccharides containing terminal alpha(1,3)-mannose residues. This condition did not affect the amount of mannosidase-resistant structures. However, this could result in more complete glycosylation of recombinant proteins in the BTI Tn5B1-4 cell line, because more structures with the potential for further processing would be produced.

Alkaline Phosphatase↗
PubMed1999

Monosaccharide compositions of Danaus plexippus (monarch butterfly) and Trichoplusia ni (cabbage looper) egg glycoproteins.

Monosaccharide compositions of eggs from Danaus plexippus (monarch butterfly) and Trichoplusia ni (cabbage looper) were analyzed. Analyses were performed mainly with high performance anion exchange chromatography (HPAEC) using crude extracts of eggs or SDS-PAGE separated and PVDF-blotted protein bands. Man and GlcN were the major components in all cases, but low levels of Gal and Fuc were possibly present in some samples. Some T. ni egg glycoproteins even contained GalN. Although a peak comigrating with Neu5Ac could be detected with HPAEC-PAD or RP-HPLC (fluorometry) after derivatization with 1,2-diamino-4,5-methylenedioxy-benzene, the quantities were too small to be significant as an integral part of the analyzed glycoproteins. These data suggests that most of glycans on the glycoproteins are pauci-Man type N-glycans, but a small portion of N-glycan may be either hybrid type or complex type.

Acetylglucosamine↗
PubMed1999

Persistent Hz-1 virus infection in insect cells: evidence for insertion of viral DNA into host chromosomes and viral infection in a latent status.

Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.

Animals↗
PubMed1998

N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines.

The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5b 1-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of alpha 1-3-linked mannose.

Alkaline Phosphatase↗
PubMed1998

Baculovirus expression of an insect gene that encodes multiple neuropeptides.

Sex pheromone production in the corn earworm, Helicoverpa zea, is regulated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosynthesis activating neuropeptide). Hez-PBAN is encoded in a preprohormone that also contains four other structurally related peptides. Two recombinant baculoviruses that contain two different sequences of Hez-PBAN cDNA under the control of a strong polyhedrin promotor were constructed. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN coding sequence. The second virus, AcBX-PBAN, contains a synthetic chimera gene encoding a bombyxin signal peptide sequence fused to a pro-Hez-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected with AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromonotropic activity was not detected in the cells infected with AcWT-PBAN. Results of chromatographic and immunochemical studies showed that some of the potential processing sites in the expressed pro-Hez-PBAN sequence were not used during posttranslational processing in the AcBX-PBAN-4-infected BTI-TN-5B1-4 cells and 4th instar T. ni larvae. However, the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN-infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immunoreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-infected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significant reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient virus, Ac-E10.

Amino Acid Sequence↗
PubMed1998

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells.

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.

Alkaline Phosphatase↗
PubMed1996

Transfection techniques for producing recombinant baculoviruses.

The production of recombinant baculoviruses usually employs cotransfection of insect tissue-culture cells with viral and transfer-plasmid DNAs. The preparation and storage of viral and plasmid DNAs suitable for optimal transfection of insect cells are discussed. Electroporation, calcium-phosphate, and lipofection transfection techniques are presented with a discussion of their relative advantages. The rates of recombinant virus formation are compared using viral infection/plasmid transfection protocols versus cotransfection of cells with transfer-plasmid and viral DNAs.

Animals↗
PubMed1996

In vitro characterization of a Trichoplusia ni single nucleocapsid nuclear polyhedrosis virus.

A Trichoplusia ni single nucleocapsid nuclear polyhedrosis virus (TnSNPV) isolate was cloned and its replication studied in the BTI-Tn-5B1-4 insect cell line. The BTI-Tn-5B1-4 cells were highly susceptible to TnSNPV infection, with 99% of the cells containing viable polyhedra by 30 h post-inoculation. Viral DNA synthesis was detected by 9 h post-infection (p.i.). Infectious budded virus (BV) was first detected at 13 h p.i. and reached an average maximum titre of 3.875 x 10(6) p.f.u./ml 27 h p.i. A total of 25 BV structural proteins having apparent molecular masses ranging from 27.5 kDa to 86 kDa were identified. Using [35S]methionine pulse-labelling, 19 virus-induced proteins with molecular masses ranging from 27 kDa to 106 kDa were detected from 4 to 28 h p.i. Host cell protein synthesis continued throughout virus replication, although at gradually decreasing rates. Thirty-two structural proteins of occlusion-derived virus ranging in apparent molecular masses from 11 kDa to 98 kDa were identified using silver staining procedures. Digestion of viral DNA with the restriction endonucleases EcoRI, HindIII and BamHI generated 31, 26 and 12 fragments, respectively. Estimates for the molecular mass of the TnSNPV genome ranged from 115.5 to 119.2 kbp. In bioassays performed with neonate T. ni larvae, the mean LD50s for the TnSNPV and Autographa californica MNPV were 1.5 (+/- 0.3) and 11.0 (+/- 4.0) polyhedra per larva, respectively.

Animals↗
PubMed1995

Intrinsic glycosylation potentials of insect cell cultures and insect larvae.

The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. The Spodoptera frugiperda (Sf21) and Trichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. Similarly, the T. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides. Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus in T. ni larvae was modified with complex oligosaccharide having sialic acid linked alpha(2-6) to galactose.

Alkaline Phosphatase↗
PubMed1993

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein.

The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinant Autographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established from Spodoptera frugiperda, Trichoplusia ni, and Mamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in the M. brassicae-derived MB0503 cell line was Endo H resistant. The commonly used S. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.

1-Deoxynojirimycin↗
PubMed1993

Comparative recombinant protein production of eight insect cell lines.

A recombinant Autographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae and Estigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, the T. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than the S. frugiperda-derived Sf9 or Sf2l cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.

Alkaline Phosphatase↗
PubMed1992

Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion.

We have devised a simple and efficient baculovirus expression vector system to evaluate insect tissue culture cells for their capacity to express, glycosylate and secrete foreign proteins. A truncated placental alkaline phosphatase (SEAP) gene was inserted into the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the transcriptional control of the polyhedrin gene promoter. Production levels, glycosylation, and secretion of the recombinant protein were examined in Trichoplusia ni (BTI-TN-5B1-4) and Spodoptera frugiperda (Sf9) cell lines. The assay for SEAP activity, which is fast, inexpensive, and quantitative to concentrations of 20 picograms per milliliter, was used to assess cell-associated and secreted SEAP activity. The proportion of SEAP which is modified with N-linked oligosaccharide can also be determined due to the difference in mobilities during SDS-PAGE between the glycosylated and nonglycosylated forms of the protein.

Alkaline Phosphatase↗
PubMed1992

Equilibrium and kinetic analysis of Autographa californica nuclear polyhedrosis virus attachment to different insect cell lines.

The kinetic and equilibrium attachment of Autographa californica nuclear polyhedrosis virus (AcMNPV) to seven insect cell lines was evaluated. Kinetic experiments revealed differences of up to 10-fold in the infection rates among cell lines. Equilibrium binding also varied between cell lines and was saturable. The Tn 5B1-4 and Tn F cell lines had the highest virus binding affinities and infection rates and exhibited diffusion-limited attachment. The rate of infection appears to be limited by the rate of attachment. For the Tn 5B1-4 cells the physical to infective particle ratio for AcMNPV was 5.3. From the Scatchard analyses, the cell lines Tn 5B1-4 and Tn F displayed affinities of 2.35 x 10(10) M-1 and 1.60 x 10(10) M-1, respectively, with 6000 and 13,700 binding sites per cell. The insect cell line Hz 1075, which is not susceptible to AcMNPV infection, displayed a much lower, but saturable, binding of AcMNPV with 900 sites/cell and an affinity of 1.1 x 10(10) M-1. Unlabelled AcMNPV, but not Lymantria dispar MNPV could compete with labelled AcMNPV for binding sites. There were 93 to 96% reductions in virus cell binding following pretreatments of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, an inhibitor of N-linked glycosylation and expression of some membrane proteins on the cell surface, reduced virus binding in a dose-dependent manner suggesting a role for glycoprotein(s) in binding. However there was no evidence for the direct involvement of oligosaccharides in attachment. Metabolic inhibitors of oligosaccharide trimming and competition binding assays using simple sugars caused no measurable reductions in virus binding. These findings suggest that AcMNPV attachment to insect cells is receptor-mediated via a glycoprotein component(s); the direct involvement of oligosaccharide moieties in binding is unlikely.

Animals↗
PubMed1992

Canine parvovirus empty capsids produced by expression in a baculovirus vector: use in analysis of viral properties and immunization of dogs.

The VP-2 genes of canine parvovirus (CPV) and a recombinant consisting of CPV and feline panleukopenia virus (FPV) sequences were cloned into baculovirus expression vectors, fused to the baculovirus polyhedrin promoter. Recombinant baculoviruses were prepared and the properties of the parvovirus proteins expressed in insect cells examined. The proteins produced were the same size as the authentic CPV VP-2 protein, and were produced late after infection; the quantity of proteins recovered from the insect cell cultures was similar to those produced in CPV infections. Parvovirus particles formed had the haemagglutination (HA), sedimentation and buoyant density properties of authentic CPV capsids. Both the CPV capsids and the CPV-FPV recombinant capsids from the baculovirus system expressed the same epitopes as those seen in the viable parvoviruses when tested with a panel of anti-parvovirus monoclonal antibodies. Lysates of recombinant baculovirus-infected cells were inoculated into dogs, giving rise to serum neutralizing and HA-inhibiting antibodies, and the immunized dogs were protected from clinical disease upon challenge with a virulent isolate of the most recent antigenic type of CPV.

Animals↗