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PubMed2002

Inhibition of transiently expressed low- and high-voltage-activated calcium channels by trivalent metal cations.

Calcium channels are important regulators of neuronal excitability and contribute to transmitter release, calcium dependent gene expression, and oscillatory behavior in many cell types. Under physiological conditions, native low-voltage (T-type)- and high-voltage-activated (HVA) currents are potently inhibited by trivalent cations. However, the presence of multiple calcium channel isoforms has hampered our ability to unequivocally assess the effects of trivalent cations on channel activity. Here, we describe the actions of nine trivalent metal ions on transiently expressed alpha1G (Cav3.1) T-type calcium channels cloned from human brain. In 2 mM external barium solution, yttrium most potently inhibited alpha1G current (IC50 = 28 nM), followed by erbium > gadolinium ~ cerium > holmium > ytterbium > neodymium > lanthanum >> scandium. With the exception of scandium, blocking affinity was loosely correlated with decreasing ionic radius. A detailed characterization of yttrium block revealed a 25-fold decrease in blocking affinity when the external concentration of charge carrier was increased from 2 mM to 20 mM. In 20 mM barium, yttrium also effectively inhibited various types of cloned HVA channels indicating that this ion is a nonselective blocker. For all calcium channels examined, yttrium preferentially inhibited inward over outward current, but block was otherwise voltage independent. In addition to peak current inhibition, P/Q- and L-type channels underwent a unique speeding of the macroscopic time course of inactivation. Whereas peak current block of alpha1A channels was highly sensitive to the external charge carrier concentration, the inactivation effects mediated by yttrium were not, suggesting that the two effects are due to distinct mechanisms. Moreover, the speeding effect was greatly attenuated by manipulations that slowed the inactivation kinetics of the channels. Thus, our evidence suggests that yttrium effects are mediated by two distinct events: peak current block likely occurring by occlusion of the pore, and kinetic speeding arising from yttrium interactions with the channel that alter the state of the inactivation gate.

Calcium Channels↗

PubMed1994

A Drosophila homolog of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells.

We have identified a Drosophila gene encoding a putative receptor tyrosine kinase by screening a genomic DNA library with a DNA probe for a Drosophila homolog of fibroblast growth factor receptors. The newly isolated gene codes for a transmembrane protein most similar in sequence to a mammalian proto-oncogene ret; thus, the gene was termed Dret. Dret mRNA is transcribed in very small amounts in the embryonic, larval, and pupal stages. Whole mount in situ hybridization experiments revealed that the mRNA is transiently expressed in neuroblasts in early embryos. In late embryos, Dret mRNA was detected in subpopulations of differentiating CNS and PNS cells. In addition, Dret expression was affected in neurogenic mutants. These results suggest that Dret can be considered as a functional homolog of mammalian ret and should play important roles in neurogenesis.

Amino Acid Sequence↗

PubMed1998

Post-transcriptional control of the level of mRNA by hepatitis B virus X gene in the transient expression system using human hepatic cells.

BACKGROUND: Hepatitis B virus (HBV) infection is closely related to the development of not only acute or chronic hepatitis, but also hepatocellular carcinoma. Among the HBV genes, the X gene has been implicated in the carcinogenicity of this virus as a major causative factor by its ability to activate viral and cellular genes in trans via protein-protein interaction with cellular factors without binding to DNA. RESULTS: To explore the possibility of other functions of the X gene, we examined the effect of X protein on the transient expression system of simian virus 40 (SV40) large T-antigen or chloramphenicol acetyltransferase (CAT) mRNA using SV40 promoter or EF-1alpha (human elongation factor 1alpha) promoter, by co-transfecting an X gene expression plasmid to human hepatic cell lines, HepG2 and Huh7. In contrast to the SV40 promoter-mediated expression, the level of both T-antigen and CAT mRNAs expressed from the EF-1alpha promoter was strikingly decreased by X protein in both hepatic cells. The nuclear run-on assay and the mRNA decay experiment using actinomycin D, indicated that the effect of X protein on the lowering of the level of chimeric mRNA was due to the degradation of mRNA, but not repression of transcriptional initiation. Moreover, this effect was dependent on the 22 bp sequence in the 5' untranslated region of mRNA derived from the EF-1alpha promoter. CONCLUSION: The present data suggest a new function of the X gene to post-transcriptionally control the stability of mRNA through the 5' untranslated region derived from the EF-1alpha promoter in human hepatic cells.

Antigens, Polyomavirus Transforming↗

PubMed1990

Transient expression of virus-specific promoters in murine resident peritoneal macrophages.

Monocyte-macrophages (MO), being non-permissive for most viruses, play an important role in resistance to virus infection. In order to establish the mechanism of abortive infection of murine resident peritoneal MO (ResPMO) by herpes simplex virus type 1 (HSV-1), it is desirable to transfect these cells with viral promoters linked to an assayable gene, for example, the bacterial chloramphenicol acetyl transferase (CAT) gene. This will facilitate studies designed to measure levels of promoter activation or repression in these specialized cells. Transient expression of CAT in ResPMO was achieved with DEAE-dextran, but not using either calcium phosphate precipitate or lipofectin. CAT expression driven by various virus-specific promoters was less efficient in ResPMO compared with Vero cells and approximately 50% of input plasmid DNA remained in Vero cells at 48 h post transfection, but only 9% was detectable in ResPMO. However, approximately 6% of ResPMO and 9% of Vero cells contained CAT-specific DNA at 24 h post transfection. In addition, 2% of cells of either cell type contained CAT-specific polypeptide at 48 h. This is therefore the first report that the non-replicating murine ResPMO can be transfected in vitro and more importantly, that these cells express the transfected gene products.

Animals↗

PubMed1997

Transient expression of the beta-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis.

The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial beta-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for beta-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the beta-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus.

Anaerobiosis↗

PubMed2004

SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors.

The Y chromosome gene Sry encodes a transcription factor required to initiate testis development. The related autosomal gene Sox9 is up-regulated shortly after the onset of Sry transcription and is thought essential for the differentiation of Sertoli cells. The lineage that gives rise to Sertoli cells has its origins within the coelomic epithelium (CE) of the genital ridge, but from cells also able to give rise to an interstitial cell type. It was not known at what point SRY acts in the derivation of this lineage or how the two genes interact. To investigate the identity of the cells expressing Sry, we designed two transgenes driven by the Sry promoter: one gives expression of a stable reporter, human placental alkaline phosphatase (hPLAP), while the second gives expression of a functional Myc-epitope tagged SRY protein (SRYMYC). Taking advantage of lasting hPLAP activity after transcription of the reporter gene has ceased, we could show that SryhPLAP was expressed exclusively in all cells fated to become Sertoli cells. SRYMYC-single-positive cells were first observed in the gonad and not in the CE. Subsequently, they became SRYMYC/SOX9-double-positive, but only for a few hours before turning into SOX9-single-positive cells. After the coelomic epithelial cells migrate into the gonad, there is first a decision to become interstitial or supporting cells, and then the transient expression of SRY in the latter determines their fate as Sertoli cells by up-regulating Sox9.

Alkaline Phosphatase↗

PubMed1992

Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding.

Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.

Animals↗

PubMedDate not supplied

Transgenic plants regenerated from hairy roots of Nicotiana benthamiana: a promising host for transient expression of foreign proteins.

Hairy root cultures of Nicotiana benthamiana have been obtained by co-cultivation of leaf explants with Agrobacterium rhizogenes strain A4 harboring a binary vector plasmid, and transgenic nature of the obtained cultures was confirmed by PCR analysis. Transgenic plants were regenerated from hairy roots. The biomass yield of transgenic plants grown in vitro was almost two-fold higher than those of wild-type N. benthamiana plants. They differed from untransformed plants by short internodes, reinforced stem, thick and wrinkled leaves and more developed root system. The level of Agrobacterium-mediated transient expression of green fluorescent protein (GFP) in the regenerated plants was similar to that of untransformed plants.

Agrobacterium tumefaciens↗

PubMed1985

Heart C-protein is transiently expressed during skeletal muscle development in the embryo, but persists in cultured myogenic cells.

The expression of cardiac and white skeletal C-protein isoforms was analyzed in developing chicken embryos and in primary skeletal muscle cell cultures by immunoblot and immunofluorescence staining using polyclonal antibodies specific for both of the two different proteins. In the embryo, cardiac C-protein was detected in the developing heart from very early stages through adulthood. In skeletal muscle, cardiac C-protein is shown to be transiently expressed between Days 3 and 15 during development. In contrast, the expression of white skeletal C-protein is gradual and progressive starting approximately from Day 15 on in development. In primary cell cultures of skeletal muscle, however, cardiac C-protein remained expressed throughout prolonged culture time, this in conjunction with white skeletal C-protein. Thus the down regulation of cardiac C-protein and the transition from cardiac C-protein to adult skeletal (white) C-protein which was observed during skeletal muscle development in vivo, does not seem to go to completion in the in vitro system.

Animals↗

PubMed1987

Transient expression of choline acetyltransferase-like immunoreactivity in Purkinje cells of the developing rat cerebellum.

The expression of choline acetyltransferase (ChAT)-like immunoreactivity was studied immunohistochemically in the cerebelli of developing rats. Brains were examined from the day of birth (postnatal day 1: P1) until adulthood. From P4 through P21, several Purkinje cells in the uvula, nodule, and flocculus of the cerebellum demonstrated ChAT-like immunoreactivity. After P23, no ChAT-positive neurons were observed in any region of the cerebellum. This finding paralleled the transient expression of acetylcholinesterase in Purkinje cells of these same cerebellar areas during development.

Acetylcholinesterase↗

PubMed2003

Transient expression of osteopontin mRNA and protein in amoeboid microglia in developing rat brain.

To investigate a potential role of osteopontin (OPN) in developing rat brain, the expression of OPN mRNA and protein in the developing rat brain relative to the distribution of brain macrophages was investigated using in situ hybridization and immunohistochemistry, and the phagocytic capability of OPN-expressing cells was accessed using rhodamine isothiocyanate (RhIc) as a tracer. OPN-expressing cells appeared from embryonic day 16. During the first week of postnatal life, OPN-labeled cells increased markedly, and peaked around P7, then declined and had completely disappeared by the end of the second postnatal week. The spatiotemporal distribution pattern of OPN mRNA closely matched that of OPN protein. Their morphology and localization were compared with those of cells expressing the established microglial marker OX-42 in adjacent sections, and double-labeling studies demonstrated that OPN was localized to the amoeboid microglia which stain with the lectin GSI-B4, another marker for microglia. Furthermore, OPN-labeled cells were confirmed to be active phagocytes emitting RhIc fluorescence indicating that the tracer into the brain tissues was engulfed by phagocytosis. Therefore, these results provide the first evidence that OPN is transiently expressed in active brain macrophages in the embryonic and early postnatal brain, and suggest that OPN may contribute to the migration and phagocytic function of brain macrophages in the developing brain.

Animals↗

PubMed2002

Effects of methylmercury on human neuronal L-type calcium channels transiently expressed in human embryonic kidney cells (HEK-293).

Methylmercury (MeHg) disrupts the function of native, high voltage-activated neuronal Ca(2+) channels in several types of cells. However, the effects of MeHg on isolated Ca(2+) channel phenotypes have not been examined. The aim of the present study was to examine the action of MeHg on recombinant, neuronal L-type voltage-sensitive Ca(2+) channels. Human embryonic kidney cells (HEK-293) were transfected with human neuronal cDNA clones of the alpha(1C-1) subunit in combination with alpha(2b) and beta(3a) Ca(2+) channel subunits and the reporter jellyfish green fluorescent protein for transient expression. Current from expressed channels (I(Ba)) and their response to MeHg applied acutely were measured using whole-cell voltage-clamp recording techniques and Ba(2+) (5 mM) as charge carrier. Amplitude of I(Ba) in these cells was reduced by the dihydropyridine (DHP), nimodipine, and enhanced by Bay K8644 [S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3 pyridine carboxylic acid methyl ester]. MeHg (0.125-5.0 microM) caused a time- and concentration-dependent reduction in amplitude of the peak and sustained current through these channels. However, even at the highest concentration of MeHg tested, reduction of current amplitude by MeHg was incomplete. Washing with MeHg-free solution could not reverse its effects. The steady-state inactivation curve was unaltered by MeHg. Increasing the stimulation frequency or the extracellular Ba(2+) concentration each attenuated slightly the reduction in amplitude of I(Ba) by MeHg. In the presence of MeHg (5.0 microM), Bay K8644 still increased the remaining current, and nimodipine (10 microM) reduced residual current that was resistant to MeHg. Thus, although MeHg reduces the amplitude of recombinant, heterologously expressed L-type channel current, a portion of current is resistant to reduction by MeHg. Furthermore, DHP agonists and antagonists retain their ability to affect L-type Ca(2+) channel current even in the presence of MeHg.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMedDate not supplied

Molecular analysis of the ependymin gene and functional test of its promoter region by transient expression in Brachydanio rerio.

Ependymins are secretory products of meningeal cells and represent the predominant glycoproteins in the cerebrospinal fluid from various orders of teleost fish. In the zebrafish, their expression starts between 48 and 72 h post-fertilization. Generally, they share characteristics with proteins involved in cell-contact phenomena. Here, we characterize the ependymin gene from Brachydanio rerio and its flanking regions. The sequence was obtained from clones generated using the polymerase chain reaction (PCR), including a variation of an "anchored" PCR. Also, clones from a conventional phage library were analyzed. We found that the transcribed portion is arranged in six exons. Transient expression of an ependymin-promoter-lacZ gene fusion in zebrafish embryos revealed that the 2.0-kb upstream regulatory region used is sufficient to direct the ependymin-specific correct temporal and spatial expression pattern of the lacZ reporter gene.

Amino Acid Sequence↗

PubMed1995

Microtubule-associated proteins in developing oligodendrocytes: transient expression of a MAP2c isoform in oligodendrocyte precursors.

The morphological differentiation of oligodendrocytes is characterized by the formation of multiple, microtubule-rich processes which endow these cells with the ability to myelinate many axons simultaneously. Since microtubule-associated proteins (MAPs) strongly influence the structure and function of microtubules, we have investigated their expression in cultured differentiating oligodendrocytes in order to gain insights into MAP function during process formation and stabilization. MAP1B has been compared with two other structural MAPs: MAP4, which is an ubiquitously expressed protein, and MAP2, which hitherto was thought to be confined to neurons and reactive astrocytes. Immunofluorescence microscopy showed that the colocalization of MAP4 with microtubules in oligodendrocyte processes is not as extensive as found previously for MAP1B (Vouyiouklis and Brophy: J Neurosci Res 35:257-267, 1993). Nevertheless, like MAP1B, the expression of MAP4 increases during oligodendrocyte differentiation. In contrast, the expression of MAP2 is transiently elevated in preoligodendrocytes but declines precipitously at the onset of terminal differentiation. Cells of the oligodendrocyte lineage exclusively express a novel isoform of MAP2c which is primarily localized in the cell bodies of preoligodendrocytes. This suggests that MAP2c assists in the initiation of process extension rather than in the stabilization of microtubules in the cytoplasm-filled membranous extensions of mature cells. MAP-tau was not expressed at any developmental stage by oligodendrocytes. The distinct subcellular localizations and patterns of developmental expression of MAP1B, MAP4, and MAP2c suggest that these MAPs have different roles in the regulation of the microtubule network during the differentiation of myelin-forming oligodendrocytes.

Amino Acid Sequence↗

PubMed2000

Transient expression of transglutaminase C during prenatal development of human muscles.

Tissue transglutaminase (TGase C, TGase II) is known to participate in cellular processes during morphogenesis, differentiation, and development of various prenatal tissues and organs. The expression of TGase C during myoblast proliferation and attachment to external laminae was examined by immunohistochemical (IH) localization at 5-12 weeks of developmental stages of prenatal human muscle in 23 embryos. IH detection using a monospecific antibody to TGase C showed a prominent expression of TGase C in muscle cells as stage- and spatial-specific patterns during an early embryonal period. The myoblasts of intervertebral, tongue, and limb muscles, attached to adjacent cartilaginous skeletons or fibrous fascia, showed a pronounced expression of TGase C at 5-6, 6-7, and 7-8 weeks after fertilization, respectively. The most intense activity of TGase C was observed in some cardiac myoblasts infiltrating into endocardial mesenchyme at 6-7 weeks after fertilization. Although weak staining was detected until 14 weeks after fertilization, the level of TGase C expression in all muscles was significantly decreased after 6-7 weeks, with the exception that the smooth muscle cells of blood vessels and gastrointestinal tract showed diffusely intense staining of TGase C between 5 and 12 weeks after fertilization. Western blotting analysis of the cellular extracts of pooled samples showed a single strong band at 80 kD at 6 weeks after fertilization. This band became weaker after 8-10 weeks of prenatal development. These findings of transient expression of TGase C, which coincides with the development of myoblast anchoring and differentiation, suggest that TGase C plays a role in myoblast attachment to the extracellular laminae during the early embryonal period.

Blotting, Western↗

PubMed1993

Feedback regulation of mammalian ornithine decarboxylase. Studies using a transient expression system.

Ornithine decarboxylase catalyzes the first step in the biosynthesis of polyamines in mammalian cells. The enzyme is subject to various control mechanisms to maintain adequate intracellular levels of polyamines. Polyamines exert a strong feedback control on ornithine decarboxylase. In a recent study [van Daalen Wetters, T., Macrae, M., Brabant, M., Sittler, A. & Coffino, P. (1989) Mol. Cell. Biol. 9, 5484-5490], it was concluded that feedback control of ornithine decarboxylase is mainly, if not exclusively, a posttranslational phenomenon. The existence of a fast-acting polyamine-stimulated component of ornithine decarboxylase degradation that acts on newly synthesized monomeric forms of the enzyme was postulated. In the present study we have used a transient expression system to test this hypothesis. The expression of ornithine decarboxylase in mock-transfected COS cells varied depending on the cellular supply of polyamines as has been found in other mammalian cells. Thus, supplementing the cells with exogenous spermidine resulted in a marked decrease in ornithine decarboxylase activity, whereas depletion of intracellular polyamines, using an ornithine decarboxylase inhibitor, gave a large increase in the cellular content of the enzyme. COS cells expressing an ornithine decarboxylase mRNA devoid of its 5' non-translated region did not exhibit any feedback control of the enzyme, neither in the presence of exogenous spermidine nor when the intracellular polyamine levels were depleted to the same extent as in the mock-transfected COS cells. The results strongly suggest that the feedback control of ornithine decarboxylase is not merely a posttranslational phenomenon.

Animals↗

PubMed2004

Transient expression of Olig1 initiates the differentiation of neural stem cells into oligodendrocyte progenitor cells.

In order to develop an efficient strategy to induce the in vitro differentiation of neural stem cells (NSCs) into oligodendrocyte progenitor cells (OPCs), NSCs were isolated from E14 mice and grown in medium containing epidermal growth factor and fibroblast growth factor (FGF). Besides supplementing the medium with oligodendrogenic factors such as Sonic Hedgehog (Shh), FGF-2, and PDGF, we attempted to initiate the gene transcription program for OPC differentiation by transfection of the Olig1 gene, a transcription factor known to be involved in the induction of oligodendrocyte lineage formation during embryogenesis. Whereas addition of Shh, FGF-2, and PDGF could induce OPC differentiation in 12% of the NSCs, the transient expression of Olig1 by use of Nucleofector gene transfection initiated OPC differentiation in 55% of the NSCs. Our results show that nonviral transfection of genes encoding for oligodendrogenic transcription factors may be an efficient way to initiate the in vitro differentiation of NSCs into OPCs.

Animals↗

PubMed1995

Transient expression of NADPH-diaphorase in the lateral geniculate nucleus of the ferret during early postnatal development.

Retinogeniculate projections in the ferret are refined during postnatal development so that inputs from the two eyes become segregated into eye-specific laminae, and each eye-specific lamina is further divided into sublaminae containing inputs from on-center or off-center afferents. Segregation into eye-specific laminae and on/off sublaminae is dependent on neuronal activity; sublamination depends on activation of N-methyl-d-aspartate (NMDA) receptors. By analogy with the suggested role of nitric oxide in NMDA-mediated long-term potentiation in the hippocampus, we investigated a possible role for nitric oxide in ferret retinogeniculate development. The expression of NADPH-diaphorase, a nitric oxide synthase, was examined histologically in the lateral geniculate nucleus of ferrets at several postnatal ages. At birth, neuropil is labeled in the nucleus, although no cell bodies are visible. After the first postnatal week, some labeled cells appear, predominantly in the C laminae. By three postnatal weeks, cell bodies are clearly labeled in all geniculate laminae. Staining reaches a peak in density at about four postnatal weeks, then declines such that by six postnatal weeks labeled cells are no longer visible. This transient expression of NADPH-diaphorase activity is consistent with a role for nitric oxide in the development of mature connections within the ferret lateral geniculate nucleus.

Animals↗