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At least 1,117 records · Page 62Linked to original sources
PubMed1989

Amelogenin gene expression in mouse incisor heterotopic recombinations.

Mouse incisor tooth organs express the genes responsible for enamel extracellular matrix formation exclusively on the labial surface of the organ. A previous investigation has suggested that lingual inner dental epithelium of mouse incisor did not contain potential ameloblasts. The present work extends our histological observations, by analyzing the presence of mouse amelogenin mRNA in heterotopic mouse incisor tissue recombinations using in situ hybridization to 35S-labelled asymmetric complementary RNA probes from a cDNA specific to the mouse Mr 26 x 10(3) amelogenin. Labial polarized ameloblasts located in front of the lingual predentin layer were associated with numberous hybridization signals indicating an increased density of amelogenin mRNA. In contrast, the lingual inner dental epithelium in contact with labial predentin never showed amelogenin hybridization signals, indicating the absence of amelogenin transcripts. These results confirm our observation that the lingual inner dental epithelium does not contain potential ameloblasts, since these cells do not transcribe nor accumulate mRNA amelogenin even when in contact with a favorable microenvironment provided by the labial predentin.

Amelogenin↗
PubMed1994

Cloning of a wheat 15-kDa grain softness protein (GSP). GSP is a mixture of puroindoline-like polypeptides.

The wheat starch 15-kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15-kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti-GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15-kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15-kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half-way through grain filling. The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16-kDa avenin and to wheat puroindoline (a lipid-binding 15-kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid-binding properties of GSP polypeptides may influence grain softness.

Amino Acid Sequence↗
PubMed1998

Secretion of GM-CSF by inflammatory cells in the lung of patients with sarcoidosis.

Several cytokines are involved in the pathophysiology of sarcoidosis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) may be one of these cytokines because its mRNA is expressed by inflammatory cells obtained from the sarcoid lung. We thus asked two questions. Is GM-CSF secreted by bronchoalveolar lavage (BAL) cells? What type of cells express GM-CSF? GM-CSF secreted by the cultured BAL cells from 19 untreated sarcoid patients was measured by an enzyme-linked immunosorbent assay. The individual cells expressing GM-CSF mRNA were identified by in situ hybridization in seven patients. Spontaneous release of GM-CSF by BAL cells was demonstrated in one patient. When stimulated, BAL cells secreted GM-CSF in all subjects studied. In situ hybridization with digoxigenin-labelled GM-CSF cRNA probe revealed that the positive cells were round in shape and larger than lymphocytes, but smaller than large alveolar macrophages that are often irregular in shape. These results strongly indicated that small alveolar macrophages secrete GM-CSF in the sarcoid lesion. This phenomenon may contribute to the enhanced immunological activities observed in sarcoidosis.

Bronchoalveolar Lavage Fluid↗
PubMed1995

D1 and D2 receptor gene expression in the rat frontal cortex: cellular localization in different classes of efferent neurons.

The dopaminergic input to the frontal cortex has an important role in motor and cognitive functions. These effects are mediated by dopamine receptors both of type D1 and of type D2, although the neural circuits involved are not completely understood. We used in situ hybridization to determine the cellular localization of D1 and D2 receptor mRNAs in the rat frontal cortex. Retrograde tracing was used in the same animals to identify the main cortical efferent populations. Fluorogold was injected into the different cortical targets of the frontal cortex and sections were hybridized with D1 and D2 35S-labelled cRNA probes. D1 and D2 mRNA-containing neurons were present in all the cortical areas investigated, with greater expression in the medial prefrontal, insular and cingulate cortexes and lower expression in the motor and parietal cortexes. Neurons containing D1 mRNA were most abundant in layer VIb; they were also present in layers VIa and V of all cortical layers and in layer II of the medial prefrontal, cingulate and insular areas. Double labelling with fluorogold demonstrated that D1 mRNA was present in corticocortical, corticothalamic and corticostriatal neurons. Neurons containing D2 mRNA were essentially restricted to layer V, but only in corticostriatal and corticocortical neurons. Neither D1 nor D2 mRNA was found in corticospinal or corticopontine neurons. The present results demonstrate that D1 and D2 receptor genes are expressed in efferent cortical populations, with higher expression for D1. In spite of an overlap in some cortical layers, the expression of D1 and D2 receptor genes is specific for different categories of pyramidal neurons.

Animals↗
PubMed2005

Inflammation alters somatostatin mRNA expression in sensory neurons in the rat.

Proinflammatory neuropeptides, such as substance P and calcitonin gene-related peptide, are up-regulated in primary afferent neurons in acute and chronic inflammation. While these neuropeptides have been intensively studied, potentially anti-inflammatory and/or anti-nociceptive neuropeptides such as somatostatin (SS) have been less widely investigated. Endogenous somatostatin is thought to exert a tonic antinociceptive effect. Exogenous SS is anti-inflammatory and antinociceptive and is thought to exert these actions through inhibition of proinflammatory neuropeptide release. In this study we have compared the expression of somatostatin in two inflammatory models: arthritis, a condition associated with increased nociception, and periodontitis, in which there is little evidence of altered nociceptive thresholds. In acute arthritis (< 24 h) SS mRNA was down-regulated in ipsilateral dorsal root ganglia (DRG; 52 +/- 7% of control, P < 0.05), and up-regulated in contralateral DRG (134 +/- 10% of control; P < 0.05). In chronic arthritis (14 days) this pattern of mRNA regulation was reversed, with SS being up-regulated ipsilaterally and down-regulated contralaterally. In chronic mandibular periodontitis (7-10 days), SS mRNA was up-regulated in only the mandibular division of the ipsilateral trigeminal ganglion (TG) (day 7, 219 +/- 9% and day 10, 217 +/- 12% of control; P < 0.02) but showed no change in other divisions of the trigeminal ganglion or in the mesencephalic nucleus. These data show that antinociceptive and anti-inflammatory neuropeptides are also regulated in inflammation. It is possible that the degree of inflammation and nociception seen may depend on the balance of pro- and anti-inflammatory and nociceptive peptide expression in a particular condition.

Animals↗
PubMed1996

The effect of sodium lauryl sulphate on the expression of cytokeratin mRNA in hamster cheek pouch epithelium.

The effect of sodium lauryl sulphate (SLS) on cytokeratin (CK) gene expression in hamster cheek pouch epithelium was studied with a hybridohistochemical technique. Using specific human anti-sense RNA probes, the plausible hamster mRNA counterparts for these human CK mRNAs were localized by detection of heterologous hybrids. In comparison with normal epithelium, the expression and distribution pattern of CK mRNAs in the hamster cheek pouch were obviously changed after application of SLS. There was a decreased expression of CK mRNAs in the hyperplastic basal layer, and increased expression in the hypertrophic granular layer. Strikingly, hybridization with the human CK 18 cRNA probe revealed an additionally expressed CK mRNA in the SLS-treated epithelium that was not found in the untreated epithelium. The present study indicates that cRNA probes for human CK mRNAs can be used successfully, not only to distinguish between different hamster CK mRNAs but also to investigate changes in CK gene expression upon the induction of non-neoplastic and neoplastic alterations in the hamster cheek pouch model. This may help elucidate the molecular changes involved in epithelial pathologies.

Animals↗
PubMed1997

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue.

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin-labeled cRNA probes (386 and 496 bp). TIMP-1 and -2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP-1 and -2 mRNA-expressing cells showed significantly different localization. TIMP-1 mRNA was broadly observed in the gingival connective tissue while TIMP-2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP-1 and TIMP-2 mRNA was detected differentially and site-specifically in periodontal diseased gingival tissue.

Aged↗
PubMed1997

The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine.

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.

Animals↗
PubMed1996

Expression and regulation of a vesicular monoamine transporter in rat stomach: a putative histamine transporter.

1. Vesicular monoamine transporters (VMATs) translocate monoamines from the cytoplasm into secretory vesicles of endocrine cells and neurones, but they have limited affinity for histamine, and the identity of the vesicular transporter for this monoamine is uncertain. The aims of the present study were to characterize VMAT representatives in rat gastric corpus, and to determine if their expression was regulated by factors that modulate histamine biosynthesis. 2. Polymerase chain reaction (PCR) cloning using oligonucleotide primers to DNA sequences conserved within the VMAT family provided evidence for VMAT2, but not VMAT1 in rat gastric corpus. Northern analysis using a VMAT2 complementary RNA probe revealed a single 4 kb mRNA species in corpus endocrine cells. 3. In rats treated for up to 5 days with the H(+)-K(+)-ATPase inhibitor omeprazole, VMAT2, histidine decarboxylase and chromogranin A mRNA abundance in gastric corpus, and plasma gastrin concentrations increased progressively. Omeprazole also elevated VMAT2 expression in rats fasted for 48 h, but fasting alone, or refeeding fasted animals had no effect. 4. The results are consistent with a role for VMAT2 in the transport of histamine into enterochromaffin-like cell secretory vesicles, and with upregulation of the transporter to accommodate the increased histamine biosynthesis and secretion that accompanies achlorhydria.

Amino Acid Sequence↗
PubMed2005

Transport of the dopamine D2 agonist pramipexole by rat organic cation transporters OCT1 and OCT2 in kidney.

We investigated the mechanism of renal tubular secretion of the dopamine D2 receptor agonist pramipexole in rats, focusing on organic cation transporters 1 and 2. The uptake of [14C]pramipexole by Xenopus oocytes injected with complementary RNA of either rat organic cation transporter (rOCT) 1 or rOCT2 was significantly higher than that by water-injected oocytes: the kinetic parameters, K(m) and V(max), of pramipexole uptake were 49.5 muM and 234 pmol/60 min/oocyte for rOCT1, and 16.9 microM and 12.8 pmol/60 min/oocyte for rOCT2. Pramipexole was taken up into kidney slices in a time- and concentration-dependent manner, and Eadie-Hofstee plots revealed the involvement of two saturable components. The kinetic parameters, K(m1) and V(max1), of the high-affinity component were 12.9 microM and 10.7 nmol/15 min/g kidney, respectively. The uptake of [14C]pramipexole by rOCT1, rOCT2, and kidney slices was inhibited by procainamide and corticosterone, which are selective inhibitors of rOCT1 and rOCT2, respectively. The IC50 values of procainamide and corticosterone for the uptake of [14C]pramipexole by rOCT1, rOCT2, and kidney slices were 7.7, 167.0, and 47.0 microM and 163.7, 10.7, and 47.7 microM, respectively. These results demonstrate that both rOCT1 and rOCT2 are involved in the renal uptake of pramipexole across the basolateral membrane of the proximal tubular epithelial cells.

Amantadine↗
PubMed2005

Involvement of uric acid transporter in increased renal clearance of the xanthine oxidase inhibitor oxypurinol induced by a uricosuric agent, benzbromarone.

Benzbromarone has been reported to increase the renal clearance of oxypurinol, an active metabolite of allopurinol. We examined the renal transport of oxypurinol to determine whether such a change in renal clearance could be explained by altered transporter-mediated reabsorption. Since the first step of reabsorption takes place at the renal epithelial apical membrane, we focused on membrane transporters. Benzbromarone is an inhibitor of reabsorption of uric acid mediated by the uric acid transporter (URAT) URAT1 (SLC22A12), which is expressed at the apical membrane of proximal tubular cells in humans. Uptake of oxypurinol by Xenopus oocytes injected with complementary RNA of URAT1 was significantly higher than that by water-injected oocytes, and the uptake was saturable, with a K(m) of about 800 microM. Moreover, benzbromarone inhibited the oxypurinol uptake by URAT1 at concentrations as low as 0.01 microM. The uptake of oxypurinol by another organic anion transporter (OAT), OAT4 (SLC22A11), which is also expressed at the apical membrane of proximal tubular epithelial cells, was negligible, whereas the uptake of [3H]estrone-3-sulfate by OAT4 was significantly inhibited by oxypurinol. Furthermore, neither the transport activity of organic cation/carnitine transporter (OCTN) 1 nor OCTN2 was affected by oxypurinol or benzbromarone. These results indicate that URAT1 is involved in renal reabsorption of oxypurinol, and the increment of renal clearance of oxypurinol upon concomitant administration of benzbromarone could be due to drug interaction at URAT1.

Animals↗
PubMed2002

Differential effects of haloperidol and clozapine on [(3)H]cAMP binding, protein kinase A (PKA) activity, and mRNA and protein expression of selective regulatory and catalytic subunit isoforms of PKA in rat brain.

The present study was undertaken to examine whether the mechanism of action of typical and atypical antipsychotics is related in their ability to regulate key phosphorylating enzyme of adenylyl cyclase-cAMP pathway, i.e., protein kinase A (PKA). For this purpose, regulatory (R) and catalytic (Cat) activities of PKA and expression of various isoforms of regulatory and catalytic subunits were examined in rat brain after single or chronic (21-day) treatment with haloperidol (HAL, 1 mg/kg) or clozapine (CLOZ, 20 mg/kg). It was observed that chronic but not acute treatment of CLOZ significantly decreased [(3)H]cAMP binding to the regulatory subunit of PKA as well as catalytic activity of PKA in particulate and cytosol fractions of the rat cortex, hippocampus, and striatum. In these fractions, CLOZ significantly decreased protein levels of selective RII alpha-, RII beta-, and Cat beta-subunit isoforms of PKA. These decreases were accompanied by decreases in their respective mRNA expression. In contrast, chronic but not acute treatment of HAL significantly increased [(3)H]cAMP binding and the catalytic activity of PKA in particulate and cytosol fractions of only the striatum brain area. In addition, chronic treatment of HAL significantly increased mRNA and protein levels of RII alpha- and RII beta-subunit isoforms in the striatum. None of the antipsychotics caused any change in the expression of the Cat alpha-, RI alpha-, or RI beta-subunit isoform. These results, thus, suggest that HAL and CLOZ differentially regulate PKA catalytic and regulatory activities and the expression of selective catalytic and regulatory subunit isoforms of PKA, which may be associated with their mechanisms of action.

Adenylyl Cyclases↗
PubMed2006

The positional influence of the helical geometry of the heteroduplex substrate on human RNase H1 catalysis.

In a companion study published in this issue (p. 83), we showed that chimeric substrates containing 2'-methoxyethyl (MOE) nucleotides inhibited human RNase H1 activity. In this study, we prepared chimeric substrates containing a central DNA region with flanking northern-biased MOE nucleotides hybridized to complementary RNA. Conformationally biased and flexible modified nucleotides were positioned at the junctions between the DNA and MOE residues of the chimeric substrates to modulate the effects of the MOE residues on human RNase H1 activity. The strong northern-biased locked-nucleic acid modification exacerbated the negative effects of the MOE modifications resulting in slower human RNase H1 cleavage rates. Enhanced cleavage rates were observed for the eastern-biased 2'-ara-fluorothymidine and bulge inducing N-methylthymidine modifications positioned at the 5'-DNA/3'-MOE junction as well as the southern-biased 2'-methylthiothymidine and conformationally flexible tetrafluoroindole (TFI) modifications positioned at the 5'-MOE/3'-DNA junction. The heterocycle of the ribonucleotide opposing the TFI deoxyribonucleotide had no effect on the human RNase H1 activity, whereas nucleotide substitutions adjacent the TFI significantly affected the cleavage rate. Mismatch base pair(s) exhibited similar effects on human RNase H1 activity as the TFI modifications. The effects of the TFI modification and mismatch base pair(s) on human RNase H1 activity were influenced by the position of the modification relative to the nucleotides interacting with the catalytic site of the enzyme rather than the juxtaposition of the modification to the MOE residues. Finally, these results provide a method for enhancing the human RNase H1 activity of chimeric antisense oligonucleotides (ASO) as well as the design of more potent ASO drugs.

Base Sequence↗
PubMed2000

Correlative association between N-methyl-D-aspartate receptor-mediated expression of period genes in the suprachiasmatic nucleus and phase shifts in behavior with photic entrainment of clock in hamsters.

Because the rapid induction of Period (Per) genes is associated with the photic entrainment of the biological clock, we examined whether N-methyl-D-aspartate (NMDA) receptors were involved in the photic induction of Per genes in the hamster suprachiasmatic nucleus (SCN). In situ hybridization observation revealed that light during the early subjective night [circadian time (CT) 13.5] or the late subjective night (CT20) caused an induction of Per1 and Per2 but not Per3 mRNA in the SCN. Photic induction of Per mRNA at CT13.5 was observed especially in the ventrolateral SCN, whereas that at CT20 was more widespread from the ventrolateral to the dorsal SCN. A noncompetitive NMDA receptor antagonist, +MK801, dose-dependently (0. 1-5.0 mg/kg) suppressed only the ventrolateral part of Per1 and Per2 mRNA induction by light at CT13.5 or CT20 in the SCN. The suppressive effects of +MK801 on Per mRNA strongly correlated with the attenuating action of this compound on phase shifts by light at both CT13.5 and CT20. A competitive NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV), also exhibited inhibitory actions on light (CT20)-induced Per1 and Per2 mRNA expression in the ventrolateral SCN. Furthermore, local injection of NMDA into the SCN resulted in the induction of Per1 and Per2 mRNA in the SCN. Among NMDA receptors, NR2B and NR2C mRNA were expressed in the ventrolateral and dorsal SCN, respectively. These results suggest that the activation of NMDA receptor is a critical step for photic induction of Per1 and Per2 transcripts in the SCN, which are linked to a photic behavioral entrainment.

Analysis of Variance↗
PubMed1964

ACTINOMYCIN D AS A PROBE FOR NUCLEIC ACID SECONDARY STRUCTURE.

The binding of actinomycin D to DNA results in an increase in the temperature at which thermal dissociation of the DNA occurs. With this enhancement of thermal stability as a criterion for binding, it is shown that complementary RNA and DNA-RNA hybrid do not bind actinomycin D. This result can be interpreted to mean that these polynucleotides have helical conformations which differ from that of DNA.

Chemical Phenomena↗
PubMed1988

Amyloid protein precursor messenger RNAs: differential expression in Alzheimer's disease.

In situ hybridization was used to assess total amyloid protein precursor (APP) messenger RNA and the subset of APP mRNA containing the Kunitz protease inhibitor (KPI) insert in 11 Alzheimer's disease (AD) and 7 control brains. In AD, a significant twofold increase was observed in total APP mRNA in nucleus basalis and locus ceruleus neurons but not in hippocampal subicular neurons, neurons of the basis pontis, or occipital cortical neurons. The increase in total APP mRNA in locus ceruleus and nucleus basalis neurons was due exclusively to an increase in APP mRNA lacking the KPI domain. These findings suggest that increased production of APP lacking the KPI domain in nucleus basalis and locus ceruleus neurons may play an important role in the deposition of cerebral amyloid that occurs in AD.

Alzheimer Disease↗
PubMed1986

Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei.

A nonrepetitive complementary RNA probe specific for an intervening sequence of the rat pro-opiomelanocortin (POMC) gene primary transcript was used to analyze the hormonal regulation of POMC gene transcription in individual cell nuclei in the rat pituitary by in situ hybridization. This probe recognized only full-length POMC heterogeneous nuclear RNA, as verified by Northern blots of pituitary RNA. When pituitary sections were hybridized with this 3H-labeled POMC intron A probe, silver grains were predominantly localized over the nuclei of cells that expressed POMC in the anterior and intermediate lobes. Adrenalectomy increased both the average grain density over corticotroph nuclei and the number of cells in the anterior pituitary with significant numbers of silver grains over their nucleus. Dexamethasone administration to intact or adrenalectomized rats results in the rapid (within 30 minutes) disappearance of silver grains over the nuclei of corticotrophs in the anterior lobe, suggesting that POMC gene transcription had been inhibited. However, adrenalectomy or dexamethasone administration did not alter the silver grain density over nuclei of intermediate lobe melanotrophs. Thus, this in situ hybridization assay utilizing an intervening sequence-specific POMC probe can measure rapid physiological changes in POMC heterogeneous nuclear RNA in individual cell nuclei.

Animals↗