Reverse Transcription-polymerase Chain Reaction Experiments Research Articles

Global Gene Expression Profile for Swarming Bacillus cereus Bacteria

Bacillus cereus can use swarming to move over and colonize solid surfaces in different environments. This kind of motility is a collective behavior accompanied by the production of long and hyperflagellate swarm cells. In this study, the genome-wide transcriptional response of B. cereus ATCC 14579 during swarming was analyzed. Swarming was shown to trigger the differential expression (>2-fold change) of 118 genes. Downregulated genes included those required for basic cellular metabolism. In accordance with the hyperflagellate phenotype of the swarm cell, genes encoding flagellin were overexpressed. Some genes associated with K(+) transport, phBC6A51 phage genes, and the binding component of the enterotoxin hemolysin BL (HBL) were also induced. Quantitative reverse transcription-PCR (qRT-PCR) experiments indicated an almost 2-fold upregulation of the entire hbl operon during swarming. Finally, BC1435 and BC1436, orthologs of liaI-liaH that are known to be involved in the resistance of Bacillus subtilis to daptomycin, were upregulated under swarming conditions. Accordingly, phenotypic assays showed reduced susceptibility of swarming B. cereus cells to daptomycin, and P(spac)-induced hyper-expression of these genes in liquid medium highlighted the role of BC1435 and BC1436 in the response of B. cereus to daptomycin.

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Up-regulation of prohibitin 1 is involved in the proliferation and migration of liver cancer cells

Although prohibitin 1 (PHB) is known to be associated with tumors, there are few studies regarding the role of PHB in hepatocellular carcinoma. An earlier glycoproteomics study of ours showed that PHB is over-expressed in MHCC97-H cells, a highly metastatic liver cancer cell line, and can be captured by wheat germ agglutinin. In the present study, western blotting and reverse transcription-PCR experiments showed an approximately 2-fold up-regulation in the expression of PHB in MHCC97-H cells. However, PHB was not significantly up-regulated in MHCC97-L cells, a low-metastatic liver cancer cell line. When PHB was over-expressed in MHCC97-L cells, cell proliferation was inhibited by 35% and migration increased about 2-fold. The results of this study show that PHB is up-regulated in MHCC97-H cells and is associated with both proliferation and migration of liver cancer cells.

Differential gene expression profile in the liver of the marine puffer fish Takifugu rubripes induced by intramuscular administration of tetrodotoxin

Marine puffer fish accumulate a high level of tetrodotoxin (TTX) in the liver and ovary, but the underlying mechanism of this toxification is unclear. To elucidate the genes related to toxification of the marine puffer fish, we examined the hepatic gene expression profile of the marine puffer fish Takifugu rubripes by suppression subtractive hybridization in response to the intramuscular administration of 0.50 mg TTX/kg body weight into the caudal muscle. The accumulation of TTX in the liver reached 68 ± 4% that of the administered dose within 12 h of administration. A total of 1048 clones from the subtracted cDNA libraries were successfully sequenced. The nucleotide sequence of 92 of the 1048 clones was identified as a hepcidin precursor. Reverse transcription-polymerase chain reaction experiments revealed that hepcidin precursors were highly expressed in the TTX-administered group. In addition, complement C3 (31 clones), serotransferrin (30 clones), apolipoprotein A-1 (14 clones), high temperature adaptation protein Wap65-2 (14 clones), complement C7 (12 clones), fibrinogen beta chain (12 clones), and 70 kDa heat-shock protein 4 (11 clones) were obtained. This study confirmed that the intramuscular administration of TTX increases the gene expression of the acute-phase response proteins in the liver of puffer fish T. rubripes.

Ataxia Telangiectasia Mutated (ATM)-mediated DNA Damage Response in Oxidative Stress-induced Vascular Endothelial Cell Senescence

Oxidative stress regulates dysfunction and senescence of vascular endothelial cells. The DNA damage response and its main signaling pathway involving ataxia telangiectasia mutated (ATM) have been implicated in playing a central role in mediating the actions of oxidative stress; however, the role of the ATM signaling pathway in vascular pathogenesis has largely remained unclear. Here, we identify ATM to regulate oxidative stress-induced endothelial cell dysfunction and premature senescence. Oxidative stress induced senescence in endothelial cells through activation/phosphorylation of ATM by way of an Akt/p53/p21-mediated pathway. These actions were abrogated in cells in which ATM was knocked down by RNA interference or inhibited by specific inhibitory compounds. Furthermore, the in vivo significance of this regulatory pathway was confirmed using ATM knock-out mice in which induction of senescent endothelial cells in the aorta in a diabetic mouse model of endothelial dysfunction and senescence was attenuated in contrast to pathological changes seen in wild-type mice. Collectively, our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.

WAVY LEAF1, an Ortholog of Arabidopsis HEN1, Regulates Shoot Development by Maintaining MicroRNA and Trans-Acting Small Interfering RNA Accumulation in Rice

In rice (Oryza sativa), trans-acting small interfering RNA (ta-siRNA) is essential for shoot development, including shoot apical meristem (SAM) formation and leaf morphogenesis. The rice wavy leaf1 (waf1) mutant has been identified as an embryonic mutant resembling shoot organization1 (sho1) and sho2, homologs of a loss-of-function mutant of DICER-LIKE4 and a hypomorphic mutant of ARGONAUTE7, respectively, which both act in the ta-siRNA production pathway. About half of the waf1 mutants showed seedling lethality due to defects in SAM maintenance, but the rest survived to the reproductive phase and exhibited pleiotropic phenotypes in leaf morphology and floral development. Map-based cloning of WAF1 revealed that it encodes an RNA methyltransferase, a homolog of Arabidopsis (Arabidopsis thaliana) HUA ENHANCER1. The reduced accumulation of small RNAs in waf1 indicated that the stability of the small RNA was decreased. Despite the greatly reduced level of microRNAs and ta-siRNA, microarray and reverse transcription-polymerase chain reaction experiments revealed that the expression levels of their target genes were not always enhanced. A double mutant between sho and waf1 showed an enhanced SAM defect, suggesting that the amount and/or quality of ta-siRNA is crucial for SAM maintenance. Our results indicate that stabilization of small RNAs by WAF1 is indispensable for rice development, especially for SAM maintenance and leaf morphogenesis governed by the ta-siRNA pathway. In addition, the inconsistent relationship between the amount of small RNAs and the level of the target mRNA in waf1 suggest that there is a complex regulatory mechanism that modifies the effects of microRNA/ta-siRNA on the expression of the target gene.

Mitomycin C Inhibits Ribosomal RNA: A NOVEL CYTOTOXIC MECHANISM FOR BIOREDUCTIVE DRUGS

Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds.

Identification and Functional Characterization of a new Sunflower Germacrene A Synthase (HaGAS3)

Sesquiterpenes and sesquiterpene lactones are major natural compounds found in linear and capitate glandular trichomes of sunflower, Helianthus annuus L. In addition to two recently identified germacrene A synthases HaGAS1 and HaGAS2, found in capitate trichome gland cells, reverse transcription-PCR experiments have now allowed identification of a third enzyme of this type, HaGAS3. Its cDNA sequence was established and its functional characterization as a germacrene A synthase was achieved through in vitro expression in engineered yeast, and by GC-MS experiments. PCR and RT-PCR experiments with cDNA from different plant organs revealed that the new enzyme is expressed independently from the other two. While these latter two were expressed in plant organs bearing capitate glandular trichomes and in roots, the new enzyme occurred in plant tissues not linked to the presence of specific trichomes (for example, cotyledons), and was absent in roots. The experiments show that independently regulated pathways for the first cyclic sesquiterpene, germacrene A, are present in sunflower.

Abstract 4250: Cigarette smoke induces expression of the putative stem cell marker ABCG2 in cultured esophageal adenocarcinoma cells

Abstract Limited information is available regarding epigenetic mechanisms by which cigarette smoke mediates initiation and progression of esophageal cancer. In order to examine this issue, esophageal adenocarcinoma cell (EsC) lines (NCI-EsC1 and NCI-EsC2) were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) under clinically relevant exposure conditions. Microarray analysis revealed that CSC exposure significantly upregulated expression of ABCG2, which encodes a xenobiotic pump protein believed to be highly expressed in cancer stem cells, and recently shown to correlate with aggressive phenotype of primary esophageal squamous cell carcinomas. Analysis revealed an average of 11 fold upregulation of ABCG2 as well as a 4 fold upregulation of another ABC transporter, ABCC3. Quantitative reverse transcription-PCR (RT-PCR), western blot, and immunohistochemistry experiments confirmed upregulation of ABCG2 in esophageal cancer cells exposed to CSC. Interestingly, basal as well as CSC-induced levels of ABCG2 were considerably higher in NCI-EsC2 derived from a smoker, relative to NCI-EsC1, which was established from a never-smoker. Time course experiments revealed that induction of ABCG2 commenced approximately 8 hours and peaked 48 hours following initiation of CSC exposure; thereafter, ABCG2 expression was sustained at these levels with continuous CSC exposure. HDAC inhibitors such as TSA and depsipeptide repressed basal levels of ABCG2 expression, and reduced CSC-mediated induction of ABCG2 in EsC cells. HDAC inhibitors in combination with verapamil (a ABCC3 inhibitor) and Depsipeptide virtually abolished basal, as well as CSC-induced ABCG2 expression in these cells. Preliminary pyrosequencing and chromatin immunoprecipitation (ChIP) experiments revealed that CSC-mediated ABCG2 induction was independent of promoter DNA demethylation, and coincided with hyperacetylation of H3K9 within the ABCG2 promoter. Flow cytometry analysis utilizing hoescht dye with and without fumitremorgin C, an ABCG2 inhibitor, revealed an increased side population in CSC-treated ESC cells relative to untreated controls. Experiments are in progress to further delineate the mechanisms by which CSC induces ABCG2 expression, and to ascertain if ABCG2 induction enhances the malignant phenotype of esophageal adenocarcinoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4250.

Dibutyryl-cAMP up-regulates nur77 expression via histone modification during neurite outgrowth in PC12 cells

An elevated level of cyclic AMP (cAMP) within cells activates gene expression through the cAMP-PKA-CREB pathway. Among the CREB target genes, some immediate early genes exist that are responsive to cAMP including the nur77 and c-fos genes. Treatment with dibutyryl-cAMP (dbcAMP) as well as nerve growth factor (NGF) induces neurite outgrowth in PC12 cells. Here, we report that acetylation of histone H3 was gradually stimulated after treatment with dbcAMP in PC12 cells and peaked 1 h after treatment. As the result of reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) experiments, both nur77 and c-fos gene expression were found to have peak 1 h after treatment. Knock-down with siRNA against nur77 mRNA inhibited the neurite outgrowth induced by dbcAMP, whereas knock-down with siRNA against c-fos mRNA did not inhibit the dbcAMP-induced neurite outgrowth. A chromatin immunoprecipitation (ChIP) assay revealed that the nur77 gene was associated with the acetylated Lys14 of histone H3 after treatment with dbcAMP. However, the amount of c-fos gene associated with acetylated histone H3 was not changed after treatment with dbcAMP. These results suggest that the expression of nur77, which is essential for the neuronal differentiation induced by dbcAMP, is up-regulated via dbcAMP-induced acetylation of the Lys14 of histone H3 in PC12 cells.

Exogenous application of 5-aminolevulinic acid increases the transcript levels of sulfur transport and assimilatory genes, sulfate uptake, and cysteine and glutathione contents in Arabidopsis thaliana

5-Aminolevulinic acid (ALA), a key precursor of porphyrin biosynthesis, promotes plant growth and crop yields. Although ALA is known to promote carbon fixation and nitrogen assimilation in plants, the effects of ALA on sulfur assimilation have not been determined. In the present study, we analyzed the effect of ALA on sulfur assimilation. We used a fusion gene construct consisting of a promoter region of the high-affinity sulfate transporter SULTR1;2 from Arabidopsis and green fluorescent protein ([GFP] P SULTR1;2 -GFP) to determine whether ALA treatment influences the expression of the sulfur transport gene. The GFP levels in P SULTR1;2 -GFP plants were significantly increased by 0.3 and 1 mmol L−1 ALA under both sulfur-sufficient and sulfur-deficient conditions. Real-time reverse transcription-polymerase chain reaction experiments revealed that these concentrations of ALA also increased the mRNA levels of other key sulfur transport and assimilatory genes, such as SULTR, adenosine 5′-phosphosulfate reductases and serine acetyl transferase. Sulfate uptake was enhanced by ALA treatment under sulfur-sufficient conditions. In addition, ALA treatment increased the accumulation of cysteine and glutathione, particularly in the shoot. Our data demonstrated that exogenously applied ALA increases the transcript levels of some sulfur assimilatory genes, sulfate uptake, and the contents of cysteine and glutathione. We propose a new role for ALA in regulating the sulfur assimilatory pathway.

Impact of female cigarette smoking on circulating B cells in vivo: the suppressed ICOSLG, TCF3, and VCAM1 gene functional network may inhibit normal cell function

As pivotal immune guardians, B cells were found to be directly associated with the onset and development of many smoking-induced diseases. However, the in vivo molecular response of B cells underlying the female cigarette smoking remains unknown. Using the genome-wide Affymetrix HG-133A GeneChip microarray, we firstly compared the gene expression profiles of peripheral circulating B cells between 39 smoking and 40 non-smoking healthy US white women. A total of 125 differential expressed genes were identified in our study, and 75.2% of them were down-regulated in smokers. We further obtained genotypes of 702 single nucleotide polymorphisms in those promising genes and assessed their associations with smoking status. Using a novel multicriteria evaluation model integrating information from microarray and the association studies, several genes were further revealed to play important roles in the response of smoking, including ICOSLG (CD275, inducible T-cell co-stimulator ligand), TCF3 (E2A immunoglobulin enhancer binding factors E12/E47), VCAM1 (CD106, vascular cell adhesion molecule 1), CCR1 (CD191, chemokine C-C motif receptor 1) and IL13 (interleukin 13). The differential expression of ICOSLG (p = 0.0130) and TCF3 (p = 0.0125) genes between the two groups were confirmed by real-time reverse transcription PCR experiment. Our findings support the functional importance of the identified genes in response to the smoking stimulus. This is the first in vivo genome-wide expression study on B cells at today's context of high prevalence rate of smoking for women. Our results highlight the potential usage of integrated analyses for unveiling the novel pathogenesis mechanism and emphasized the significance of B cells in the etiology of smoking-induced disease.

Distribution of Cepacian Biosynthesis Genes among Environmental and Clinical Burkholderia Strains and Role of Cepacian Exopolysaccharide in Resistance to Stress Conditions

The genus Burkholderia includes strains pathogenic to animals and plants, bioremediators, or plant growth promoters. Genome sequence analyses of representative Burkholderia cepacia complex (Bcc) and non-Bcc strains for the presence of the bce-I gene cluster, directing the biosynthesis of the exopolysaccharide (EPS) cepacian, further extended this previously described cluster by another 9 genes. The genes in the bce-II cluster were named bceM to bceU and encode products putatively involved in nucleotide sugar precursor biosynthesis and repeat unit assembly, modification, and translocation across the cytoplasmic membrane. Disruption of the B. cepacia IST408 bceQ and bceR genes, encoding a putative repeat unit flippase and a glycosyltransferase, respectively, resulted in the abolishment of cepacian biosynthesis. A mutation in the bceS gene, encoding a putative acyltransferase, did not affect EPS production yield significantly but decreased its acetylation content by approximately 20%. Quantitative real-time reverse transcription-PCR experiments confirmed the induction of genes in the bce-I and bce-II clusters in a Burkholderia multivorans EPS producer clinical isolate in comparison to the level for its isogenic EPS-defective strain. Fourier Transform infrared spectroscopy analysis confirmed that the exopolysaccharide produced by 10 Burkholderia isolates tested was cepacian. The ability of Burkholderia strains to withstand desiccation and metal ion stress was higher when bacteria were incubated in the presence of 2.5 g/liter of cepacian, suggesting that this EPS plays a role in the survival of these bacteria by contributing to their ability to thrive in different environments.

Genome-Wide Characterization of the SloR Metalloregulome in Streptococcus mutans

Streptococcus mutans is the primary causative agent of human dental caries, a ubiquitous infectious disease for which effective treatment strategies remain elusive. We investigated a 25-kDa SloR metalloregulatory protein in this oral pathogen, along with its target genes that contribute to cariogenesis. Previous studies have demonstrated manganese- and SloR-dependent repression of the sloABCR metal ion transport operon in S. mutans. In the present study, we demonstrate that S. mutans coordinates this repression with that of certain virulence attributes. Specifically, we noted virulence gene repression in a manganese-containing medium when SloR binds to promoter-proximal sequence palindromes on the S. mutans chromosome. We applied a genome-wide approach to elucidate the sequences to which SloR binds and to reveal additional "class I" genes that are subject to SloR- and manganese-dependent repression. These analyses identified 204 S. mutans genes that are preceded by one or more conserved palindromic SloR recognition elements (SREs). We cross-referenced these genes with those that we had identified previously as SloR and/or manganese modulated in microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) experiments. From this analysis, we identified a number of S. mutans virulence genes that are subject to transcriptional upregulation by SloR and noted that such "class II"-type regulation is dependent on direct SloR binding to promoter-distal SREs. These observations are consistent with a bifunctional role for the SloR metalloregulator and implicate it as a target for the development of therapies aimed at alleviating S. mutans-induced caries formation.

Complex Phosphatase Regulation of Ca2+-activated Cl− Currents in Pulmonary Arterial Smooth Muscle Cells

The present study was undertaken to determine whether the two ubiquitously expressed Ca(2+)-independent phosphatases PP1 and PP2A regulate Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) elicited by 500 nM [Ca(2+)](i) in rabbit pulmonary artery (PA) myocytes dialyzed with or without 3 mM ATP. Reverse transcription-PCR experiments revealed the expression of PP1alpha, PP1beta/delta, PP1gamma, PP2Aalpha, PP2Abeta, PP2Balpha (calcineurin (CaN) Aalpha), and PP2Bbeta (CaN Abeta) but not PP2Bgamma (CaN Agamma) in rabbit PA. Western blot and immunofluorescence experiments confirmed the presence of all three PP1 isoforms and PP2A. Intracellular dialysis with a peptide inhibitor of calcineurin (CaN-AIP); the non-selective PP1/PP2A inhibitors okadaic acid (0.5, 10, or 30 nM), calyculin A (10 nM), or cantharidin (100 nM); and the selective PP1 inhibitor NIPP-1 (100 pM) potently antagonized the recovery of I(Cl(Ca)) in cells dialyzed with no ATP, whereas the PP2A-selective antagonist fostriecin (30 or 150 nM) was ineffective. The combined application of okadaic acid (10 nM) and CaN-autoinhibitory peptide (50 microM) did not potentiate the response of I(Cl(Ca)) in 0 ATP produced by maximally inhibiting CaN or PP1/PP2A alone. Consistent with the non-additive effects of either classes of phosphatases, the PP1 inhibitor NIPP-1 (100 pM) antagonized the recovery of I(Cl(Ca)) induced by exogenous CaN Aalpha (0.5 microM). These results demonstrate that I(Cl(Ca)) in PA myocytes is regulated by CaN and PP1 and/or PP2A. Our data also suggest the existence of a functional link between these two classes of phosphatases.

Widespread Estrogen-Dependent Repression of microRNAs Involved in Breast Tumor Cell Growth

Altered expression of microRNAs (miRNA), an abundant class of small nonprotein-coding RNAs that mostly function as negative regulators of protein-coding gene expression, is common in cancer. Here, we analyze the regulation of miRNA expression in response to estrogen, a steroid hormone that is involved in the development and progression of breast carcinomas and that is acting via the estrogen receptors (ER) transcription factors. We set out to thoroughly describe miRNA expression, by using miRNA microarrays and real-time reverse transcription-PCR (RT-PCR) experiments, in various breast tumor cell lines in which estrogen signaling has been induced by 17beta-estradiol (E(2)). We show that the expression of a broad set of miRNAs decreases following E(2) treatment in an ER-dependent manner. We further show that enforced expression of several of the repressed miRNAs reduces E(2)-dependent cell growth, thus linking expression of specific miRNAs with estrogen-dependent cellular response. In addition, a transcriptome analysis revealed that the E(2)-repressed miR-26a and miR-181a regulate many genes associated with cell growth and proliferation, including the progesterone receptor gene, a key actor in estrogen signaling. Strikingly, miRNA expression is also regulated in breast cancers of women who had received antiestrogen neoadjuvant therapy. Overall, our data indicate that the extensive alterations in miRNA regulation upon estrogen signaling pathway play a key role in estrogen-dependent functions and highlight the utility of considering miRNA expression in the understanding of antiestrogen resistance of breast cancer.

The mRNA distribution of C7orf24, a gamma-glutamyl cyclotransferase, in rat tissues.

The putative protein C7orf24 is encoded by Homo sapiens chromosome 7 open reading frame 24. C7orf24 was first identified as a 21-kDa cytochrome c-releasing factor detected in the cytosolic fraction of human leukemia U937 cells after treatment with geranylgeraniol. C7orf24 protein was recently identified as a gamma-glutamyl cyclotransferase, an enzyme in the gamma-glutamyl cycle. However, the exact localization of C7orf24 mRNA in normal tissues remains unknown. The present study examined the distribution pattern of C7orf24 mRNA in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. The RT-PCR experiments demonstrated that C7orf24 and a variant C7orf24 mRNA were expressed in various tissues. Quantitative RT-PCR analysis revealed significantly high levels of both C7orf24 mRNAs in the liver and kidney, compared with other tissues examined. In situ hybridization histochemistry localized C7orf24 mRNA to hepatocytes in the liver and renal tubules in the kidney. The present results thus implicated an important role for C7orf24 in liver and kidney.

Up-regulation in the expression of renin gene by the influence of aluminium

The excretion of aluminium in urine was significantly increased after intake of analgesics containing aluminium, confirming increased absorption and hence exposure to aluminium with such medication. The effect of aluminium on the kidney was further investigated by study of gene expression in mice. After a single dose of aluminium, an up-regulation of renin gene was found by DNA sequencing of the products of differential display analysis. The up-regulation of renin was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments in the dose dependent treatments and the time course observation after aluminium citrate injection. The up-regulation of the renin expression by aluminium is a strong indication of the influence of aluminium on the renin-angiotensin-aldosterone-system, resulting in possible induction of essential hypertension.

Syncytin-2 Plays an Important Role in the Fusion of Human Trophoblast Cells

Human endogenous retrovirus (HERV)-encoded Syncytin-1 has been suggested to play a major role in trophoblast cell fusion and thereby placenta development. However, recent studies have strongly suggested that other HERV envelope proteins could also be implicated in this process. Based on this premise, herein we compared the expression and functional implication of Syncytin-1 with the more recently described Syncytin-2 protein in various trophoblast cells. Real-time reverse transcription PCR and Western blot analyses in differentiating primary trophoblast cells first indicated a direct correlation between mRNA and protein levels of Syncytin-2 and cell fusion, while an inverse correlation for Syncytin-1 was noted. Similar reverse transcription PCR experiments and promoter studies showed that cell fusion-inducing agents in the trophoblastic BeWo cell line increased the expression of Syncytin-1 but, more importantly, augmented Syncytin-2 expression. Confocal microscopy experiments further revealed that in BeWo cells and in freshly isolated primary human trophoblast cells, Syncytin-1 was present as a cytoplasmic punctuated structure in proximity to regions of cell-to-cell contact. On the other hand, Syncytin-2 presented an inducible signal, which mainly localized to the cytoplasmic membrane. Experiments with siRNA (small interfering RNA)-transfected BeWo and primary human trophoblast cells demonstrated an important diminution in the number of cell fusion events upon repression of Syncytin-2 expression, whereas transfection experiments with Syncytin-1-specific siRNA resulted in a more modest effect. Overall, these results highlight the importance of Syncytin-2 in BeWo and primary human trophoblast cell fusion.

Novel gene clusters involved in arsenite oxidation and resistance in two arsenite oxidizers: Achromobacter sp. SY8 and Pseudomonas sp. TS44

This study describes three gene clusters involved in arsenic redox transformation of two arsenite oxidizers: Achromobacter sp. SY8 and Pseudomonas sp. TS44. A 17.5-kb sequence containing the arsenite oxidase (aox) gene cluster (aoxX-aoxS-aoxR and aoxA-aoxB-aoxC-aoxD) was isolated from SY8 using a fosmid library approach. Similarly, a 14.6-kb sequence including the aox cluster (arsD-arsA-aoxA-aoxB) and the arsenic resistance (ars) gene cluster (arsC1-arsR-arsC2-ACR3-arsH-dual specificity phosphatase (DSP)-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-major facilitator superfamily (MFS)) was obtained from TS44 by inverse polymerase chain reaction (PCR). According to reverse transcription (RT) PCR experiments, SY8 aoxXSR and aoxABCD transcribed as two different transcripts in opposite directions, and TS44 aox and ars clusters transcribed as a single transcript in their respective cluster. All of these genes were found to be upregulated by the addition of arsenite [As(III)], arsenate [As(V)], and antimonite [Sb(III)], except that TS44 arsC1-arsR appeared to be expressed constitutively. The SY8 aox cluster was predicted to be regulated by a two-component signal transduction system and a potential regulatory model was proposed. The TS44 aox cluster is unusual since it contains structural genes only and arsDA in its upstream. The TS44 ars cluster includes several genes previously identified not associated with arsenic resistance or transformation. This study showed novel structures and arrangements of arsenic gene clusters associated with bacterial As(III) oxidation and As(V) reduction.