Microarray expression profile analysis of circular RNAs and their potential regulatory role in bladder carcinoma.

Dysregulated circular RNAs (circRNAs) often contribute to the occurrence and development of various tumors; however, the function and mechanism of circRNAs are largely unknown in human bladder cancer (BC). In the present study, dysregulated circRNAs between BC and adjacent non­neoplastic bladder tissues were analyzed by circRNA microarray. We randomly selected 10 upregulated and five downregulated circRNAs for validation by quantitative real­time PCR. Bioinformatics analysis was further conducted to investigate the potential function of these differentially expressed circRNAs, with the differential expression of hsa_circRNA_100876, mir­136­5p, and mRNA­chromobox 4 (CBX4) subsequently verified. A total of 512 differentially expressed circRNAs were identified after scanning and normalization (340 upregulated and 172 downregulated circRNAs), with pathway and Gene Ontology analyses revealing their association with multiple significant cancer pathways. Construction of a circRNA­microRNA­mRNA network suggested additional potential roles of these circRNAs. The expression of hsa_circRNA_100876 and CBX4 was significantly negatively correlated with the expression of miR­136­5p. Additionally, hsa_circRNA_100876 was highly positively correlated with CBX4 expression. The results revealed that hsa_circRNA_100876 inhibition suppressed BC cell proliferation and it was associated with advanced T stage and lymphatic metastasis, and poor overall survival of BC patients. In conclusion, these differentially expressed circRNAs offer novel insights into potential biological markers or new therapeutic targets for the treatment of BC. Furthermore, hsa_circRNA_100876 may increase the expression of CBX4 by competing with miR­136­5p, ultimately promoting the malignant biological behavior of BC. Aberrantly expressed hsa_circRNA_100876 could be used as a potential non­invasive biomarker for the early detection and screening of BC.

Microbead-based ligase detection reaction assay using a molecular beacon probe for the detection of low-abundance point mutations.

A microbead-based ligase detection reaction (LDR) assay using a molecular beacon probe was developed for the facile and rapid detection of point mutations present in low copy numbers in a mixed population of wild-type DNA. Biotin-tagged ligation products generated in the LDR were captured on the surface of streptavidin-modified magnetic beads for purification and concentration. The resulting product-tethered microbeads were combined with a molecular beacon probe solution, and the suspension was directly flowed into a capillary. The microbeads were accumulated in a confined space within the capillary using a bar magnet. The packed bead sample was then scanned by a fluorescence scanning imager to detect the presence of any mutations. With the developed methodology, we were able to successfully detect one cancer mutation in a mixture of 400 wild-type templates (t test at 95% confidence level). Furthermore, the post-LDR processing, typically the most laborious and time-consuming step in LDR-based mutation detection assays, could be carried out much more rapidly (approximately 20 min). This was enabled by the simple bead and fluid manipulations involved in the present assay.

Structures and mechanisms of the mycothiol biosynthetic enzymes.

In the past decade, the genes encoding all four enzymes responsible for the biosynthesis of mycothiol in Mycobacterium tuberculosis have been identified. Orthologs of each of these have been stably expressed and structurally characterized. The chemical mechanisms of all the four have also been studied. Because of the unique phylogenetic distribution of mycothiol, and the enzymes responsible for its biosynthesis, these enzymes represent interesting potential targets for antimycobacterial agents.

A coupled spectrophotometric assay for l-cysteine:1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase and its application for inhibitor screening.

Most actinomycetes, including Mycobacterium tuberculosis, do not produce glutathione but make an alternative thiol, mycothiol, which has functions similar to those of glutathione. A key step in mycothiol biosynthesis is the ATP-dependent ligation of Cys to GlcN-Ins catalyzed by MshC to produce Cys-GlcN-Ins, AMP, and PP(i). MshC is essential for growth of M. tuberculosis and is therefore a potential target for drugs directed against tuberculosis. A coupled-enzyme assay for MshC was developed using pyrophosphatase to convert pyrophosphate to phosphate and spectrophotometric detection of the latter via the phosphomolybdate complex with malachite green. The assay was readily adapted for use in a 96-well microtiter plate format. A secondary high-performance liquid chromatography assay measuring Cys-GlcN-Ins production was used to validate potential hits. Preliminary testing on a library of 2,024 compounds predicted to inhibit ATP-dependent enzymes identified many promiscuous and pyrophosphatase inhibitors of MshC and a single validated inhibitor with IC(50) approximately 100 microM.

Downregulation of Cap43 gene by von Hippel-Lindau tumor suppressor protein in human renal cancer cells.

We previously identified 9 genes (i.e., thymosin beta4, secreted protein acidic and rich in cysteine, Cap43, ceruloplasmin, serum amyloid A, heat shock protein 90, LOT1, osteopontin and casein kinase Igamma) that are more highly expressed in cancerous regions than in noncancerous regions in human renal cancers. In our study, we considered the possibility that the von Hippel-Lindau (VHL) tumor suppressor gene might be able to affect the expression of these 9 genes in renal cancer cells. We first established 2 VHL-positive cell lines, 786/VHL-1 and 786/VHL-2, after the introduction of wild-type VHL into VHL-negative renal cancer 786-O cells. Of these 9 genes, expression of the Cap43 gene was specifically downregulated by VHL. Expression of Cap43 was also much lower in 4 other VHL-positive renal cancer cell lines than in VHL-negative 786-O cells. Cap43 promoter assays with several deletion or mutation constructs demonstrated that the Sp1 site in the element from -286 base pairs (bp) to -62 bp was partly responsible for VHL-induced suppression of the Cap43 gene. Immunostaining analysis with human specimens of renal cancers demonstrated that the Cap43 protein was expressed in most cancer cells and macrophages. We also observed a marked and specific increase of Cap43 mRNA levels in response to hypoxia or nickel in all VHL-positive cell lines. Cellular expression of Cap43 mRNA in response to hypoxia or nickel thus is closely associated with VHL gene expression in renal cancer cells. Although the function of the Cap43 protein remains unclear, the expression of Cap43 protein could be a molecular marker closely associated with VHL in renal cancer.

Molecular detection of von Hippel-Lindau gene mutations in urine and lymph node samples in patients with renal cell carcinoma: potential biomarkers for early diagnosis and postoperative metastatic status.

PURPOSE: Organ confined renal cell carcinoma can be cured in the majority of patients, whereas more extensive lesions have a poor prognosis. Therefore, the development of a useful biomarker for early diagnosis as well as postoperative metastatic status would contribute to the appropriate therapy for renal cell carcinoma. To diagnose renal cell carcinoma preoperatively we developed a novel urinary test and detected occult lymph node micrometastasis using a molecular approach. MATERIALS AND METHODS: Urine samples were obtained preoperatively from 27 patients with renal cell carcinoma and von Hippel-Lindau (VHL) gene mutations in the tumors, and were analyzed for VHL gene mutations using a nested single strand conformational polymorphism analysis. Lymph nodes without evidence of histological metastasis were obtained from 15 patients with renal cell carcinoma and VHL gene mutations, and analyzed for VHL gene mutations using mutation specific nested reverse transcription polymerase chain reaction method. RESULTS: In urine samples 5 of 27 VHL gene mutations (18.5%) were found and each mutation pattern was the same as that detected in each renal cell carcinoma. One lymph node micrometastasis was found. CONCLUSIONS: These data indicate the presence of detectable levels of tumor derived DNA in the urine of patients with renal cell carcinoma and suggest that nested single strand conformational polymorphism analysis of VHL gene of urine samples provides a possible tool for the early detection of renal cell carcinoma. Furthermore, mutation specific nested reverse transcription polymerase chain reaction is useful to detect occult lymph node micrometastasis and may predict patients at risk for local recurrence. These 2 combined approaches using VHL gene mutations may contribute to the total therapy for and prognosis of renal cell carcinoma.

Preliminary findings of quorum signal molecules in clinically stable lung allograft recipients.

BACKGROUND: Infection with bacteria such as Pseudomonas is common in lung allograft recipients, particularly during chronic rejection. Analysis of sputum samples from patients with cystic fibrosis infected with Pseudomonas aeruginosa or Burkholderia cepacia has indicated the presence of bacterial N-acylhomoserine lactones (AHLs) quorum sensing signalling molecules. AHLs not only control the expression of bacterial virulence genes but are also involved in stimulating the maturation of antibiotic resistant biofilms and host chemokine release. It was hypothesised that AHLs may be detected even in clinically stable lung transplant recipients free of clinical infection or rejection. METHODS: Three 60 ml samples of bronchoalveolar lavage (BAL) fluid were taken from nine stable lung transplant recipients 3-12 months after transplantation. Detection of AHLs was carried out on dichloromethane extracted supernatants using the bioluminescence based AHL reporter plasmid pSB1075. This responds to the presence of AHLs with long acyl chains (C10-C14), generating light. Synthetic AHLs were included as positive controls. RESULTS: Five of the nine BAL fluid supernatants exhibited AHL activity, suggesting the presence of AHLs with long N-acyl chains. There was no correlation between the levels of AHLs detected or their absence and BAL fluid microbiology or diagnosis before transplantation. CONCLUSIONS: This is the first evidence for the presence of AHL quorum sensing signals in human lung allograft recipients, even in subjects with no rejection or apparent infection. Further longitudinal follow up of these preliminary findings is required to elucidate potential links with infection, rejection, and allograft deterioration.

Absence of mutations in the VHL gene but frequent loss of heterozygosity at 3p25-26 in non-small cell lung carcinomas.

In this study we have examined 79 primary non-small cell lung tumours for the presence of mutations of the VHL gene as well as for allelic imbalance at the gene surrounding loci. While allelic imbalance was found in 83% of specimens, frequently affecting the whole 3p25-p26 region, no mutations were detected in the VHL coding region. The fractional regional loss (FRL) was significantly higher in squamous cell carcinomas (0.746) than adenocarcinomas (0.493) (Wilcoxon P=0.002). This is the first investigation of the VHL gene mutational status in primary lung tumours. Our results indicate that mutation is not a common means of VHL inactivation in NSCLC.

RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway.

Oocyte meiosis and early mitotic divisions in developing embryos rely on the timely production of cell cycle regulators and their clearance via proteasomal degradation. Ret Finger Protein-Like 4 (Rfpl4), encoding a RING finger-like protein with a B30.2 domain, was discovered during an in silico search for germ cell-specific genes. To study the expression and functions of RFPL4 protein, we performed immunolocalizations and used yeast two-hybrid and other protein-protein interaction assays. Immunohistochemistry and immunofluorescence showed that RFPL4 accumulates in all growing oocytes and quickly disappears during early embryonic cleavage. We used a yeast two-hybrid model to demonstrate that RFPL4 interacts with the E2 ubiquitin-conjugating enzyme HR6A, proteasome subunit beta type 1, ubiquitin B, as well as a degradation target protein, cyclin B1. Coimmunoprecipitation analyses of in vitro translated proteins and extracts of transiently cotransfected Chinese hamster ovary (CHO)-K1 cells confirmed these findings. We conclude that, like many RING-finger containing proteins, RFPL4 is an E3 ubiquitin ligase. The specificity of its expression and these interactions suggest that RFPL4 targets cyclin B1 for proteasomal degradation, a key aspect of oocyte cell cycle control during meiosis and the crucial oocyte-to-embryo transition to mitosis.

The E3 ubiquitin ligase GREUL1 anteriorizes ectoderm during Xenopus development.

We have identified a family of RING finger proteins that are orthologous to Drosophila Goliath (G1, Gol). One of the members, GREUL1 (Goliath Related E3 Ubiquitin Ligase 1), can convert Xenopus ectoderm into XAG-1- and Otx2-expressing cells in the absence of both neural tissue and muscle. This activity, combined with the finding that XGREUL1 is expressed within the cement gland, suggests a role for GREUL1 in the generation of anterior ectoderm. Although GREUL1 is not a direct inducer of neural tissue, it can activate the formation of ectopic neural cells within the epidermis of intact embryos. This suggests that GREUL1 can sensitize ectoderm to neuralizing signals. In this paper, we provide evidence that GREUL1 is an E3 ubiquitin ligase. Using a biochemical assay, we show that GREUL1 catalyzes the addition of polyubiquitin chains. These events are mediated by the RING domain since a mutation in two of the cysteines abolishes ligase activity. Mutation of these cysteines also compromises GREUL1's ability to induce cement gland. Thus, GREUL1's RING domain is necessary for both the ubiquitination of substrates and for the conversion of ectoderm to an anterior fate.

A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro.

A gene encoding a putative guanosine 3',5'-bispyrophosphate (ppGpp) synthase-degradase, designated Cr-RSH, was identified in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. The encoded Cr-RSH protein possesses a putative chloroplast-targeting signal at its NH2-terminus, and translocation of Cr-RSH into chloroplasts isolated from C.reinhardtii was demonstrated in vitro. The predicted mature region of Cr-RSH exhibits marked similarity to eubacterial members of the RelA-SpoT family of proteins. Expression of an NH2-terminal portion of Cr-RSH containing the putative ppGpp synthase domain in a relA, spoT double mutant of Escherichia coli complemented the growth deficits of the mutant cells. Chromatographic analysis of 32P-labeled cellular mononucleotides also revealed that expression of Cr-RSH in the mutant bacterial cells resulted in the synthesis of ppGpp. SpoT, which catalyzes (p)ppGpp degradation, is dispensable in E.coli only if cells also lack RelA, which possesses (p)ppGpp synthase activity. The complementation analysis thus indicated that Cr-RSH possesses both ppGpp synthase and degradase activities. These results represent the first demonstration of ppGpp synthase-degradase activities in a eukaryotic organism, and they suggest that eubacterial stringent control mediated by ppGpp has been conserved during evolution of the chloroplast from a photosynthetic bacterial symbiont.

Identification and characterization of UEV3, a human cDNA with similarities to inactive E2 ubiquitin-conjugating enzymes.

Recent studies have shown that ubiquitination is an essential factor in endosomal sorting and virus assembly. The human TSG101 gene has been demonstrated to belong to a group of genes coding for apparently inactive E2 ubiquitin-conjugating enzymes, which exert regulatory effects on E2 activity in cellular ubiquitination processes. In this study, a novel human cDNA (UEV3) encoding a putative protein of 379 amino acids was isolated from a human placenta library that may represent a partial paralogue of human TSG101. The predicted protein contains an N-terminal domain homologous to the catalytic domain of ubiquitin-conjugating enzymes (Ubc), which is fused to a sequence showing significant homology to members of the lactate dehydrogenase protein family. The UEV3 gene is located on chromosome 11 closely adjacent to TSG101 and LDH-C. Northern blot and UEV3-specific reverse transcription/polymerase chain reaction (RT/PCR) analyses of various colon carcinoma cell lines as well as both normal and tumor samples from colon revealed an expression of the UEV3 cDNA in all tested samples.

Parkin localizes to the Lewy bodies of Parkinson disease and dementia with Lewy bodies.

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.

The fission yeast COP9/signalosome is involved in cullin modification by ubiquitin-related Ned8p.

BACKGROUND: The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. A recent report by Lyapina et al. shows that the COP9/signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p. Disruption of caa1/csn1, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex. RESULTS: We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to approximately 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN. CONCLUSIONS: Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.

Aberrant expression of the NF-kappaB and IkappaB proteins in B cells from viable motheaten mice.

In viable motheaten mice, a mutation in the gene encoding the phosphatase, SHP1, causes severe immunodeficiency and autoimmunity. A defective phosphatase may result in modified phosphorylation of proteins involved in gene regulation. Since the NFkappaB/IkappaB proteins are regulated through phosphorylation, we wished to understand if the expression of these proteins was altered by the SHP1 defect. Splenic B cells from viable motheaten mice were isolated and assessed for purity by flow cytometry. Levels of each protein in isolated B cells were examined by Western blot analyses. Measurement of RNA levels for each protein was assessed by semi-quantitative RT-PCR. Western blots revealed that, in me(v) whole cell lysates, there were reduced levels of RelA and RelB proteins and increased levels of p50 and c-Rel. Furthermore, we analyzed the protein levels of IkappaBalpha and found that, in me(v), this inhibitor was significantly reduced, while the level of another member of the IkappaB family, IkappaBbeta, was not. To determine if these findings in me(v) were secondary to the autoimmune process, we evaluated NF-kappaB/IkappaB expression in the BXSB murine model of autoimmunity. Unlike me(v), B cells from BXSB/Yaa mice had NF-kappaB complexes composed of the RelA submit, and IkappaBalpha was readily detected. In addition, RNA for the RelA and IkappaBalpha proteins in me(v) and control littermates was detected by RT-PCR, indicating that the reduced amounts of these proteins was not exclusively due to transcriptional defects. We conclude that the differences in NF-kappaB/IkappaB proteins that we have described in me(v) are likely a consequences of the SHP1 defect and could contribute to the clinical disorder that characterizes me(v) mice.

p53-mediated repression of nuclear factor-kappaB RelA via the transcriptional integrator p300.

The p53 tumor suppressor gene plays an instrumental role in transcriptional regulation of target genes involved in cellular stress responses. p53-dependent transactivation and transrepression require its interaction with p300/CBP, a coactivator that also interacts with the RelA subunit of nuclear factor-kappaB. We find that p53 inhibits RelA-dependent transactivation without altering RelA expression or inducible kappaB-DNA binding. p53-mediated repression of RelA is relieved by p300 overexpression and the increased RelA activity conferred by p53-deficiency is counteracted by either transactivation domain-deficient p300 fragments that bind RelA or a transdominant mutant of IkappaB alpha. Our results suggest that p53 can regulate diverse kappaB-dependent cellular responses.

Immunolocalization of the ubiquitin-protein ligase Nedd4 in tissues expressing the epithelial Na+ channel (ENaC).

The epithelial Na+ channel (ENaC) was previously shown to be expressed in several Na(+)- and fluid-absorbing epithelia, particularly those of the kidney, colon, and lung. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein with ENaC and demonstrated that Nedd4 binds by its WW domains to the proline-rich PY motifs of ENaC. These PY motifs were recently shown to be deleted/mutated in patients afflicted with Liddle's syndrome, a hereditary form of systemic renal hypertension. Such mutations cause elevated channel activity by an increase in channel number/stability at the plasma membrane and by increased channel opening. We then proposed that Nedd4, by regulating channel stability/ degradation, may be a suppressor of ENaC. To test whether Nedd4 is localized to those tissues/regions that express ENaC, we performed immunocytochemical analysis of rat Nedd4 (rNedd4) distribution in rat kidney, colon, and lung tissues. Our results show that, in the kidney, rNedd4 is primarily localized to the cortical collecting tubules and outer and inner medullary collecting ducts. These tubular segments were previously shown to express ENaC. The epithelium lining medullary calyxes was also intensely stained, and microvillar borders of proximal convoluted tubules expressed variable amounts of rNedd4. In the lung, rNedd4 was mainly expressed in the epithelia lining the airways, in the submucosal glands and ducts, and in the distal respiratory epithelium. These sites resemble the pattern of ENaC expression. In contrast, in the distal colon, rNedd4 was strongly expressed in the epithelia lining the crypts but not in the ENaC-expressing surface epithelium. Low-salt diet (to elevate serum aldosterone levels) had no effect on rNedd4 distribution in the kidney or colon. Thus Nedd4 is coexpressed and likely colocalizes with ENaC in specific regions within the kidney and lung but not in the colon. We speculate this difference in colocalization may reflect differences in the regulation of channel stability in those tissues.

Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography.

A non-radiochemical assay procedure for CTP synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays. The assay was linear with time and protein concentration, although at low protein concentration a lag phase was observed. An amount of 2 x 10(6) cells was already sufficient to determine the specific activity of CTP synthetase in HL-60 cells, lymphocytes and in lymphoblasts obtained from pediatric patients suffering from acute lymphoblastic leukemia.

Reduction of bleomycin-induced acute DNA injury in the rat lung by the 21-aminosteroid, U-74389G.

OBJECTIVE: To determine whether pretreatment with a 21-aminosteroid, U-74389G, can prevent subsequent DNA injury in bleomycin-exposed lungs. SUBJECTS: Thirty-six adult male Sprague-Dawley rats. DESIGN: Controlled animal laboratory investigation of DNA injury in vivo. INTERVENTIONS: Animals were treated with 21-aminosteroid (10 mg/kg) or vehicle and subsequently received intratracheal instillation of bleomycin (1.75 U) or normal saline. MEASUREMENTS AND MAIN RESULTS: Twenty-four hours after bleomycin exposure, the 21-aminosteroid-treated animals had decreased evidence of DNA injury, expressed as percentage of DNA fragmentation normalized to the control group (113.5 +/- 6 [SEM] vs. 132 +/- 3.9%, p < or = .05), and activity of the DNA repair enzyme poly ADP-ribose synthetase (3.4 +/- 0.2 vs. 5.6 +/- 0.9 pmol nicotinamide adenine dinucleotide/min/mg protein, p < or = .05). Only bleomycin-exposed (+ vehicle) animals demonstrated significant evidence of increased DNA injury vs. the intratracheal saline-exposed control groups. CONCLUSIONS: The 21-aminosteroid pretreatment decreases subsequent pulmonary DNA injury induced by bleomycin exposure. This finding is likely due to the 21-aminosteroid's iron-chelating and cell-permeating abilities, and suggests that these agents may be effective in other diseases where iron-dependent free radical reactions occur.