Validation of common reference genes stability in exosomal mRNA-isolated from liver and breast cancer cell lines.

Accurate relative gene expression analysis by reverse transcription-quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well-known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes-isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK-BR-3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes-isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

Reference gene selection and validation for mRNA expression analysis by RT-qPCR in murine M1- and M2-polarized macrophage.

Murine bone marrow-derived macrophages (M0) and M1- and M2-polarized macrophages are being widely used as a laboratory model for polarized macrophages related molecular mechanism analysis. Gene expression analysis based on reference gene normalization using RT-qPCR was a powerful way to explore the molecular mechanism. But little is known about reference genes in these cell models. So, the goal of this study was to identify reference genes in these types of macrophages. Candidate reference genes in murine bone marrow-derived and polarized macrophages were selected from microarray data using Limma linear model method and evaluated by determining the stability value using five algorithms: BestKeeper, NormFinder, GeNorm, Delta CT method, and RefFinder. Finally, the selected stable reference genes were validated by testing three important immune and inflammatory genes (NLRP1, IL-1ß, and TNF-α) in the cell lines. Our study has clearly shown that Ubc followed by Eef1a1 and B2m respectively were recognized as the three ideal reference genes for gene expression analysis in murine bone marrow-derived and polarized macrophages. When three reference genes with strong different stability were used for validation, a large variation of a gene expression level of IL-1ß, TNF-α and NLRP1 were obtained which provides clear evidence of the need for careful selection of reference genes for RT-qPCR analysis. Normalization of mRNA expression level with Ubc rather than Actb or Gusb by qPCR in macrophages and polarized macrophages is required to ensure the accuracy of the qPCR analysis.

The polyubiquitin gene MrUBI4 is required for conidiation, conidial germination, and stress tolerance in the filamentous fungus Metarhizium robertsii.

The polyubiquitin gene is a highly conserved open reading frame that encodes different numbers of tandem ubiquitin repeats from different species, which play important roles in different biological processes. Metarhizium robertsii is a fungal entomopathogen that is widely applied in the biological control of pest insects. However, it is unclear whether the polyubiquitin gene is required for fungal development, stress tolerance, and virulence in the entomopathogenic fungus. In the present study, the polyubiquitin gene (MrUBI4, MAA_02160) was functionally characterized via gene deletion in M. robertsii.Compared to the control strains, the MrUBI4 deletion mutant showed delayed conidial germination and significantly decreased conidial yields (39% of the wild-type 14 days post-incubation). Correspondingly, the transcript levels of several genes from the central regulatory pathways associated with conidiation, including brlA, abaA, and wetA, were significantly downregulated, which indicated that MrUBI4 played an important role in asexual sporulation. Deletion of MrUBI4 especially resulted in increased sensitivity to ultraviolet (UV) and heat-shock stress based on conidial germination analysis between mutant and control strains. The significant increase in sensitivity to heat-shock was accompanied with reduced transcript levels of genes related to heat-shock protein (hsp), trehalose, and mannitol accumulation (tps, tpp, nth, and mpd) in the MrUBI4 deletion mutant. Deletion of MrUBI4 has no effect on fungal virulence. Altogether, MrUBI4 is involved in the regulation of conidiation, conidial germination, UV stress, and heat-shock response in M. robertsii.

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3.

BACKGROUND: Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1A/1B-light chain 3 (LC3) and perineural invasion (PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI. AIM: To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level. METHODS: Two sets of gene expression profiles of pancreatic cancer/normal tissue (GSE16515 and GSE15471) were collected from the Gene Expression Omnibus. Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network. RESULTS: Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition, 65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene, which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy. CONCLUSION: Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Ubiquitin C decrement plays a pivotal role in replicative senescence of bone marrow mesenchymal stromal cells.

Human bone marrow-mesenchymal stromal cells (hBM-MSCs) undergo cellular senescence during in vitro culture. In this study, we defined this replicative senescence as impaired proliferation, deterioration in representative cell characteristics, accumulated DNA damage, and decreased telomere length and telomerase activity with or without genomic abnormalities. The UBC gene expression gradually decreased during passaging along with the reduction in series of molecules including hub genes; CDK1, CCNA2, MCM10, E2F1, BRCA1, HIST1H1A and HIST1H3B. UBC knockdown in hBM-MSCs induced impaired proliferation in dose-dependent manner and showed replicative senescence-like phenomenon. Gene expression changes after UBC knockdown were similar to late passage hBM-MSCs. Additionally, UBC overexpession improved the proliferation activity of hBM-MSCs accompanied by increased expression of the hub genes. Consequently, UBC worked in higher-order through regulation of the hub genes controlling cell cycle and proliferation. These results indicate that the decrement of UBC expression plays a pivotal role in replicative senescence of hBM-MSCs.

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

Ubiquitin levels: the next target against gynecological cancers?

Ubiquitylation is a tightly regulated process that is essential for appropriate cell survival and function, and the ubiquitin pathway has shown promise as a therapeutic target for several forms of cancer. In this issue of the JCI, Kedves and colleagues report the identification of a subset of gynecological cancers with repressed expression of the polyubiquitin gene UBB, which renders these cancer cells sensitive to further decreases in ubiquitin production by inhibition of the polyubiquitin gene UBC. Moreover, inducible depletion of UBC in mice harboring tumors with low UBB levels dramatically decreased tumor burden and prolonged survival. Together, the results of this study indicate that there is a synthetic lethal relationship between UBB and UBC that has potential to be exploited as a therapeutic strategy to fight these devastating cancers.

Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant regardless of aortic valve morphology, aortic diameter and other factors associated with aortopathy. Here, we identified an appropriate set of HK genes to be used as endogenous reference for quantifying gene expression in ascending aortic tissue using a spin column-based RNA extraction method. Ascending aortic biopsies were collected intra-operatively from patients undergoing aortic valve and/or ascending aortic surgery. These patients had BAV or tricuspid aortic valve (TAV), and the aortas were either dilated (≥4.5cm) or undilated. The cohort had an even distribution of gender, valve disease and hypertension. The expression stability of 12 reference genes were investigated (ATP5B, ACTB, B2M, CYC1, EIF4A2, GAPDH, SDHA, RPL13A, TOP1, UBC, YWHAZ, and 18S) using geNorm software. The most stable HK genes were found to be GAPDH, UBC and ACTB. Both GAPDH and UBC demonstrated relative stability regardless of valve morphology, aortic diameter, gender and age. The expression of B2M and SDHA were found to be the least stable HK genes. We propose the use of GAPDH, UBC and ACTB as reference genes for gene expression studies of BAV aortopathy using ascending aortic tissue.

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth.

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2(E301K)) suppressed the ubi4Δ growth defect. The rpt2(E301K) mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2(E301K) mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells.

Activation of Cre recombinase alone can induce complete tumor regression.

The Cre/loxP system is a powerful tool for generating conditional genomic recombination and is often used to examine the mechanistic role of specific genes in tumorigenesis. However, Cre toxicity due to its non-specific endonuclease activity has been a concern. Here, we report that tamoxifen-mediated Cre activation in vivo induced the regression of primary lymphomas in p53-/- mice. Our findings illustrate that Cre activation alone can induce the regression of established tumors.

Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control? / Estudo de expressão transcricional no sistema nervoso central de ratos: qual gene deve ser usado como controle interno?

Objective A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). Methods Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. Results Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. Conclusion Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene. .

Objetivo Um número crescente de artigos publicados relaciona a expressão de genes específicos com diferentes padrões de comportamento em ratos. Os níveis de transcritos de ácido ribonucleico mensageiro são geralmente analisados por transcrição reversa, seguida de reação em cadeia da polimerase, e quantificados após a normalização com um controle interno ou gene de referência (gene housekeeping). No entanto, os genes housekeeping exibem expressão diferencial no sistema nervoso central, dependendo das condições fisiológicas e da área do cérebro a ser estudada. A escolha de um bom gene de controle interno é essencial para a obtenção de resultados confiáveis. Este estudo avaliou a expressão de três genes housekeeping (beta-actina, ciclofilina A e ubiquitina C) em diferentes áreas do sistema nervoso central de ratos (bulbo olfatório, hipocampo, estriado e córtex pré-frontal). Métodos Foram usadas ratas Wistar (fêmeas virgens, n=6) durante o período de diestro. O ácido ribonucleico total foi extraído a partir de cada região do cérebro; o ácido desoxirribonucleico complementar foi sintetizado por transcrição reversa e amplificado por reação em cadeia da polimerase quantitativo em tempo real utilizando SYBR® Green e primers específicos para cada um dos genes de referência. A estabilidade de expressão foi determinada utilizando NormFinder. Resultados A beta-actina foi o gene mais estável no hipocampo e estriado, enquanto a ciclofilina A e a ubiquitina C apresentaram maior estabilidade no córtex pré-frontal e no bulbo olfatório, respectivamente. Conclusão Com base em nosso trabalho, estudos posteriores de expressão gênica utilizando ratos como modelos animais devem levar ...

Topography of lymph node metastases in prostate cancer patients undergoing radical prostatectomy and extended lymphadenectomy: results of a combined molecular and histopathologic mapping study.

BACKGROUND: To determine the anatomic extent of pelvic lymph node dissection (PLND) in prostate cancer (PCa) patients at the time of radical prostatectomy (RP), knowledge about the topography of lymph node (LN) metastases is required. OBJECTIVE: Because small-volume LN metastases may be missed by standard histopathologic examination, we performed an anatomic mapping study combining molecular and histopathologic LN examination in PCa patients treated with RP and extended PLND (ePLND). DESIGN, SETTING, AND PARTICIPANTS: A total of 52 patients with intermediate- (n=15) and high-risk (n=37) PCa underwent RP and ePLND without neoadjuvant treatment. ePLND included dissection of the obturator fossa and the external, internal, and common iliac vessels. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: LNs ≥3 mm in diameter were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) for prostate-specific antigen (PSA) expression and by standard histopathology. Topography of positive LNs was determined descriptively. RESULTS AND LIMITATIONS: Of 1469 dissected LNs (median: 27 LNs per patient), 1186 LNs were ≥3 mm. Molecular LN analysis was positive in 127 LNs of 27 patients (52%) including 32 LNs of 12 patients (23%) with histopathologic positive LNs. Molecular examination was negative in 3 of 35 histopathologic positive LNs (9%). Combining both molecular and histopathologic findings, positive LNs were located in the standard PLND field defined by obturator fossa and external iliac vessels in 71%, along the internal iliac vessels in 16%, and along the common iliac vessels in 13%. Of LN-positive patients, 63% had LN metastases outside the standard PLND field. The internal iliac field was involved in 48% and the common iliac field in 37% of node-positive patients. Notably, internal and common iliac vessels were the only positive regions in 7% and 11% of node-positive patients, respectively. A limitation is the small number of patients included. CONCLUSIONS: These findings underline the enhanced sensitivity of qRT-PCR in comparison with standard histopathology for detection of small-volume LN metastases in PCa patients. Our results support an ePLND including the common iliac vessels, at least up to the ureteral crossing, to optimise nodal staging and to remove LNs potentially harbouring metastases.

Brain site-specific proteome changes in aging-related dementia.

This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains (for example, Alzheimer's disease (AD)). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD.

Disruption of polyubiquitin gene Ubc leads to defective proliferation of hepatocytes and bipotent fetal liver epithelial progenitor cells.

We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We confirmed that Ubc(-/-) embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc(-/-) cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid-gestation lethality of Ubc(-/-) embryos.

Genetic modification of cancer cells using non-viral, episomal S/MAR vectors for in vivo tumour modelling.

The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into "immune deficient" mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome.

Identification of suitable reference genes for gene expression measurement in uterine sarcoma and carcinosarcoma tumors.

OBJECTIVE: To date no suitable reference genes have been identified in carcinosarcomas and non-epithelial malignant tumors of corpus uteri for normalizing real-time RT-PCR (qRT-PCR) assays. The purpose of this study was to select appropriate references for gene expression studies in these tumors. MATERIALS AND METHODS: We used RNA extracts from 75 tissue samples, representing 50 tumors and 25 fragments of normal uterine tissues obtained from 50 patients treated for mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. qRT-PCR for five potential reference (housekeeping) genes, namely B2M, HMBS, HPRT1, TBP and UBC, was performed. The expression stability of these genes was assayed using geNorm software application. RESULTS: The analysis of gene expression data with geNorm identified HPRT1 as the most stable reference gene, followed by UBC and HMBS, for all the investigated tissues. When stratified by disease, the results still pointed at HPRT1 as the gene that retained the greatest robustness in mixed tumors as well as in smooth muscle and stromal sarcomas. CONCLUSIONS: Our work is the first report on reference gene selection for qRT-PCR applications in mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. A ranking of candidate genes' stability values for the three types of tumors is provided and might serve as a valuable guide for future gene expression studies of these rare entities.

Selection of housekeeping genes for gene expression studies in a rat model of irinotecan-induced mucositis.

BACKGROUND/AIMS: Mucositis is the term used to describe damage caused by chemotherapy to mucous membranes of the alimentary tract. RT-PCR has recently been utilised to determine the molecular events that occur in mucositis. As this method relies on the use of a validated endogenous control, this study aims to validate commonly used housekeeping genes in an irinotecan-induced mucositis model. METHODS: Rats were administered irinotecan and sacrificed at different time points, in particular 1, 24, 72 and 144 h following treatment. Histopathological damage was assessed by haematoxylin and eosin staining. RT-PCR was used to evaluate the expression of 11 housekeeping genes. Expression stability was determined by the Normfinder program. Matrix metalloproteinase 2 was used as a target gene to validate the appropriateness of the top-ranking housekeeping gene. RESULTS: For normalisation to multiple housekeeping genes, the most stable combination across all time points in the jejunum was Ywhaz/UBC and in the colon UBC/ß-actin. SDHA and GAPDH were the most variable genes in the jejunum and colon where they were 4.4 and 3.2 fold upregulated following irinotecan, respectively. CONCLUSIONS: For normalisation of irinotecan-induced mucositis gene expression studies, a combination of Ywhaz/UBC and UBC/ß-actin should be used in the jejunum and colon, respectively. UBC is the most favourable if restricted to a single housekeeping gene across all time points.

Impaired skin regeneration and remodeling after cutaneous injury and chemically induced hyperplasia in taps-transgenic mice.

Recently, we identified an AP-1-dependent target gene in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated mouse back skin, which encodes a retroviral-like aspartic proteinase (Taps/Asprv1). Taps expression was detected almost exclusively in stratified epithelia of mouse embryos and adult tissues, and enhanced protein levels were present in several non-neoplastic human skin disorders, implicating a crucial role for differentiation and homeostasis of multilayered epithelia. Here, we generated a mouse model in which Taps transgene expression is under the control of the human ubiquitin C promoter (UBC-Taps). Although no obvious phenotype was observed in normal skin development and homeostasis, these mice showed a significant delay in cutaneous wound closure compared with control animals. Shortly after re-epithelialization, we found an increase in keratinocytes in the stratum granulosum, which express Filaggrin, a late differentiation marker. A hypergranulosum-like phenotype with increased numbers of Filaggrin-positive keratinocytes was also observed in UBC-Taps mice after administration of TPA. In summary, these data show that aberrant Taps expression causes impaired skin regeneration and skin remodeling after cutaneous injury and chemically induced hyperplasia.

Optimal promoter usage for lentiviral vector-mediated transduction of cultured central nervous system cells.

Lentiviral vectors transduce both dividing and non-dividing cells and can support sustained expression of transgenes. These properties make them attractive for the transduction of neurons and other neural cell types in vitro and in vivo. Lentiviral vectors can be targeted to specific cell types by using different promoters in the lentiviral shuttle vector. Even with identical constructs, however, levels of expression can vary significantly in different types of neurons and different culture preparations; expression levels in the same neuronal subtypes can be very different in primary cell culture and in vivo. We systematically assessed the ability of different promoters to direct expression of foreign transgenes in primary murine neocortical neurons, cerebellar granule cells and in undifferentiated and differentiated neuroblastoma cells. In primary cortical neurons, constructs using the ubiquitin C promoter directed the highest level of transgene expression; the phosphoglycerate kinase (PGK) promoter also directed robust transgene expression, while the cytomegalovirus (CMV) and MND (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer) promoters resulted in the expression of the transgenes in only limited number of neurons. In contrast, in cerebellar granule cells and in differentiated SH-SY5Y neuroblastoma cultures, the CMV promoter directed the most robust transgene expression. There was similar variability in transgene expression directed by these promoters in primary cultures of oligodendrocytes and astrocytes. These findings may prove useful in the design of lentiviral vectors for use in cell culture models of the nervous system.

The structure of the UbcH8-ubiquitin complex shows a unique ubiquitin interaction site.

Ubiquitin-mediated proteolysis utilizes a series of three key enzymes (E1, E2, and E3) to transfer and then covalently modify a substrate with ubiquitin. E2 conjugating enzymes are central proteins in this pathway responsible for the acceptance of a ubiquitin from the E1 enzyme and association with an E3 protein. All E2 enzymes covalently bind ubiquitin through a thiolester linkage between a conserved active-site cysteine on E2 and the C-terminal glycine on ubiquitin. It is not known whether E2 enzymes utilize similar surfaces and residues to coordinate a ubiquitin molecule and how this might contribute to any substrate specificity. In this work, we determined the structure of the human E2 enzyme UbcH8 (UBE2L6) covalently bound to ubiquitin by NMR spectroscopy. A disulfide bond mimicking the short-lived thiolester was formed between the two proteins providing a stable complex. Overall, the structure of UbcH8 does not undergo a significant conformational change upon forming a complex with ubiquitin. Chemical shift perturbation and cross-saturation experiments were used to identify contacts between UbcH8 and ubiquitin and those contacts used as inputs for HADDOCK molecular docking to produce the structure of the UbcH8-ubiquitin complex. An ensemble of 16 structures (root-mean-square deviation of 0.83 A) showed that ubiquitin interacts with the linker region prior to the alpha5 helix as well as residues near the catalytic site. This region corresponds to an area of negative potential on the UbcH8 surface and is considerably different from other E2-ubiquitin interaction sites. Our findings indicate the positioning of ubiquitin on UbcH8 would still allow interaction with E1 and E3 enzymes. Together, the results suggest the UbcH8-ubiquitin complex may provide an additional level of specificity in the ubiquitination pathway.