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Results for “Transient expression”

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PubMed1996

A membrane-bound heparan sulfate proteoglycan that is transiently expressed on growing axons in the rat brain.

Monoclonal antibodies were raised to membrane-bound proteoglycans derived from rat brain and three monoclonal antibodies that recognized a 200-kDa heparan sulfate proteoglycan (designated H5-PG) with a core glycoprotein of 140 kDa were obtained. The expression of H5-PG was spatially and temporally regulated in the central nervous system. In the cerebellar cortex, H5-PG was associated mainly with the actively growing parallel fibers of granule cells. The expression was abruptly down-regulated in parallel with the formation of synapses on dendrites of Purkinje cells. In the cerebral cortex, the proteoglycan was widely distributed throughout the cortex. The temporal pattern of expression was similar to that in the cerebellar cortex; the peak level of expression was observed during the period from postnatal days 0 to 20 when neuritogenesis and synaptogenesis occur most extensively in the rat cerebral cortex. H5-PG in the central nervous system disappeared prior to adulthood except in the olfactory bulb. High-level expression was recognized on the olfactory nerves and glomeruli, where the renewal of both axons and synapses is occurring constantly. The data suggest that H5-PG is a glycoconjugate on axonal surface that is involved in axonal outgrowth and/or synaptogenesis.

Animals↗

PubMed1996

Kainic acid activates transient expression of tenascin-C in the adult rat hippocampus.

Kainic acid-induced limbic seizures enhance expression of tenascin-C (TN) in the hippocampus of adult rats. TN mRNA was detectable by in situ hybridization in many granule cells in the dentate gyrus 4.5 hr after kainic acid injection but not in saline-injected animals (controls) or in animals killed 2 or 24 hr after injection. Thirty days after kainic acid injection, TN mRNA was detectable only in pyramidal cells of CA3 and CA1. At the protein level, TN was detectable by immunocytochemistry in control animals in the strata oriens and lacunosum moleculare of CA1, in the molecular layer, and within a narrow area at the inner surface of the granule cell layer in the dentate gyrus. Twenty-four hours after kainic acid injection, TN immunoreactivity was enhanced in these areas and throughout the granule cell layer. Thirty days after kainic acid injection, TN immunoreactivity was downregulated in these areas, while it was prominent in the stratum oriens and in clusters of immunoreactivity in the stratum lucidum of CA3. Western blot analysis of the hippocampus showed a peak of TN expression 24 hr after kainic acid injection. These observations show that TN expression is upregulated in predominantly neuronal cells already by 4.5 hr after kainic acid injection, coincident with activation of granule cells and sprouting of axon terminals, whereas the remaining TN expression 30 days after injection relates to pyramidal cells in CA1 and CA3, coincident with an astroglial response, as marked by a strong expression of glial fibrillary acidic protein.

Animals↗

PubMed1997

Relax, a novel rat bHLH transcriptional regulator transiently expressed in the ventricular proliferating zone of the developing central nervous system.

A number of basic helix-loop-helix containing proteins have been shown to be required for neural development at different sites or times. Here, Relax, a novel rat basic helix-loop-helix transcriptional regulator, has been isolated and characterized. Analysis of the temporal and spatial distributions shows that the Relax transcripts are detected exclusively in the central nervous system, in discrete regions from embryonic day 11.5 to 18.5. Most strikingly, Relax is expressed along two major boundaries that define the longitudinal axis in the spinal cord and the hindbrain and is a marker of the anterior tip of this axis in the forebrain. Relax-expressing cells are strictly localized in the ventricular zone of the neural tube, where neural progenitors originate. This unique pattern of expression suggests that Relax is involved in neural fate determination.

Animals↗

PubMed2000

Transient expression of the glial glutamate transporters GLAST and GLT in hippocampal neurons in primary culture.

The extracellular glutamate concentration is kept low by glutamate transporters in the plasma membranes. Here we have studied the expression of the glutamate transporters GLAST, GLT and EAAC during the in vitro development of embryonic hippocampal neurons grown in a defined (serum free) medium. Immunochemistry studies showed that both the GLAST and GLT proteins are expressed in a subpopulation of neurons at the early, but not at the later stages of the cultures. Glial cells expressing the GLAST and GLT proteins were found at all stages. EAAC was only detected in neurons. This is one of the first evidence of a neuronal ability to express GLAST.

ATP-Binding Cassette Transporters↗

PubMed2000

Intermediate filament protein synemin is transiently expressed in a subset of astrocytes during development.

Synemin, a developmentally regulated protein first described in muscle cells, has recently been recognized as an intermediate filament (IF) protein. Because IF proteins are invaluable markers of cell origin within the nervous system, we were interested in determining the expression pattern of synemin in the brain. Our results show that, during development of the rat cortex, synemin is expressed only in a subpopulation of astrocytic cells expressing GFAP as well as vimentin and nestin. Unlike GFAP, however, synemin is not expressed in mature astrocytes and, unlike vimentin and nestin, synemin is not present in astrocytic precursors before GFAP expression. Taken together with morphological evidence, the time course of synemin expression, as determined by Western blotting, suggests that synemin is expressed in radial glial cells undergoing morphological transformation into astrocytes. Studies of synemin expression in vitro demonstrate that, early in primary culture, the majority of polygonal astrocytes are derived from synemin(+) radial glial cells. With time in culture, however, polygonal astrocytes either stop expressing synemin or are overgrown by cells not expressing synemin. The unique pattern of synemin expression, both in vivo and in vitro, suggests that the use of synemin as a marker will add a new dimension to studies of astrocytic differentiation.

Animals↗

PubMed1999

Transient expression of a mitochondrial gene cluster including rps4 is essential for the phase-shift of Dictyostelium cells from growth to differentiation.

Using synchronized Dictyostelium discoideum Ax-2 cells and the differential display method, a mitochondrial gene cluster (referred to as differentiation-associated gene 3; dia3) was isolated as one of the genes expressed specifically during the transition of Ax-2 cells from growth to differentiation. The dia3 gene encodes for a mitochondrial protein cluster (NADH dehydrogenase (NAD) subunit 11, 5, ribosomal protein S4 (RPS4), RPS2, and NAD4L). Northern blot analysis using nonsynchronized Ax-2 cells has shown that the dia3 RNA of about 8 kb is scarcely expressed during the vegetative growth phase, and the maximal expression was attained at 2 h after starvation. To analyze the gene function of dia3, we tried inactivation of rps4 by means of homologous recombination and obtained several transformed clones showing mitochondrial DNA heteroplasmy. The transformed cells grew normally in nutrient medium, but their development after starvation was greatly impaired, thus resulting in the failure of many cells to differentiate. In this connection, the cAMP receptor 1 (car1) expression, which is one of the earliest markers of differentiation, was found to be markedly reduced in the rps4-inactivated cells.

Animals↗

PubMed1995

Transient expression of somatostatin receptors in the brain during development.

The study of somatostatin receptors by means of autoradiography in tissue sections revealed high densities of binding sites in the immature central nervous system. In rat cerebral cortex, the receptors are present in the intermediate zone and in association with cells migrating through the cortical plate. Somatostatin receptors in the intermediate zone of fetuses and in the cortical plate of postnatal rats exhibit high and low affinities respectively for the somatostatin analogue MK 678. In the rat cerebellum, the external granule cell layer, a germinal matrix containing interneuron precursors, contains a high density of receptors. These receptors exhibit high affinity for MK 678 throughout the period of cell multiplication. In granule cell cultures from eight-day-old rats, MK 678, octreotide and somatostatin are able to inhibit cAMP formation induced by forskolin or pituitary adenylyl cyclase-activating polypeptide. Somatostatin reduces the intracellular Ca2+ concentration in cultured granule cells; this response desensitizes rapidly. These results suggest that the somatostatin receptors in the external granule cell layer are type 2 receptors (sstr2). A low density of receptors with low affinity for MK 678 was also detected in the external granule cell layer and in the granule cell layer of neonatal rats. In adult rats the cerebellum is devoid of somatostatin receptors. These observations indicate that somatostatin probably exerts morphogenetic activities through different receptor types in several structures of the central nervous system.

Animals↗

PubMed1994

Transient expression of type II collagen and tissue mobilization during development of the scleral ossicle, a membranous bone, in the chick embryo.

Development of the chick scleral ossicle was studied with respect to expression of various collagen types, cartilage matrix molecules, and osteoblastic cell surface antigens. The extra-cellular matrix of the scleral ossicle primordium of stage 35.5 chick sclera and the mesenchyme beneath the conjunctival epithelium was immunoreactive with anti-type II collagen antibody, giving the impression that certain materials and/or cell clusters surrounded by reactive matrix were descending from the epithelial-mesenchymal interface to the scleral ossicle primordium. In stage 37 embryos, type II collagen immunoreactivity was restricted to the bone matrix of the scleral ossicles, and persisted through stage 39. However, at stage 41, virtually no type II collagen was detected. In contrast, strong immunostaining of type I collagen was first detected in the developing scleral ossicle at stage 37, coinciding with the formation of mineralized bone matrix. Following the extensive accumulation of type I collagen in bone matrix, type XII collagen was detected at the surface of the bone; both type I and type XII collagen immunostainings then remained. By stage 37, immunoreactivity with a pre-osteoblastic cell surface marker was detected on cells of the scleral ossicle, and typical osteocytes were subsequently identified by both morphological and specific immunostaining techniques. Antibodies other than for type II collagen, specific to chondrogenic mesenchyme or cartilage matrix, never reacted with the scleral ossicle and its primordium during development. Taken together, these observations indicate that the scleral ossicle is a membranous bone, whose development may not require overt chondrogenesis. Implications of type II collagen distribution during the positioning of scleral ossicles and their early bone matrix formation are discussed with respect to the origin and evolution of endoskeletons in vertebrate animals.

Animals↗

PubMed2002

Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells.

The transduction of auditory signals by cochlear hair cells depends upon the integrity of hair cell stereociliary bundles. Stereocilia contain a central core of actin filaments, cross-linked by actin bundling proteins. In the cochlea, the two proteins described to date as responsible for the spatial arrangement of actin filaments in sterocilia are fimbrin and the recently discovered espin. Fimbrin (the chick homolog of human I-plastin) belongs to the plastins/fimbrin family that includes two additional isoforms of plastins, T- and L-plastin. In the present study, we used isoform specific antibodies to investigate the presence of the T- and L-isoforms of plastin/fimbrin in the adult and developing rat cochlea. We found that T-plastin, but not L-plastin, is expressed in the rat cochlea. During postnatal development of the rat organ of Corti, T-plastin can be detected in the core of stereocilia from early stages of hair cell differentiation, and its expression gradually increases in stereocilia as hair cells mature. However, as opposed to other actin-binding proteins expressed in stereocilia, T-plastin is absent from the stereocilia of mature hair cells. Such temporally restricted expression strengthens the idea of functional differences between plastins isoforms, and suggests that T-plastin could have a specific role in stereocilia formation.

Aging↗

PubMed2001

Transient expression of synaptic zinc during development of uncrossed retinogeniculate projections.

The transition metal zinc is an essential dietary constituent that is believed to serve an important intercellular signaling role at certain excitatory synapses in the central nervous system. In the present study, we used histochemical techniques to investigate the distribution of synaptic zinc during postnatal development of retinogeniculate projections in rats. From postnatal day (P) 1 until P-21, the pattern of zinc histochemical staining in the dorsal lateral geniculate nucleus (LGNd) precisely matched the distribution of axon terminals from the ipsilateral eye that were labeled by anterograde transport of horseradish peroxidase. Regions of the LGNd that contained only crossed axons were devoid of zinc staining. Abnormalities in the distribution of uncrossed retinogeniculate projections in albino versus pigmented rats were paralleled by identical variations in localization of synaptic zinc. Unilateral enucleation on P-10 was followed within 5 days by loss of zinc staining in the LGNd ipsilateral to the removed eye without affecting staining in the contralateral nucleus. Finally, the ability to detect zinc histochemically in the LGNd ceased at approximately P-24. These findings provide evidence that zinc is sequestered within synaptic boutons of a subpopulation of retinal ganglion cells whose axons terminate on the ipsilateral side of the brain. The duration of zinc staining overlaps with the major period of axonal remodeling in the LGNd, suggesting that synaptically released zinc may play a role in postnatal refinement of retinogeniculate projections.

Age Factors↗

PubMed2005

Low-density lipoprotein receptor-related protein (LRP)-2/megalin is transiently expressed in a subpopulation of neural progenitors in the embryonic mouse spinal cord.

The lipoprotein receptor LRP2/megalin is expressed by absorptive epithelia and involved in receptor-mediated endocytosis of a wide range of ligands. Megalin is expressed in the neuroepithelium during central nervous system (CNS) development. Mice with homozygous deletions of the megalin gene show severe forebrain abnormalities. The possible role of megalin in the developing spinal cord, however, is unknown. Here we examined the spatial and temporal expression pattern of megalin in the embryonic mouse spinal cord using an antibody that specifically recognizes the cytoplasmic part of the megalin molecule. In line with published data, we show expression of megalin in ependymal cells of the central canal from embryonic day (E)11 until birth. In addition, from E11 until E15 a population of cells was found in the dorsal part of the developing spinal cord strongly immunoreactive against megalin. Double labeling showed that most of these cells express vimentin, a marker for immature astrocytes and radial glia, but not brain lipid binding protein (BLBP), a marker for radial glial cells, or glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. These findings indicate that the majority of the megalin-positive cells are astroglial precursors. Megalin immunoreactivity was mainly localized in the nuclei of these cells, suggesting that the cytoplasmic part of the megalin molecule can be cleaved following ligand binding and translocated to the nucleus to act as a transcription factor or regulate other transcription factors. These findings suggest that megalin has a crucial role in the development of astrocytes of the spinal cord.

Animals↗

PubMed1992

Immunohistochemical evidence for transient expression of fibronectin in the developing dorsal lateral geniculate nucleus of the ferret.

In recent years, the important role of the extracellular matrix in neural development has been increasingly recognized. In order to begin to examine what role might be played by the extracellular matrix in the developing dorsal lateral geniculate nucleus (dLGN), the present study used immunocytochemistry to assess the distribution of a recognized extracellular matrix molecule, fibronectin (FN), during postnatal development of the ferret dLGN. Prior to the segregation of cell layers, no clear pattern of FN distribution can be distinguished within the dLGN. By P16, when layers A and A1 are separated by an interlaminar space, FN is localized in that space. By P24, FN is present not only between layers A and A1, but also within layers A and A1 as bands parallel to the laminar borders. These bands appear to correspond spatially and temporally to the development of sublaminar boundaries in the ferret (Hahm and Sur, Neurosci Abstr 14:460, 1988). By the end of the first postnatal month, immunoreactivity is diminished but still present. FN is no longer present at P44 or in the adult. Adjacent sections were incubated with antiserum to glial fibrillary acid protein (GFAP). When a laminar pattern of GFAP can be distinguished, it coincides with the interlaminar distribution of FN. The findings described here are consistent with a role for FN in the development of a laminar organization in the dLGN.

Animals↗

PubMed1995

Early postmitotic neurons transiently express TOAD-64, a neural specific protein.

To identify proteins involved in the early development of the mammalian cerebral cortex, we previously used two-dimensional gels to compare proteins synthesized at different stages in corticogenesis in the embryonic rat at embryonic day 14 (E14), E17, and E21. During this period, the cortex develops from a morphologically homogeneous population of proliferative precursor cells into a complex structure containing a diverse array of terminally differentiated neurons. Several proteins are up-regulated coincident with the generation of postmitotic neurons. Here we describe the purification, partial amino acid sequencing, and characterization of one of these proteins, TOAD-64 (Turned On After Division; 64 kDa), using polyclonal antisera to two synthetic peptides from the protein. This analysis reveals that TOAD-64 is a 64,000 Da protein that increases in abundance over the period of corticogenesis and then subsequently decreases to very low levels in the adult. The protein is neural specific and is expressed by postmitotic neurons as they begin their migration out of the ventricular zone into the developing cortical plate. It is expressed in advance of most other neuronal proteins. Progenitor cells do not express TOAD-64. Therefore, this protein is a marker for postmitotic cells that have made a commitment to a neuronal phenotype. The extremely early expression, the relative abundance in newly born neurons, as well as the restriction in expression to the period of initial neuronal differentiation suggest that TOAD-64 may be a key structural protein for early neuronal function.

Amino Acid Sequence↗

PubMed2002

Transient expression of TIP60 protein during early chick heart development.

Screening of an embryonic chick cDNA library revealed a gene product termed chick TIP60 (cTIP60) due to its homology with human TIP60, a founding member of the "MYST" family of proteins that possess functional motifs, including chromo, zinc finger, and histone acetyltransferase domains. cTIP60 expression was assessed during early chick embryogenesis, at the RNA level by using reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level by using Western blotting and immunohistochemistry. RT-PCR indicated that cTIP60 transcripts in whole embryos are present as early as Hamburger-Hamilton (HH) stage 5, diminishing after HH10. Western blotting of total embryonic protein revealed that cTIP60 was present in uniform quantities between HH3 and HH25. By contrast, Western blotting of protein from isolated hearts revealed that cTIP60 protein was strongly expressed at the earliest stages of heart development (HH11-13), diminishing thereafter. This finding was corroborated by immunohistochemistry, which revealed that cTIP60 protein was selectively expressed at high levels in the myocardium between HH 10-14. Considered in the context of its functional domains, these findings suggest that cTIP60 modulates transcriptional processes which regulate terminal cell differentiation, proliferation, or both, during early myocardial development.

Acetyltransferases↗

PubMed2006

Identification of novel mutations in the human ornithine transcarbamylase (OTC) gene of Korean patients with OTC deficiency and transient expression of the mutant proteins in vitro.

The urea cycle plays key roles to prevent the accumulation of toxic nitrogenous compound and synthesize arginine de novo. Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea cycle, which is inherited in an X-linked manner. This study was undertaken to characterize molecular defects in Korean patients with OTC deficiency. With direct sequence analysis of OTC gene of 26 unrelated Korean patients with OTC deficiency, 23 different mutations were identified. Among these mutations, eleven were novel mutations. The novel mutations were p.Leu9X, p.Arg26Pro, p.Gly100Arg, p.Met205Thr, p.Lys221Asn, p.Asp249Gly, p.Phe281Ser, p.Val323Met, c.571delC, c.853delC, and c.796-805del. All the novel mutations in this study were tested in 100 normal alleles. In vitro expression study of some of novel missense mutations elucidated the correlation of genotype and phenotype of the OTC deficiency.

Adolescent↗

PubMed1987

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.

Carcinoma, Hepatocellular↗

PubMed1990

A mouse zinc finger gene which is transiently expressed during spermatogenesis.

Zinc finger proteins are polypeptides with sequence-specific, nucleic acid-binding properties. Substantial evidence has established them as a class of trans-acting molecules with regulatory roles in cellular growth and differentiation. We have screened an 11.5 day post coitum urogenital ridge cDNA library with an oligonucleotide encoding a sequence conserved between a variety of zinc finger proteins. By cDNA cloning and sequencing we show that a novel mouse gene, Zfp-35, encodes a protein with a block of 18 zinc finger domains and an N-terminal region rich in acidic residues. The 2.4 kb mRNA encoding this polypeptide is selectively expressed in adult testis, by comparison with other organs. We have analysed Zfp-35 expression in whole testes of sex-reversed mice, whole testes of prepuberal XY animals, germ cell fractions from XY adult testes and by in situ hybridization to sections from adult XY testes. Our studies show that a considerable increase in expression is restricted to spermatocytes at the pachytene stage of meiotic prophase. These experiments suggest that Zfp-35 may act to control gene activity during this particular stage of spermatogenesis.

Amino Acid Sequence↗

PubMed1994

Transient expression of prostaglandin endoperoxide synthase-2 during mouse macrophage activation.

We investigated regulation of macrophage prostaglandin production during activation by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). An in vitro model was established using the mouse macrophage-like cell line RAW 264.7. Cells were cultivated in the presence of IFN-gamma and LPS for up to 48 h and changes in the secretion of nitric oxide (NO.) and tumor necrosis factor alpha (TNF-alpha) were observed as activation markers. Under these conditions a prompt and strong increase in PGE2 production was found in the first 8 h followed by nearly constant generation of PGE2 during the next 40 h. In contrast, the activity of prostaglandin endoperoxide synthase (PGHS), measured as PGE2 production of microsomal protein fractions, was also increased, but reached a clear maximum at 24 h. Recently a second form of PGHS was cloned (PGHS-2) and specific antibodies and mRNA probes for both isoforms are available. PGHS-2 enzyme was expressed maximally after 24 h of activation whereas PGHS-1 was not influenced. In the presence of IFN-gamma and LPS, PGHS-2 mRNA expression reached a maximum at 8 h but PGHS-1 mRNA was not induced during the whole time period. These data indicate that changes in PG synthesis following macrophage activation are due to regulation of PGHS-2 expression.

Animals↗