Transient expression of alkaline phosphatase over HeLa cell surfaces.
Explore the source record for details and available documents.
Alkaline Phosphatase↗
SEARCH · PubMed Health
Results for “Transient expression”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Transient expression of transferrin receptors and localisation of iron in amoeboid microglia in postnatal rats.
The expression of transferrin receptors marked by the monoclonal antibody OX-26 and the localisation of iron were studied in amoeboid microglial cells in postnatal rats. Transferrin receptors were vigorously expressed in amoeboid microglia in rats ranging from 1 to 10 d of age but were undetectable in older rats. Thin serial sections showed that the OX-26 positive amoeboid microglial cells were also immunoreactive for OX-42 and ED1. Using Perls' medium, this study showed the presence of a considerable amount of iron in amoeboid microglial cells in 1-10 d rats. Most iron-containing cells were round but their number had diminished by 2 and 3 wk of age, when the iron was localised instead in some branched cells which were identified as either oligodendrocytes or ramified microglia cells. There has been much speculation on the functional significance of transferrin receptors on amoeboid microglia in postnatal rats. It is suggested that the receptors facilitate the acquisition of iron necessitated for various functions of amoeboid microglia in the developing brain. The presence of iron in some oligodendrocytes suggests their involvement in mediating iron mobilisation and storage. Its localisation in some ramified microglia in older rats indicates the possible role of these cells in sequestration and detoxification of iron in the central nervous system.
Aging↗
Transient expression of SPARC in the dorsal axis of early Xenopus embryos: correlation with calcium-dependent adhesion and electrical coupling.
Our comprehension of the molecular mechanisms underlying embryogenesis has been greatly enhanced by the identification and characterization of associated extracellular matrix macromolecules. Using Xenopus laevis as a model, we investigated the expression and distribution of SPARC (Secreted Protein, Acidic, Rich in Cysteine; also called osteonectin and BM-40) during early embryonic development. SPARC has been found to be enriched in tissues undergoing rapid morphological development, differentiation, and remodeling. In Xenopus, SPARC transcripts are first expressed by primordial cells which give rise to the first embryonic tissues, the notochord and somites. SPARC RNA levels remained high throughout the rapid morphological development and differentiation phase of these tissues, and then rapidly decreased. Of particular interest, SPARC protein began to accumulate within the intersomitic clefts at the onset of trunk myotome contraction. The intersomitic enrichment of SPARC remained high as long as the myotomes remained electrically coupled, principally by gap junctions. As myotomes became innervated, SPARC expression decreased dramatically within the somites. SPARC was also found to be enriched within other tissues, such as the neural tube and epidermis. In addition, the selective spatial-temporal enrichment of SPARC suggests it makes important calcium-dependent contributions to early morphological development.
Animals↗
A transient expression of alpha B-crystallin in the developing rat retinal pigment epithelium.
PURPOSE: To investigate the expression of alpha B-crystallin in the developing rat retina by immunohistochemical techniques. METHODS: Rat eyes were enucleated on embryonic day 18 and on postnatal days 1, 4, 9, 15, 19, and 90. The avidin-biotin-peroxidase technique was applied to show alpha B-crystallin immunostaining. After the cornea and lens were removed, the developing rat eyeballs were solubilized by 1% sodium dodecyl sulfate. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to detect alpha B-crystallin in the extracts. RESULTS: From postnatal day 4 to postnatal day 9, alpha B-crystallin was present in retinal pigment epithelium, but it disappeared after postnatal day 15. alpha A-crystallin immunoreactivity was negative in retina and retinal pigment epithelium throughout development. The results of Western blot analysis and ELISA coincided with those of the immunostaining. CONCLUSIONS: These results show that retinal pigment epithelium in mammalian rat retina demonstrates alpha B-crystallin expression in the early developmental stages but loses it after 15 days of age.
Animals↗
Strong transient expression of the type I interferon-induced MxA protein in hepatitis A but not in acute hepatitis B and C.
The human MxA protein is a new specific marker for type I interferon activity both in vitro and in vivo. In the study presented here, this interferon-induced marker, as well as the 2',5'-oligoadenylate synthetases, was measured in circulating mononuclear cells from 21 patients with acute hepatitis A, 20 patients with acute hepatitis B and 14 patients with acute hepatitis C for determination of the activation of the interferon system in these viral diseases. In acute hepatitis A a strong expression (10 of 10 patients) of the MxA protein and the 2',5'-oligoadenylate synthetase activity in peripheral-blood mononuclear cells was observed during the first 2 wk after onset of clinical symptoms. In this period the MxA protein concentrations reached levels similar to those measured in patients treated with up to 5 x 10(6) IU interferon-alpha three times a week. Beyond wk 3, in eight of eight patients with hepatitis A no increased MxA protein levels were found. In contrast, peripheral-blood mononuclear cells from patients with acute hepatitis B contained either no measurable MxA protein or only slightly higher levels of the MxA protein, as did those of most patients (12 of 14) with acute hepatitis C. The MxA protein levels of both hepatitis B and C patients were significantly lower (p < 0.05) than those found in hepatitis A patients. Furthermore, sera from 6 of 10 patients with hepatitis A, but none of 10 patients with acute hepatitis B and C, contained measurable MxA protein. This serum MxA protein may originate from interferon-exposed and subsequently damaged liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)
2',5'-Oligoadenylate Synthetase↗
Transient expression of tagged prion protein (PrP) in a neuronal cell line.
Explore the source record for details and available documents.
Animals↗
MN20, a D2 cyclin, is transiently expressed in selected neural populations during embryogenesis.
Although the regulation of proliferation and differentiation during brain development has long been considered to be interrelated, the mechanisms that coordinate the control of cell division and histogenesis are poorly understood. The cell cycle is a dynamic process that is governed by the concerted action of numerous cell cycle regulatory proteins in response to signals both intrinsic and extrinsic to the cell. Thus, proteins that regulate the cell cycle are well suited to provide a link between processes that control neuroblast proliferation and differentiation. We reported previously the isolation from brain of a message form of D2 cyclin, one of several cyclin proteins known to promote the progression from G1 to S phase. This MN20/D2 cyclin mRNA is expressed in highly restricted neural populations at embryonic (E) day 15 and postnatal (P) day 6 in the mouse. To gain insight into the role(s) this cyclin may serve in brain formation, the spatial and temporal pattern of MN20/D2 cyclin expression was examined by in situ hybridization at 48 hr intervals from E10.5 to P8. MN20 mRNA was detected in developing cerebellum, dorsal mesencephalon, cerebral cortex, and epithalamus, but not hippocampus, striatum, or thalamus. Comparison with 5-bromodeoxyuridine labeling of cells in S phase indicated that MN20 expression in embryonic cerebellum and cerebral cortex was most pronounced in young neurons that recently had become postmitotic. Although expressed in other embryonic cerebellar neurons, MN20 was detected in granule precursors only postnatally, after their migration from the rhombic lip to the external germinal layer. This indicates that MN20/D2 cyclin is induced in cerebellar granule precursors as they become competent to differentiate. The spatial distribution of MN20 expression in the developing brain suggests that regional differences in cell cycle regulation depend in part on the selective use of cyclin proteins. Moreover, detection of MN20 mRNA in postmitotic neural cells indicates that cyclin D2 expression has effects beyond promoting cell cycle progression and may also have a role in the response of the neural precursor to terminal differentiation signals as the cells exits from proliferation.
Animals↗
Trans-species gene transfer for analysis of glucocorticoid-inducible transcriptional activation of transiently expressed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes.
Interindividual variation in the spontaneous and in the glucocorticoid-or rifampicin-inducible expression of the CYP3A cytochromes P450, the dominant froms of this supergene family that catalyze the oxidation of numerous drugs and environmental chemicals in human liver, remains largely unexplained, due in part to the lack of a validated animal model. We analyzed the 5'-flanking sequences of CYP3A genes from the rat (CYP3A23, CYP3A2), rabbit (CYP3A6), and human (CYP3A4, CYP3A5, CYP3A7) and found variable regions separated by three areas (consensus I, II, and III) of sequence homology immediately upstream of their respective promoters. We used trans-species gene transfer in cellulo as a new approach for determining the basis for qualitative differences among species in liver expression of different forms of CYP3A. When we transfected into cultured rat hepatocytes vectors containing 5'-flanking DNA from CYP3A23, CYP3A4, or CYP3A6 genes, we found that CAT activity was induced on treatment with dexamethasone or pregnenolone-16 alpha-carbonitrile only if consensus II sequences were included. Rifampicin treatment had no effect. When the same constructions containing consensus II were transfected into rabbit hepatocytes, increased activity was observed on treatment of the cells with dexamethasone or with rifampicin but not with pregnenolone-16 alpha-carbonitrile. These results suggest that the host cellular environment rather than the structure of the gene dictates the pattern of CYP3A inducibility. The application of this new model system will provide a unique technique for identifying mechanisms of induction and advancing the development of appropriate toxicological models for human safety assessment.
Animals↗
Transient expression of an atypical D1-like dopamine receptor system during avian retina differentiation.
Intact cultured retina cells from chick embryos at stage E9C5 (cultures initiated with retinae from 9-day old embryos followed by 5 days in culture), preincubated with 2 nM unlabelled SCH 23390 (R(+)-7- chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) for 20 to 60 min at 37 degrees C and then washed 5 to 25 times (approximately 1.5 min/wash) with 2 ml SCH 23390-free medium, responded to dopamine with cAMP accumulation that corresponded to 30-50% of the dopamine-promoted cAMP accumulation observed in untreated cells or in cells exposed to the inactive isomer of SCH 23390. Therefore, 50 to 70% of the dopamine response of SCH 23390-pretreated cells was inhibited after extensive washings of the cultures. At E9C12 the fraction of the dopamine response that remained inhibited by SCH 23390 after the washings declined to 30% of the control cultures or the cultures exposed to the SCH 23390 enantiomer. Cultures at stage E9C5 treated with SCH 23390 followed by extensive washings as above and then used for measuring the number of [3H]-SCH 23390 specific binding sites revealed that 60% of the sites did not interact with the tritiated compound when compared to untreated cultures or to cultures preincubated with the inactive isomer of SCH 23390. When E9C12 cultures were subjected to the same experimental protocol less than 10% of D1-like sites did not interact with [3H]-SCH 23390 after the cells had been exposed to the unlabelled compound. Dissociated cells prepared from intact retinae obtained from 12-13-day old embryos also displayed a subpopulation of D1-like sites that interacted irreversibly with SCH 23390 in a stereospecific way. These sites corresponded to 25% of the total number of D1-like sites present in the retina at this developmental stage. In retina cells obtained from one-day old posthatched chicks these sites were no longer detected. These data show that cultured retina cells as well as cells obtained from retina developing in ovo display two populations of D1-like receptors. One interacts irreversibly with SCH 23390 and is present only in the undifferentiated tissue or in cells at the early stages of culture and the other has a lower affinity for SCH 23390 with which its interaction follows reversible kinetics. These sites are present throughout the differentiation stages studied.
Animals↗
[DNA vaccination and gene therapy based on transient expression of nucleic acids in human and animal somatic cells].
Explore the source record for details and available documents.
Animals↗
Stable expression of a cDNA encoding a human beta 1 --> 3galactosyltransferase responsible for lacto-series type 1 core chain synthesis in non-expressing cells: variation in the nature of cell surface antigens expressed.
Transient expression of a human colonic adenocarcinoma Colo 205 cell derived cDNA in cell lines which ordinarily express only neolacto-series glycolipids has resulted in the expression of a beta 1 --> 3galactosyltransferase gene responsible for synthesis of glycolipids based upon the lacto-series type 1 core chain. Calcium phosphate transfected cells were panned on anti-IgM coated plates after initial treatment with a combination of monoclonal antibodies specific for type 1 chain terminal structures (TE-3) and a very broadly specific antibody reactive with multiple type 1 chain derivatives (TE-2). Adherent cells after panning were capable of efficiently transferring Gal in beta 1 --> 3-linkage to the acceptor glycolipid Lc3. Using these reagents, clones of stably transfected human colonic adenocarcinoma HCT-15 cells were produced and isolated. Parental HCT-15 cells do not express type 1 chain based antigens. The nature of the type 1 chain based antigens produced in each of these clones was analyzed by solid phase antibody binding assays. Three types of behavior were observed. Formation of type 1 terminal structures that were either exclusively sialylated or fucosylated, or a mixture of sialylated and fucosylated determinants occurred. In contrast, no difference in type 2 antigen expression between any clone and the parental cells was observed. These data suggest that coordination of subsequent reactions capable of modifying type 1 chain structures is not the same in all clones. The relationship of these results to aspects of cellular regulation of carbohydrate biosynthesis is discussed.
Adenocarcinoma↗
Distinct viral sequence elements are necessary for expression of Tomato golden mosaic virus complementary sense transcripts that direct AL2 and AL3 gene expression.
Transient expression studies using Nicotiana benthamiana protoplasts and plants have identified sequences important for transcription of complementary sense RNAs derived from Tomato golden mosaic virus (TGMV) DNA component A that direct expression of AL2 and AL3. Transcription of two complementary sense RNAs, initiating at nucleotides 1,935 (AL1935) and 1,629 (AL1629), is directed by unique sequences located upstream of each transcription initiation site. One element is located between 28 and 124 nucleotides (nt) upstream of the AL1935 transcription start site, which differs from a second element located 150 nt downstream, between 129 and 184 nt upstream of the AL1629 transcription start site. Transcription initiation at nucleotide 1,935 is lower than that at nucleotide 1,629 as determined by run-on transcription assays, and the resulting transcript is only capable of expressing AL3. The transcript initiating at nucleotide 1,629 is capable of directing expression of both AL2 and AL3, although expression of AL3 is up to fourfold greater than that for AL2. Nuclear factors purified from tobacco suspension cells bind to sequences upstream of both AL1935 and AL1629, correlating with the ability of these sequences to direct gene expression. Thus, in tobacco, regulatory sequences direct transcription of two unique TGMV messenger RNAs that differentially express AL2 and AL3.
Begomovirus↗
Ephemerally expressed wild-type and mutant steroid hormone receptors are equally able to influence expression of transient or resident templates.
We have tested transiently expressed mutant and chimeric glucocorticoid receptors (GR) for their ability to influence transcription of either a co-transfected or a stably integrated reporter gene. To the latter purpose we have generated a cell line harbouring 2 chromosomally anchored copies of the well-characterized mouse mammary tumor virus (MMTV) promoter/enhancer region fused to the bacterial beta-galactosidase gene (LacZ). We were particularly interested in verifying whether some earlier characterized dominant negative GR mutants would still act the same way on chromosomal targets. We show that trans-regulation (activation/-repression) of the chromosomally anchored reporter is qualitatively and quantitatively indistinguishable from trans-regulation obtained with transient co-transfection. In parallel, we also tested ephemerally expressed wild-type progesterone receptor (PR) and androgen receptor (AR) for their capacity of acting on either transient or resident MMTV reporter templates. Also in this case we show that activation of chromosomally anchored or transiently co-transfected reporter by both these steroid hormone receptors is qualitatively and quantitatively indistinguishable. These results outline that newly expressed trans-effectors may exert their specific function independently of the precise structural organization of their responsive genes.
Animals↗
The 5'-flanking region of the human P-450(SCC) gene shows responsiveness to cAMP-dependent regulation in a transient gene-expression system of Y-1 adrenal tumor cells.
The chronic effect of cAMP-dependent regulation on adrenocortical steroidogenesis is known to be revealed in the stimulation of the biosynthesis of steroidogenic enzymes. P-450(SCC), one of the enzymes, catalyzes the first and the rate-limiting reaction in steroidogenesis from cholesterol and its synthesis is regulated by cAMP. In order to investigate cis-acting DNA elements of this gene in response to cAMP-dependent regulation, we have constructed a fusion gene (pSCC5.4k) by ligating the 5'-flanking and the upstream untranslated region (5.4 kb) of the human P-450(SCC) gene to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected it into various culture cells including Y-1 (mouse adrenal tumor), L929 (mouse fibroblast), HTC (rat hepatoma) and Hepa-1 (mouse hepatoma). Only Y-1 cells transfected with pSCC5.4k were found to express transiently the enhanced CAT activity in response to the cAMP analogue, cyclic dibutyryl-AMP (Bt2cAMP). Primer-extension analysis of RNA prepared from the cells treated with or without Bt2cAMP showed that the enhanced CAT activity was due to an increase in the CAT mRNA and that the transcription start site, determined here with the human P-450 gene in the adrenal cortex, was correctly utilized with the fusion gene in the transient expression system. Forskolin and cholera toxin, activators of adenylate cyclase, also increased the expression of the CAT activity in the Y-1 cells. It has been demonstrated, therefore, that the cAMP-dependent regulation of the P-450(SCC) gene in adrenal cortex is faithfully reflected in the transient expression system using Y-1 cells and the fusion gene and that a cis-acting DNA element(s) in response to cAMP is present within the 5'-flanking sequence (5.4 kb) of the P-450(SCC) gene.
Adenylyl Cyclases↗
The role of p16 in the E2F-dependent thymidine kinase regulation.
The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein.
Carrier Proteins↗
Thyroid hormone regulates the mouse thyrotropin beta-subunit gene promoter in transfected primary thyrotropes.
In TtT 97 cells, a thyrotropin-producing mouse pituitary tumor, thyroid hormone rapidly inhibits the transcription rate of both the thyrotropin alpha- and beta-subunit (TSH beta) genes, and this closely parallels the increase in nuclear thyroid hormone receptor occupancy. In this study, we have identified regions of the mouse TSH beta gene which are involved in mediating tissue-specific and thyroid hormone-regulated expression. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the firefly luciferase gene. Following transfection by electroporation, efficient expression of TSH beta 5'-flanking luciferase constructs occurred only in cells derived from TtT 97 tumors which express the endogenous TSH beta gene. Deletion analysis demonstrated that the region of the 5'-flanking DNA between positions -271 and -80 relative to the major transcriptional start site is important for TSH beta promoter activity in thyrotropes. No expression was measurable in mouse L cells, a fibroblast line, whereas a low level of expression was seen in MGH 101A cells derived from a thyrotropic tumor which no longer expresses the TSH beta gene. Reduced expression of TSH beta constructs was also found in GH3 and GH4 pituitary tumor lines. Addition of thyroid hormone effectively inhibited the level of transient TSH beta promoter activity in TtT 97 cells in a dose-dependent manner. The inhibitory effect was more pronounced and more accurately reflected the transcription rate data when transfected cells were derived from tumors treated with thyroid hormone for 5 days prior to transfection. Deletion of all but 46 base pairs of TSH beta gene 5'-flanking DNA and 3 base pairs of the first exon had no effect on thyroid hormone inhibition. This indicates that signals sufficient for transcriptional regulation of the TSH beta gene by thyroid hormone reside in the vicinity of the proximal promoter and may act by interfering with basal transcriptional factors.
Animals↗
Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants.
We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B" and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with alpha-amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.
Arabidopsis↗
Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression.
The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.
3T3 Cells↗