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At least 847 records · Page 47Linked to original sources
PubMed1996

Expression of mu-, delta- and kappa-opioid receptors in baculovirus-infected insect cells.

The mu-, delta- and kappa-opioid receptors have been expressed in Sf9 and 'High Five' insect cells using the baculovirus expression system. In both cell lines highest receptor levels (pmol/mg membrane protein) were observed 48 h after infection. Concomitant exposure to the narcotic antagonist naloxone (1 microM) enhanced the production of each receptor type. However, "High Five' cells differed from Sf9 cells in a 2-3-fold higher receptor density in the cell membrane and were therefore employed for receptor characterization. In membranes of 'High Five' cells opioid receptor levels ranged from 1.0 +/- 0.2 pmol/mg protein for the kappa-opioid receptor, 1.7 +/- 0.2 pmol/mg for the delta-opioid receptor to 2.1 +/- 0.5 pmol/mg for the mu-opioid receptor. The mu-, delta- and kappa-opioid receptor agonists [D-Ala2,N-methyl-Phe4-Gly-ol5]enkephalin ([3H]DAMGO), [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) and (5 alpha, 7 alpha, 8 beta)-(+)-N-methyl-N-(7-(1-pyrrolidinyl-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide ([3H]U69,563) bound to the opioid receptors with Kd values of 3.4 +/- 0.3 nM, 4.5 +/- 0.1 nM and 1.2 +/- 0.3 nM, respectively, resembling those reported for opioid receptors expressed in mammalian cells. Testing the functionality of the receptors in 'High Five' cells, we found that high affinity agonist binding was strongly reduced in the presence of GTP gamma S/sodium, indicating their coupling to G proteins. Furthermore, activation of the three receptor types inhibited forskolin-stimulated cAMP formation. The results presented here suggest that the 'High Five' cell/baculovirus system provides a convenient method for high level expression of functionally intact opioid receptors as judged by receptor binding studies, their G-protein coupling and inhibition of adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1997

24(24(1))[Z]-dehydroamarasterone B, a phytoecdysteroid from seeds of Leuzea carthamoides.

A new phytoecdysteroid, 24(24(1))[Z]-dehydroamarasterone B, has been isolated from seeds of Leuzea (Rhaponticum) carthamoides. It has been unambiguously identified by CIMS, 13C NMR and 1H NMR spectroscopy. The biological activity of the ecdysteroid has been determined in the Drosophila melanogaster BII bioassay. The ED50 (5.2 x 10(-7) M) is 70-fold higher than that for 20-hydroxyecdysone (7.5 x 10(-9) M).

Animals↗
PubMed1998

Assembly of Drosophila lamin Dm0 and C mutant proteins studied with the baculovirus system.

Despite extensive knowledge of the in vitro polymerization properties of nuclear lamins, it is still not well understood how the nuclear lamina assembles in vivo. To learn more about the relationship between in vitro and in vivo polymerization of nuclear lamins, we expressed Drosophila lamin Dm0, mutant proteins, having well defined alterations of their in vitro polymerization properties, in Sf9 cells using the baculovirus system. All lamin Dm0 mutants assembled into fibrillar aggregates indistinguishable in morphology from those assembled by the wild-type protein. However, in contrast to wild-type lamin Dm0, mutant proteins were extracted with buffers of physiological ionic strength and pH containing Triton X-100. These results indicate that various types of lamin dimer-dimer interactions can be disrupted without affecting the morphology of the lamin Dm0 polymer. However, all types of dimer-dimer interactions tested appear to be important for full polymer stability. In addition, we analyzed the polymer formation of two Drosophila lamin C mutants and found that a segment in the carboxy-terminal tail domain is required for assembly of lamin C paracrystals at the nuclear lamina.

Animals↗
PubMed1997

Expression of individual members of a tRNA(Gly)1 multigene family in vivo follows the same pattern as in vitro.

Individual members of a tRNA(Gly)1 multigene family from Bombyx mori are transcribed to different levels in vitro in homologous nuclear extracts but their transcription status in vivo is not known. Two sets of tRNA(Gly)1 belonging to the extreme groups of highly transcribed and barely transcribed copies have been examined for their expression patterns in vivo in B. mori-derived cell lines following transfection. We have developed a sensitive and reliable method for directly quantifying the transcription levels of transfecting tRNA genes without relying on the biological activity of the transcript. The strategy involved the insertion of synthetic oligodeoxyribonucleotide sequences into the coding region of the transfecting gene and monitoring the transcripts in an RNase protection assay using an antisense probe that clearly distinguished them from the endogenous tRNAs. The oligonucleotide insertion did not significantly affect the transcriptional status of the genes, even though the distance between the A and B boxes was enhanced by 10-15 nt. In vivo also the transcription of tRNA(Gly)1 reached very high levels, whereas the transcripts arising from tRNA(Gly)1-6:7 accounted for only 2-5% of the former, closely resembling the transcription patterns in vitro. These individual gene copies having identical coding sequences and consequently the same internal conserved regions, differed only in their flanking sequences which modulate their transcription levels.

Animals↗
PubMedDate not supplied

Continuous cell lines and immune ascitic fluid pools in arbovirus detection.

Successive experiments led us to use two cellular systems, MOS61 (Aedes pseudoscutellaris cells) and Vero cells, among the continuous cell lines recommended by the WHO Collaborating Center for systematic research and isolation of arboviruses. Virus detection in cell cultures is carried out with 7 mixtures containing 10 hyperimmune ascitic fluids made with the reference viruses. This technique enables the detection of 70 of the 80 arboviruses transmitted by mosquitoes in Africa and very easily detects arbovirus associations by using either monospecific or monoclonal immune ascitic fluids (dengue-1-2-3-4 and yellow fever viruses) used in the indirect immunofluorescence technique.

Aedes↗
PubMed2003

Characterization of RNA interference in an Anopheles gambiae cell line.

Introduction of double stranded RNA into invertebrate cells often results in posttranscriptional silencing of target genes through a mechanism termed RNA interference (RNAi). Double-stranded RNA is cleaved by an RNAse III-like enzyme, termed dicer, to small interfering RNAs (siRNAs). In Drosophila, these siRNAs are incorporated in the RNA induced silencing complex (RISC) and mediate degradation of target mRNA. The RISC complex contains members of Argonaute (Ago) family of proteins. We show here that RNAi in a hemocyte cell line of Anopheles gambiae, the principal malaria vector in Africa, requires expression of dicer-2, Ago2 and Ago3 proteins. Furthermore, we demonstrate that RNAi in the mosquito does not spread outside of the target region, suggesting that RNA dependent RNA polymerase mediated transitive amplification is absent in the mosquito.

Animals↗
PubMed1998

Basis for selective action of a synthetic molting hormone agonist, RH-5992 on lepidopteran insects.

The non-steroidal ecdysone agonist, RH-5992, induces a precocious incomplete molt in lepidopteran insects but is refractory to insects of other orders. We used two lepidopteran cell lines, FPMI-CF-203 (CF-203) and IPRI-MD-66 (MD-66) and two dipteran cell lines, DM-2 and Kc, to investigate the lepidopteran specificity of RH-5992. The mRNAs for hormone receptor 3 homologues cloned from Drosophila (DHR3) and Choristoneura (CHR3) are directly induced by 20-hydroxyecdysone (20E) and serve as suitable markers for studying ecdysone action. Dose response experiments showed that 10(-7) M 20E induced CHR3 mRNA in CF-203 cell and DHR3 mRNA in DM-2 cells. Concentrations of RH-5992 as low as 10(-10) M induced CHR3 mRNA in CF-203 cells, whereas concentrations as high as 10(-6) M induced only very low levels of DHR3 mRNA in DM-2 cells. Studies using 14C-RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than the dipteran cell lines (DM-2 and Kc). The clearance of RH-5992 from DM-2 cells was temperature dependent and was blocked by 10(-5) M ouabain, an inhibitor of Na+, K(+)-ATPase suggesting that the efflux was due to active transport.

Animals↗
PubMed1984

Isolation of bluetongue and related viruses from Culicoides spp. in the Sudan.

Infection of domestic ruminants with bluetongue virus (BTV) is widespread in the Sudan but there are no records of vector species of Culicoides in that country. Therefore, light-trap collections of Culicoides for virus isolation procedures were made in the Khartoum and Um Benein areas of the Sudan during September-October 1982. Two virus isolates were made from pools of unengorged, female Culicoides. An isolate from a pool of C. kingi (schultzei gp) is a member of the Epizootic Haemorrhagic Disease (EHD) serogroup. The other isolate from a pool of C. imicola, a known BTV vector in other parts of Africa, is type-5 BTV. In laboratory experiments, the North American vector of BTV, C. variipennis, supported replication of both Sudanese isolates to a high titre and transmission occurred after 10 days' incubation. This paper records the first isolation in the Sudan of arboviruses from Culicoides, with the identification of a BTV serotype and the presence of a member of the EHD (genus orbivirus, family Reoviridae) serogroup.

Animals↗
PubMed2000

Fringe forms a complex with Notch.

The Fringe protein of Drosophila and its vertebrate homologues function in boundary determination during pattern formation. Fringe has been proposed to inhibit Serrate-Notch signalling but to potentiate Delta-Notch signalling. Here we show that Fringe and Notch form a complex through both the Lin-Notch repeats and the epidermal growth factor repeats 22-36 (EGF22-36) of Notch when they are co-expressed. The Abruptex59b (Ax59b) and AxM1 mutations, which are caused by missense mutations in EGF repeats 24 and 25, respectively, abolish the Fringe-Notch interaction through EGF22-36, whereas the l(1)N(B) mutation in the third Lin-Notch repeat of Notch abolishes the interaction through Lin-Notch repeats. Ax mutations also greatly affect the Notch response to ectopic Fringe in vivo. Results from in vitro protein mixing experiments and subcellular colocalization experiments indicate that the Fringe-Notch complex may form before their secretion. These findings explain how Fringe acts cell-autonomously to modulate the ligand preference of Notch and why the Fringe-Notch relationship is conserved between phyla and in the development of very diverse structures.

Animals↗
PubMed2003

SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development.

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes.

Amino Acid Sequence↗
PubMed2006

Functional analysis of recombinant mutants of maxadilan with a PAC1 receptor-expressing melanophore cell line.

Maxadilan, a 61-amino-acid vasodilatory peptide, was initially isolated from the salivary glands of the sand fly Lutzomyia longipalpis. Although its primary sequence has no homology to that of pituitary adenylate cyclase-activating peptide, maxadilan is an agonist for the PAC1 receptor. A total of 58 substitution and deletion mutants was engineered in an effort to determine which residues were important for receptor activation. The mutants were characterized functionally using an assay based on pigment granule translocation in PAC1-expressing Xenopus laevis melanophores. Substitution of charged residues and proline 43 could alter (but not eliminate) the agonist activity of the mutants. In contrast, we found that several multiple substitution mutants of the predicted beta-strand threonine residues became antagonists at the PAC1 receptor. The results suggest that these threonine residues are cooperatively involved in PAC1 activation.

Amino Acid Sequence↗
PubMed1998

Structural analysis of Drosophila merlin reveals functional domains important for growth control and subcellular localization.

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila. As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane- associated NH2-terminal 350 amino acids of Merlin. Removal of a seven-amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene.

Animals↗
PubMed2003

Stable and heritable gene silencing in the malaria vector Anopheles stephensi.

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles . This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.

Animals↗
PubMed1989

Sequence, transcription and translation of a late gene of the Autographa californica nuclear polyhedrosis virus encoding a 34.8K polypeptide.

A 1.4 kb region downstream of the DNA polymerase gene of Autographa californica nuclear polyhedrosis virus was sequenced. Two open reading frames (ORFs) were identified of 927 and 474 bases in length. The 927 base ORF encodes a 34.8K protein as determined by in vitro translation of both hybrid-selected RNA and RNA synthesized in vitro from a 927 base ORF template. The predicted amino acid sequence of the 34.8K polypeptide (p34.8) reveals a hydrophobic N terminus, two potential N-glycosylation sites, and potential sites for phosphorylation by casein kinase I and protein kinase C. The p34.8 gene has a strong codon usage bias which is strikingly different from that of the polyhedrin gene. The two 5' ends of the 927 base ORF transcripts initiate from an ATAAG sequence and a GTAAG sequence 11 and 87 bases upstream of the ATG codon respectively. A short upstream reading frame is present in the leader sequence of the longer RNA. The transcripts have multiple 3' ends; the most proximal endpoint correlates with a polyadenylation signal overlapping the translational termination codon of the 927 base ORF. Transcripts of the latter were not observed early in the infection cycle but appeared 6 h after infection and were maximally expressed at 12 to 24 h post-infection. The late nature of these transcripts was confirmed by their sensitivity to aphidicolin and cycloheximide, inhibitors of DNA replication and protein synthesis respectively. Attempts to construct viral mutants carrying a deletion of the p34.8 gene and fusion with the beta-galactosidase gene suggest that the former gene is essential for viral replication.

Amino Acid Sequence↗
PubMed1995

Toward a physical map of Aedes aegypti.

Labelled recombinant cosmids were used as in situ hybridization probes to Aedes aegypti metaphase chromosomes. The cosmid probes yielded paired signals, one on each arm of sister chromatids, and they were ordered along the three chromosomes. In total, thirty-seven different probes were mapped to the three chromosomes of Ae. aegypti (2n = 6): twenty-eight to chromosome 1, six to chromosome 2, and six to chromosome 3. These results represent an initial stage in the generation of a physical map of the Ae. aegypti genome.

Aedes↗
PubMed1979

Baculovirus replication in a mosquito (dipteran) cell line.

The baculovirus from the lepidopteran host Autographa californica (alfalfa looper) was shown to replicate in a dipteran cell line without the production of characteristic polyhedral inclusion bodies. The low level of replication could not be detected by 50% tissue culture infective dose titrations, but was apparent by [3H]thymidine labeling of the viral genome. Immunoprecipitation of the radioactive product confirmed baculovirus production.

Aedes↗
PubMed1997

Replication of flock house virus RNAs from primary transcripts made in cells by RNA polymerase II.

To develop vector systems that combine high transcription activity with biologically safe delivery vehicles, we have explored the use of RNA replication to amplify mRNAs, by using flock house virus (FHV) as a model system. The FHV RNA replicase is encoded in the larger of the two segments that comprise the viral positive-sense RNA genome. A cDNA copy of this self-replicating RNA was precisely positioned between a promoter site for cellular RNA polymerase II and a cDNA encoding a self-cleaving ribozyme from hepatitis delta virus. Transfection of this plasmid into cultured BHK cells resulted in prolonged, autonomous FHV RNA replication in the cytoplasm and substantial amplification of the RNA replicon. The replicase also amplified RNA transcribed from a second plasmid of similar design that contained a cDNA copy of the other FHV genome segment. These results constitute a significant step toward the harnessing of nodaviral RNA replication as the basis of a versatile vector system.

Animals↗