Semi-quantitative RT-PCR Experiments Research Articles

Molecular and biological characteristics of a peach latent mosaic viroid PC isolate in peach from China: base mutations in hairpin stems and implications for symptomatology

Peach latent mosaic viroid (PLMVd) infects peach trees in China and induces a conspicuous albino phenotype (peach calico, PC) that is closely associated with variants containing a 12–14 nucleotide hairpin insertion capped by a U-rich loop. Initially, PC disease distribution was limited to parts of Italy, and it was first detected in the field in China in 2019. To explore the molecular and biological characteristics of PLMVd PC isolates in peach in China, we conducted a comprehensive analysis of disease phenotype development, and investigated the data-associated pathogenicity and in vivo dynamics of Chinese isolate PC-A2 using slash-inoculated into GF-305 peach seedlings. Inoculated seedlings displayed PC symptoms much earlier following topping treatment, and PLMVd infectivity was further assessed using bioassay and semiquantitative RT-PCR experiments. Evolutionary analysis showed that the PC isolate and its progeny variants clustered into a single phylogroup distinct from reference PC-C40 isolates from Italy, and PC-K1 and PC-K2 from South Korea. Some PC-A2 progeny variants from green leaves of PC-expressing seedlings showed unbalanced point mutations in hairpin stems compared with the PC-C40 reference sequence, and constituted a new stem insertion type. The results reveal associations between the recessive phenotypes of peach albino symptoms and base variation in hairpin stem insertions relative to the PC-C40/chloroplastic heat shock protein 90 reference sequence.

Identification of novel indole based heterocycles as selective estrogen receptor modulator

In the present study, we have designed and synthesized indole derivatives by coalescing the indole nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were selected from series I and II after sequential combinatorial library generation, docking, absorption, distribution, metabolism and excretion (ADME) filtering, anti-proliferative activity, cytotoxicity, and ER-α competitor assay kit by utilizing estrogen receptor-α (ER-α) dominant T47D BC cells line and PBMCs (Peripheral Blood Mononuclear Cells). Cell imaging experiment suggested that both the compounds successfully cross cellular biomembrane and accumulate in nuclear, cytoplasmic and plasma membrane region. Semiquantitative RT-PCR and Western blotting experiments further supported that both compounds reduced the expression of mRNA and receptor protein of ER-α, thereby preventing downstream transactivation and signaling pathway in T47D cells line. Current findings imply that 5c and 6d represent novel ER-α antagonists and may be used in the development of chemotherapy for the management of BC.

Identification and Comparison of Candidate Olfactory Genes in the Olfactory and Non-Olfactory Organs of Elm Pest Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) Based on Transcriptome Analysis.

The leaf beetle Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) is a predominant forest pest that causes substantial damage to the lumber industry and city management. However, no effective and environmentally friendly chemical method has been discovered to control this pest. Until recently, the molecular basis of the olfactory system in A. quadriimpressum was completely unknown. In this study, antennae and leg transcriptomes were analyzed and compared using deep sequencing data to identify the olfactory genes in A. quadriimpressum. Moreover, the expression profiles of both male and female candidate olfactory genes were analyzed and validated by bioinformatics, motif analysis, homology analysis, semi-quantitative RT-PCR and RT-qPCR experiments in antennal and non-olfactory organs to explore the candidate olfactory genes that might play key roles in the life cycle of A. quadriimpressum. As a result, approximately 102.9 million and 97.3 million clean reads were obtained from the libraries created from the antennas and legs, respectively. Annotation led to 34344 Unigenes, which were matched to known proteins. Annotation data revealed that the number of genes in antenna with binding functions and receptor activity was greater than that of legs. Furthermore, many pathway genes were differentially expressed in the two organs. Sixteen candidate odorant binding proteins (OBPs), 10 chemosensory proteins (CSPs), 34 odorant receptors (ORs), 20 inotropic receptors [1] and 2 sensory neuron membrane proteins (SNMPs) and their isoforms were identified. Additionally, 15 OBPs, 9 CSPs, 18 ORs, 6 IRs and 2 SNMPs were predicted to be complete ORFs. Using RT-PCR, RT-qPCR and homology analysis, AquaOBP1/2/4/7/C1/C6, AquaCSP3/9, AquaOR8/9/10/14/15/18/20/26/29/33, AquaIR8a/13/25a showed olfactory-specific expression, indicating that these genes might play a key role in olfaction-related behaviors in A. quadriimpressum such as foraging and seeking. AquaOBP4/C5, AquaOBP4/C5, AquaCSP7/9/10, AquaOR17/24/32 and AquaIR4 were highly expressed in the antenna of males, suggesting that these genes were related to sex-specific behaviors, and expression trends that were male specific were observed for most candidate olfactory genes, which supported the existence of a female-produced sex pheromone in A. quadriimpressum. All of these results could provide valuable information and guidance for future functional studies on these genes and provide better molecular knowledge regarding the olfactory system in A. quadriimpressum.

An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors.

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues.

190 AST-120 (SPHERICAL CARBON ADSORBENT) IN COVERT HEPATIC ENCEPHALOPATHY: RESULTS OF THE ASTUTE TRIAL

fold; p,0.001 increase in protein secretion following a 2h or 4h stimulation, respectively. The upregulation of IL-6 expression by adenosine was inhibited by the non-specific adenosine receptor antagonist, CGS-15943. Semi-quantitative RT-PCR experiments demonstrated that A2b was the primary adenosine receptor expressed by H69 cells, and expression of A2b protein was confirmed by confocal immunofluorescence. Thus, we directly tested the role of A2b in IL-6 upregulation using pharmacologic and molecular methods. The specific A2b receptor antagonist MRS-1754 completely blocked adenosine-sensitive IL-6 upregulation; moreover, H69 cells transfected with A2b-specific siRNA, but not an siRNA control, did not upregulate IL-6 in response to adenosine. Since A2b receptors are known to be Gscoupled, we demonstrated that adenosine induced an upregulation of cAMP (6.6-fold; p,0.001). In summary, adenosine, acting via the A2b receptor, activates cholangiocyte IL6 release. Since IL-6 is a critical survival signal, we propose that this signaling pathway is important in the maintenance of liver survival in injury. 1. Cressman, D.E., et al., Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science, 1996. 274(5291): p. 1379-83. 2. Ezure, T., et al., The development and compensation of biliary cirrhosis in interleukin-6-deficient mice. Am J Pathol, 2000. 156(5): p. 1627-39. 3. Chan, E.S., et al., Adenosine A(2A) receptors play a role in the pathogenesis of hepatic cirrhosis. Br J Pharmacol, 2006. 148(8): p. 1144-55. 4. Chouker, A., et al., In Vivo Hypoxic Preconditioning Protects From Warm Liver Ischemia-Reperfusion Injury Through the Adenosine A2B Receptor. Transplantation, 2012. 94(9): p. 894-902.

Heterogeneous cell population derived from human ovarian follicular liquid: morphological studies and molecular screening

The origin of oocytes and primary follicles in ovaries of adult mammalian females is still a matter of dispute [1]. The components of new primary follicles, primitive granulosa and germ cells, differentiate sequentially and de novo from mesenchymal progenitor cells residing in the ovarian tunica albuginea (TA). It appears that mesenchymal progenitor cells contribute to the generation of epithelial cells similar to granulosa cells (GCs). The multipotency of a subset of granulosa cells was also established by in vitro differentiation into other cell types [2]. Up to now, luteinizing GCs were considered to be terminally differentiated, unavoidably becoming apoptotic a few days after ovulation. Previously, we have provided evidence for the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitro fertilization techniques [3]. These cells grow in minimal medium condition, without any growth factor (i.e. LIF), that is considered essential according to other procedures [4]. Using immunocytochemistry and flow cytometry we showed that these cells are positive for several mesenchymal stemness markers, including CD90, CD73, CD44, CD105. However, morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial- like and neural-like shapes. These observations are also supported by the identification of cells expressing specific neural markers, such as neurofilaments and PGP9.5, in addition to vimentin and cytocheratin positive cells. All these data are suggestive of the presence of different cell populations in follicular fluids. To verify this hypothesis we select a panel of markers specific for the different cell populations previously identified and we plan a molecular screening to follow their expression in the follicular fluid derived cells at different times of minimal culture conditions in vitro. Bone marrow derived MSCs were used as a control. For each sample we performed semiquantitative RT-PCR experiments normalizing the cDNAs used as templates on the basis of the number of pseudo-mesenchymal cells morphologically identified in the sample. For this purpose OCT-4 was selected as a stem marker to follow the mesenchymal stem cell population, while FSH-R was used to identify granulosa derived cells; CNTF and beta-3-tubuline were used to discriminate between neural and neuronal cells populations; epithelial and hematopoietic cells were followed using cytokeratin (CK8 and CK10) and CD45 markers, respectively. GAPDH and β-actin specific primers were used on all samples for normalization. Here we compare the results of this molecular screening with the previously obtained immunocytochemical and morphological data to confirm the presence of these different cytotypes in the samples purified from the follicular liquid and their persistence, loss or amplification at different times of in vitro minimal culture conditions.

Temporal expression of caffeic acid 3-O-methyltransferase (COMT) in mango (Mangifera indica var. Dashehari) fruit ripening

In the present study, evidence has been provided for the involvement of caffeic acid 3-O-methyltransferase (COMT) in mango fruit ripening at enzyme/protein and gene levels. A comt gene cloned from a cDNA library of ripe mango pulp exhibited 98% identity with the higher plant comt genes including substantial identity (81%) with flavonoid O-methyltransferase. Semi-quantitative RT-PCR experiments indicated that the comt transcript was present at low levels during the early stages of ripening but the relative expression augmented at ripened stage. Western blotting revealed a consistently coordinated increase in expression patterns of COMT protein in fully-ripe stage as compared to unripe stage. Use of the well established ripening delayer and an enhancer modulated COMT activity suggesting its role in mango ripening. The phenylpropanoid pathway has traditionally been associated with secondary metabolism and lignification, but, the present study suggests the involvement of comt in mango ripening and its possible role in flavonoid biosynthesis.

Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL‐2 knockdown in human malignant neuroblastoma SK‐N‐DZ cells

The potent antiapoptotic molecule Bcl-2 is markedly up-regulated in a majority of cancers, including neuroblastoma. Genistein is an isoflavone with antitumor properties. The present study sought to elucidate the molecular mechanism of genistein-induced apoptosis and also to examine the effect of genistein in increasing apoptosis during Bcl-2 knockdown in human malignant neuroblastoma SK-N-DZ cells. The cells were transfected with Bcl-2 siRNA plasmid vector, treated with 10 microM genistein, or the combination, and subjected to TUNEL staining and FACS analysis. Semiquantitative and real-time RT-PCR experiments were performed for examining expression of Fas ligand (FasL), tumor necrosis factor-alpha (TNF-alpha), Fas-associated death domain (FADD), and TNFR-1-associated death domain (TRADD). The cell lysates were analyzed by Western blotting for levels of molecules involved in both receptor- and mitochondria-mediated apoptotic pathways. Treatment with the combination of Bcl-2 siRNA and genistein resulted in more than 80% inhibition of cell proliferation. TUNEL staining and FACS analysis demonstrated apoptosis in 70% of cells after treatment with the combination of both agents. Apoptosis was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and activation of caspases through the mitochondria-mediated apoptotic pathway. Genistein triggered the receptor-mediated apoptotic pathway through upregulation of TNF-alpha, FasL, TRADD, and FADD and activation of caspase-8. Combination of Bcl-2 siRNA and genistein triggered a marked increase in cleavage of DFF45 and PARP that resulted in enhanced apoptosis. Our study demonstrates that Bcl-2 knockdown during genistein treatment effectively induced apoptosis in neuroblastoma cells. Therefore, this strategy could serve as a potential therapeutic regimen to inhibit the growth of human malignant neuroblastoma.

Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A2 and investigation of its potential contribution to hepatic fibrosis

A gene encoding a homologue of phospholipase A2 was identified from the Clonorchis sinensis adult cDNA plasmid library. The deduced amino acid sequence including a signal peptide that has 28–46% identity with secretory phospholipase A2, group III (group III sPLA2) of other species. It also has typical features of group III sPLA2s including 10 cysteines, the key residues of the Ca2+ loop and catalytic site. The recombinant protein encoded by this gene expressed in Escherichia coli showed a product of about 34kDa in SDS-PAGE. Prediction of signal peptide and Western blot analysis indicated the group III secretory phospholipase A2 of C. sinensis (CsGIIIsPLA2) was an excretory–secretory product (ES product). The enzyme activity of the recombinant protein was determined using phosphatidylcholine as substrates. The result revealed that the protein was a Ca2+-dependent PLA2. Both MTT test and cell cycle analysis of LX-2 showed a higher percentage of cells are in proliferation phase. Semi-quantitative RT-PCR experiments demonstrated an up-regulated expression of collagen III in these cells after incubation with the recombinant protein. We also identified that the recombinant CsGIIIsPLA2 could bind to some membrane proteins on LX-2 cells specifically by immunofluorescence, thus there might be receptors of CsGIIIsPLA2 on the LX-2 cell membrane. Our results suggest that CsGIIIsPLA2 might play an important role in the initiation and development of hepatic fibrosis caused by C. sinensis.

Adenosine deaminase-related genes: Molecular identification, tissue expression pattern and truncated alternative splice isoform in adult zebrafish ( Danio rerio)

Adenosine deaminase (ADA) is responsible for cleaving the neuromodulator adenosine to inosine. Two members of ADA subfamilies, known as ADA1 and ADA2, were described and evidence demonstrated another similar protein group named ADAL (adenosine deaminase “like”). Although the identification of ADA members seems to be consistent, the expression profile of ADA1, ADA2 and ADAL genes in zebrafish has not yet been reported. The aim of the present study was to map the expression pattern of ADA-related genes in various tissues of adult zebrafish ( Danio rerio). An extensive search on zebrafish genome followed by a phylogenetic analysis confirmed the presence of distinct ADA-related genes ( ADA1, ADAL and two orthologous genes of ADA2). Specific primers for each ADA member were designed, optimized semi-quantitative RT-PCR experiments were conducted and the relative amount of transcripts was determined. The tissue samples (brain, gills, heart, liver, skeletal muscle and kidney) were collected and the expression of ADA1, ADAL and ADA2 genes was characterized. ADA1 had a similar expression pattern, whereas ADAL was less expressed in the heart. The highest relative amount of ADA2-1 transcripts was observed in the brain, liver and gills and it was less expressed in the heart. RT-PCR assays revealed that the other ADA2 form ( ADA2-2) was expressed ubiquitously and at comparable levels in zebrafish tissues. The strategy adopted also allowed the identification of an ADA2-1 truncated alternative splice isoform ( ADA2-1/T), which was expressed at different intensities. These findings demonstrated the existence of different ADA-related genes, their distinct expression pattern and a truncated ADA2-1 isoform, which suggest a high degree of complexity in zebrafish adenosinergic system.

Identification of differentially expressed genes from contaminant and thermal exposed goldfish Carassius auratus in Gaobeidian Lake in Beijing, China

Gaobeidian Lake, located in Beijing, China, derives its water mainly from the effluent of the Gaobeidian Wastewater Treatment Plant, which is moderately polluted. Additionally, as this water is used as a coolant in the nearby thermal power plant, the water of this lake has an elevated temperature. To screen differential gene expression in Gaobeidian Lake, suppressive subtractive hybridization (SSH) methodology was performed on RNA in goldfish Carassius auratus hepatic tissues from the lake, using Huairou Reservoir as reference site. A total of 768 candidate clones were selected to perform differential screening. Of these, 264 clones were differentially expressed between the two sites, 124 of which were then subjected to DNA sequencing. Consequently, 36 different genes with known functions were obtained, and some of these differential genes were further confirmed by semiquantitative RT-PCR experiments. Many genes related to detoxification, stress and immune response, and metabolism, such as glutathione S-transferase (GST), cytochrome P450, family 2, subfamily b, polypeptide 10 (CYP2B10), CYP2X10, alpha-1-antitrypsin, and apolipoprotein A-I (Apo-AI), had higher expression levels in goldfish hepatic tissue from Gaobeidian Lake than those from the reference site. A set of nine genes with known functions were downregulated in Gaobeidian Lake compared to the reference site. The results provided evidence that organisms inhabiting Gaobeidian Lake were suffering a complex stress process and showing metabolism changes and disturbance of homeostasis.

Tissue Expression and Genomic Sequences of Rat N-acetyltransferases rNat1, rNat2, rNat3, and Functional Characterization of a Novel rNat3*2 Genetic Variant

Human arylamine N-acetyltransferases NAT1 and NAT2 are highly polymorphic genes that modify individual susceptibility to cancers caused by exposure to arylamine procarcinogens. Strong similarities exist between rat Nats and human NATs, and rat Nat2 polymorphisms result in slow acetylator phenotype. Recently, a third rat Nat, rNat3*1, was reported. Although in vivo toxicological and carcinogenic studies are often conducted in rats, relatively little is known about Nat sequences among available inbred rat strains. We report here that rNat1 and rNat2 open reading frames (ORFs) in 12 inbred rat strains (ACI, BN, BUF, CDF, COP, DA, LEW, LOU/M, MW, PVG, SHR, WF) corresponded to reference rNat1*13 and rNat2*20. While 10 of the 12 strains had reference rNat3*1 ORFs, strains ACI and COP had a variant rNat3*2 ORF characterized by a G619>T transversion (A207S). The rNat3*2 single nucleotide polymorphism reduced Nat3 protein levels and N- and O-acetyltransferase activity when recombinantly expressed in bacteria. Recombinant expression of rNat3 1 and rNat3 2 in COS-1 cells yielded equivalent protein levels but undetectable catalytic activities. Relative tissue expressions of rNat1, rNat2, and rNat3 mRNAs were assessed in liver and 12 extrahepatic tissues (lung, spleen, kidney, heart, esophagus, stomach, urinary bladder, prostate, colon, duodenum, jejunum, ileum) from male F344 rats exsanguinated prior to sacrifice. Semiquantitative RT-PCR experiments demonstrated that the relative expression of the rNat transcripts in liver and 12 extrahepatic tissues was rNat1 > rNat2, while rNat3 transcripts were not detected. This study concludes that rNat1 and rNat2 are primarily responsible for acetylation phenotype in rats.

Immunomodulatory effects of CMDB, the component of Cordyceps militaris mycelium, by the enhanced production of cytokines in macrophages (B64)

Abstract Cordyceps militaris (CM) have been reported to modify the immune and inflammatory responses both in vivo and in vitro. Macrophages play important roles in the innate immunity through the phagocytosis of antigens. This study examined the effects of CM on the activation of murine macrophage RAW264.7 cells. The filtered components of the draff broth (DB) cultured with mycelium of CM, CMDB, CMDBW, CMDB5P, CMDB25P, were purified by propyl alcohol extraction and HP 20 column chromatography. All the components of CM produced significant amounts of nitric oxide (NO). In particular, CMDB produced much more NO in RAW264.7 cells and primary macrophages than the other fractions of CM. CMDB increased significantly the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 dose-dependently in RAW264.7 cells. To examine whether the CMDB effects on cytokine production were attributable to its influence on the expression of mRNAs, semi-quantitative RT-PCR experiments were performed for the transcripts. Examination of the gene expression level showed that the enhanced production of cytokines was correlated with the up-regulation of i-NOS expression, cycloxygenase (COX)-2 expression, IL-1β and IL-6 expression, and TNF-α gene expression. Western blot analysis also confirmed that CMDB enhances the expression level of these cytokines. These results suggest that CMDB has immunostimulatory effects on macrophages through the enhanced production of cytokines.

Impaired activity of volume‐sensitive Cl− channel is involved in cisplatin resistance of cancer cells

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.

Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.

Identification of histological markers for malignant glioma by genome-wide expression analysis: dynein, α-PIX and sorcin

Glioblastoma multiforme (GBM), the most malignant class of glial neoplasm (grade IV in WHO criteria), carries the worst clinical prognosis among primary brain tumors in adults. To identify a set of genes involved in the tumorigenesis of GBM, we evaluated expression profiles of GBM tissues from 11 patients using a cDNA microarray representing 25,344 human genes. By comparing the profiles with those of normal brain tissue, we identified a number of differentially expressed genes: 54 with increased expression and 45 with reduced expression in GBMs. Semi-quantitative RT-PCR experiments with 6 of those genes confirmed higher expression of DNCH2, ARHGEF6, NPM1 and SRI and lower expression of NRGN and TM4SF2 in GBM tumors. Immunohistochemical staining for 3 of the respective gene products, dynein (product of DNCH2), alpha-PIX (product of ARHGEF6), and sorcin (product of SRI) indicated that this technique might be useful for histological grading of glial tumors. To establish criteria for this diagnostic approach, we scored glial tumor tissues of different histological grades according to the staining results; the scores were significantly higher in anaplastic astrocytomas and GBMs than in diffuse astrocytomas or normal brain tissues. These findings indicated that levels of these three proteins might serve as histological markers for malignant glioma classification.

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

The genome of the acidophilic, proteobacterium Acidithiobacillusferrooxidans, contains linked but divergently oriented genes, termed afel and afeR, whose predicted protein products are significantly similar to the LuxI and LuxR families of proteins. A possible promoter and Lux box are predicted upstream of afel. A cloned copy of afel, expressed in E. coli, encodes an enzyme that catalyzes the production of a diffusible compound identified by gas chromatography and mass spectrometry as an unsubstituted N-acyl homoserine lactone (AHL) of chain length C14. This AHL can be detected by a reporter strain of Sinorhizobium meliloti Rm41 suggesting that it is biologically active. The reporter strain also responds to extracts of the supernatant of A. ferrooxidans grown to early stationary phase in sulfur medium indicating that a diffusible AHL is produced by this microorganism. Semi-quantitative RT-PCR experiments indicate that afeI and afeR are expressed maximally in early stationary phase and are more expressed when A. ferrooxidans is grown in sulfur--rather than iron-containing medium. Given the predicted amino acid sequence and functional properties of AfeI and AfeR it is proposed that A. ferrooxidans has a quorum sensing system similar to the LuxI-LuxR paradigm.

Comprehensive gene expression profiling of anaplastic thyroid cancers with cDNA microarray of 25 344 genes

Little is known about the genetic mechanisms of anaplastic thyroid cancer (ATC). This is the most virulent of all human malignancies, and it is believed to result from transformation of differentiated thyroid cancers. To identify a set of genes involved in the development of ATC, we investigated expression profiles of 11 cell lines derived from ATC using a cDNA microarray representing 25 344 genes. Semi-quantitative RT-PCR experiments carried out for some genes that had shown altered expression on the microarray verified frequent over-expression of destrin, HSPA8, stathmin, LDH-A, ATP5A1, PSMB6, B23, HDP-1 and LDH-B, and frequent under-expression of thyroglobulin, PBP and c-FES/FPS genes among the cell lines and also among ten primary ATCs. In addition to mRNA expression studies, up-regulation of GDI2, destrin and stathmin were confirmed with immunohistochemical analysis. The extensive list of genes identified provides valuable information towards understanding the development of ATC, and provides a source of possible biomarkers for diagnosis and/or molecular targets for the development of novel drugs to treat ATC.

Expression profiling to predict postoperative prognosis for estrogen receptor-negative breast cancers by analysis of 25,344 genes on a cDNA microarray.

Estrogen receptor (ER) status is an essential determinant of clinical and biological behavior of human breast cancers. While ER-positive breast cancers respond well to adjuvant hormone therapy, ER-negative tumors are generally resistant. To date, no attempts have succeeded in finding molecular markers for classifying ER-negative breast cancers with respect to postoperative prognosis. To identify a set of prognostic markers for this type of cancer, we used a cDNA microarray consisting of 25,344 human genes to investigate expression profiles of ten primary breast cancers from patients who had died of breast cancer within 5 years after surgery (5y-D) and 10 from patients who had survived disease-free for more than 5 years (5y-S). Sets of genes characterizing each group were identified by Mann-Whitney and random-permutation tests. We documented 71 genes with higher expression in the 5y-D group than in the 5y-S group, and 15 with higher expression in the 5y-S group than in the 5y-D group. Semi-quantitative RT-PCR experiments were carried out to confirm the results of the microarray analysis. We established a scoring system for predicting postoperative prognosis of ER-negative breast cancers on the basis of aberrant gene expression. The list of genes reported here provides valuable information with regard to progression of breast cancer and is a source of possible target molecules for development of novel drugs to treat patients with ER-negative breast cancers.