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PubMed1971

Evidence for and localization of vegetative viral DNA replication by autoradiographic detection of RNA-DNA hybrids in sections of tumors induced by Shope papilloma virus.

The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis.

Animals↗
PubMed1983

Different mRNAs have different nuclear transit times in Dictyostelium discoideum aggregates.

Nuclear processing of mRNA precursors in differentiating multicellular Dictyostelium discoideum aggregates is markedly slower than in growing amoebae. Thus, we have been able to determine the time of nuclear processing of individual mRNA species in postaggregating cells by following the incorporation of 32PO4 into nuclear and cytoplasmic RNA complementary to cloned cDNAs. Precursors of mRNAs synthesized during both growth and differentiation remain in the nucleus for about 25 to 60 min. By contrast, typical mRNAs which are synthesized only by postaggregative cells have nuclear processing times between 50 and 100 min. Depending on the particular mRNA, between 20 and 60% of nuclear transcripts are converted into cytoplasmic mRNA. A third class of mRNAs are transcribed from a set of repetitive DNA segments and are expressed predominantly during differentiation. Nuclear precursors of these mRNAs are extensively degraded within the nucleus or very rapidly after transport to the cytoplasm. Those sequences that are stable in the cytoplasm exit from the nucleus only after a lag of over 2 h. Thus, mRNAs encoded by different genes that are subject to different types of developmental controls display different times of transit to the cytoplasm and different efficiencies of nuclear processing. Differential nuclear processing may contribute to the regulation of the level of individual cytoplasmic mRNAs.

Biological Transport↗
PubMed1996

Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum.

Release of Ca2+ stored in endoplasmic reticulum is a ubiquitous mechanism involved in cellular signal transduction, proliferation, and apoptosis. Recently, sphingolipid metabolites have been recognized as mediators of intracellular Ca2+ release, through their action at a previously undescribed intracellular Ca2+ channel. Here we describe the molecular cloning and characterization of a protein that causes the expression of sphingosyl-phosphocholine-mediated Ca2+ release when its complementary RNA is injected into Xenopus oocytes. SCaMPER (for sphingolipid Ca2+ release-mediating protein of endoplasmic reticulum) is an 181 amino acid protein with two putative membrane-spanning domains. SCaMPER is incorporated into microsomes upon expression in SO cells or after translation in vitro. It mediates Ca2+ release at 4 degrees C as well as 22 degrees C, consistent with having ion channel function. The EC50 for Ca2+ release from Xenopus oocytes is 40 microM, similar to sphingosyl-phosphocholine-mediated Ca2+ release from permeabilized mammalian cells. Because Ca2+ release is not blocked by ryanodine or La3+, the activity described here is distinct from the Ca2+ release activity of the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. The properties of SCaMPER are identical to those of the sphingolipid-gated Ca2+ channel that we have previously described. These findings suggest that SCaMPER is a sphingolipid-gated Ca2+-permeable channel and support its role as a mediator of this pathway for intracellular Ca2+ signal transduction.

Amino Acid Sequence↗
PubMed1988

Adenovirus transcriptional complexes contain EIa encoded tumour antigens physically bound to cellular proteins.

Adenovirus type 12 transcriptional complexes were isolated from cells during the early phase of infection. Sedimentation analysis identified a fast sedimenting complex type I and a slow sedimenting complex type II. Both complexes made virus specific RNA complementary to all the early genes and both contained viral DNA, which in type II but not in type I had nucleosome like configuration. Analysis of the proteins of the complexes with antiserum against Ad 12 EIa-beta-galactosidase fusion protein expressed in E. coli demonstrated the following: (a) type I complex contained EIa 45 K protein, which co-precipitated with cellular proteins of mol. wt. 42, 58, and 60 K, (b) type II complex contained EIa 47 K protein, which co-precipitated with major cellular proteins of 35, 40-46 K and minor proteins of 58, 60, 68, 76, 86, and 120-150 K. Association of EIa specific and cellular proteins to transcriptional complexes was sensitive to both 1 M NaCl and DNAse I indicating the DNA binding nature of these proteins. Treatment of transcriptional complexes with 1 M NaCl or DNase I released EIa proteins, which still remained strongly bound to cellular proteins. These findings suggested that EIa proteins bind to viral DNA and that this binding is probably mediated by cellular proteins.

Adenovirus Early Proteins↗
PubMed2005

[Antiviral effects of dual-target antisense rna: an experimental study with hepatitis B virus transgenic mice].

OBJECTIVE: To investigate the curative effects of dual-target antisense RNA targeting the X and P regions in the genome of hepatitis B virus (HBV). METHODS: Retrovirus vector pLXSN was used to construct 4 kinds of recombinant vector plasmids expressing dual-target antisense RNA complementary to the X and P regions in the genome of HBV, namely, pLXSN-asX, pLXSN-asP, pLXSN-asXP, and pLXSN-seX. 48 HBV transgenic mice were randomly divided into 6 equal groups: pLXSN-asX group, pLXSN-asX group, pLXSN-asX group, pLXSN-asX group, and blank plasmid blank (pLXSN) group, to be injected into the caudal vein with corresponding plasmids thrice for every other day, and blank control group. Venous blood samples were collected before, 1 day and 3 days, and 2, 4, and 8 weeks after the injection to undergo detection of serum HBV DNA and HBsAg. Eight weeks later the mice were killed and immunohistochemistry was used t examine the HBsAg and HBcAg in the tissues. Pathological examination of the tissues was performed. RESULTS: The serum HBsAg concentrations 4 and 8 weeks after injection were significantly lower than that before injection in the.pLXSN-asX and pLXSN-asXP groups (all P <0.05) with an inhibition rate of 24% and 27% respectively. In comparison with that before injection, the HBV DNA expression level 2 weeks after injection was significantly lower in the pLXSN-asX group (P < 0.05) with an inhibition rate of 58%. In comparison with that before injection, the HBV DNA expression level 8 weeks after injection was significantly lower in the pLXSN-asP group (P <0.05) with an inhibition rate of 58%. In comparison with that before injection, the HBV DNA expression level in the pLXSN-asXP group showed 2 times of significant decrease 1 week and 8 weeks after injection (both P < 0.05) with the inhibition rates of 66% and 77% respectively. HBsAg and HBcAg were expressed in liver were significantly lower in the pLXSN-asX, pLXSN-asP, and pLXSN-asXP groups than in other groups (P < 0.05). No significant abnormality was found in the tissues in all groups. CONCLUSION: Dual-target antisense RNA targeting the X and P regions in the genome of HBV inhibits the replication and expression of HBV, significantly stronger than single-target antisense-RNA.

Animals↗
PubMed1997

Initiation of vesicular stomatitis virus mutant polR1 transcription internally at the N gene in vitro.

The vesicular stomatitis virus (VSV) polymerase is thought to initiate transcription of its genome by first copying a small leader RNA complementary to the 3' end of the template. The polR VSV mutants, in contrast to wild-type virus, frequently read through the leader termination site during transcription in vitro. To shed light on polymerase termination and reinitiation events at the crucial leader-N gene junction, we employed RNase protection assays to precisely measure molar ratios of leader, N, and readthrough transcript accumulation in vitro. Wild-type virus synthesized essentially equimolar amounts of leader and N transcripts, but, unexpectedly, the polR1 mutant yielded about twice as much N mRNA as leader (ratio of 1.9 +/- 0.1). Primer extension assays ruled out an increase in abortive N transcript synthesis for polR1. Transcription entailed multiple rounds of synthesis, with transcript ratios remaining the same after 0.5 or 2 h of synthesis, ruling out a significant contribution from polymerases "pre-positioned" at the N gene. No significant degradation of either leader or N transcripts was observed after incubating purified products with virions. Our data lead us to conclude that transcription can initiate internally at the N gene, at least in the case of polR1 VSV. We propose, however, that productive internal initiation of transcription is a fundamental property of the VSV polymerase and that of related viruses. A model postulating two distinct polymerase complexes, one for leader synthesis and one for internal initiation, is presented.

Gene Expression Regulation, Viral↗
PubMed1986

Cyclic adenosine nucleotides and growth hormone-releasing factor increase cytosolic growth hormone messenger RNA levels in cultured rat pituitary cells.

The cellular mechanisms involved in GH biosynthesis have been investigated by the measurement of steady-state levels of cytosolic GH messenger RNA (mRNA) in primary cultures of rat pituitary cells using an RNA-complementary DNA (cDNA) hybridization assay. Growth hormone mRNA-cDNA hybridization increased in a linear manner with increasing cytosol concentration. Cellular GH mRNA levels rose by an average of 2.4-fold (range, 1.6-3.3; n = five experiments) after exposure to GH-releasing factor (GRF(1-40); 10 nmol/l) for 3 days. Treatment with GRF increased the release of GH into the culture medium, and depleted the cellular GH content by 40%. Total GH (in the medium plus cells) after GRF treatment increased by between 1.5- and 3.8-fold, a magnitude similar to the increase in GH mRNA levels. Treatment of cells with dibutyryl adenosine 3':5'-cyclic monophosphate (1 mmol/l) or forskolin (5 mumol/l) increased the levels of cytosolic GH mRNA by between 1.6- and 4.7-fold. These agents increased GH release into the medium, depleted cellular GH content and increased total GH in the system to the same extent as GRF (10 nmol/l). These data demonstrate that cyclic adenosine nucleotides may mediate the GRF induction of GH gene transcription. In addition, we have shown that increases in the levels of cellular GH mRNA are reflected by increased GH biosynthesis, suggesting that the regulation of hormone gene transcription is one cellular site for the control of hormone biosynthesis and, ultimately, hormone available for release.

Animals↗
PubMed1991

Repression of integrin beta 1 subunit expression by antisense RNA.

A quail cell line (QT6-c) was co-transfected with pTEX vector expressing RNA complementary to chicken integrin beta 1 subunit mRNA (Anti-Int) and pRSVneo vector by a calcium phosphate method. Transfectants showing reduced expression of quail integrin beta 1 subunit were selected with an immunoblot assay, and a few positive clones were examined in detail. Northern blot and immunoblot analyses revealed that the Anti-Int caused a clear reduction of the transcript encoding integrin beta 1 subunit depending on culture conditions. The number of cell surface integrins also decreased in proportion to the decrement of the total amount of integrin beta 1 subunits. When one transfectant (QA23) was cultured in a serum-free medium, cell shape changed from fibroblast-like to neuron-like morphology accompanied by a low growth rate, and the cells did not form focal contact on fibronectin. A similar morphological change occurred in QT6-c cells when the cells were infected with Rous Sarcoma virus, which could produce the Anti-Int. The QA23 cells did not attach to fibronectin as efficiently as did the original QT6-c cells. These data suggest that reduced expression of integrin beta 1 subunit affects cell growth as well as cell morphology by disordering the interaction between integrins and matrix proteins and/or cytoplasmic proteins.

Animals↗
PubMed2003

Adenovirus-mediated ribonucleotide reductase R1 gene therapy of human colon adenocarcinoma.

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1). Recombinant viruses were constructed by FLP-mediated site-specific recombination and demonstrated high infectivity of a human colon carcinoma cell line (Colo320 HRS), as assessed by expression of a viral encoded beta-Gal gene (rAd5-LacZ). R1mRNA and protein were overexpressed in Colo320 HRS cells infected with rAd5-R1 compared with untreated or rAd5-LacZ-infected cells. Infection with rAd5-R1 inhibited Colo320 HRS cell proliferation, in vitro, in a time- and dose-dependent manner. When Colo320 HRS cells were treated with rAd5-R1, before injection into CD-1 mice, there was complete inhibition of tumor growth compared with treatment with rAd5-LacZ. Furthermore, intratumoral injection of rAd5-R1 into Colo320 HRS tumor xenografts inhibited tumor growth in CD-1 mice compared with rAd5-LacZ treated mice (P = 0.0001). These results demonstrate gene-specific antitumor effects of R1 and suggest that rAd5-R1 gene therapy has the potential to improve currently available treatments for colon cancer.

Adenocarcinoma↗
PubMed1994

Alpha calcium/calmodulin-dependent protein kinase II selectively expressed in a subpopulation of excitatory neurons in monkey sensory-motor cortex: comparison with GAD-67 expression.

In situ hybridization histochemistry and immunocytochemistry, including double immunofluorescence, were used to study the populations of neurons expressing the alpha subunit of type II calcium/calmodulin-dependent protein kinase (CAM II kinase-alpha) or glutamic acid decarboxylase (GAD) in the somatic sensory and motor areas of the macaque monkey cerebral cortex. Sections were subjected to in situ hybridization using radioactive, complementary RNA probes specific for monkey CAM II kinase-alpha or 67 kDa GAD mRNAs. Others were stained immunocytochemically for CAM II kinase-alpha and/or GABA. CAM II kinase-alpha and GAD-67 are expressed in different populations of cells, with no colocalization. CAM II kinase-alpha is expressed in pyramidal cells of layers II-VI, especially layers II and III, as well as in certain small nonpyramidal cells of layer IV in areas 3a, 3b, 1, and 2 and of middle regions of area 4. Both cell types produce excitatory amino acid transmitters. Therefore, as in subcortical regions, CAM II kinase-alpha will be found on the presynaptic side of excitatory synapses but on the postsynaptic side only when these synapses occur on excitatory neurons in the sensory-motor cortex. Quantitative examination showed that CAM II kinase-alpha immunoreactive cells form, on average, approximately 50% of the total neuronal population in each area, while GABA immunoreactive or GAD cRNA hybridized cells form approximately 25-30%. Thus, CAM II kinase-alpha expressing cells cannot account for the total population of non-GABAergic cortical cells, and a certain proportion of the pyramidal cells probably do not express it. In other cortical areas, gene expression for the two molecules is regulated by afferent activity. Therefore, the present results form a necessary basis for studies aimed at determining the role of activity-dependent changes in the balance of excitation and inhibition as a mechanism underlying plasticity of representational maps in the primate sensory-motor cortex.

Animals↗
PubMed1997

[Transfer and expression of antisense genes of hepatitis B virus (HBV) and their anti-HBV effects].

OBJECTIVE: To observe the transfer and expression of HBV antisense genes and their anti-HBV effects in 2.2.15 cells mediated by retroviral vector-packaging cell line system. METHODS: The recombinant retroviral vectors mediating expression of HBV antisense RNA complementary to 1402-2906 fragment (preC/C) or 2839-1986 fragment (preS/S) of HBV DNA were used to transduce into NIH 3T3 cells and 2.2.15, respectively. Then, the total RNAs of tranduced NIH 3T3 cells were extracted and hybridized with HBV DNA probe. The HBsAg and HBeAg in the supernatants of cultured 2.2.15 cells were assayed with RIA method and the DNAs were hybridized with HBV DNA probe by dot blot. RESULTS: The results showed that the HBV antisense genes could be transfered into and expressed in the NIH 3T3 cells mediated by recombinant retroviral vector packaging cell line (PA317 cell). The inhibitory effects on the expression of HBV antigens by antisense RNAs appeared as early as on the day 3 after transduced, reached peak level on the day 5, and persisted at least for eleven days. The inhibitory rates of HBsAg and HBeAg were 71% and 23% by antisense preS/S, and 23% and 59% by antisense preC/C on the day 5 after transduction. In comparison to blank control or sense--gene expressing vectors, the inhibitory effects of HBV antisense vectors were highly significant (P < 0.01). HBV DNA level in the supernatant of the 2.2.15 cells transduced with either antisense preS/S or preC/C in comparison to the blank control vectors was also reduced on the day 5 after transduction as detected by DNA dot blot assays, but the viability of transduced 2.2.15 cells was not affected as detected by MTT assays. CONCLUSIONS: HBV antisense genes can be transfered and expressed in the eukaryotic cells and the expression of HBV antisense RNAs in 2.2.15 cells can inhibit the replication and expression of HBV.

3T3 Cells↗
PubMed1977

Rate of divergence of cellular sequences homologous to segments of Moloney sarcoma virus.

The RNA genome of the Moloney isolate of murine sarcoma virus (M-MSV) consists of two parts--a sarcoma-specific region with no homology to known leukemia viral RNAs, and a shared region present also in Moloney murine leukemia virus RNA. Complementary DNA was isolated which was specific for each part of the M-MSV genome. The DNA of a number of mammalian species was examined for the presence of nucleotide sequences homologous with the two M-MSV regions. Both sets of viral sequences had homologous nucleotide sequences present in normal mouse cellular DNA. MSV-specific sequences found in mouse cellular DNA closely matched those nucleotide sequences found in M-MSV as seen by comparisons of thermal denaturation profiles. In all normal mouse cells tested, the cellular set of M-MSV-specific nucleotide sequences was present in DNA as one to a few copies per cell. The rate of base substitution of M-MSV nucleotide sequences was compared with the rate of evolution of both unique sequences and the hemoglobin gene of various species. Conservation of MSV-specific nucleotide sequences among species was similar to that of mouse globin gene(s) and greater than that of average unique cellular sequences. In contrast, cellular nucleotide sequences that are homologous to the M-MSV-murine leukemia virus "common" nucleotide region were present in multiple copies in mouse cells and were less well matched, as seen by reduced melting profiles of the hybrids. The cellular common nucleotide sequences diverged very rapidly during evolution, with a base substitution rate similar to that reported for some primate and avian endogenous virogenes. The observation that two sets of covalently linked viral sequences evolved at very different rates suggests that the origin of M-MSV may be different from endogenous helper viruses and that cellular sequences homologous to MSV-specific nucleotide sequences may be important to survival.

Animals↗
PubMed1997

Endocrine profile and neuroendocrine challenge tests in transgenic mice expressing antisense RNA against the glucocorticoid receptor.

A transgene expressing antisense RNA complementary to a fragment of the glucocorticoid receptor cDNA was incorporated into the mouse genome and resulted in a transgenic animal that has decreased glucocorticoid receptor function. The transgenic mice showed basal plasma ACTH and corticosterone levels similar to those of the normal control animals. We have further investigated changes in HPA axis regulation by use of different neuroendocrine challenge tests including a dexamethasone suppression test (DST). In comparison to normal mice, a tenfold higher dose of dexamethasone (i.e. 20 micrograms/100 g body weight) was required to suppress the basal corticosterone levels of transgenic mice. Dexamethasone (2 micrograms/100 g body weight) produced a long-lasting suppression of plasma ACTH and corticosterone levels in control mice, whereas in transgenic animals only a short-lasting decrease in ACTH levels was apparent. Corticotropin-releasing hormone (CRH) administration resulted in an enhanced response in plasma ACTH levels in transgenic mice, whereas the corticosterone response was markedly reduced. The discrepancy between ACTH and corresponding corticosterone secretions in transgenic mice could be attributed, in part, to a reduced sensitivity of the adrenal gland to stimulation by ACTH. Pituitaries of transgenic mice contained about 50% less proopiomelanocortin (POMC) mRNA than those of control animals. No significant differences were noted in the ACTH or protein contents of normal and transgenic mice pituitary glands although a slight increase in protein content of the transgenic mouse adrenal gland was apparent. In conclusion, transgenic mice with impaired GR function show major disturbances in HPA axis regulation which seem to be caused by the primary defect in conjunction with secondary modifications in, amongst others, pituitary CRH receptor system(s), sympathetic output and adrenal development. This mouse is therefore a useful model to study the consequences of life-long defective GR function and HPA axis regulation in general.

Adrenal Glands↗
PubMed1980

The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei.

We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.

Animals↗
PubMed1999

Laminar and cellular distribution of AMPA, kainate, and NMDA receptor subunits in monkey sensory-motor cortex.

In situ hybridization histochemistry and immunocytochemistry were used to examine lamina- and cell-specific expression of glutamate receptor (GluR) mRNAs and polypeptide subunits in motor and somatosensory cortex of macaque monkeys. Radioactive complementary RNA (cRNA) probes were prepared from cDNAs specific for alpha-amino-3-hydroxy-5-methylisoxozolepropionate (AMPA)/kainate (GluR1-GluR4), kainate (GluR5-GluR7), and N-methyl-D-aspartate (NMDA; NR1, NR2A-NR2D) receptor subunits. AMPA/kainate and NR1, NR2A, and NR2B receptor transcripts show higher expression than other transcripts. All transcripts show lamina-specific patterns of distribution. GluR2 and GluR4 mRNAs show higher expression than do GluR1 and GluR3 mRNAs. GluR6 transcript expression is higher than that of GluR5 and GluR7. NR1 mRNA expression is much higher than that of NR2 mRNAs. NR2C subunit expression is very low except for a very distinct band of high expression in layer IV of area 3b. Immunocytochemistry, using subunit-specific antisera and double labeling for calbindin, parvalbumin, or alpha type II Ca2+/calmodulin-dependent protein kinase (CaMKII-alpha), allowed identification of cell types expressing different subunit genes. GluR1 and GluR5/6/7 immunoreactivity is found in both pyramidal cells and gamma-amino butyric acid (GABA) cells; GluR2/3 immunoreactivity is preferentially found in pyramidal cells, whereas GluR4 immunoreactivity is largely restricted to GABA cells; NMDA receptor subunit immunoreactivity is far greater in excitatory cells than in GABA cells. The density of expression of AMPA/kainate, kainate, and NMDA receptor subunit mRNAs differed within and across the architectonic fields of sensory-motor cortex. This finding and the lamina- and cell-specific patterns of expression suggest assembly of functional receptors from different arrangements of available subunits in specific neuronal populations.

Animals↗
PubMed2000

Expression of NMDA receptor subunit mRNAs in neurochemically identified projection and interneurons in the human striatum.

N-methyl-D-aspartate (NMDA) receptors are composed of subunits from two families: NR1 and NR2. We used a dual-label in situ hybridization technique to assess the levels of NR1 and NR2A-D messenger ribonucleic acid (mRNA) expressed in projection neurons and interneurons of the human striatum. The neuronal populations were identified with digoxigenin-tagged complementary RNA probes for preproenkephalin (ENK) and substance P (SP) targeted to striatal projection neurons, and somatostatin (SOM), glutamic acid decarboxylase 67 kD (GAD(67)), and choline acetyltransferase (ChAT) targeted to striatal interneurons. Intense NR1 signals were found over all striatal neurons. NR2A signals were high over GAD(67)-positive neurons and intermediate over SP-positive neurons. ENK-positive neurons displayed low NR2A signals, whereas ChAT- and SOM-positive neurons were unlabeled. NR2B signals were intense over all neuronal populations in striatum. Signals for NR2C and NR2D were weak. Only ChAT-positive neurons displayed moderate signals, whereas all other interneurons and projection neurons were unlabeled. Moderate amounts of NR2D signal were detected over SOM- and ChAT-positive neurons; GAD(67)- and SP-positive striatal neurons displayed low and ENK-positive neurons displayed no NR2D hybridization signal. These data suggest that all human striatal neurons have NMDA receptors, but different populations have different subunit compositions that may affect function as well as selective vulnerability.

Aged↗
PubMed1996

Ontogenic and glucocorticoid-accelerated expression of rat 14 kDa bile acid-binding protein.

BACKGROUND: A 14 kDa protein is a major rat ileal cytosolic bile acid-binding protein (14 kDa I-BABP). This report describes the normal and glucocorticoid-accelerated postnatal expression of 14 kDa I-BABP. METHODS. 14 kDa I-BABP and its mRNA were detected by antiserum and antisense cRNA probe, respectively, in ileum and ovary from 7-, 14-, 21-, 28-, and 35-day-old rats. RESULTS: A positive histochemical reaction with 14 kDa I-BABP antiserum was found in cytosol of ileal enterocytes and ovarian luteal cells at 21 and 35 days of age, respectively. Likewise, Northern blot analysis indicated that the cRNA probe hybridized to a single transcript of 500 bp in total cellular RNA prepared from the ileum of 21-day-old and ovary of 35-day-old rats. Corticosteroid treatment resulted in a histochemical reaction in ileum of 14-day-old animals as compared to the appearance of this reaction in 21-day-old control littermates. Similarly, Northern blot analysis detected the earlier appearance of mRNA levels in corticosteroid-treated animals (11 days vs. 17 days in control animals). CONCLUSIONS: 14 kDa I-BABP and its mRNA are normally expressed in rat ileum at 17 days of postnatal life. 14 kDa I-BABP is expressed considerably later in rat ovary. Corticosteroid treatment results in precocious expression of ileal 14 kDa I-BABP.

Age Factors↗
PubMed1999

Developmental changes in distribution of death-associated protein kinase mRNAs.

Death-associated protein kinase (DAP-kinase) is Ca(2+)/calmodulin-dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon-gamma-induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP-kinase, the tissue distribution and developmental changes in mRNA expression of DAP-kinase were investigated by Northern blot and in situ hybridization analyses. DAP-kinase mRNA was predominantly expressed in brain and lung. In brain, DAP-kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP-kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP-kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP-kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP-kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP-kinase and neuronal cell death.

Animals↗