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Results for “Transient expression”

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PubMed1993

Genes expressed during the differentiation of the pupal wings are also transiently expressed during the larval moult in Bombyx mori.

By a differential screening of cDNA libraries made with RNAs present in differentiating imaginal wing discs of Bombyx mori, we have isolated clones whose expression is dramatically modified during the formation of the pupal wings. The RNAs corresponding to all but one of these clones are also transiently accumulated in the wing primordia during the last larvo-larval moult and in response to 20-hydroxyecdysone injections. If the injections are made to juvenilized larvae the expression of the same genes is stimulated but the range of the stimulation is reduced. It is suggested that in Bombyx, cells of the wings reach the pupal stage of differentiation by steps corresponding to the phases during which high levels of ecdysteroids are present.

Animals↗

PubMed1999

Human desmocollin 1a transiently expressed in COS-7 cells and NIH 3T3-3 cells is reacted by IgG4 antibodies in a pemphigus foliaceus serum.

Human desmocollin (Dsc) 1 is an autoantigen in the subcorneal pustular dermatosis type of IgA pemphigus. Moreover, Dscs, particularly bovine Dscs, are recognized by IgG antibodies in certain sera of various types of pemphigus by immunoblotting. In this study, 10 pemphigus sera were examined by immunofluorescence for IgG antibodies to human Dsc1a, Dsc2a and Dsc3a transiently expressed in COS-7 and NIH 3T3-3 cells using two different transfection methods. IgG antibodies in a number of sera showed a strong background staining with untransfected COS-7 and NIH 3T3-3 cells. Using COS-7 cells, a pemphigus foliaceus serum, which did not stain untransfected COS-7 cells, was found to contain IgG antibodies reactive exclusively with Dsc1a. This serum showed the same reactivity in studies using NIH 3T3-3 cells. Moreover, this pemphigus foliaceus serum contained IgG4, but not IgG1 antibodies, to Dscla expressed in the NIH 3T3-3 cells. These results indicate that autoantibody response in pemphigus foliaceus might be more heterogenous than hitherto supposed.

3T3 Cells↗

PubMed1995

Disruption of the blood-brain interface in neonatal rat neocortex induces a transient expression of metallothionein in reactive astrocytes.

Exposure of the adult rat brain parenchyma to zinc induces an increase in the intracerebral expression of the metal-binding protein, metallothionein, which is normally confined to astrocytes, ependymal cells, choroid plexus epithelial cells, and brain endothelial cells. Metallothionein is expressed only in diminutive amounts in astrocytes of the neonatal rat brain, which could imply that neonatal rats are devoid of the capacity to detoxify free metals released from a brain wound. In order to examine the influence of a brain injury on the expression of metallothionein in the neonatal brain, PO rats were subjected to a localized freeze lesion of the neocortex of the right temporal cortex. This lesion results in a disrupted blood-brain interface, leading to extravasation of plasma proteins. From 16 h, reactive astrocytosis, defined as an increase in the number and size of cells expressing GFAP and vimentin, was observed surrounding the neocortical lesion site. Astrocytes and pial cells situated adjacent to the area of injury also became positively stained for metallothionein. At 3-6 days post-lesion, the highest level of reactive astrocytes expressing metallothionein was observed. Neo-Timm staining revealed that histochemically reactive zinc had disappeared from the lesion site. Extracellular albumin and metallothionein-positive astrocytes were absent approximately 2 weeks after the lesion, whereas reactive astrocytosis was still observed. These results show that a lesion of the neonatal rat brain induces a transient expression of metallothionein in reactive astrocytes, probably as a response to metals released from the site of the brain injury.

Animals↗

PubMed2003

Testing the efficiency of dsRNAi constructs in vivo: a transient expression assay based on two fluorescent proteins.

Double-stranded RNA interference technology has recently been shown to be a powerful tool to silence gene expression in various organisms, including plants. Sustained double-stranded RNA interference mediated gene silencing is normally triggered by hairpin RNAs generated by in vivo transcription of inverted repeat DNA constructs. To test the efficiency of inverted repeat constructs for their in vivo gene silencing capability, we developed an assay that is based on transient expression in single cells using two fluorescent reporter proteins with different emission spectra. We co-expressed one fluorescent protein as marker for transfected cells and the second as translational fusion with the target gene. Co-transfection of a vector mediating expression of inverted repeats of the target gene resulted in a specific decrease or undetectable fusion protein fluorescence in the majority of transfected cells.

Gene Expression Regulation, Plant↗

PubMed1989

Transient expression of insulin-like growth factor I immunoreactivity by vascular cells during angiogenesis.

The present study was undertaken to investigate whether vascular cells show insulin-like growth factor I (IGF-I; somatomedin C) immunoreactivity under normal conditions and/or during angiogenesis in humans and animals, as the trophic peptide IGF-I is considered important for cell growth and differentiation. In adult animals normal blood vessels, i.e., arteries, veins, and capillaries, did not show any IGF-I immunoreactivity. In newborn animals every vascular cell showed IGF-I immunoreactivity; the frequency and intensity thereafter decreased and eventually vanished as the animals approached maturity. Injury of a tissue or organ rapidly induced extensive blood vessel formation and such new blood vessels transiently expressed IGF-I immunoreactivity. Endothelial cells in budding capillaries showed distinct cytoplasmic IGF-I immunoreactivity, as did endothelial cells, smooth muscle cells, and fibroblast in newly formed arteries and veins. In biopsies of human tissue, transient IGF-I immunoreactivity was evident in vascular cells during angiogenesis after injury, as it also was in granulation tissue, skin wounds, and scar capsules around implants. Increased IGF-I immunoreactivity was further demonstrated in vascular cells in biopsies from patients with other changes involving blood vessel formation, e.g., nasal polyps, and in specimens from patients with arteritis, tendonitis, synovitis, Wegener's granulomatosis, idiopathic midline destructive disease, neurofibromatosis (von Recklinghausen's disease), and muscular dystrophy. It is concluded that during angiogenesis, obviously irrespective of inducing factors and mechanisms, vascular wall cells transiently show IGF-I immunoreactivity.

Animals↗

PubMedDate not supplied

GABA(A) receptor immunoreactivity is transiently expressed in the developing outer retina.

Extensive evidence has suggested a trophic role of gamma-aminobutyric acid (GABA) on developing cone photoreceptors in postnatal retina. In a previous study, we showed that GABA raises intracellular calcium levels in the developing cones via activation of GABA(A) receptors. Using confocal microscopy in conjunction with immunocytochemistry, we have now demonstrated that (1) GABA(A) receptor subunits are localized on cone cell bodies as well as on cone pedicles, indicating that GABA has a direct, rather than indirect, effect on cones and (2) the temporal expression of GABA(A) receptor subunits coincides with the developmental effects of GABA on cone synaptogenesis. An antibody against the beta 2/3 subunits of the GABA(A) receptor and a specific cone marker peanut-agglutinin lectin (PNA) were used to double-label wholemount neonatal retinal preparations. Results show that GABA(A) receptors are transiently expressed on cone photoreceptors in the early stages of postnatal retinal development. GABA (A)receptor immunoreactivity is clearly present on cone cell bodies and their processes and on other--as yet unidentified--elements (horizontal cells?) in the outer plexiform layer. Immunoreactivity decreases within cone photoreceptor somata after postnatal day 5, but persists in the processes of the outer plexiform layer until day 7. Our results provide support for the hypothesis that GABA acts as an important developmental regulator of cone photoreceptor maturation.

Animals↗

PubMed1996

Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

3T3 Cells↗

PubMed1992

Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells.

In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.

Amino Acid Sequence↗

PubMed1999

Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel (delta, epsilon) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda.

Atypical protein kinase (PK)C isoforms, zeta and lambda, have been reported to be activated by insulin via phosphoinositide 3-kinase, and have been suggested to be required for insulin-stimulated glucose transport. Here, we have examined the effects of transiently expressed wild-type (WT), constitutively active (Constit) and kinase-inactive (KI) forms of atypical PKCs, zeta and lambda, on haemagglutinin antigen (HAA)-tagged glucose transporter 4 (GLUT4) translocation in rat adipocytes, and compared these effects with each other and with those of comparable forms of conventional (alpha, beta) and novel (delta, epsilon) PKCs, which have also been proposed to be required for insulin-stimulated glucose transport. KI-PKC-zeta evoked consistent, sizeable (overall mean of 65%) inhibitory effects on insulin-stimulated, but not basal or guanosine-5'-[gamma-thio]triphosphate-stimulated, HAA-GLUT4 translocation; moreover, inhibitory effects of KI-PKC-zeta were largely reversed by co-transfection of WT-PKC-zeta. Like KI-PKC-zeta, KI-PKC-lambda inhibited insulin-stimulated HAA-GLUT4 translocation by approx. 40-60%, and the combination of KI-PKC-zeta and KI-PKC-lambda caused nearly complete (85%) inhibition. Of particular interest is the fact that inhibitory effects of KI forms of PKC-zeta and PKC-lambda were largely reversed by the opposite WT forms, i.e. PKC-lambda and PKC-zeta respectively. In contrast with KI forms of atypical PKCs, KI forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon had little or no effect on insulin-stimulated HAA-GLUT4 translocation. Concerning the question of sufficiency, overexpression of WT-PKC-zeta enhanced insulin effects on HAA-GLUT4 translocation, whereas WT forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon did not affect GLUT4 translocation; furthermore, Constit PKC-zeta evoked increases in HAA-GLUT4 translocation approaching those of insulin, but Constit forms of PKC-alpha and PKC-beta2 were without effect. Our findings suggest that, among PKCs, the atypical PKCs, zeta and lambda, appear to be specifically, but interchangeably, required for insulin effects on HAA-GLUT4 translocation.

Adipose Tissue↗

PubMed1998

Transient expression of a translation initiation factor is conservatively associated with embryonic gene activation in murine and bovine embryos.

In the present study the abundance of mRNAs for eukaryotic translation initiation factors eIF-1A (formerly known as eIF-4C), -2alpha, -4A, -4E, and -5 was examined in in vivo-derived mouse embryos throughout preimplantation development using a semiquantitative reverse transcription-polymerase chain reaction assay. Although the mRNA profile for each gene is unique, only mRNA for eIF-1A transiently increases during embryonic gene activation (EGA) at the 2-cell stage, and this was confirmed by an independent hybridization-based assay. In in vitro-developed bovine embryos, mRNA for eIF-1A was transiently detected at the 8-cell stage, when the major activation of the genome occurs in this species. As in the mouse, detection in 8-cell bovine embryos was sensitive to the transcriptional inhibitor alpha-amanitin. It was also observed at the same time relative to cleavage in embryos cultured in defined medium under a reduced oxygen environment, and in medium supplemented with serum and somatic cells in 5% CO2 in air. Neither the chronology of early cleavage divisions nor the yield of bovine blastocysts differed in these culture media. Our results suggest that transient expression of eIF-1A in the mouse and cow is a conserved pattern of gene expression associated with EGA in mammals.

Amanitins↗

PubMed2002

Visible transient expression of EGFP requires intranuclear injection of large copy numbers.

For the development of human artificial chromosomes (HACs) as a gene transfer vehicle we need to assess the efficiency of de novo chromosome formation depending on the type and the copy number of transferred DNA constructs. In order to check transient EGFP expression as a reporter to immediately detect presence of transfected DNA, we microinjected approximately 1 to 10(5) copies of pEGFP-N1 plasmid into the nucleus of various cell types. Whether using primary, immortalized, or tumor cells, at least 10(3)-10(4) copies were required to generate a visible green signal in the majority of the 50-90% of cells surviving injection. Generally, the cells showed relatively constant, copy number-dependent signals. 10(3) copies resulted in faint and 10(5) in bright fluorescence under the microscope. In addition, the different copy number groups contained a small fraction of cells showing much stronger fluorescence, indicating activation or lack of suppression which facilitates detection of as few as 10(2) transferred copies in rare instances. Thus, transient expression from single copies is not sufficient to reliably detect presence of DNA in the nucleus. The result is relevant for the development of low copy HAC transfer protocols.

Chromosomes, Artificial, Human↗

PubMed2002

Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant.

Potential contamination of animal-derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule. In continuation of our previous work [Ruggiero et al. (2000) FEBS Lett. 469, 132-136], here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H). This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through co-expression with an animal cell-derived modifying enzyme. We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37 degrees C.

Agrobacterium tumefaciens↗

PubMed1997

NO synthase-II is transiently expressed in embryonic mouse olfactory receptor neurons.

Among three NO synthase (NOS) isoforms only the inducible NOS-II was localized in developing olfactory receptor neurons of the mouse. First NOS-II immunoreactive receptor cells including their processes were detected by embryonic day 11 when the olfactory pit starts to invaginate. Cellular staining lasted until embryonic day 16, and was reduced during the next few days. At embryonic day 20 no reactivity was found in the olfactory epithelium, whereas centripetal nerve fibers remained positive. This transient expression of NOS-II implies a role for the differentiation of early olfactory receptor neurons and synaptic plasticity.

Animals↗

PubMed1993

Functional analysis of Arabidopsis thaliana rRNA gene and spacer promoters in vivo and by transient expression.

In eukaryotes, RNA polymerase I transcription is controlled by DNA elements located within the spacers that separate the tandemly arranged rRNA genes. Unlike rRNA coding sequences, the intergenic spacers evolve rapidly and have little sequence similarity even among closely related species. Nonetheless, the arrangement of functional elements, such as spacer promoters and enhancers, is thought to be highly conserved. Here, we identify spacer promoters in the plant Arabidopsis thaliana, thereby demonstrating their existence in both the plant and animal kingdoms. We also use an Arabidopsis transient expression system to perform transcriptional analysis of the ribosomal gene promoter. Spacer promoters share sequence similarity with the gene promoter from -91 to +22 relative to the transcription start site, +1. Deletion analysis shows that sequences required for RNA polymerase I transcription reside within these boundaries. Spacer sequences upstream of the gene promoter have only a small positive effect on transcription in transfected protoplasts but can increase transcription from a Xenopus ribosomal gene promoter in injected frog oocytes. This trans-kingdom enhancer effect further suggests that the functional elements within eukaryotic ribosomal genes are highly conserved.

Animals↗

PubMed1993

Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs.

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

Animals↗

PubMed1991

N-methyl-D-aspartate and aspartate raise the cytosolic free calcium concentration by acting upon receptors transiently expressed on immature cerebellar Purkinje cells.

N-Methyl-D-aspartate (NMDA) or aspartate (Asp) increased the cytosolic free calcium concentration ([Ca]in) in some populations of Purkinje cells dissociated from immature rat cerebellum. The NMDA- and Asp-induced rise in [Ca]in was affected only a little by adding glycine or NMDA antagonists, but was reduced either by adding Mg2+, Gallopamil hydrochloride (D-600) and gamma-amino-butyric acid, or by removing external Na+. The results suggest that stimulation of the NMDA-sensitive receptors transiently expressed on immature Purkinje cell soma results in a rise in [Ca]in through the activation of voltage-dependent Ca2+ channels.

2-Amino-5-phosphonovalerate↗

PubMed2006

In vitro functional test of two subclasses of an anti-RhD antibody produced by transient expression in COS cells.

For over 35 years hemolytic disease of the fetus and newborn (HDFN) due to RhD has been effectively prevented by anti-RhD antibodies obtained from alloimmunized women or deliberately immunized men. However, due to the reduced number of immunized women and for ethical reasons it is foreseen that other sources of anti-RhD will be needed. One such source is recombinant human antibodies. Here we describe the construction of plasmids encoding two subclasses (IgG1 and IgG3) of an anti-RhD antibody, their transient expression in COS cells, and subsequent functional characterization of the antibodies with regard to specificity and ability to mediate a respiratory burst. The recombinant anti-RhD antibodies were specific for the RhD antigen and were able to mediate a respiratory burst. Thus these antibodies might be of use as future rhesus prophylaxis.

Animals↗

PubMed1991

Transient expression of opioid receptors in defined regions of developing brain: are embryonic receptors selective?

The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective mu-opioid ligand [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)OH [( 3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the delta-selective peptide Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) shows higher affinity of the delta opioid to P1 membranes than to P60 membranes, though the number of delta receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 +/- 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗