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PubMed1994

Kinetic microphotometric evaluation of in situ hybridization for mRNA of slow myosin heavy chain in type I and C fibres of rabbit muscle.

The present study was undertaken in order to test the possibility of microphotometric evaluation of in situ hybridizations. The histochemical detection of mRNA specific to the slow myosin heavy chain (HCI), in fibre cross sections of normal and transforming rabbit muscles with a digoxigenin-labelled complementary RNA (cRNA) probe was used as a model. Scanning densitometry of Northern blot hybridizations showed that the detection of cRNA/mRNA hybrids by a staining reaction catalysed by alkaline phosphatase coupled to an anti-digoxigenin antibody occurs in a concentration-dependent manner and follows a linear time course. These findings were the basis for elaborating a comparative microphotometric evaluation of in situ hybridization in tissue sections by measuring the reaction rate of the alkaline phosphatase-catalysed formazan production. Relative amounts of HCI mRNA were thus determined by comparing reaction rates instead of by single point microphotometry. This method was applied to studies on the distribution of HCI mRNA in different fibre types of normal rabbit muscles and and muscles undergoing fast-to-slow fibre transformation in response to low-frequency stimulation. The different fibre types were identified by histochemical staining for myofibrillar actomyosin ATPase (mATPase) in cross sections adjacent to the sections processed for in situ hybridization. On the average, type I fibres displayed 2.3-fold higher reaction rates than the mean value recorded for C fibres. According to the pronounced scattering of the values measured in single C fibres, these fibres represented a heterogeneous population in the transforming muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels.

The localization of mRNA encoding preproatrial natriuretic peptide (ANP) was investigated in cultured human umbilical vein endothelial cells (HUVEC) and tissue preparations of umbilical vein and artery. The techniques used were in situ hybridization and in situ hybridization combined with immunocytochemistry, using 32P-radiolabelled and non-radioactive digoxigenin labelled complementary RNA probes. Human ANP mRNAs are mainly localized in the endothelial cells of the umbilical vein and, to a lesser extent, in the endothelial cells of the umbilical artery. The autoradiographic labelling and the intensity of digoxigenin staining were significantly reduced by treatment with RNase before in situ hybridization. This study provides unequivocal evidence for the expression of the ANP gene in the endothelial cells of human umbilical vessels, confirming that these endothelial cells have the ability to synthesize this peptide. The functional significance of the presence of the ANP mRNA in the endothelial cells of human umbilical vessels is discussed.

Atrial Natriuretic Factor↗
PubMed1981

The chromosomal distribution of repetitive DNA sequences within the human beta globin gene cluster.

The chromosomal distribution and degree of repetitiveness of the sequences within the human beta globin gene cluster has been studied by in situ hybridisation. A genomic recombinant, H beta G1, which contains 15.9 thousand base pairs (kb) of DNA inserted into lambda charon 4A, was used as a template for the [3H] complementary RNA used for hybridisation. The inserted DNA contains sequences 4.7 kb to the 5' side of the delta gene and continues through the delta and beta genes to a site 2.8 kb to the 3' side of the beta gene. Two highly repetitive sequences, which are distributed evenly over all the chromosomes, have been identified within this DNA. Another somewhat less repetitive sequence has been identified between the beta and delta genes.

Base Sequence↗
PubMed1976

The chromosomal localisation of satellite DNA in Ptyas mucosus (Ophidia, Colubridae).

Ptyas mucosus male DNA has a repetitious DNA satellite (p = 1.700 g cm-3) constituting 5% of the haploid genome. In situ hybridisation of radioactive complementary RNA (cRNA) has revealed that satellite sequences are located in the centromeric region of one pair of macrochromosomes and in the terminal region of 8 pairs of microchromosomes. These regions are constitutively heterochromatic as revealed by C-banding. The possibility of involvement of satellite rich microchromosomes in nucleolus organisation is discussed.

Animals↗
PubMed1994

Localization of follistatin, an activin-binding protein, in bone tissues.

We have previously reported that activin-A has a mitogenic effect on osteoblast-like MC3T3-E1 cells and that these cells produce an activin-binding protein, follistatin (Hashimoto, M., Shoda, A., Inoue, S., Yamada, R., Kondo, T., Sakurai, T., Ueno. N., and Muramatsu, M. (1992) J. Biol. Chem. 267, 4999-5004). Activin-A was also detected abundantly in bone matrix tissues and was found to act as an osteoclast differentiation factor. Here, we studied the expression of follistatin in bone tissues by in situ hybridization with a follistatin cRNA probe. Follistatin transcript was detected in the osteoblasts and a fraction of osteocyte population in the developing mouse mandible. Immunohistochemistry using anti-follistatin antibody supported this observation at the protein level. Follistatin mRNA was also detected in the callus of repairing bone after fracture. These findings suggest that activin and follistatin may play significant roles in the bone system in vivo.

Animals↗
PubMed1976

Satellite DNA and evolution of sex chromosomes.

The satellite DNA (SATELLITE III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (delta equals 1.700 g cm-3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.

Animals↗
PubMed1979

Localization of satellite DNAs in the chromosomes of the guinea pig.

The in situ hybridization method has been used to investigate the localization of each of the three satellite DNAs present in the genome of the guinea pig. Purified fractions of the satellite DNAs were utilized as templates for synthesis of 3H-labeled complementary RNA (cRNA) by E. coli RNA polymerase, then each cRNA was hybridized to metaphase spreads of embryonic guinea pig cells. The cRNAs of all three satellite DNAs hybridized predominantly to the centromeric region of the chromosomes. The cRNAs of satellite DNAs II and III hybridized to all chromosomes except the Y chromosome. The cRNA of satellite DNA I did not hybridize to the Y chromosome nor to two pairs of small acrocentric chromosomes. Satellite II cRNA hybridized to the telomeric region of chromosomes 3 and 4.

Animals↗
PubMed1976

The fluorescence localization of mouse satellite DNA in interphase nuclei.

Nuclei from various mouse tissues exhibit a pattern of fluorescence characteristic of the cell type when stained with the fluorescent compound Hoechst 33258. When such preparations are hybridized in situ with 3H-RNA complementary to the A-T rich satellite of mouse, it is clearly seen that only the fluorescent regions of the nuclei contain the satellite DNA. Thus Hoechst 33258 allows the precise localization of satellite DNA at all stages of the mouse cell cycle.

Animals↗
PubMed1983

Expression of a prokaryotic gene in yeast: isolation and characterization of mutants with increased expression.

The Escherichia coli Tn9 derived chloramphenicol resistance gene (camr) is functionally expressed in the yeast Saccharomyces cerevisiae. This gene was introduced into yeast cells as part of a hybrid yeast/E. coli shuttle plasmid. A number of plasmid associated yeast mutants overproducing the camr gene product, chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-0-acetyltransferase, E.C. 2.3.1.28) were isolated. One of the plasmid mutants was analyzed in some detail. Even though this mutant showed a 1,000 fold overproduction of chloramphenicol acetyltransferase in the yeast host the level of RNA complementary to the camr gene was not increased. A deletion of 127 base pairs in the region immediately upstream from the 5' end of the camr gene appeared to be responsible for the "up" phenotype of this mutant. This mutation affected the expression of the camr gene in E. coli in a "down" fashion, in contrast to its effect in yeast.

Acetyltransferases↗
PubMed1978

Denaturation of mouse satellite DNA upon melting of chromatin in solution.

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.

Animals↗
PubMed1997

Expression of MDR1/p-glycoprotein and multidrug resistance-associated protein in childhood solid tumours.

We evaluated the expression of MDR1/p-glycoprotein in paediatric tumours using reverse transcriptase polymerase chain reaction (RT-PCR), RNA dot blot analysis, and immunohistochemistry on formalin fixed paraffin-embedded material with JSB-1 and C-219 monoclonal antibodies, and compared these three techniques. The expression of multidrug resistance-associated protein (MRP) gene was examined by RT-PCR assay. We studied MDR1/p-glycoprotein and MRP expression in 13 samples from 10 neuroblastoma patients, 11 samples from 10 nephroblastoma patients, 2 rhabdomyosarcomas, 1 adrenocortical carcinoma and 10 benign tumours or tumour-like lesions. Eleven of 13 neuroblastomas, 7 of 11 nephroblastomas, 2 rhabdomyosarcomas, 1 adrenocortical carcinoma, and 7 of 10 benign tumours or tumour-like lesions showed MDR1 PCR products. By RNA dot blot analysis, MDR1 transcripts were detectable in 11 of 34 specimens. Immunohistochemically, we detected positive reaction products for JSB-1 in 26 of 36 samples. There was a significant correlation between the immunoreactivity for JSB-1 and the expression of MDR1 mRNA expression by RT-PCR (P = 0.0001). However, the presence of p-glycoprotein immunostaining does not correlate with the MDR1 expression shown by RT-PCR in every case. As for MRP mRNA expression, 9 of 13 neuroblastomas and 10 of 11 nephroblastomas revealed PCR products.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1980

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid.

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicol-resistant (CAPR) human x mouse hybrids and one CAPR cybrid derived from CAPR HeLa cells and CAPS mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with 32P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [32P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited "reverse" chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human-mouse hybrid is to retain human mitochondrial DNA.

Animals↗
PubMed1996

Regulation of stress-induced transcriptional changes in the hypothalamic neurosecretory neurons.

Transcriptional changes in corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) gene expression were studied by in situ hybridization histochemistry using cRNA probes directed against intronic sequences. Acute ether stress resulted in a rapid induction of CRF and a delayed activation of vasopressin heteronuclear (hn)RNA in the parvocellular neurosecretory neurons within the paraventricular nucleus (PVN) of the hypothalamus. To explore possible molecular mechanisms regulating stress-related neuropeptide expression in vivo, the time-courses of stress-induced activation of different transcription factor classes were compared to that of changes in neuropeptide transcription. The peak of CRF transcription was parallel to that of cAMP response-element binding protein (CREB) phosphorylation but preceded the induction of c-fos and NGFI-B mRNAs and Fos protein. In contrast, AVP expression occurred in step with immediate-early gene (IEG) responses, suggesting involvement of different mechanisms underlying stress-induced neuropeptide responses. The interference of glucocorticoid hormones with stress-induced neuropeptide and transcription-factor responses has also been revealed in rats acutely or chronically pretreated with glucocorticoids. Acute dexamethasone injection did not prevent neuropeptide and transcription factor responses to either inhalation, whereas chronic corticosterone administration completely blocked IEG and neuropeptide induction in the stress-related neurosecretory neurons.

Afferent Pathways↗
PubMed2004

Gene expression profiles during beta cell maturation and after IL-1beta exposure reveal important roles of Pdx-1 and Nkx6.1 for IL-1beta sensitivity.

AIM/HYPOTHESIS: Maturation of the beta cells in the islets of Langerhans is dependent upon sequential activation of different transcription factors such as Pdx-1 and Nkx6.1. This maturation is associated with an acquired sensitivity to cytokines and may eventually lead to type 1 diabetes. The aims of this study were to characterise changes in mRNA expression during beta cell maturation as well as after interleukin-1beta (IL-1beta) exposure. METHODS: Transcriptome analyses were performed on two phenotypes characterised as a glucagon-producing pre-beta-cell phenotype (NHI-glu), which matures to an IL-1beta-sensitive insulin-producing beta cell phenotype (NHI-ins). Beta cell lines over-expressing Pdx-1 or Nkx6.1, respectively, were used for functional characterisation of acquired IL-1beta sensitivity. RESULTS: During beta cell maturation 98 fully annotated mRNAs changed expression levels. Of these, 50 were also changed after 24 h of IL-1beta exposure. In addition, 522 and 197 fully annotated mRNAs, not affected by maturation, also changed expression levels following IL-1beta exposure of the beta cell and the pre-beta-cell phenotype, respectively. Beta cell maturation was associated with an increased expression of Nkx6.1, whereas both Pdx-1 and Nkx6.1 expression were decreased following IL-1beta exposure. Over-expression of Nkx6.1 or Pdx-1 in cell lines resulted in a significantly increased sensitivity to IL-1beta. CONCLUSIONS/INTERPRETATION: These results suggest that the final beta cell maturation accompanied by increased IL-1beta sensitivity is, in part, dependent upon the expression of genes regulated by Pdx-1 and Nkx6.1. Future classification of the genes regulated by these transcription factors and changed during beta cell maturation should elucidate their role in the acquired sensitivity to IL-1beta and may be helpful in identifying new targets for intervention/prevention strategies.

Animals↗
PubMed1997

A kinetic model with ordered cytoplasmic dissociation for SUC1, an Arabidopsis H+/sucrose cotransporter expressed in Xenopus oocytes.

To elucidate the kinetic properties of the Arabidopsis H+/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range -180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H+ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (iHmax, iSmax for H+ and sucrose) and of the ligand concentrations eliciting half-maximal currents (KHm, KSm). The iHmax and iSmax exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the KHm and KSm decreased with increasingly negative membrane potential. Furthermore, at any given voltage, iSmax increased and KSm decreased as a function of the external H+ concentration. Eight six-state carrier models-which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier-were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton.

Animals↗
PubMedDate not supplied

Effects of hyperoxia on tumor necrosis factor alpha and Grobeta expression in newborn rabbit lungs.

Chemokines have been implicated in the pathogenesis of many inflammatory processes, including bronchopulmonary dysplasia in mechanically ventilated premature infants. We hypothesized that early expression of the proinflammatory cytokine, tumor necrosis factor alpha (TNFalpha), would be followed by later expression of the downstream chemokine, Grobeta, in the oxygen-injured newborn lung. Reverse transcriptase-polymerase chain reaction (RT-PCR) and ribonuclease protection assay (RPA) were used to assess TNFalpha and Grobeta mRNA expression in lung RNA samples from newborn rabbits exposed to > 95% O2 for 8-9 days, followed by 60% O2 for a further 2-4 weeks or from control rabbits exposed to air. Four lung samples per condition were collected every 2 days from day 0 to day 14, and at days 22 and 36. Rabbit alveolar macrophages (AM) stimulated in vitro with bacterial lipopolysaccharide served as positive controls ( n = 8). Grobeta mRNA expression in rabbit lung samples increased with oxygen exposure until day 8, then returned toward baseline levels. This corresponded to previously described elevations in neutrophil number in the lungs. TNFalpha mRNA expression in lung samples was below the limit of detection by RPA and showed no upregulation in hyperoxic lung samples by RT-PCR. TNFalpha activity was assessed in lung lavage ( n = 2 samples per condition per time) using an L929 cell line bioassay and was not increased in hyperoxic animals. The expression of Grobeta mRNA without antecedent or concurrent TNFalpha mRNA expression or activity makes it unlikely that Grobeta in the hyperoxic newborn rabbit lung is elaborated in response to a stimulus by TNFalpha.

Animals↗
PubMed2003

Expression of glutamate transporters in human and rat retina and rat optic nerve.

l-Glutamate is the major excitatory transmitter in the vertebrate retina and plays a central role in the transmission of the various retinal neurons. Glutamate is removed from the extracellular space by at least five different glutamate transporters. The cellular distribution of these has been studied so far mainly using immunocytochemistry. In the present study non-radioactive in situ hybridisation using complementary RNA probes was applied in order to identify the cell types of rat retina and optic nerve expressing generic GLT1, GLT1 variant (GLT1v or GLT1B), GLAST and EAAC1. The results were compared with immunocytochemical data achieved using affinity-purified antibodies against transporter peptides. In the immunohistochemical studies the human retina was included. The study showed that in the rat retina GLT1v and EAAC1 were coexpressed in various cell types, i.e. photoreceptor, bipolar, horizontal, amacrine, ganglion and Müller cells, whereas GLAST was only detected in Müller cells and astrocytes. In the rat optic nerve GLT1v and EAAC1 were preferentially expressed in oligodendrocytes, whereas GLAST was revealed to be present mainly in astrocytes. Generic GLT1 could not be detected in the retina or optic nerve. The cellular distribution of glutamate transporters (only immunocytochemistry) in the human retina was very similar to that of the rat retina. Remarkable results of our studies were that generic GLT1 was not detectable in the rat (and human) retina and that GLT1v and EAAC1 were demonstrable in most cell types of the retina (including photoreceptor cells and their terminals).

Amacrine Cells↗
PubMedDate not supplied

Detection of mRNAs encoding peroxisomal proteins by non-radioactive in situ hybridization with digoxigenin-labelled cRNAs.

We have used a non-radioactive in situ hybridization (ISH) protocol for the detection of mRNAs encoding proteins localized in peroxisomes. In this presentation the literature on detection of "peroxisomal mRNAs" is reviewed and the results obtained by application of the non-radioactive method are compared with those obtained by hybridization with radioactive probes. Moreover, the special processing conditions and the application of the method for the specific visualization of mRNAs coding for several peroxisomal proteins with different abundance levels and distinct tissue distributions are presented. The combination of the following technical details in the ISH procedure were found to be essential for obtaining optimal sensitivity and good histological quality of the preparations: (a) perfusion-fixation with a fixative containing 4% depolymerized paraformaldehyde/0.05% glutaraldehyde, (b) the use of paraffin embedding instead of frozen sections, (c) specific proteinase K-digestion time for each tissue, and (d) the use of digoxigenin-labelled cRNA probes (hydrolyzed to a length of about 200 bases) for detection. By using this technique, we were able to localize several peroxisome-specific mRNAs with different degrees of abundance: (1) high-level (catalase and urate oxidase) and (2) low-level (all beta-oxidation enzymes and the 70-kDa peroxisomal membrane protein) in rat liver and kidney. The specificity of the method was confirmed by the negative results obtained with corresponding sense controls and the distinct positive staining patterns obtained for albumin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. All transcripts for mRNAs encoding peroxisomal proteins were localized to the cytoplasm of hepatocytes, with all nuclei as well as epithelial cells of bile ducts and sinusoidal cells remaining negative. In rat kidney, the catalase transcripts were confined to proximal tubular epithelial cells, which is consistent with the high abundance of peroxisomes in this part of the nephron. In contrast, no transcripts for urate oxidase were present in the kidney, corresponding to the absence of that protein in this organ. The transcripts for GAPDH on the other hand were localized in proximal and distal tubular epithelial cells as well as in collecting ducts. The application of this technique to the rat adrenal gland and testis in recent unpublished studies have revealed exclusive localization of catalase transcripts to the adrenal cortex and to interstitial cells of Leydig, which are known to be rich in microperoxisomes. These observations demonstrate the suitability of this technique for accurate localization of mRNAs encoding peroxisomal proteins and for the analysis of alterations in the expression of the corresponding genes under different experimental conditions.

Animals↗