Sense RNA Probes Research Articles

First Report of Raspberry leaf blotch virus in Raspberries in Finland.

Raspberry (Rubus idaeus L.) is a valuable and widely grown softfruit that is a host for 40 viruses and virus-like agents, of which many are not characterized at the molecular level. Recently, Raspberry leaf blotch virus (RLBV, putative emaravirus species) was described from raspberries (cv. Glen Ample) in the United Kingdom and Serbia. Plants displayed conspicuous yellow blotches on leaves and abnormal development of leaf hairs in the corresponding areas of the abaxial side (3). Similar symptoms were observed in 'Glen Ample' grown in protective plastic tunnels and open fields in the main berry growing area in eastern Finland in June 2011. In three farms, leaves were sampled from symptomatic and symptomless plants of 'Glen Ample' and also cv. Polka displaying no symptoms. Total RNA was extracted using CTAB reagent. Equal amounts of RNA were pooled from 13 samples and subjected to small-RNA (sRNA) deep sequencing (Fasteris SA, Plan-les-Ouates, Switzerland) to detect viruses without advance information (2). Contigs were built on 21- to 24-nt sRNA reads using Velvet. Contigs larger than 50 nt were used to search homologous sequences in GenBank by BLAST and significant similarity (up to 99%) was observed with RLBV RNA3 and RNA4. Mapping sRNA reads to the genome of RLBV (1) by MAQ resulted in significant coverage of RNA1 (16%), RNA2 (37%), RNA3 (46%), RNA4 (65%), and RNA5 (27%). cDNA was synthesized on RNA of one symptom-expressing plant using random hexamer primers and the cDNA tested by PCR with a forward primer (RLBV-F 5'-TCAAATCCACTTGCATAGAACC-3', nt 723 to 744) and reverse primer (RLBV-R1 5'-CCTCAAACCTTGCAAACACA-3', nt 1,318 to 1,337) designed according to the nucleocapsid (NP) gene of the Scottish RLBV isolate (3). The sequence of the amplified partial NP gene (576 nt; GenBank Accession No. JQ684678) was 92.8% and 94.8% identical to the Scottish isolate at nt and amino acid levels, respectively. The forward primer RLBV-F and a new reverse primer (RLBV-R2 5'-GCCGAAAGTCAAACCTGGTG-3', nt 943 to 962) were used to test additional plants for RBLV and to make a probe (198 nt) to detect RLBV using digoxigen-labeled sense and antisense RNA probes, as described for European mountain ash ringspot associated virus (1). RLBV was detected in all tested plants of 'Glen Ample' with yellow leaf blotch symptoms in the three farms, but not in any symptomless plants of 'Glen Ample' and 'Polka.' The sense probes gave strong signals, in contrast to the antisense probes, which gave only weak or no detectable signals in the virus-positive plants, consistent with the negative RNA strand of RLBV being encapsidated in virus particles. The results show RLBV is associated with severe, distinct, and characteristic symptoms in raspberries of cv. Glen Ample grown in plastic tunnels and open fields in Finland and has an apparent negative impact on plant growth and yield. Our observations in 2011 also suggest that the incidence of diseased plants is much higher in plastic tunnels than in open fields, perhaps because the conditions for the vector of RLBV (raspberry leaf and bud mite, Phyllocoptes gracilis Nalepa) (1) are more favorable in plastic tunnels. These results clarify the etiology of raspberry leaf blotch disease in Finland and emphasize the need to inspect raspberry planting materials for RLBV for better control of the disease.

Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei

The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

Acis-Encoded Antisense Small RNA Regulated by the HP0165-HP0166 Two-Component System Controls Expression ofureBinHelicobacter pylori

Expression of urease is essential for gastric colonization by Helicobacter pylori. The increased level of urease in gastric acidity is due, in part, to acid activation of the two-component system (TCS) consisting of the membrane sensor HP0165 and its response regulator, HP0166, which regulates transcription of the seven genes of the urease gene cluster. We now find that there are two major ureAB transcripts: a 2.7-kb full-length ureAB transcript and a 1.4-kb truncated transcript lacking 3' ureB. Acidic pH (pH 4.5) results in a significant increase in transcription of ureAB, while neutral pH (pH 7.4) increases the truncated 1.4-kb transcript. Northern blot analysis with sense RNA and strand-specific oligonucleotide probes followed by 5' rapid amplification of cDNA ends detects an antisense small RNA (sRNA) encoded by the 5' ureB noncoding strand consisting of ∼290 nucleotides (5'ureB-sRNA). Deletion of HP0165 elevates the level of the truncated 1.4-kb transcript along with that of the 5'ureB-sRNA at both pH 7.4 and pH 4.5. Overexpression of 5'ureB-sRNA increases the 1.4-kb transcript, decreases the 2.7-kb transcript, and decreases urease activity. Electrophoretic mobility shift assay shows that unphosphorylated HP0166 binds specifically to the 5'ureB-sRNA promoter. The ability of the HP0165-HP0166 TCS to both increase and decrease ureB expression at low and high pHs, respectively, facilitates gastric habitation and colonization over the wide range of intragastric pHs experienced by the organism.

Distribution of goblet cells and MUC5AC mRNA in the canine nictitating membrane

This study evaluated the distribution of goblet cells and the expression of MUC5AC mRNA in the canine nictitating membrane. The distribution of goblet cells in the nictitating membrane and temporal bulbar conjunctiva of beagle dogs was examined by histochemical analysis of impression cytology specimens and frozen sections. MUC5AC mRNA was detected by the reverse transcription polymerase chain reaction (RT-PCR). The distribution of MUC5AC mRNA was also examined by in situ hybridization using digoxigenin-labeled antisense and sense RNA probes. Histochemical analysis showed that the canine nictitating membrane epithelium contained many more periodic acid-Schiff positive goblet cells, particularly on the palpebral side, compared with the temporal bulbar conjunctiva. RT-PCR revealed that MUC5AC was expressed in both the nictitating membrane and in conjunctival tissue. When the distribution of MUC5AC mRNA was assessed by in situ hybridization, its expression was high on the palpebral side of the nictitating membrane and low in the temporal bulbar conjunctiva. MUC5AC mRNA expression corresponded with the distribution of goblet cells by histochemical examination. In conclusion, there were numerous goblet cells in the canine nictitating membrane epithelium, particularly on the palpebral side, and MUC5AC mRNA was expressed in the nictitating membrane epithelium at locations corresponding to the goblet cells.

Distribution of vasoactive intestinal peptide and substance P messenger ribonucleic acid in intestinal nerve of Remak of chicken

The intestinal nerve of Remak (INR) is unique to birds. The exact distributions and secretions of the peptide transmitters in the INR that innervate the intestine, and the patterns of their projections to the nerve plexus of the intestinal wall, are still unclear. In the present study, the distributions of chicken vasoactive intestinal peptide (VIP) mRNA and substance P (SP) mRNA in the chicken INR were demonstrated by means of in situ hybridization histochemistry, using sense and antisense digoxigenin-labeled RNA probes. The in situ hybridization histochemistry results suggested that VIP mRNA-positive neurons were mainly distributed in ganglions, whereas some of the INR fibers were weakly positive. The proportion of VIP mRNA-positive cells among total neurons was 75% in the juxta-jejunoileum portion and 87% in the juxta-rectal portion of the INR. Most SP mRNA-positive cells were sporadically distributed in layers or small groups within the ganglions, as well as in nerve trunks between ganglions. Moreover, the proportion of positive cells among total neurons was 83% in the juxta-jejunoileum portion and 98% in the juxta-rectal portion of the INR, which suggests that both VIP and SP mRNA are coexpressed in the chicken INR. Our results provide an approach to further study of the locations of VIP and SP in nerve tissue.

The Expression Patterns of Cdc25A, Cdc25B, Sox2 and Mnb in Central Nervous System in Early Chicken Embryos

The sense and antisense digoxigenin-labeled RNA probes of four genes, Cdc25A, Cdc25B, Sox2 and Mnb, were produced by using SP6 and T7 RNA polymerases, respectively, and in vitro transcription. Expression patterns of the four genes were detected by in situ hybridization in HH (Hamburger and Hamilton) stage 10 chick embryos. In general, expression patterns of the four genes were similar. mRNA of the four genes was mostly restricted to the entire CNS (central nervous system). All were confined to an identical region, neural tube, neural groove and caudal neural plate, corresponding to the notochord or spinal cord, but there was some distinction in specific region or in concentration, for example in somites. The overlap in expression at the same developmental stage in the CNS suggests that the four genes may be functional similar or related in CNS development. Expression patterns of the four genes support specific roles of these regulators in the developing CNS.

Detection of hepatitis C virus RNA by in situ hybridization.

Persistent infection with hepatitis C virus (HCV) is associated with chronic hepatitis and cirrhosis which may eventually develop into primary hepatocellular carcinoma. The mechanism of pathogenesis is ill-defined and nothing is known of the distribution, frequency or type of infected cell in the liver of HCV-infected individuals. In this study we have examined liver tissue taken at autopsy from 2 anti-HCV-positive patients by in situ hybridization for the presence of HCV RNA. Viral RNA was detected by autoradiography after hybridization with 125I-labelled riboprobes, representing approximately 35% of the HCV genome. Only a few positive cells were identified in the HCV-infected liver samples, but not in a normal liver sample. Hybridization with an unrelated probe was negative in all samples. The HCV RNA-positive cells were detected with anti-sense but not sense RNA probes, suggesting that they contained a high ratio of genomic:antigenomic RNA. The appearance and distribution of the HCV RNA-positive cells suggested that they were not hepatocytes and were more likely to be lymphocytes or macrophages.

Specific regional distribution of gec1 mRNAs in adult rat central nervous system

GEC1 protein shares high identity with GABARAP (GABA A Receptor-Associated Protein), interacts with tubulin and GABA A receptors and is potentially involved in intracellular transport processes. Recently, using quantitative real time PCR, we have reported the gec1 mRNA expression in different rat brain areas. In the present study, we investigated the cell types expressing gec1 in rat brain. Sense and anti-sense gec1 RNA probes, corresponding to the 3′-untranslated region, were generated. In northern blotting experiments, the anti-sense probe revealed only the 1.75 kb gec1 mRNAs. On the other hand, in immunohistochemistry experiments, GEC1 polyclonal antibodies did not discriminate between GEC1 and GABARAP proteins. Therefore, we used digoxigenin-labeled RNA probes for in situ hybridization (ISH) experiments to map the gec1 expression. Using the anti-sense probe, we detected the gec1 mRNAs specifically in neurons throughout the rostrocaudal extent of the brain as well as in the spinal cord. Although a majority of neurons expressed the gec1 mRNAs, different intensities of labeling were observed depending on the areas: the strongest labeling was observed in the isocortex, hippocampus, basal telencephalon, some thalamic and most of hypothalamic nuclei, cerebellum, and numerous brainstem nuclei. Furthermore, the gec1 mRNAs were intensely expressed in neurons involved in somatomotor and neuroendocrine functions and weakly expressed in sensory and reticular structures. These results corroborate the putative role of the GEC1 protein in the trafficking of receptor GABA A.

Expression of the polymeric Immunoglobulin Receptor (pIgR) in mucosal tissues of common carp ( Cyprinus carpio L.)

The mucosal immune system seems to be an important defence mechanism for fish but the binding of IgM in mucosal organs is poorly described in fish. In this study the gene encoding the polymeric Immunoglobulin Receptor (pIgR) in carp has been isolated and sequenced from a liver cDNA-library and aligned with other species. The pIgR of carp consists of 2 Ig domains, a transmembrane and an intracellular region, together 327 amino acids. In situ hybridisations with sense and anti-sense DIG-labelled pIgR RNA probes were performed on liver, gut and skin of common carp ( Cyprinus carpio L.) and in these organs only anti-sense probes were found to hybridise. In liver the majority of hepatocytes was stained around the nucleus. In gut and skin, staining could be detected around the nucleus of the epithelial cells, but in gut also a subpopulation of lymphoid cells was stained in epithelium and lamina propria. The specific in situ hybridisation of the epithelia and hepatocytes coincides with the in situ binding of FITC-labelled carp IgM to the same cells. RT-PCR results indicate the expression of the pIgR gene in all lymphoid organs of carp, but not in muscle. Macrophages/neutrophils enriched by adherence or sorted B cells (MACS) did not show expression of the pIgR gene and are excluded as the pIgR expressing lymphoid cells in the intestine. The relevance of pIgR staining and gene expression in mucosal organs is discussed.

Expression and Regulation of the Osteoarthritis-associated Protein Asporin

Asporin (ASPN) is a small leucine-rich proteoglycan that is involved in pathological processes of osteoarthritis. Previously, we showed that asporin can inhibit transforming growth factor-beta1 (TGF-beta1)-mediated expression of cartilage matrix genes and chondrogenesis in vitro (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). However, details about regulation of asporin itself are not yet known. Here, we examined ASPN expression in skeletal tissue and potential regulation of ASPN by TGF-beta. In situ hybridization revealed the presence of ASPN mRNA in the perichondrium/periosteum of long bones, but its absence in articular cartilage and growth plates. Immunohistochemical analysis also showed ASPN protein expression predominantly in the perichondrium/periosteum. TGF-beta1 induced endogenous ASPN mRNA expression over time in vitro, and this induction was suppressed by the TGF-beta type I receptor kinase inhibitor SB431542. Inhibition of Smad3 significantly reduced TGF-beta1-induced ASPN expression, whereas overexpression of Smad3 augmented the induction. Characterization of the human ASPN promoter region revealed a region from -126 to -82 that is sufficient for full promoter activity; however, TGF-beta1 failed to increase activity through the ASPN promoter. Our findings indicate that TGF-beta1 induces ASPN through Smad3 but that this induction is indirect.

Inactivation of the UNC5C Netrin-1 Receptor Is Associated With Tumor Progression in Colorectal Malignancies

The UNC5H netrin-1 receptors (UNC5H1-3 also called UNC5A-C) belong to the functional dependence receptors family, which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer a tumor-suppressor activity. Indeed, cells harboring these receptors are thought to be dependent on ligand availability for their survival, thereby inhibiting uncontrolled tumor cell proliferation. We investigate here whether UNC5C acts as a tumor suppressor in colorectal malignancies. The level of UNC5C was analyzed in a panel of 86 primary sporadic colorectal carcinomas. Loss of heterozygosity in the UNC5C locus and epigenetic alterations in the UNC5C promoter were also analyzed. Intestinal tumor progression was monitored in mice bearing both UNC5C and APC1638N mutations, and apoptosis was measured in intestinal tumors developed in UNC5C/APC1638N mutant mice. We show here that UNC5C expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, in mice, inactivation of UNC5C is associated with increased intestinal tumor progression and a decrease in tumor cell apoptosis. The loss of UNC5C expression observed in human colorectal cancer is a selective advantage for tumor progression, in agreement with the dependence receptor hypothesis. Thus, the UNC5C dependence receptor is a tumor suppressor that regulates sporadic colorectal cancer.

Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation.

Hyphal Invasion of Candida albicans Inhibits the Expression of Human β-Defensins in Experimental Oral Candidiasis

Oral epithelium reacts to microbial challenges by eliciting a defensive response that includes the production of antimicrobial peptides. This study investigated the expression of human beta-defensins-1, 2, and 3 in reconstituted human oral epithelia during experimental oral infections with six different Candida species, and a wild-type C. albicans isolate and five of its mutants. The expression of defensins was induced after 12 hours infection with the wild-type C. albicans, but this response was not seen for the noninvasive hyphal mutants nor the secreted aspartyl proteinase mutants. Furthermore, defensin expression was not detected after 48 hours in epithelia infected with either C. albicans wild-type isolate or its invasive hyphal and proteinase mutants. Most of the non-albicans Candida were capable of inducing the expression of defensins in epithelia after 24 or 48 hours of infection. These Candida-host interaction patterns suggest that the oral epithelia possess mechanisms for sensing the early invasion of C. albicans through recognition of the presence of hyphae and proteinases of Candida and respond to the insult by producing antimicrobial peptides. This hyphal-invasion-dependent inhibition of defensin expression in oral epithelium that undermines the host surveillance system represents a hitherto undescribed novel pathogenic mechanism of C. albicans.

Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists

The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor-1 (IGF-1). The liver is the main source of circulating IGF-I. The authors have used rodent primary hepatocytes for studies on pharmacological intervention of IGF-I mRNA expression. A 96-well nonradioactive IGF-1 mRNA quantification assay was developed, based on the hybridization of sense and antisense RNA probes, to replicate membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GH-receptor binding compounds were evaluated. Two compounds were found to down-regulate IGF-I mRNA. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate potential unspecific transcriptional effects, the mRNA levels of the housekeeping genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochrome P450 2C12 (CYP2C12) and a rat homologue to the human alpha1B-glycoprotein (A1BG), were quantified by RNase protection assays and found to be down-regulated, confirming the antagonistic property of 1 compound. In conclusion, a direct filter hybridization assay of hepatocyte lysates using nonradioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds.

Contribution of Transmembrane Tumor Necrosis Factor to Host Defense against Mycobacterium bovis Bacillus Calmette-Guerin and Mycobacterium tuberculosis Infections

To study the specific role of transmembrane tumor necrosis factor (TmTNF) in host defense mechanisms against bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis infections, we compared the immune responses of TNF/lymphotoxin (LT)-alpha(-/-) mice expressing a noncleavable transgenic TmTNF (TmTNF tg) to those of TNF/LT-alpha(-/-) and wild-type mice. Susceptibility of TNF/LT-alpha(-/-) mice to BCG infection was associated with impaired induction of systemic RANTES but not of monocyte chemoattractant protein 1 (MCP-1), the development of excessive local and systemic Th1-type immune responses, and a substantially reduced inducible nitric oxide synthase (iNOS) activity. Resistance of TmTNF tg mice to BCG infection was associated with efficient activation of iNOS in granulomas and with the regulated release of local and systemic chemokines and Th1-type cytokines. However, M. tuberculosis infection of TmTNF tg mice resulted in longer survival and enhanced resistance compared to TNF/LT-alpha(-/-) mice but higher sensitivity than wild-type mice. TmTNF tg mice exhibited reduced pulmonary iNOS expression and showed an exacerbated cellular infiltration in the lungs despite a modest bacillary content. Our data thus indicate a role for TmTNF in host defense against mycobacteria by contributing to induction and regulation of Th1-type cytokine and chemokine expression leading to development of bactericidal granulomas expressing iNOS, which critically determines susceptibility versus resistance of the host to mycobacterial infections.

Expression of neurokinin a receptor mRNA in human nasal mucosa

In airway tissues, it has been suggested that tachykinins act as the transmitter for afferent sensory nerves which respond to various irritants and may be involved in airway allergic reactions. Three classes of tachykinin receptor have been recognized, denoted NK1, NK2 and NK3, which exhibit preferential affinity for substance P, neurokinin A and neurokinin B, respectively. We used molecular probes to study the gene expression and distribution of NK2 receptor in human nasal mucosa. Total RNA was isolated from human nasal mucosa and NK2 receptor mRNA was detected in these tissues using reverse transcriptase polymerase chain reaction (RT-PCR). For an in situ hybridization study of human nasal mucosa, we utilized the PCR directly to incorporate a T7 RNA polymerase promoter sequence onto the NK2 receptor cDNA, and these PCR products were used as the DNA template for producing digoxigenin-labeled antisense and sense RNA probes. These studies revealed that NK2 receptor mRNA was expressed in blood vessels. The results suggest a primary role for neurokinin A in the form of vascular responses in the upper respiratory tract.

Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure, expression pattern, and ligand binding.

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5'-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.

Effect of acoustic stress on glucocorticoid receptor mRNA in the cochlea of the guinea pig

As a first step toward elucidating the mechanism underlying the therapeutic effect of glucocorticoids on acute noise-induced hearing loss, we used semiquantitative reverse transcription polymerase chain reaction to study the level of glucocorticoid receptor (GR) mRNA in the cochlea of the guinea pig after acoustic overstimulation. The cochleas were dissected and divided into three portions (lateral portion, medial portion and modiolus). In the lateral portion, the glucocorticoid receptor mRNA level was significantly decreased 2 h after exposure to a 120 dB SPL sound and both 2 and 6 h after exposure to a 130 dB SPL sound. To determine where in the cochlea the changes in glucocorticoid receptor mRNA levels occur, in situ hybridization histochemistry was performed with digoxigenin-labeled sense and antisense RNA probes complementary to guinea pig glucocorticoid receptor mRNA. Glucocorticoid receptor mRNA was demonstrated in the spiral ligament, stria vascularis, spiral limbus and spiral ganglion. Moreover, the glucocorticoid receptor mRNA level was decreased in the spiral ligament, especially in the spiral prominence of the basal turn, 2 h after exposure to the 130 dB SPL sound. These results could imply that the therapeutic effect of glucocorticoids in the cochlea might be mediated in the spiral ligament, particularly in the spiral prominence.

Loss of expression of the putative tumor suppressor NES1 gene in biopsy-proven ductal carcinoma in situ predicts for invasive carcinoma at definitive surgery

Purpose The loss of expression of NES1, a novel putative tumor suppressor gene, is an early marker of breast tumorigenesis. NES1 is expressed in normal breast tissue and ductal hyperplasia but is absent or markedly diminished in invasive cancer. In cases of ductal carcinoma in situ (DCIS), NES1 expression has been shown previously to be present in approximately 50% of specimens. This study examined the expression level of NES1 in diagnostic biopsy samples found to contain pure DCIS. These data were then correlated with the pathologic findings found at definitive local surgery. Methods and materials Twenty-nine cases with initial biopsy showing DCIS without invasive carcinoma followed by subsequent reexcision were discovered and archived. Formalin-fixed tissue specimens were obtained for analysis. Each biopsy specimen was subjected to hematoxylin-eosin staining and reviewed by two pathologists to confirm the diagnosis of pure DCIS. NES1 cDNA (1069 bp), including 238 bp of 5′ and 3′ untranslated region and the entire protein-coding region, was cloned into a vector. To generate the antisense and sense RNA probes, the plasmid was linearized and the transcription reaction was carried out with polymerases T7 and T3, respectively. The detection of in situ hybridization probes was performed using an mRNA locator-Biotin Kit. Staining was characterized as negative (0/1+) or positive (2+/3+). Subsequent to an initial biopsy diagnosis of DCIS, all cases had a definitive surgical procedure. Detailed sectioning of the resultant tissue was performed and subjected to hematoxylin-eosin staining to determine the presence or absence of invasive carcinoma. Results The initial diagnostic biopsy specimens showed that 17 of 17 high-grade, 3 of 7 intermediate-grade, and 3 of 5 low-grade DCIS specimens were negative for NES1 expression. Of the 6 cases of DCIS found to be positive for NES1 expression, none (0%) were subsequently found to have invasive carcinoma at definitive surgery. In contrast, the loss of NES1 expression in the initial diagnostic biopsy was associated with a 40% incidence of invasive carcinoma at definitive surgery. Additional stratification by nuclear grade showed invasive carcinoma in 5 (83%) of 6 NES1-negative, low- to intermediate-grade DCIS ( p ≤0.01) and 4 (24%) of 17 NES1-negative, high-grade DCIS ( p ≤0.05). Conclusion These results show that a lack of NES1 expression in DCIS identified at the diagnostic biopsy predicts for a high risk of invasive cancer in the definitive surgical specimen. The predictive value of NES1 expression appears to be particularly relevant for low- and intermediate-grade DCIS.