DNA Array Studies Research Articles

Reinforcement of barrier function and scalp homeostasis by Senkyunolide A to fight against dandruff.

Senkyunolide-A (SENKY) can be isolated from Apium graveolens seed oil obtained using supercritical CO2 extraction. SENKY and its parent compounds, the N-butyl phthalides, have been demonstrated to protect cells from CO poisoning, to prevent diabetes mellitus and to decrease cancer cell proliferation. This study was undertaken to evaluate in vitro and in vivo the effect of SENKY on epidermal function improvement, Malassezia effect control, scalp soothing and dandruff reduction via skin protection-related pathways. DNA-array and proteomic studies were performed on human keratinocytes, sebocytes and skin explants to demonstrate SENKY activities. Two clinical evaluations were performed under dermatologist control on 106 volunteers, with greasy or dry scalp, experiencing dandruff, itching and redness. Volunteers tested a shampoo followed, or not, by a leave-on, containing SENKY, or their placebos. Dandruff severity and redness were scored on the scalp. Moisturization and sebum release were recorded using relevant measuring apparatus. Itching and scratching evaluations came from volunteers' self-declarations. DNA-array studies on keratinocytes showed a clear regulation of skin barrier functions and epidermis defence pathways. Upregulation of epidermal differentiation complex genes was observed. These preliminary observations were reinforced by immunocytochemistry and immunohistochemistry studies showing a significant increase of involucrin, filaggrin, loricrin, SPRR, LC3B and ceramide 2 productions. Tight-junctions and corneodesmosomes were significantly reinforced both in keratinocyte cultures (corneodesmosin, claudin, ZO-1) and in skin explants (desmoglein). DNA-array studies also demonstrated upregulation of genes involved in detoxification and anti-inflammation pathways. Proteomic studies revealed that hBD2 production was increased in keratinocytes in contact with SENKY, whereas IL-8, PGE-2 and TLR-9 releases were repressed as well as sebocyte lipid production. Clinical evaluations confirmed that after 3 weeks, SENKY significantly reduced dandruff intensity, redness, itching and scalp histamine content compared to placebo and beginning of treatment. For the first time, SENKY has been shown to promote scalp homoeostasis by reinforcing barrier and defence functions at both gene and protein levels. It reduces irritation and redness in promoting detoxification and anti-inflammation pathways while controlling the niche of Malassezia. Applied on scalp, SENKY significantly reduces the formation of dandruff and soothes the scalp.

Role of Glutamate Decarboxylase-like Protein 1 (GADL1) in Taurine Biosynthesis

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Abstract A85: Mutant P53 as a potential target of chemopreventive isothiocyanates.

Abstract Studies in animals and humans support isothiocyanates (ITCs) as a promising class of cancer chemopreventive agents. It is believed that apoptosis induction and cell cycle arrest are important mechanisms underlying their chemopreventive effects. While several signaling pathways have been shown to be associated with their activities, little is known about the molecular targets of ITCs until recent years. Evidence obtained in this study supports a role of mutant p53 as a potential target of ITCs. We showed ITCs can selectively deplete mutant p53, but not the wild type, in various human cancer cells in a structure-dependent manner, as phenethyl ITC (PEITC) and benyl ITC (BITC) are potent mutant p53 depletors, whereas sulforaphane (SFN) is not. The SAR studies showed 2,2-diphenyl ITC, a synthetic compound, is the most potent mutant p53 depletor. We found the potencies of depletion coincide with their mutant p53 binding affinities, providing circumstantial evidence for a mechanism involving its direct binding by ITCs. The depletion of mutant p53 in cells by ITCs appears to involve protein aggregation. Nine pairs of isogenic cells with mutant p53 and shRNA knockdown were generated. Overall, cells with mutant p53 appear to grow faster than the corresponding cells with mutant p53 knockdown, consistent with its role in gain-of-function. However, no significant difference was noted in PEITC-induced apoptosis between the cell pairs examined so far, suggesting targets other than mutant p53 in these cancer cells may also be involved. DNA array studies using PEITC-treated SW480 colon cancer cells provide potential new lead targets. Furthermore, the role of mutant p53 depletion in preventing lung carcinogenesis by ITCs is also examined in mutant p53 transgenic animal models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A85.

A genomic and ecotoxicological perspective of DNA array studies in aquatic environmental risk assessment

Ecotoxicogenomics is developing into a key tool for the assessment of environmental impacts and environmental risk assessment for aquatic ecosystems. This review aims to report achievements and drawbacks of this technique and to explore potential conceptual and experimental procedures to improve future investigations. Ecotoxicogenomic literature evidences the ability of genomic technologies to characterize toxicant specific gene transcriptome patterns that can be used to identify major toxicants affecting aquatic species. They also contribute decisively to the development of new molecular biomarkers and, in many cases, to the determination of new possible gene targets. Primary transcriptomic responses obtained after short exposures provided more information of putative gene targets than secondary responses obtained after long, chronic exposures, which in turn are usually more accurate to describe actual environmental impacts in natural populations. Several problems need to be addressed in future investigations: the lack of studies (and genomic information) on key ecological species and taxa, the need to better understand the different transcriptomic responses to high and low doses and, especially, short and long exposures, and the need to improve experimental designs to minimize false transcriptome interpretations of target genes.

Circadian transcription in liver

Circadian rhythms regulate a wide range of cellular, physiological, metabolic and behavioral activities in mammals. The complexity of tissue- and day-time specific regulation of thousands of clock controlled genes (CCGs) suggests that many transcriptional regulators are involved. Our bioinformatic analysis is based on two published DNA-array studies from mouse liver. We search overrepresented transcription factor binding sites in promoter regions of CCGs using GC-matched controls. Analyzing a large set of CCG promoters, we find known motifs such as E-boxes, D-boxes and cAMP responsive elements. In addition, we find overrepresented GC-rich motifs (Sp1, ETF, Nrf1), AT-rich motifs (TBP, Fox04, MEF-2), Y-box motifs (NF-Y, C/EBP) and cell cycle regulators (E2F, Elk-1). In a subset of system-driven genes, we find overrepresented motifs of the serum response factor SRF and the estrogen receptor ER. The analysis of published ChIP data reveals that some of our predicted regulators (C/EBP, E2F, HNF-1, Myc, MEF-2) target relatively many clock controlled genes. Our analysis of CCG promoters contributes to an understanding of the complex transcriptional regulation of circadian rhythms in liver.

Identification of proteins of Neisseria meningitidis induced under iron‐limiting conditions using the isobaric tandem mass tag (TMT) labeling approach

Isobaric labeling reagents such as Tandem Mass Tags (TMT(R)) enable the genome-wide quantification of protein expression levels under different conditions using a gel-free MS/MS-based approach. Here, we applied a TMTduplex approach with two isobaric tags to study the response of the human pathogen Neisseria meningitidis to deprivation of iron, a condition met in the human body. In total, 609 proteins were identified in samples of three independent growth experiments, in which we compared cultures grown in the presence and absence of iron. Expression of 35 proteins was found to be induced or repressed under iron-limiting conditions, including 11 proteins whose ORFs were not previously identified in DNA array studies as being regulated by iron availability at the transcriptional level. These 11 proteins include proteins likely involved in iron metabolism.

The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins

YhaK is a protein of unknown function found in low abundance in the cytosol of Escherichia coli. DNA array studies have revealed that YhaK is strongly up-regulated by nitroso-glutathione (GSNO) and also displays a 12-fold increase in expression during biofilm growth of E. coli 83972 and VR50 in human urine. We have determined the YhaK crystal structure and demonstrated that in vitro YhaK is a good marker for monitoring oxidative stresses in E. coli. The YhaK protein structure shows a bicupin fold where the two cupin domains are crosslinked with one intramolecular disulfide bond (Cys10 to Cys204). We found that the third cysteine in YhaK, Cys122, is oxidized to a sulfenic acid. Two chloride ions are found in the structure, one close to the reactive Cys122, and the other on a hydrophobic surface close to a symmetry-related molecule. There are major structural differences at the N-terminus of YhaK compared with similar structures that also display the bicupin fold (YhhW and hPirin). YhaK showed no quercetinase and peroxidase activity. However, reduced YhaK was very sensitive to reactive oxygen species (ROS). The complete, functional E. coli glutaredoxin or thioredoxin systems protected YhaK from oxidation. E. coli thioredoxin reductase and NADPH produced ROS and caused oxidation and oligomerization of reduced YhaK. Taken together, we propose that YhaK is the first of a new sub-class of bicupins that lack the canonical cupin metal-binding residues of pirins and may be involved in chloride binding and/or sensing of oxidative stress in enterobacteria.

The Etiology of Steroid Cataract

Steroid-induced posterior subcapsular cataracts (PSCs) exhibit three main distinctive characteristics: (i) association only with steroids possessing glucocorticoid activity, (ii) involvement of aberrant migrating lens epithelial cells, and (iii) a central posterior location. The first characteristic suggests a key role for glucocorticoid receptor activation and subsequent changes to the transcription of specific genes. Glucocorticoid receptor activation is associated in many cell types with proliferation, suppressed differentiation, a reduced susceptibility to apoptosis, altered transmembrane transport, and enhancement of reactive oxygen species activity. Glucocorticoids may be capable of inducing changes to the transcription of genes in lens epithelial cells that are related to many of these cellular processes. This review examines the various mechanisms that have been proposed to account for the development of PSC in the context of recent DNA array studies. Additionally, given that the glucocorticoid receptor can also engender wide-ranging indirect activities, glucocorticoids could also indirectly affect the lens through the responses of other cells within the ocular compartment and/or through effects on cells at more remote locations. These indirect mechanisms, which, for example, could be mediated through alterations to the intraocular levels of growth factors that normally orchestrate lens development and maintain lens homeostasis, are also discussed. Although the mechanism of steroid cataract induction remains unknown, glucocorticoid-induced gene transcription events in lens epithelial cells, and also other intraocular or systemic cells, likely interact to generate steroid cataracts. Finally, although evidence for glucocorticoid-protein adduct formation in the lens is inconclusive, the generation of such adducts cannot yet be discounted as a contributing factor and must necessarily be retained in discussions of the etiology of steroid cataract.

Ectopic expression and oncogenesis of Mer tyrosine kinase in E2A-PBX1 expressing B-cell acute lymphoblastic leukemia

9561 Background: Mer is a receptor tyrosine kinase that belongs to a tyrosine kinase subfamily including Axl and Tyro-3. Mer is expressed in monocytes/macrophages, dendritic cells and platelets, but not in lymphocytes or thymocytes. However, prior DNA array studies have suggested an aberrant expression of Mer in B-cell ALL (B-ALL) expressing the E2A-PBX1 fusion protein associated with t(1;19). We investigated the potential oncogenic role of Mer in E2A-PBX1 expressing B-ALL. Methods: Mer expression was evaluated in diagnostic bone marrow samples in pediatric patients with E2A-PBX1 B-ALL (EP+) and non-E2A-PBX1 B-ALL (EP-) by quantitative RT-PCR, Western blot, and flow cytometry. Using the REH B-ALL cell line with a zinc-inducible E2A-PBX1, induction of Mer expression was evaluated by Western blot. A mouse model was developed by retroviral infection of E2A-PBX1 fusion protein into Mer wild-type (WT), transgenic (TG), and knock-out (KO) mouse bone marrow cells followed by transplantation of these cells into sublethally irradiated WT recipients. Evaluation was done for E2A-PBX1 expression in peripheral blood and bone marrow cells were analyzed by flow cytometry for the development of leukemia. Results: Thirteen EP+ and 14 EP- patient samples were processed. All 13 EP+ samples had Mer protein expression by flow cytometry and/or Western blot. Conversely, 1/14 EP- samples was weakly positive for Mer protein expression by flow cytometry and Western blot. Quantitative RT-PCR was performed on 4 EP+ samples and 6 EP- samples. There was a 7–324 fold increase in Mer expression in EP+ samples when compared to the mean Mer expression of EP- samples. In our mouse transplant model, the median time to develop leukemia from Mer TG/E2A-PBX1 and Mer KO/E2A-PBX1 mice was 92 days and 117 days, respectively, with one KO mouse still surviving disease free at 140 days post-transplant. Conclusions: Mer is ectopically expressed in E2A-PBX1 expressing B-ALL. Mer TG/E2A-PBX1 mice develop leukemia sooner than Mer KO/E2A-PBX1 mice, suggesting cooperativity between the oncogenes. Mer may be an attractive therapeutic target for patients with E2A-PBX1 expressing ALL. No significant financial relationships to disclose.

JASN Moves On

Beginning July 1, 2007, JASN will come under the direction of a new editor-in-chief, Dr. Eric Neilson, and a new editorial team selected by him. Reflecting that changeover, next month JASN will appear in a new style and format with new categories, new features, and new directions. It is time for

Interleukin (IL)‐12 mediates the anti‐osteoclastogenic activity of CpG‐oligodeoxynucleotides

Bacterial DNA activates the innate immune system via interactions with Toll-like receptor 9 (TLR9). This receptor recognizes CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the CpG dinucleotides in certain sequence contexts characterizing this DNA. Most studies have shown increased osteoclast differentiation by TLR ligands. We found that activation of TLRs (specifically TLR4 and TLR9) in early osteoclast precursors results in inhibition of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Our objective is to identify the mechanism leading to this inhibitory effect of a TLR ligand. Since both RANKL-RANK and CpG-ODN-TLR9 interactions result in NF-kappaB activation, p38 and ERK phosphorylation, and TNF-alpha synthesis (all implicated in osteoclastogenesis), we hypothesized that CpG-ODN (but not RANKL) in addition induces the synthesis of an anti-osteoclastogenic factor. Control osteoclast precursors, and cells treated with RANKL, CpG-ODN, or their combination were studied using DNA arrays (GEArray Q Series Mouse NF-kappaB Signaling Pathway Gene Array, MM-016, SuperArray). We found a marked increase in the mRNA levels of the osteoclastogenesis inhibitor interleukin-12 (IL-12) in osteoclast precursors treated with CpG-ODN and CpG-ODN + RANKL. Northern and Western analyses, together with ELISA, confirmed the DNA array studies. In correlation with these findings, IL-12 inhibited RANKL-induced osteoclast differentiation and specific anti-IL-12-antibodies inhibited the anti-osteoclastogenic effect of CpG-ODN. In conclusion, activation of TLR9 by its ligand, CpG-ODN, results in synthesis and release of IL-12 opposing RANKL-induced osteoclast differentiation.

Gene Array Studies in Renal Neoplasia

Renal cell carcinoma (RCC) is comprised of several distinct histologic subtypes many of which have characteristic cytogenetic abnormalities. The molecular pathogenesis of some of these neoplasms is beginning to be elucidated. Yet renal cell carcinoma is often discovered at an advanced clinical stage and effective pharmacologic therapies for this disease remain to be discovered. For these reasons, renal cell carcinoma is ideally suited to the genome scale investigation made possible by DNA microarrays. A number of DNA array studies of renal cell carcinoma have been published. Renal cell carcinomas have also been studied by array based comparative genomic hybridization. The purpose of this review will be to summarize these studies, to compare the results of the different studies, and to suggest future areas of investigation with a particular emphasis on clinically relevant advances.

Molekularbiologische Klassifikation von Hochrisiko-Patientinnen beim primären Mammakarzinom durch Genexpressionsanalysen

Objective: Many tumors are subclassified with genetic markers to improve therapy and follow-up. A goal is to use DNA arrays as a tool to understand and classify tumors on the basis of gene expression patterns. Global determination of cellular transcriptional activity may identify gene expression signatures that predict the clinical behavior of tumors. Methods: We performed DNA array studies and subsequent cluster analyses to establish a molecular profile of 73 breast cancer specimens (T1-4,N0-1.M0-1). Results: Our analyses identified a group of transcriptionally related specimens. Fourteen of these 16 tumors were node positive. This group had an accumulation of patients with distant metastases at the time of diagnosis (25% vs. 4%). Conclusions: Differentially expressed marker genes in conjunction with sample clustering algorithms provide a novel molecular classification system for breast cancers. This may help identify patients at high risk of recurrence.

DNA Array Studies Demonstrate Convergent Regulation of Virulence Factors by Cph1, Cph2, and Efg1 in Candida albicans

Candida albicans, normally a human commensal, can cause fatal systemic infections under certain circumstances. Its unique ability to switch from yeast to hyphal growth in response to various environmental signals is inherent to its pathogenicity. Filamentation is regulated by multiple pathways including a Cph1-mediated mitogen-activated protein kinase pathway, an Efg1-mediated cAMP/PKA pathway, and a Cph2 pathway. To gain a general picture of how these various signaling pathways regulate differential gene expression during filamentation, we have constructed a partial C. albicans DNA array of 7,000 genes and used it to study the gene expression profiles using various mutants and growth conditions. By combining this novel technology with a new liquid medium in which cph1/cph1 is defective in filamentation, previously identified differentially expressed genes (ECE1, HWP1, HYR1, RBT1, SAPs5-6, and RBT4) are found to be regulated by all three pathways. In addition, two novel genes, DDR48 and YPL184, have been found to be differentially regulated during hyphal development and by all three pathways. This suggests that distinct filamentation signaling pathways converge to regulate a common set of differentially expressed genes. As one of the mechanisms for the observed convergence, we find that the transcription of a key regulator, TEC1, is regulated by Efg1 and Cph2. Importantly, most of the genes regulated by multiple filamentation pathways encode known virulence factors. Perhaps, C. albicans utilizes converging pathways to regulate its vital virulence factors to ensure its survival and pathogenicity in various host environments.

DNA arrays and functional genomics in neurobiology

Publisher Summary This chapter provides a background discussion of the approaches and techniques underlying DNA arrays and the application of this form of study. DNA arrays are used for purposes other than expression profiling, for example, for DNA sequencing or single nucleotide polymorphism detection. This chapter focuses on the use of these arrays in expression analysis. The chapter highlights early work suggesting that DNA arrays may offer significant advantages to the study of development and function of the central nervous system (CNS). The rapid development of new methodologies for RNA isolation, array preparation, and data analysis/ retrieval is highly likely, given the exponential growth in array studies. Given some of the special issues regarding expression profiling in neurobioiogy, it seems likely that a subdiscipline of functional neurogenomics may eventually develop. Perhaps the most exciting aspect of DNA array studies is the tremendous integrative and collaborative nature of such work. This approach is highly suited to neuroscience, a discipline long versed in the value of integrative science.

Expression profiling of neural cells reveals specific patterns of ethanol-responsive gene expression.

Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.