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PubMed1992

Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.

Animals↗

PubMed1987

Induction of transiently expressed genes in PC-12 pheochromocytoma cells.

Rat PC-12 pheochromocytoma cells, in response to nerve growth factor (NGF), stop proliferating and differentiate into cells resembling sympathetic neurons. This model of cell differentiation was used to investigate the expression of a previously isolated collection of mitogen-induced primary response sequences cloned from murine 3T3 cells; the TIS (tetradecanoyl phorbol acetate-induced sequences) genes (Lim et al., 1987). The TIS cDNAs were used to probe RNA isolated from PC-12 cells stimulated with NGF and other agents. Six of these messages were rapidly and transiently induced by NGF, tetradecanoyl phorbol acetate (TPA), or epidermal growth factor (EGF). Expression of these TIS genes generally resembled the NGF-stimulated induction of c-fos. In contrast, one TIS gene (TIS 10), induced by mitogens in 3T3 cells, was not induced by NGF, TPA, or EGF in PC-12 cells. Like c-fos, these TIS genes induced by NGF could also be superinduced by the combined administration of NGF and benzodiazepine. Elevated potassium ion, which leads to the induction of c-fos in PC-12 cells via activation of a voltage-dependent Ca2+ channel, also induces all TIS genes, with the notable exception of TIS 10. The induction of this family of genes may be involved in the general transduction of extracellular signals into biological responses.

Animals↗

PubMed2003

Transient expression of E1A and Ras oncogenes causes downregulation of c-fos gene transcription in nontransformed REF52 cells.

Stable transformation of rat embryo fibroblast (REF) cells with E1A and cHa-ras oncogenes leads to downmodulation of c-fos gene transcription. This repression is provided in part by the association of Elk-1 transcription factor with histone deacetylases mediated through effects of Ras on MAP-kinase cascades. Here, we focus on the primary effects of E1A and Ras displayed in transient transfection assay on the transactivating capability of Elk-1, which is a key transcription factor of c-fos gene regulation. Our data show that E1A is able to suppress serum- and Ras-induced stimulation of Gal-luc reporter activity by a full-length Gal-Elk1-428 fusion protein as well as the expression of c-fos promoter-driven luciferase constructs (fos-luc). The repression can be relieved by trichostatin A, a histone deacetylase (HDAC) inhibitor, implying the involvement of HDACs and an inactive chromatin structure formed due to underacetylation of nucleosomal histones. Thus, upon transient transfection of E1A and Ras oncogenes in REF52 cells or their stable expression in E1A+cHa-ras cells, E1A contributes to the formation of inactive chromatin structure through association with p300/CBP histone acetyltransferases at c-fos promoters, whereas Ras mediates its effect through constitutive activation of the MAP/ERK kinase cascade, thereby promoting the recruitment of HDAC1 to the Elk-1 transcription factor. As a result, downregulation of c-fos gene transcription revealed in established E1A+Ras transformants is unlikely to be a consequence of cell transformation itself, but follows from primary effects of E1A and Ras on chromatin remodeling factors.

Adenovirus E1A Proteins↗

PubMed2005

High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions.

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.

Agrobacterium tumefaciens↗

PubMed1988

Duck hepatitis B virus (DHBV) particles produced by transient expression of DHBV DNA in a human hepatoma cell line are infectious in vitro.

Transfection of the human hepatocellular carcinoma cell line HuH7 with a plasmid containing a tandem copy of the duck hepatitis B virus DNA sequence resulted in transient replication of the virus. Viral particles secreted by transfected HuH7 cells exhibited physical properties similar to those of serum-derived duck hepatitis B virus and were infectious in primary duck hepatocyte cultures.

Animals↗

PubMed1998

Transient expression of the vesicular monoamine transporter during development in the rat thalamus and cortex.

The postnatal developmental pattern of the central vesicular monoamine transporter-2 (VMAT2) was analyzed in the rat brain by means of quantitative autoradiography with a specific and high affinity ligand [3H]dihydrotetrabenazine ([3H]TBZOH). We show a dense expression of VMAT2 in the cortex (especially area 17) and thalamus (particularly the dorsal lateral geniculate nucleus) at postnatal days 1 and 8. This pattern of VMAT2 distribution was transient since it was no longer observed at day 20 or in the adult rat brain where VMAT2 density was weak and uniform in these regions. These data suggest that monoamine vesicular storage participates in the early postnatal maturation of thalamus and cortex.

Animals↗

PubMed1987

A morphogenic role for transiently expressed acetylcholinesterase in developing thalamocortical systems?

Experiments in infant rats have revealed patterns of intense acetylcholinesterase (AChE) activity in primary sensory thalamic nuclei and thalamocortical recipient zones of primary sensory areas of cerebral cortex. The distribution and appearance of AChE staining indicates that the transient AChE in these areas is characteristic of thalamocortical neurons. The patterns of AChE are detectable during the first postnatal week of life, reach peak intensity during the second postnatal week, and decline to normal adult levels by the end of the third postnatal week. These temporal parameters correlate well with the time of thalamocortical axon growth into the cerebral cortex. These data suggest that the transient AChE may be playing a morphogenic role in the development of thalamocortical connections.

Acetylcholinesterase↗

PubMed2002

Transient expression of juvenile-type neurocan by reactive astrocytes in adult rat brains injured by kainate-induced seizures as well as surgical incision.

Neurocan is one of the major chondroitin sulfate proteoglycans expressed in nervous tissues. The expression of neurocan is developmentally regulated, and full-length neurocan is detected in juvenile brains but not in adult brains. In the present study, we demonstrated by western blot analysis that full-length neurocan transiently appeared in adult rat hippocampus when it was lesioned by kainate-induced seizures. Immunohistochemical studies showed that neurocan was detected mainly around the CA1 region although the seizure resulted in neuronal cell degeneration in both the CA1 and CA3 regions of the hippocampus. Double-labeling for neurocan mRNA and glial fibrillary acidic protein demonstrated that many reactive astrocytes expressed neurocan mRNA. The re-expression of full-length neurocan was also observed in the surgically injured adult rat brain. In contrast, the expression of other nervous tissue chondroitin sulfate proteoglycans, such as phosphacan and neuroglycan C, was not intensified but rather was either reduced in the kainate-lesioned hippocampus or in the surgically injured cerebral cortex. These observations indicate that induction of neurocan expression by reactive astrocytes is a common phenomenon in the repair process of adult brain injury, and therefore, it can be postulated that juvenile-type neurocan plays some roles in brain repair.

Animals↗

PubMed1998

XCIRP (Xenopus homolog of cold-inducible RNA-binding protein) is expressed transiently in developing pronephros and neural tissue.

The pronephros functions in the amphibian larval stage. It differentiates in certain presumptive regions of the amphibian embryo. The study of molecules functioning during pronephrogenesis is important for understanding the mechanism of kidney formation. Herein, we report a gene expressed during differentiation of the pronephros and neural tissues that we isolated by differential hybridization using our pronephros in-vitro induction system. The gene, XCIRP, is 887bp in length, and encodes a putative protein composed of 163 amino acid residues. The deduced protein contains two CS-RBDs (consensus sequence RNA-binding domain) and a glycine-rich domain, and is 74% identical to homologs from other species (mouse, rat and human). The expression of XCIRP increased rapidly during gastrulation, and XCIRP localization was seen in the presumptive pronephros and neural tissues. These findings suggest that XCIRP may play important roles in pronephrogenesis and neurogenesis.

Amino Acid Sequence↗

PubMed2000

The product of the rice myb7 unspliced mRNA dimerizes with the maize leucine zipper Opaque2 and stimulates its activity in a transient expression assay.

myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its target b32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu-Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription.

Cell Nucleus↗

PubMed1988

Transient expression of pentameric IgM on the surface of NZB B cells.

In contrast to normal B lymphocytes, splenic B cells from autoimmune NZB mice express on their surface greater amounts of IgM than IgD. We have found that the predominant mu chain species present on these cells is derived from pentameric secretory IgM which can be radioiodinated during transient association with the cell membrane. If the contribution from 19S IgM is discounted, then the surface IgM:IgD ratio of NZB mice is only moderately higher than that of B cells from normal strains.

Animals↗

PubMed2001

Transient expression of CYP1A1 in rat epithelial cells cultured in suspension.

Suspension of human epidermal cells in methylcellulose-containing medium induces CYP1A1 by a mechanism requiring functional Ah receptor (AhR). In present work CYP1A1 mRNA was induced in a variety of cultured rat epithelial cells by suspension, but the induction was transient, with CYP1A1 mRNA reaching maximal levels by 5 h and disappearing by 12 h. Though the methylcellulose itself contained no detectable ligand, (a) suspension activated the AhR, as judged by mobility shift assays, (b) the AhR competitive inhibitor alpha-naphthoflavone inhibited suspension-mediated induction, and (c) induction was dependent upon dioxin responsive transcriptional elements in the CYP1A1 promoter. The rapid disappearance of CYP1A1 mRNA after 5 h of suspension was unaffected by the addition of TCDD but was prevented by the inclusion of the protein synthesis inhibitor cycloheximide. Thus the downregulation appears to be mediated by a novel short-lived protein induced or activated by suspension.

Animals↗

PubMed1994

Transient expression of [D-Ala2] deltorphin I-like immunoreactivity in prenatal rat small intestine.

We studied the distribution of immunoreactive elements for [D-Ala2] deltorphin I (DADTI), a delta-opioid receptor ligand, in fetal and postnatal rat small intestine. DADTI-like immunoreactive cells were detected transiently on embryonic Days 20 and 21. Electron microscopic examination revealed that positive staining occurred in mucous epithelial cells, either mature goblet cells or undifferentiated cells containing only a few mucous granules. Positive immunoreaction products in mature goblet cells were confined in their apical cytoplasm to the luminal parts of mucous granule aggregates. The result suggests that a DADTI-like molecule(s) is synthesized in rat intestinal goblet cells and is secreted in a diacrine fashion into the intestinal lumen at a late fetal period. The molecule(s) thus secreted may be important for the intestine of rats just before birth, because DADTI-like immunopositive goblet cells are no longer seen at any postnatal period.

Amino Acid Sequence↗

PubMed2002

V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein.

V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the alpha and beta subunits of the capping protein (CP, alternatively called CapZ or beta-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1.CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 x 10(-7) m and a molecular stoichiometry of approximately 1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development.

Actin Depolymerizing Factors↗

PubMed2003

Transient expression of human herpesvirus-8 (Kaposi's sarcoma-associated herpesvirus) ORF50 enhances HIV-1 replication.

OBJECTIVE: Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma, the most common neoplasm in patients with acquired immunodeficiency syndrome. Current evidence indicates that activation of viral replication may be critical to the development of the disease. A key factor in the induction of HHV-8 lytic replication is ORF50, an immediate-early gene encoding a transactivating protein necessary for viral reactivation. We recently reported that ORF50 synergizes with HIV-1 TAT at a post-transcriptional level. To study the effects of these molecular interactions upon HIV replication and biology, cell lines of different origin were transiently transfected with ORF50 and subsequently infected with HIV. METHODS: Jurkat, BCBL-1 and A172 cells were transfected with ORF50 and subsequently infected with different MOI of HIV. The development of infection was evaluated by analyzing p24 antigen release, production of infectious HIV particles and the presence and transcription of HIV proviral DNA. RESULTS: ORF50 induced increased levels of HIV replication and production in CD4+ Jurkat T cells. Transfection of ORF50 into nonsusceptible B and glial cells (BCBL-1 and A172, respectively) increased cell susceptibility to infection and resulted in transient permissiveness to HIV replication. CONCLUSIONS: HIV replication can be significantly affected by the presence of HHV-8. Expression of ORF50 increases the efficiency of HIV infection in different cell types. This potentially could result in enhanced HIV spread within the infected organism and faster progression of the disease.

B-Lymphocytes↗

PubMed1997

The Escherichia coli stpA gene is transiently expressed during growth in rich medium and is induced in minimal medium and by stress conditions.

The transcriptional regulation of the stpA gene, encoding the Escherichia coli H-NS-like protein StpA, has been studied as a function of a variety of environmental conditions, and its response to trans-acting factors has been characterized. Chromosomally located stpA is expressed primarily from a promoter immediately upstream of the gene which is severely repressed by the homologous nucleoid-associated protein H-NS. However, we show here that even in a strain containing functional H-NS, stpA is transiently induced during growth of a batch culture in rich medium. It can also be induced strongly by osmotic shock and, to a lesser extent, by an increase in growth temperature. Moreover, when cells are grown in minimal medium, we observe a more sustained induction of stpA which is dependent on the leucine-responsive regulatory protein (Lrp). This enhanced level of stpA transcription is virtually abolished in an H-NS-independent manner when the culture undergoes carbon starvation. A sensitivity of the stpA promoter to DNA topology may contribute to some of these responses. Results reported here show that cloned fragments of the stpA promoter region can confer H-NS and Lrp responsiveness upon a lacZ reporter gene and suggest that several hundred base pairs of DNA upstream of the transcriptional start may be required for regulation by these two proteins.

Bacterial Outer Membrane Proteins↗

PubMed1998

Identification and characterization of tenp, a gene transiently expressed before overt cell differentiation during neurogenesis.

The initiation of cellular differentiation in the vertebrate central nervous system is a cascade of regulated gene expression events involving both intrinsic and extrinsic factors. Currently, the molecular events underlying the developmental transition from the cessation of cell proliferation to the onset of cell differentiation during neurogenesis are still poorly understood. We have identified a gene, tenp, which is likely to play a role during the transition. tenp mRNA was detected in the developing retina and brain, but not in the heart, kidney, or liver. Anatomically, cells expressing tenp formed a narrow strip at the boundary between the ventricular zone (consisting of proliferating cells) and the intermediate zone (occupied by postmitotic, differentiating cells). Further analysis showed that they were bromodeoxyuridine negative and thus postmitotic, yet without apparent differentiation such as the expression of microtubule-associated protein (MAP2) and neurofilament (NF68). When expressed in chicken embryonic fibroblast cells through transfection, Tenp protein was immunodetected in membrane fractions, implying that Tenp might be a membrane protein as predicted by a computer analysis of its primary sequence. Our data suggested that tenp might be involved in an early neurogenic event that existed transiently after terminal mitosis, yet before the overt differentiation of neural precursor cells.

Amino Acid Sequence↗

PubMed1993

Transient expression of calbindin-D28k immunoreactivity in layer V pyramidal neurons during postnatal development of kitten cortical areas.

Calbindin-D28k is a 28 kDa calcium binding protein that has been shown to colocalize with a specific subpopulation of gamma-aminobutyric acid inhibitory interneurons in mammalian neocortex. We have examined the ontogeny of calbindin in neonatal kitten cortex in areas 17,18,19,7, medial and lateral suprasylvian visual areas, splenial visual area and cingulate cortex from the day of birth (P0) through maturation of the brain (P101). Transient staining of immature layer V pyramidal cells was seen in kittens six weeks old and younger. This transient staining of pyramidal cells was most intense and the stained neurons were most numerous in cingulate cortex. Apical dendrites of pyramidal cells in cingulate cortex were prominently stained and could be followed to layer I, where they were seen to branch extensively. There were very few calbindin immunoreactive pyramidal cells in primary cortical areas postnatally. Transient staining in extrastriate visual cortical areas disappeared first from the lateral suprasylvian areas, and persisted longest in area 7. Pyramidal neurons in the cingulate gyrus expressed calbindin longest, but calbindin expression by pyramidal neurons ceased by the sixth postnatal week in all areas of the brain.

Aging↗