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PubMed2003

N-acetylglucosamine-6-O-sulfotransferase-1: production in the baculovirus system and its applications to the synthesis of a sulfated oligosaccharide and to the modification of oligosaccharides in fibrinogen.

N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the C-6 position of non-reducing GlcNAc. Human GlcNAc6ST-1 was expressed as a fusion protein with protein A in an insect cell line (Tn 5 cells) using the baculovirus system. The recombinant enzyme was purified to homogeneity by IgG Sepharose column chromatography. The substrate specificity and the kinetic properties of the enzyme were similar to those of the enzyme expressed in the mammalian system. The purified recombinant enzyme was used to synthesize 6-sulfo GlcNAcbeta1-3Galbeta1-4Glc, which was identified by time of flight mass spectrometry. This sulfated trisaccharide served as a better substrate for microsomal galactosyltransferase from the mouse colon compared to 6-sulfo GlcNAc. The purified recombinant enzyme was also used to sulfate oligosaccharide chains on fibrinogen after enzymatic desialylation and degalactosylation to expose nonreducing GlcNAc residues. It is known that desialylation greatly increases the rate of clotting of fibrinogen after the addition of thrombin. Subsequent sulfation of desialylated and degalactosylated fibrinogen slightly decreased the rate of clotting. The recombinant GlcNAc6ST-1 is a useful reagent for 6-sulfate exposed GlcNAc residues both in oligosaccharides and in glycoproteins.

Animals↗
PubMed2002

Methods for maintaining insect cell cultures.

Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains.

Animals↗
PubMed2004

Degenerative evolution and functional diversification of type-III secretion systems in the insect endosymbiont Sodalis glossinidius.

Sodalis glossinidius, a maternally transmitted endosymbiont of tsetse flies, maintains two phylogenetically distinct type-III secretion systems encoded by chromosomal symbiosis regions designated SSR-1 and SSR-2. Although both symbiosis regions are closely related to extant pathogenicity islands with similar gene inventories, SSR-2 has undergone novel degenerative adaptations in the transition to mutualism. Notably, SSR-2 lacks homologs of genes found in SSR-1 that encode secreted effector proteins known to facilitate the host cell cytoskeletal rearrangements necessary for bacterial entry and uptake into eukaryotic cells. Also, as a result of relaxed selection, SSR-2 has undergone inactivation of genes encoding components of the type-III secretion system needle substructure. In the current study, we used quantitative PCR to determine the expression profiles of ysaV (SSR-1) and invA (SSR-2) transcripts when S. glossinidius infects an insect cell line, and we used an invasion assay to characterize the phenotype of an S. glossinidius mutant that lacks the ability to produce an OrgA protein that is required for function of the SSR-2 secretome. Whereas SSR-1 is required for bacterial invasion of host cells and ysaV is expressed when bacteria contact host cells, SSR-2 is required for bacterial proliferation after entry, and invA is only expressed in the intracellular stage of infection. These results demonstrate that degenerative genetic adaptations in SSR-2 have promoted functional diversification of the Sodalis SSR-2 type-III secretion system.

Animals↗
PubMed1995

cDNA cloning, expression, and chromosomal localization of a human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase.

Oligonucleotide primers derived from the cDNA encoding a full-length bovine UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) [Homa, F. L., Hollander, T., Lehman, D. J., Thomsen, D. R., and Elhammer, A. P. (1993) J. Biol. Chem. 268, 12609-12616], were used for PCR to isolate sequences encoding a homologous enzyme from human salivary gland cDNA. Comparison of the human and bovine nucleotide sequences reveals 94.8% sequence identity in their coding regions and 87% identity in their 3-untranslated regions. The translation of the human GalNAc-transferase coding region predicts an amino acid sequence which is nearly identical (99.6%) to that of the bovine counterpart; there are five conservative and one non-conservative amino acid substitutions between the two enzymes. Expression of the bovine and human cDNAs in the insect cell line, Sf9, resulted in the synthesis of proteins which appeared identical on SDS-PAGE and which had similar enzymatic properties. Screening of a somatic cell human/rodent hybrid panel with a probe derived from the human GaLNAc-transferase cDNA sequence indicated that the human GalNAc-transferase gene is localized to chromosome 18.

Amino Acid Sequence↗
PubMed1998

Recruitment of the TATA-binding protein to the HIV-1 promoter is a limiting step for Tat transactivation.

OBJECTIVES: To examine the functional interaction between HIV-1 Tat protein and the TATA-binding protein (TBP). DESIGN: HIV long terminal repeat reporter plasmids were cotransfected into mammalian and Drosophila insect cells with expression vectors encoding Tat and vectors encoding TBP either alone or linked to an heterologous DNA-binding domain. METHODS: The activity of the different reporters was compared in the presence or absence of Tat or TBP, or both, upon cotransfections into human and Drosophila insect cell lines. RESULTS: Tat protein is unable to transactivate enhancerless HIV-1 minimal promoter bearing only the TATA box and TAR. Artificial recruitment of human TBP (hTBP) to the enhancerless HIV minimal promoter was found to trigger gene expression and coexpression of Tat resulted in a marked synergy. Tat protein cooperated with DNA-bound hTBP by inducing high levels of processive viral transcripts. Synergy between Tat and hTBP was also observed when both factors were targeted to a promoter DNA template. The functional cooperation between TBP and Tat was further demonstrated using the Drosophila Schneider SL2 cells. In these cells Tat protein alone was ineffective; however, coexpression of Drosophila TBP and Tat resulted in a trans-activating response region-dependent synergistic transactivation of basal transcription. CONCLUSION: The strong synergy between TBP and Tat in the absence of any DNA-bound activator suggests that Tat stimulates transcription in an activator-independent manner most likely by a functional interaction with general transcription factors that occurs after TBP recruitment. Thus, efficient recruitment of TBP represents a limiting step for Tat transactivation.

Animals↗
PubMed2003

Human genetic variations in the 5HT2A receptor: a single nucleotide polymorphism identified with altered response to clozapine.

OBJECTIVE: to determine if the agonist serotonin and antagonists loxapine and clozapine have an altered potency for four allelic variants (T25N, I197V, A447V, and H452Y) of the human 5HT2A receptor when compared to the wild-type allele. METHODS: The receptor or its variants are studied in an in-vitro functional assay system consisting of a Sf9 insect cell line that is stably transformed with the human wild-type and mutant alleles. This assay system measures release of calcium stores due to receptor activation by agonists and inhibition of this agonist stimulated response by antagonists. RESULTS: Both loxapine and clozapine exhibit non-competitive antagonism of serotonin stimulation of the human 5HT2A receptor signal transduction system and loxapine is the more potent inhibitor. This study shows that the I197V allele requires a two-fold higher concentration of the atypical neuroleptic clozapine to inhibit serotonin stimulation compared to the wild-type receptor (P = 0.036). The I197V mutation does not affect the inhibition of serotonin stimulation by the typical neuroleptic loxapine nor does it alter the activation of the receptor by serotonin. It is also significant that the results of this study indicate that the T25N, A447V, and H452Y mutations in the human 5HT2A receptor do not significantly alter the response of the receptor to the agonist serotonin or the antagonists loxapine and clozapine.

Animals↗
PubMed1990

Characteristically distinct isolates of the nuclear polyhedrosis virus from Spodoptera litura.

More than 100 isolates were plaque-purified to examine the genetic variations in four wild stocks of Spodoptera litura nuclear polyhedrosis virus (NPV) collected in Japan. These isolates were characterized by their in vitro host range in three established insect cell lines, growth characteristics, polyhedral protein, DNA restriction endonuclease pattern and DNA hybridization. The isolates were separated into four distinct groups: (I) isolates corresponding to Autographa californica NPV, (II and IV) two different groups of isolates of S. littoralis NPV which had been previously characterized and (III) isolates with no correspondence to any reported virus group. Of the S. litura NPV wild stocks, two were mixtures of more than two different groups of NPVs. We have discussed the advantage of having a mixture of different NPV groups in the same wild virus stocks.

Animals↗
PubMed1996

Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo.

The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) has high potential for development as a bio-insecticide for control of the beet armyworm (S. exigua). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in Se-UCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNA from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases XbaI, BamHI, Bg/II, PstI, SstI, HindIII and SpeI. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12.9 and 32.3.

Animals↗
PubMed1996

Baculovirus infection of Spodoptera exigua larvae: lacZ expression driven by promoters of early genes pe38 and me53 in larval tissue.

To follow the progression of infection of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) within tissues of its larval host, we have constructed AcMNPV recombinants carrying lacZ reporter genes under the control of the early virus promoters pe38 and me53, in addition to the authentic genes. The early promoter-lacZ gene cassettes were located upstream of the very late polyhedrin gene. In infected insect cell lines, pe38 transcription is initiated at an early promoter, while me53 transcripts start from both early and late sites. Transcriptional mapping of the duplicated me53 and pe38 promoters driving lacZ expression showed that they initiated at the same start sites as in the authentic genes. Expression of lacZ by these recombinants was compared to a recombinant driving beta-glucuronidase expression from the very late p10 promoter and lacZ expression from the constitutive heat shock protein 70 promoter of Drosophila melanogaster. After infection of Spodoptera exigua larvae with the different recombinants, we followed reporter gene expression and polyhedron formation in different tissues using immunohistochemistry and electron microscopy. LacZ expression, indicative of early viral transcriptional activity, was detected in nearly all larval tissues during the course of infection. In most tissues these early events were followed by pathophysiological changes associated with late and very late gene expression. However, p10 transcription and polyhedron formation were not observed in midgut goblet cells, Malpighian tubules and salivary glands. These results suggest that expression of early virus genes, such as me53 and pe38, is not restricted to larval tissues that are permissive for AcMNPV replication.

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↗
PubMed1988

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro.

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.

Aedes↗
PubMed2005

Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells.

The polydnavirus Toxoneuron nigriceps bracovirus (TnBV) is an obligate symbiont associated with the braconid wasp T. nigriceps, a parasitoid of Heliothis virescens larvae. Previously, to identify polydnavirus genes that allow parasitization by altering the host immune and endocrine systems, expression patterns of TnBV genes from parasitized H. virescens larvae were analysed and cDNAs were obtained. To study the function of the protein from one such cDNA, TnBV1, overexpression of the protein was attempted by using the baculovirus Autographa californica multicapsid nucleopolyhedrovirus. Recovery of stable recombinant virus was unsuccessful, with the exception of recombinants with deletions/mutations within the TnBV1 gene. It was hypothesized that TnBV1 expression was cytotoxic to the Spodoptera frugiperda (Sf21) insect cells that were used to produce the recombinants. Therefore, the Bac-to-Bac system was used to create recombinant baculoviruses maintained in Escherichia coli expressing either TnBV1 (Ac-TnBV1) or an initiator-methionine mutant [Ac-TnBV1(ATG-)]. Microscopy revealed substantial cell death of Sf21 and High Five cells from 48 h post-infection with Ac-TnBV1, but not with the Ac-TnBV1(ATG-) recombinant virus. Ac-TnBV1-infected Sf21 cells, but not those with parental virus infection, showed an increased caspase-3-like protease activity, as well as increased terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) for breaks in host genomic DNA. Although indicative of apoptosis, blebbing and apoptotic bodies were not observed in infected cells. Transiently expressing TnBV1 alone caused TUNEL staining in High Five cells. These data suggest that TnBV1 expression alone can induce apoptosis-like programmed cell death in two insect cell lines. Injection of Ac-TnBV1 budded virus, compared with parental virus, did not result in an alteration of virulence in H. virescens larvae.

Animals↗
PubMed1988

Comparison of the in vivo and in vitro activity of the delta-endotoxin of Bacillus thuringiensis var. morrisoni (HD-12) and two of its constituent proteins after cloning and expression in Escherichia coli.

The insecticidal crystal delta-endotoxin of Bacillus thuringiensis var. morrisoni HD-12 contains at least five polypeptides in the range 126-140 kDa. Immune blotting revealed that individual proteins in this complex share homology with a range of other B. thuringiensis delta-endotoxins. In vivo the native HD-12 crystal killed a lepidopteran larva (Pieris brassicae) and a dipteran larva (Anopheles gambiae), but not the related dipteran Aedes aegypti. In vitro the solubilized activated crystal lysed Choristoneura fumiferana cells (lepidopteran) and dipteran cells derived from Anopheles gambiae and Culex quinquefasciatus but not those from Aedes aegypti. An intragenic probe derived from a B. thuringiensis var. sotto lepidoptera-specific delta-endotoxin gene hybridized with one of six plasmids extracted from HD-12. When cloned into pUC18 two HindIII fragments from this plasmid (pEG1 and pEG2) were shown to encode polypeptides cross-reacting with HD-12 antiserum. Escherichia coli lysates containing pEG2 were toxic in vivo to lepidoptera and diptera larvae and in vitro to a broader range of insect cell lines than the native crystal. E. coli cells containing pEG3, a subclone derived from pEG1, synthesised large amounts of a 140-kDa protein in the cytoplasm as inclusion bodies. The cytotoxicity of the protein encoded by pEG3 was restricted to C. fumiferana and A. gambiae cell lines.

Animals↗
PubMed2001

Interaction of hydrophobic anions with the rat skeletal muscle chloride channel ClC-1: effects on permeation and gating.

Permeation of a range of hydrophobic anions through the rat skeletal muscle chloride channel, rClC-1, expressed in Sf-9 (a Spodoptera frugiperda insect cell line) cells has been studied using the whole-cell patch-clamp technique. Bi-ionic reversal potentials measured with external application of foreign anions gave the following permeability sequence: Cl- (1) > benzoate (0.15) > hexanoate (0.12) > butyrate (0.09) > propionate (0.047) approximately formate (0.046). Anions with larger hydrophobic moieties were more permeant, which suggested that ClC-1 selectivity for hydrophobic anions is dominated by their interaction with a hydrophobic region in the external mouth of the pore. All anions studied when applied from outside show an apparently paradoxical voltage-dependent block of inward currents; this voltage-dependent block could be qualitatively described by a discrete-state permeation model with two binding sites and three barriers. Effects of the external anions with aliphatic side-chains on the apparent open probability (Po) suggested that they are unable to gate the channel, but can modulate ClC-1 gating, probably, by changing Cl- affinity to the gating site. Effects of internal application of benzoate, hexanoate or propionate mimicked those of increasing internal pH, and similarly depended on the channel protonation from the external side. Results for internal benzoate support the concept of a negatively charged cytoplasmic particle being involved in the ClC-1 gating mechanism sensitive to the internal pH.

Animals↗
PubMed1989

Effects of ultraviolet light on thymidine incorporation and DNA chain elongation in photoreactivable insect cells.

An insect cell line, IAL-PID2, was exposed to UV and analyzed for its ability to incorporate [3H]-thymidine and to elongate replicon-sized DNA fragments. After exposure to 5 or 10 J/m2 UV, the cells exhibited a rapid and prolonged depression in the rate of thymidine incorporation. Photoreactivation reduced this depression but did not entirely reverse it. For exposures of 5 J/m2 or above, full recovery did not occur until 18 h after exposure. The blockage of fork progression after UV exposure was fluence-dependent, with replication segments after exposure to 20 J/m2 being shorter than those observed after exposure to 10 J/m2. Immediately after exposure to either 10 or 20 J/m2, photoreactivation reversed blockage of fork progression, indicating that the (5-6) cyclobutyl pyrimidine dimer is responsible for blockage. This also indicates that blockage of fork progression may not be the only factor responsible for the prolonged depression seen in thymidine incorporation. Three hours after exposure to either 10 or 20 J/m2, replication segments were still significantly shorter than control segments. Photoreactivation completely reversed blockage after exposure to 10 J/m2, but did not completely reverse blockage after exposure to 20 J/m2, indicating that at such fluences, other lesions may play a role in UV-induced blockage of fork progression.

Animals↗
PubMed1992

Electrical coupling between cells of the insect Aedes albopictus.

1. Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used to study the electrical properties of intercellular junctions. A double voltage-clamp approach was adopted to control the voltage gradient between the cells and measure the intracellular current flow. 2. Determinations of junctional conductance (gj) revealed two types of intercellular contacts, gap junctions and cytoplasmic bridges. Identification occurred by means of functional criteria, i.e. the dependency of gj on (i) junctional membrane potential, (ii) non-junctional membrane potential, and (iii) heptanol. 3. In cell pairs with putative gap junctions, gj was dependent on the junctional membrane potential (Vj). When determined at the beginning of voltage pulses, gj was insensitive to Vj; when determined at the end of 15 s pulses, it depended on Vj in a bell-shaped manner (70% decrease for a change in Vj of +/- 75 mV). 4. These cell pairs also showed a dependency of gj on the non-junctional membrane potential (Vm). When determined immediately after changing the non-junctional membrane potential in both cells, gj was not affected by Vm; when determined 30 s later, gj was modified by Vm in a S-shaped fashion (100% decrease when Vm was depolarized to +50 mV). 5. Exposure to 3 mM-heptanol gave rise to complete and reversible block of gj in cell pairs with putative gap junctions. 6. Cell pairs susceptible to uncoupling by heptanol revealed junctional currents indicative of the operation of gap junction channels. The single-channel conductance, determined at a Vm of -50 to -70 mV, was 133 pS. 7. In the case of putative cytoplasmic bridges, gj was insensitive to the junctional and non-junctional membrane potential. In addition, it was not affected by 3 mM-heptanol. 8. While most cell pairs showed functional properties characteristic of gap junctions or cytoplasmic bridges, few cell pairs exhibited junctional currents compatible with the co-existence of both junctional structures.

Aedes↗
PubMed1997

Shaker B K+ conductance in Na+ solutions lacking K+ ions: a remarkably stable non-conducting state produced by membrane depolarizations.

1. Shaker B K+ channels, expressed in the insect cell line Sf9, were studied in zero K+, Na+ or N-methyl-D-glucamine (NMG)-containing solutions. In the absence of K+ ions on both sides of the membrane, the K+ conductance collapsed with the delivery of short depolarizing pulses that activated the channels. The collapse of the conductance was fully prevented when the channels were kept closed at a holding potential of -80 mV. 2. The fall in K+ conductance had the notable characteristic of being strikingly stable. At -80 mV or more negative holding potentials, the conductance never recovered (cells observed for up to 1 h). 3. The extent of collapse of the K+ conductance depended on the number of depolarizing activating pulses applied in zero K+ solutions. For moderate to low frequencies of pulsing (1 to 0.002 Hz), the extent of the collapse did not depend on the frequency. 4. K+, Rb+, Cs+ and NH4+ added to the external Na+ solution impeded the fall in K+ conductance. 5. TEA added to the external, zero K+, Na(+)-containing solution also precluded the fall of the conductance. The protection by TEA paralleled its block of the outward K+ currents recorded with standard recording solutions. 6. The fall in K+ conductance was prevented by depolarized holding potentials. 7. The K+ conductance that was thought to be irreversibly lost at -80 mV or more negative holding potentials was fully recovered, however, after a prolonged (tens of seconds to minutes) change in the holding potential to depolarized values (above -50 mV). Full recovery could be obtained at any time after the former halt of the conductance.

Animals↗