Antisense Oligonucleotide Probes Research Articles

Chimeric Peptide-Based Biomolecular Constructs for Versatile Nucleic Acid Biosensing.

Nucleic acid biomarkers hold great potential as key indicators for the diagnosis and monitoring of diseases. Herein we design and implement bifunctional chimeric biomolecules composed of a solid-binding peptide (SBP) domain that specifically adsorbs onto solid sensor surfaces and a peptide nucleic acid (PNA) moiety that facilitates anchoring of antisense oligonucleotide (ASO) probes for the detection of nucleic acid targets. A gold-binding peptide, AuBP1, previously selected by directed evolution to specifically bind to gold, served as the basis for immobilizing nucleic acid probes onto gold substrates. Using surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) analyses, we demonstrate the sequential biomolecular assembly of the heterofunctional solid-binding peptide-antisense oligomer (SBP-ASO) construct onto a sensor surface and the subsequent detection of DNA in an aqueous environment. The effect of steric hindrance on optimal probe assembly is observed, establishing that less packing density results in greater target capture efficacy. In addition, an adsorbed layer of chimeric solid-binding peptide-peptide nucleic acid (SBP-PNA) undergoes viscoelastic changes at the solid-liquid interface upon probe immobilization and DNA target capture, whereby the rigid biofunctional layer becomes more flexible. The dual nature of the chimeric construct is highly amenable to a variety of platforms allowing for both specific recognition and probe immobilization on the sensor surface, while the modular design of the solid-binding peptide-antisense oligonucleotide provides facile functionalization of a wide diversity of solid substrates.

LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway.

Long noncoding RNA (LncRNA) is a new type of regulatory RNA. LncRNA HOX antisense intergenic RNA (HOTAIR), as an oncogene in non-small cell lung cancer (NSCLC), is one of the key determinants of tumor progression. However, its possible molecular mechanism and the immunomodulatory pathway involved in NSCLC are still unclear. This study aims to explore whether HOTAIR promotes proliferation, migration and invasion of the NSCLC cells by inhibiting the expression of C-C Motif Chemokine Ligand 22 (CCL22). We collected 30 clinical samples of cancer and adjacent normal tissues from the patients with NSCLC, using real-time quantitative polymerase chain reaction (RT-qPCR) to detect the LncRNA HOTAIR and CCL22 mRNA expression in tissues. Immunohistochemistry was used to detect the protein expression of CCL22 in cancer and adjacent normal tissues. Cell experiments were conducted to verify that LncRNA HOTAIR regulates the expression of CCL22 and participates in the progress of NSCLC. The antisense oligonucleotide (ASO) probe interfering with LncRNA HOTAIR and the interference fragment of CCL22 (si-CCL22) were constructed. A549 cells were co-transfected with ASO-HOTAIR and si-CCL22. We used RT-qPCR to detect the expression of LncRNA HOTAIR and CCL22 mRNA in the cells, enzyme-linked immunosorbent assay (ELISA) used to detect the CCL22 protein level in the cell supernatant. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was applied to detect cell proliferation, the Flow cytometry to detect cell apoptosis. Finally, the Transwell test was utilized to detect cell migration and invasion. In conclusion, this study suggests that HOTAIR may promote proliferation, migration and invasion of the NSCLC cells by inhibiting CCL22 expression, which may play a key role in NSCLC cell immunity.

Radiosynthesis of a novel antisense imaging probe targeting LncRNA HOTAIR in malignant glioma

BackgroundLong non-coding RNA (LncRNA) HOTAIR was amplified and overexpressed in many human carcinomas, which could serve as a useful target for cancer early detection and treatment. The 99mTc radiolabeled antisense oligonucleotides (ASON) could visualize the expression of HOTAIR and provide a diagnostic value for malignant tumors. The aim of this study was to evaluate whether liposome-coated antisense oligonucleotide probe 99mTc-HYNIC-ASON targeting HOTAIR can be used in in vivo imaging of HOTAIR in malignant glioma xenografts.MethodsThe ASON targeting LncRNA HOTAIR as well as mismatched ASON (ASONM) were designed and modified. The radiolabeling of 99mTc with two probes were via the conjugation of bifunctional chelator HYNIC. Then probes were purified by Sephadex G25 and tested for their radiolabeling efficiency and purity, as well as stability by ITLC (Instant thin-layer chromatography) and gel electrophoresis. Then the radiolabeled probes were transfected with lipofectamine 2000 for cellular uptake test and the next experimental use. Furthermore, biodistribution study and SPECT imaging were performed at different times after liposome-coated 99mTc-HYNIC-ASON/ASONM were intravenously injected in glioma tumor-bearing mice models. All data were analyzed by statistical software.ResultsThe labeling efficiencies of 99mTc-HYNIC-ASON and 99mTc-HYNIC-ASONM measured by ITLC were (91 ± 1.5) % and (90 ± 0.6) %, respectively, and both radiochemical purities were more than 89%. Two probes showed good stability within 12 h. Gel electrophoresis confirmed that the oligomers were successfully radiolabeled no significant degradation were found. Biodistribution study demonstrated that liposome-coated antisense probes were excreted mainly through the kidney and bladder and has higher uptake in the tumor. Meanwhile, the tumor was clearly shown after injection of liposome coated 99mTc-HYNIC-ASON, and its T/M ratio was higher than that in the non-transfection group and mismatched group. No tumor was seen in mismatched and blocking group.ConclusionThe liposome encapsulated 99mTc-HYNIC-ASON probe can be used in the in vivo, real-time imaging of LncRNA HOTAIR expression in malignant glioma.

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics

To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers’ RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication.

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.

Transcript specific mRNP capture from Drosophila egg-chambers for proteomic analysis

mRNA binding proteins (RBPs) play a major role in post-transcriptional control of gene expression. To understand the complex regulatory processes regulating a specific mRNA during its life-time, a comprehensive view of the bound RBPs is essential. Here, we describe a method for transcript-specific isolation of endogenous ribonucleoprotein complexes (RNPs) from Drosophila egg-chambers. The method, which is based on in-solution hybridization of short biotinylated antisense DNA oligonucleotide probes to multiple segments of a transcript of interest allows unbiased identification of associated proteins by quantitative proteomics.

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA's family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That's the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA's family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That's the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest. This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

Cloning and characterization of a bone morphogenetic protein homologue of Schistosoma japonicum

Bone morphogenetic proteins (BMPs) are known to play an important role in the regulation of cell proliferation, survival, differentiation and apoptosis in many vertebrates and invertebrates through the TGF-β signaling pathway. Although the TGF-β signaling pathway exists in schistosomes, BMP homologue, a ligand of TGF-β in Schistosoma japonicum, has not yet been identified. In this study, a BMP homologue of S. japonicum was cloned and characterized. The full length SjBMP cDNA is 3,020bp and encodes 928 amino acids, which include a TGF-β superfamily conserved domain at the C-terminus. BLAST analysis showed that, SjBMP has 68%, 51% and 43% homology with BMP from Schistosoma mansoni, Schmidtea mediterranea and Dugesia japonica at the amino acid level, respectively. According to data from real-time PCR, SjBMP was expressed in lung-stage schistosomula, 21-day liver-stage schistosomula, 50-day adult worms (the male and female), and eggs. The PCR data also indicated that, there was a ∼27- and ∼37-fold increase of SjBMP transcripts in the lung-stage schistosomula and eggs, respectively, and that there was relatively more SjBMP transcript in the adult male worm than in the adult female, in which the hepatic schistosomula was set as the calibrator for calculation. In situ hybridization based on FITC-labeled specific antisense oligonucleotide probes showed that SjBMP mRNA localized to the ovary of female worms and the integument and epithelium of female and male worms. After treatment with double-stranded RNA (dsRNA) at a concentration of 8×10−2μg/ml, which was added to the culture medium every other day for a week, the level of SjBMP mRNA in the cultured adult mixed-sex S. japonicum decreased at a range of ∼25–98% within 7days compared with the level of SjBMP mRNA in the blank control group. On the 2nd day, the number of eggs produced per pair of worms decreased 28.7%, and the percent of normal eggs also decreased (12.7% vs. 4.3%) in the SjBMP dsRNA-treated group when compared with the eggs laid by the blank control group. No difference was detected between the two groups on the 7th day of treatment, because the eggs of the untreated worms were also mostly abnormal, similar to the eggs laid by the treated group. In addition, no significant difference in the morphological structure of the adult worms was observed. Thus, the preliminary in vitro experiment indicated that SjBMP may be involved in the oviposition behavior of S. japonicum, and further studies based on the recombinant virus vector-induced steady knockdown of SjBMP or in vivo experiments are required for more in-depth investigation.

Detection of central single-nucleotide mismatches in short duplex DNAs on hyper-branched amine surfaces

High capability to distinguish single-nucleotide mismatches of genes using short oligonucleotide probes is essential in diagnostic methods for identification of point mutations and single nucleotide polymorphisms. To investigate the feasibility of using an aziridine-treated surface containing hyper-branched amine groups to discriminate single-nucleotide mismatches in a human gene, target probes for exons 5–8 of the p53 gene from liver cancer cells were hybridized with four types of surface-bound capture probes, one for perfect match and three for central single-nucleotide mismatches. The aziridine slide with high DNA-loading capacity exhibited greater ability to detect single-nucleotide mismatch than did the generic amine slide. When a T30 tether was linked to the capture probe, the mismatch discrimination capability increased when using a chemical cross-linker, but decreased when using UV irradiation for cross-linking. DNA duplexes had lower melting temperatures when the single-nucleotide mismatch was in the central region than when it was in the terminal region regardless of the type of mismatched nucleotide. Our results suggest that capture probes attached to the aziridine surface can effectively identify point mutations in a genomic sequence or and can estimate the affinity of gene-specific antisense oligonucleotide probes.

Saturation of the Human Phenome

The phenome is the complete set of phenotypes resulting from genetic variation in populations of an organism. Saturation of a phenome implies the identification and phenotypic description of mutations in all genes in an organism, potentially constrained to those encoding proteins. The human genome is believed to contain 20-25,000 protein coding genes, but only a small fraction of these have documented mutant phenotypes, thus the human phenome is far from complete. In model organisms, genetic saturation entails the identification of multiple mutant alleles of a gene or locus, allowing a consistent description of mutational phenotypes for that gene. Saturation of several model organisms has been attempted, usually by targeting annotated coding genes with insertional transposons (Drosophila melanogaster, Mus musculus) or by sequence directed deletion (Saccharomyces cerevisiae) or using libraries of antisense oligonucleotide probes injected directly into animals (Caenorhabditis elegans, Danio rerio). This paper reviews the general state of the human phenome, and discusses theoretical and practical considerations toward a saturation analysis in humans. Throughout, emphasis is placed on high penetrance genetic variation, of the kind typically asociated with monogenic versus complex traits.

Distribution and neuronal expression of phosphatidylinositol phosphate kinase IIγ in the mouse brain

The role of cellular phosphatidylinositol 5-phosphate (PtdIns5P), as a signalling molecule or as a substrate for the production of small, compartmentalized pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], may be dependent on cell type and subcellular localization. PtdIns5P levels are primarily regulated by the PtdIns5P 4-kinases (type II PIP kinases or PIP4Ks), and we have investigated the expression and localization in the brain of the least-studied PIP4K isoform, PIP4Kγ. In situ hybridization and immunohistochemistry, using antisense oligonucleotide probes and a PIP4Kγ-specific antibody, revealed that this isoform has a restricted CNS expression profile. The use of antibodies to different cell markers showed that this expression is limited to neurons, particularly the cerebellar Purkinje cells, pyramidal cells of the hippocampus, large neuronal cell types in the cerebral cortex including pyramidal cells, and mitral cells in the olfactory bulb and is not expressed in cerebellar, hippocampal formation, or olfactory bulb granule cells. In neurons expressing this enzyme, PIP4Kγ has a vesicular distribution and shows partial colocalization with markers of cellular compartments of the endomembrane trafficking pathway. The PIP4Kγ isoform expression is established after day 7 of postnatal development. Overall, our data suggest that PIP4Kγ may have a role in neuron function, specifically in the regulation of vesicular transport, in specific regions of the developed brain. J. Comp. Neurol. 517:296–312, 2009. © 2009 Wiley-Liss, Inc.

The ratio of matrix metalloproteinase to E‐cadherin expression: A pilot study to assess mRNA and protein expression among African American prostate cancer patients

We assessed the expression of Matrix Metalloproteinase (MMP) to E-cadherin (M/E ratio) to determine the correlation of gene expression with pathologic variables and outcome in a cohort of African American (AA) prostate cancer patients. Tumors from formalin-fixed, paraffin embedded RP specimens were examined. Gleason scores were 6, 7, and >or=8 in 7, 16, 13 tumors, respectively. Pathologic stage was organ confined (pT2) in 18 and advanced (>pT2) in 18 tumors. A colorimetric mRNA in situ hybridization (ISH) assay was performed using biotinylated anti-sense oligonucleotide probes for MMP 2 and 9, as well as for E-cadherin gene transcripts. Immunohistochemistry (IHC) was performed utilizing specific monoclonal antibodies to detect the above genes. Image analysis was performed to determine the intensity of both mRNA and protein expression. Two reviewers analyzed ISH gene expression independently. The M/E expression ratio was significantly increased at the invasive edge (but not the center) of tumors of higher Gleason score (P = 0.02 and 0.0008) and pathologic stage (P = 0.0001 and <0.0001) when examined by both ISH and IHC. Significant variability in ISH staining interpretation was noted within and among the two study reviewers. An M/E ratio >2.5 was associated with biochemical recurrence after radical prostatectomy in addition to tumor pathologic stage subsequent to univariate statistical analysis. The M/E ratio characterizes an important aspect of the molecular phenotype associated with the histologic progression of prostate cancer among African American prostate cancer patients. A larger comparative study is required to determine potential racial variation and prognostic significance of gene expression.

Biogenesis of Short Intronic Repeat 27-Nucleotide Small RNA from Endothelial Nitric-oxide Synthase Gene

Endothelial nitric-oxide synthase (eNOS) is a constitutively expressed gene in endothelium that produces NO and is critical for vascular integrity. Previously, we reported that the 27-nucleotide (nt) repeat polymorphism in eNOS intron 4, a source of 27-nt small RNA, which inhibits eNOS expression, were associated with cardiovascular risk and expression of the eNOS gene. In the current study, we investigated the biogenesis of the intron 4-derived 27-nt small RNA. Using Northern blot, we showed that the eNOS-derived 27-nt short intronic repeat RNA (sir-RNA) expressed only in the eNOS expressing endothelial cells. Cells containing 10 x 27- or 5 x 27-nt repeats produced higher levels of 27nt sir-RNA and lower levels of eNOS mRNA than the cells with 4 x 27-nt repeats. The 27nt sir-RNA was mostly present within the endothelial nuclei. When the splicing junctions of the 27-nt repeat containing intron 4 in the full-length eNOS cDNA vector were mutated, 27nt sir-RNA biogenesis was abolished. Suppression of Drosha or Dicer diminished the biogenesis of the 27nt sir-RNA. Our study suggests that the 27nt sir-RNA derived through eNOS pre-mRNA splicing may represent a new class of small RNA. The more eNOS is transcribed or higher number of the 27-nt repeats, the more 27nt sir-RNA is produced, which functions as a negative feedback self-regulator by specifically inhibiting the host gene eNOS expression. This novel molecular model may be responsible for quantitative differences between individuals carrying different numbers of the polymorphic repeats hence the cardiovascular risk.

Expressions of Steroidogenic Enzymes mRNA in the Testes of Japanese Black Bears (Ursus thibetanus japonicus) as Revealed by in situ Hybridization.

The Japanese black bear (Ursus thibetanus japonicus), which is a subspecies of the Asiatic black bear (Ursus thibetanus) and inhabits Japan, is a seasonal breeder with the mating season in June to July. Male bears exhibit full spermatogenesis and high concentrations of testosterone in blood in the pre-mating season. Molecular mechanism of spermatogenesis, complicatedly regulated by factors such as sex steroid hormones and cytokines, is known to be different among species. Up to date, few report of steroidogenic enzymes detection at mRNA level in the testes of Carnivora has been published, in spite of many reports on immunolocalization of them. Especially, 17βHSD family has been recently analyzed for its structure, so its expression remains to be elucidated, although it is the key enzyme concerning testosterone synthesis in the testes. To investigate steroidogenesis in the testes of the Japanese black bear, we utilized the testes samples taken from 4 adult male bears in mating season. Partial mRNA sequence of five steroidogenic enzymes (P450scc, 3βHSD, P450c17, 17βHSD3 and P450arom) expressing in the Japanese black bear testes were determined and their expression was detected by means of in situ hybridization with 35S labeled 45-mer antisense oligonucleotide probes specific to each enzyme. Partial mRNA codes of P450scc, 3βHSD, P450c17, 17βHSD3, and P450arom i487bp, 229bp, 512bp, 719bp and 460bp, respectivelyj showed 91.1~95.7% homology with those of the dog or cat. All of 4 testes samples used in this study showed full spermatogenesis. Expressions of P450scc, 3βHSD and P450c17 mRNA were intense in the interstitial tissue, suggesting that pregnenolone, progesterone, and androstenedione were synthesized in the interstitial tissue. Signal of 17βHSD3 mRNA appeared at the margin of the seminiferous tubules. This is a remark that testosterone is synthesized inside the seminiferous tubules. P450arom mRNA expressed weakly inside almost entire parts of the seminiferous tubules. Estrogen would be synthesized not only in the somatic cells but also in the germ cells in bears, as reported in the mouse, rat, human, etc. These results were mostly in accordance with past reports on immunohistological localization of steroidogenic enzymes. Conclusion is that the interstitial tissue, probably Leydig cells, are the biosynthetic sites of steroid hormones such as pregnenolone, progesterone and androstenedione, and germ cells have capability for synthesizing estrogen in the Japanese black bear testes. Moreover, in bears, this report is the first detection of 17βHSD3, which is the isozyme specifically expressing in testes, and indicating that testosterone is synthesized inside the seminiferous tubules.

Isx Participates in the Maintenance of Vitamin A Metabolism by Regulation of β-Carotene 15,15′-Monooxygenase (Bcmo1) Expression

Isx (intestine specific homeobox) is an intestine-specific transcription factor. To elucidate its physiological function, we generated Isx-deficient mice by knocking in the beta-galactosidase gene (LacZ) in the Isx locus (IsxLacZ/LacZ mice). LacZ staining of heterozygous (IsxLacZ/+) mice revealed that Isx was expressed abundantly in intestinal epithelial cells from duodenum to proximal colon. Quantitative mRNA expression profiling of duodenum and jejunum showed that beta-carotene 15,15'-monooxygenase (EC1.14.99.36 Bcmo1) and the class B type I scavenger receptor, which are involved in vitamin A synthesis and carotenoid uptake, respectively, were drastically increased in IsxLacZ/LacZ mice. Although mild vitamin A deficiency decreased Isx expression in duodenum of wild-type (Isx+/+) mice, severe vitamin A deficiency decreased Isx mRNA expression in both duodenum and jejunum of Isx+/+ mice. On the other hand, vitamin A deficiency increased Bcmo1 expression in both duodenum and jejunum of Isx+/+ mice. However, Bcmo1 expression was not increased in duodenum of IsxLacZ/LacZ mice by mild vitamin A deficiency. These data suggest that Isx participates in the maintenance of vitamin A metabolism by regulating Bcmo1 expression in the intestine.

Vesicular Glutamate Transport at a Central Synapse Limits the Acuity of Visual Perception in Zebrafish

The neural circuitry that constrains visual acuity in the CNS has not been experimentally identified. We show here that zebrafish blumenkohl (blu) mutants are impaired in resolving rapid movements and fine spatial detail. The blu gene encodes a vesicular glutamate transporter expressed by retinal ganglion cells. Mutant retinotectal synapses release less glutamate, per vesicle and per terminal, and fatigue more quickly than wild-type in response to high-frequency stimulation. In addition, mutant axons arborize more extensively, thus increasing the number of synaptic terminals and effectively normalizing the combined input to postsynaptic cells in the tectum. This presumably homeostatic response results in larger receptive fields of tectal cells and a degradation of the retinotopic map. As predicted, mutants have a selective deficit in the capture of small prey objects, a behavior dependent on the tectum. Our studies successfully link the disruption of a synaptic protein to complex changes in neural circuitry and behavior.

Role of protein kinase B in breast cancer

Breast cancer is the most common cancer in women and is increasing in both the developed and developing countries. There is an urgent need to understand the precise mechanisms of tumour development in breast cancer, to develop new treatment strategies and to identify predictive markers for tumour aggressiveness and therapy resistance. A protein called protein kinase B (PKB, also called Akt) is frequently elevated in breast cancers and has been implicated as a key player in breast cancer development and progression. The activation level of PKB is also thought to correlate with patient outcome. However, the function of the three isoforms of PKB in mediating the crucial responses is unknown. We have developed a set of antisense phosphorothioate oligonucleotide probes that target antisense-active regions in PKB and that enable >90% knockdown of all three known PKB isoforms (alpha, beta and gamma), either individually or in various combinations, including removal of all three isoforms together. We have demonstrated that these agents specifically and potently prevent the growth of breast cancer cells. Application of these antisense agents offers a unique opportunity to understand how PKB works and contributes to breast cancer, and to provide insight into the role of signalling by individual PKB isoforms in breast cancer cells. Such work may also identify clinically relevant markers of disease, thereby enabling better predictors of patient outcome, and provide the necessary intellectual framework for the development of PKB-isoform selective inhibitors (for example, antisense oligonucleotides, small chemical inhibitors) as novel therapeutic agents.