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At least 937 records · Page 52Linked to original sources
PubMed2004

Cloning and expression of human UDP-glucuronosyltransferase 1A4 in Bac-to-Bac system.

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from uridine diphosphate-glucuronic acid (UDP-GA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGT1A4, the Bac-to-Bac expression system was used to express the recombinant UGT1A4 with His-tag on the C-terminal. The His-tagged recombinant UGT1A4 expressed in Spodoptera frugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14+/-15pmol/min/mg protein (n=3) with 0.5mM imipramine by HPLC, but was not detectable in blank Sf9 cells. It paved the way for the further studies for drug glucuronidation by UGT1A4. The purification of the UGT1A4 can be done by Ni-resin. This is helpful to do research on the structure of the UFT1A4.

Animals↗
PubMed2005

Development of a bi-cistronic baculovirus expression vector by the Rhopalosiphum padi virus 5' internal ribosome entry site.

A bi-cistronic baculovirus transfer vector was constructed based on the 5'UTR internal ribosome entry site (IRES) of the Rhopalosiphum padi virus (RhPV). Recombinant baculoviruses containing the red fluorescent protein gene and green fluorescent protein gene flanking the RhPV 5'UTR IRES can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. Quantization by fluorescence spectrophotometry of the fluorescent proteins produced in Sf21 cells indicated that the translational efficacy of the RhPV 5'UTR IRES was about 3-fold weaker than cap-dependent translation. We also demonstrated that recombinant baculoviruses containing the human interferon-gamma gene (IFN-gamma) and green fluorescent protein gene flanking the RhPV 5'UTR IRES can produce IFN-gamma proteins as well as green fluorescent proteins. These results suggest that the RhPV IRES can be used in the development of bi-cistronic baculovirus expression vectors for production of heterologous multiprotein complexes or can be combined with selection markers to facilitate applications of baculovirus expression systems.

5' Untranslated Regions↗
PubMed2005

CYP2K6 from zebrafish (Danio rerio): cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme.

A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.

Aflatoxin B1↗
PubMed2006

Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices.

Single cell analytics is a key method in the framework of proteom research allowing analyses, which are not subjected to ensemble-averaging, cell-cycle or heterogeneous cell-population effects. Our previous studies on single cell analysis in poly(dimethylsiloxane) microfluidic devices with native label-free laser induced fluorescence detection [W. Hellmich, C. Pelargus, K. Leffhalm, A. Ros, D. Anselmetti, Electrophoresis 26 (2005) 3689] were extended in order to improve separation efficiency and detection sensitivity. Here, we particularly focus on the influence of poly(oxyethylene) based coatings on the separation performance. In addition, the influence on background fluorescence is studied by the variation of the incident laser power as well as the adaptation of the confocal volume to the microfluidic channel dimensions. Last but not least, the use of carbon black particles further enhanced the detection limit to 25 nM, thereby reaching the relevant concentration ranges necessary for the label-free detection of low abundant proteins in single cells. On the basis of these results, we demonstrate the first electropherogram from an individual Spodoptera frugiperda (Sf9) cell with native label-free UV-LIF detection in a microfluidic chip.

Animals↗
PubMed2006

Interaction of rice dwarf virus outer capsid P8 protein with rice glycolate oxidase mediates relocalization of P8.

Yeast two-hybrid and coimmunoprecipitation assays indicated that P8, an outer capsid protein of Rice dwarf phytoreovirus (RDV), interacts with rice glycolate oxidase (GOX), a typical enzyme of peroxisomes. Confocal immunofluorescence microscopy revealed that P8 was colocalized with GOX in peroxisomes. Time course analysis demonstrated that the localization of P8 in Spodoptera frugiperda cells changed from diffuse to discrete, punctuate inclusions during expression from 24 to 48 h post inoculation. Coexpression of GOX with P8 may target P8 into peroxisomes, which serve as replication sites for a number of viruses. Therefore, we conclude that the interaction of P8 with the GOX of host cells leads to translocation of P8 into peroxisomes and we further propose that the interaction between P8 and GOX may play important roles in RDV targeting into the replication site of host cells. Our findings have broad significance in studying the mechanisms whereby viruses target appropriate replication sites and begin their replication.

Alcohol Oxidoreductases↗
PubMed2005

Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis.

Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.

Animals↗
PubMed2004

Biological activity of phenolic compounds from Alchornea glandulosa.

From the isopropyl acetate fraction obtained by partition of the leaves ethanolic extract of Alchornea glandulosa, six phenolic compounds were isolated and identified. General lethality of the extract, fractions and compounds were assayed with Brine Shrimp Test (BST) and with Antifeedant Activity Bioassay (AAB) against neonate larvae of Spodoptera frugiperda. Aqueous fraction and ethyl gallate, one of the major constituents of the polar fraction, have shown the highest toxicity in BST. Ethanolic extract, isopropyl acetate fraction and gallic acid reduced significantly larval growth of S. frugiperda neonates.

Animals↗
PubMed2004

Insecticidal activity of certain medicinal plants.

The methanol extracts of eight species of medicinal plants were tested for insecticidal activity in third instar larvae of Egyptian cottonworm (Spodoptera littoralis). All extracts showed a certain degree of larval toxicity. The extracts of Ocimum basilicum, Origanum majorana and Salvia officinalis appeared to be highly toxic. The extracts significantly affected the growth indexes [relative growth rate (RGR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD)].

Animals↗
PubMed2006

Insecticidal activity of Vitex mollis.

Organic extracts from the leaves of Vitex mollis were assessed for their toxic effect on fall armyworm neonate larvae (Spodoptera frugiperda), an important insect pest of corn. The extracts showed insecticidal and insect growth regulatory activity, being CHCl(3)-MeOH (1:1) extract the most active, also found to be toxic in the Artemia salina test.

Animals↗
PubMed2004

The role of carboxyl, guanidine and imidazole groups in catalysis by a midgut trehalase purified from an insect larvae.

A trehalase (EC 3.2.1.28) of 67 kDa was purified to homogeneity from the midgut of Spodoptera frugiperda (Lepidoptera) larvae. The enzyme is inhibited by toxic beta-glucosides produced by plants (amygdalin, prunasin, salicin and phlorezin) and by their aglycones (mandelonitrile, phloretin). From kcat and Km values determined in different pHs, the pKa values of catalytic essential groups were calculated (pKa = 4.5 and pKa = 8.0). These pKa values agree with the ones determined from enzyme chemical in activation with carbodiimide and phenyl glyoxal, respectively, indicating that the enzyme has a carboxyl group that act as a nucleophile and a guanidine group that is the proton donor during the catalytic cycle. The enzyme has two putative subsites for glucose binding. Based on the protection afforded by ligands against chemical modification, the roles of the subsites were inferred. Thus, the one that binds the competitive inhibitors, methyl alpha-glucoside (MalphaGlu) and mandelonitrile, contains the catalytic carboxyl, whereas the other having the catalytic Arg residue binds the competitive inhibitor Tris. Diethyl pyrocarbonate is ineffective except in the presence of MalphaGlu, when it decreases trehalase activity and changes the pKa value of the catalytic Arg residue. This suggests that the pKa value of the Arg residue is modulated by a His residue located near the active site. This also indicates that the enzyme molecule changes its conformation when the subsite containing the carboxyl group is occupied. The increase in trehalase inactivation by phenyl glyoxal in the presence of MalphaGlu agrees with the last observation.

Animals↗
PubMed2005

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells.

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.

Animals↗
PubMed2004

The deletion of the pif gene improves the biosafety of the baculovirus-based technologies.

Our goal was to improve the biosafety of baculovirus-based technologies by deleting the pif (per os infectivity factor) gene from baculovirus expression vectors. Such a deletion would block transmission in nature without disturbing protein production. A pif deletion mutant of Autographa californica multiplecapsid nucleopolyhedrovirus (AcMNPV) was constructed and its infectivity to two host species was tested by oral or intrahemocoelic inoculation. Virus replication after oral inoculation was monitored using PCR. Oral inoculations with a mixture of the wild type and the pif deletion viruses were carried out. The pif deletion blocked oral infection but it did not hamper infectivity in cell culture. The blockage took place early after inoculation and could not be overcome by mixed inoculations with the wild type. The cat gene was inserted under the control of the polyhedrin promoter in the deletion mutant and the wild type CAT yield was measured in Spodoptera frugiperda insect cells (Sf9) infected with either recombinant. The pif deletion did not hamper CAT production. This deletion significantly improved CAT yields early in the infection. Hence, expression vectors lacking pif may produce higher quality protein. The pif deletion is a simple measure that dramatically reduces the chances of virus spread or gene transfer in nature.

Animals↗
PubMed2005

Protein response of insect cells to bioreactor environmental stresses.

Protein expression of Spodoptera frugiperda (Sf9) insect cells was characterized upon exposure to environmental stresses typically present in bioreactors including heat shock, oxygen deprivation, shear stress, change of pH, and salinity or ethanol shock. This study fills the void in knowledge as to how bioreactor hydrodynamics, anoxia, small changes in pH as well as salinity alterations due to pH control or exposure to ethanol used in asepsis treatments affect protein expression in Sf9 cells. Heat shock at 43 degrees C induced proteins at 83 kDa, 68-78 kDa and six small heat shock proteins (hsps) at 23-15.5 kDa. Anaerobic conditions in CO2 atmosphere reduced significantly the normal protein synthesis and induced a small subset of heat shock proteins at 70 kDa. Oxygen deprivation in nitrogen atmosphere transiently induces the 70 kDa proteins and had minor effects on the normal protein synthesis. Exposure to increased salinity or ethanol concentration failed to trigger the stress response, but may extensively inhibit the induction of normal proteins even though there was a negligible change in cell viability. Shear stress that had a major reducing effect on cell viability did not change the protein synthesis profile of Sf9 cells. Both long and short term exposures to small pH changes had negligible effects on protein synthesis.

Animals↗
PubMed2006

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization.

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.

Animals↗
PubMed2005

The isolation and purification of biologically active recombinant and native autoantigens for the study of autoimmune disease.

The expression of recombinant, biologically active mouse myeloperoxidase (MPD) and the recombinant non-collagenous (NC1) domain of mouse collagen alpha 3 Type IV was achieved for the first time in Sf21 cells (Spodoptera frugiperda ovarian insect cells) using a baculovirus expression system. Following purification, the proteins were identified by reducing and non-reducing SDS-PAGE electrophoresis. Recombinant mouse MPO has a molecular weight of approximately 90 kDa and mouse alpha3(IV)NC1 approximately 32 kDa. In addition, milligram quantities of native mouse myeloperoxidase were purified from 32Dcl3 cells. Both native and recombinant myeloperoxidase were biologically active. This study also demonstrated that the immunization of myeloperoxidase deficient (Mpo-/-) mice with purified recombinant mouse myeloperoxidase induced a significant antibody response to native myeloperoxidase.

Animals↗
PubMed2004

Stage-dependent expression of Chelonus inanitus polydnavirus genes in the host and the parasitoid.

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid of Spodoptera littoralis (Noctuidae). Along with the egg it also injects polydnaviruses (CiV) and venom, which are prerequisites for successful parasitoid development. CiV protects the parasitoid from encapsulation by the host's immune system and induces a developmental arrest in the prepupal stage. The polydnavirus genome consists of several double-stranded circular DNA segments. Proviral DNA is integrated in the wasp's genome and virus replication is restricted to the wasp's ovary. Here, the analysis of eight CiV genes located on five different segments revealed four patterns of expression in the course of parasitization: early, late, persistent but variable, and early and late. The comparison between parasitized and CiV/venom only containing hosts indicated that the presence of the parasitoid larva modulates transcript levels. Haemocytes, fat body and nervous tissue contained viral transcripts, values being highest in haemocytes. Small amounts of CiV transcripts were also observed in parasitoid larvae and pupae, suggesting transcription from the proviral integrated form of viral DNA. This is the first comparative analysis of the expression patterns of several viral genes in both parasitized and CiV/venom only containing hosts over the entire period of parasitization, and it reveals intricate interactions between the parasitoid, the polydnavirus and the host.

Animals↗
PubMed2005

Stage-dependent strategies of host invasion in the egg-larval parasitoid Chelonus inanitus.

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid which lays its eggs into eggs of Spodoptera littoralis (Noctuidae); the parasitoid larva then develops in the haemocoel of the host larva. Host embryonic development lasts approx. 3.5 days while parasitoid embryonic development lasts approx. 16 h. All stages of host eggs can be successfully parasitized, and we show here that either the parasitoid larva or the wasp assures that the larva eventually is located in the host's haemocoel. (1) When freshly laid eggs, up to almost 1-day-old, are parasitized, the parasitoid hatches while still in the yolk and enters the host either after waiting or immediately through the dorsal opening. (2) When 1-2-day-old eggs are parasitized, the host embryo has accomplished final dorsal closure and is covered by an embryonic cuticle when the parasitoid hatches; in this case the parasitoid larva bores with its moving abdominal tip into the host. (3) When 2.5-3.5-day-old eggs are parasitized, the wasp oviposits directly into the haemocoel of the host embryo; from day 2 to 2.5 the embryo is still very small and the wasps, after probing, often restrain from oviposition for a few hours.

Animals↗