Oligonucleotide Microarray Technology Research Articles

Prognosis Is Genetically Fixed In Polycythemia Vera and Definable by Gene Expression Profiling

AbstractAbstract 1987Polycythemia vera (PV) is a chronic, clonal, stem cell disorder whose natural history is usually indolent but an undefined fraction of patients have a clinical course characterized by extramedullary hematopoiesis, myelofibrosis, massive splenomegaly, bone marrow failure, and a propensity to develop spontaneous acute leukemia or early death. Recently, a prognostic scoring system was developed to determine the survival of such patients after the onset of bone marrow failure (Blood, 2008; 111:3383-7). However, at that point, for most such patients, only palliative therapy is available. We have been conducting a gene expression profiling (GEP) study in PV peripheral blood (pb) CD34+ cells using oligonucleotide microarray technology, confirmed by QPCR, and now report that in patients who develop an aggressive form of PV, the associated genetic abnormalities are time independent, providing an opportunity to identify patients at risk before bone marrow failure ensues. Our observations were obtained in a cross-sectional study of 19 PV patients (8 men and 11 women) from whom pb CD34+ cells were harvested for GEP in comparison to immunophenotypically similar G-CSF mobilized pb CD34+cells from normal individuals (3 men and 3 women). The median age (55 years; range 33–71), disease duration (11.5 years; range 1–33) and JAK2 V617F allele burden (92%: range 50–100) of the men at the time of study were not significantly different from the women (median age 51 years; range 42–70); disease duration, (8 years; range 1–13) and JAK2 V617F allele burden (100%; range 49–100);16 patients were homozygous for the mutation. Gender-specific differences in PV clinical features (Haematologica 2010; 95:1090-7) were mirrored by gender-specific differences in GEP between the men and women patients, allowing us to identify a core set of 89 genes (31 down regulated and 58 up regulated) shared in common. Using this core gene set and unsupervised hierarchical clustering, the 19 PV patients could be segregated into a small homogeneous group (6 patients; 3 men and 3 women) and a larger heterogeneous group(5 men and 8 women), which did not differ with respect to median age (64.5 vs 67 years); median disease duration (10.5; range 5–20, vs 7 years; range 1–33); JAK2 V617F allele burden (100% vs 88%), or leukocyte and platelet counts, but did differ with respect to their clinical characteristics with the smaller group having a lower median hemoglobin level (10.7 gm% vs 13.1 gm%; p= 0.01), a greater number of thrombotic events (p = 0.017), greater spleen size, (p= 0.01), a need for splenectomy (p= 0.017), a need for chemotherapy (p =0.009), and a shorter median survival time (17 years vs 25 years; log rank p value 0.02). Since the median disease duration of the two groups did not differ, the underlying genetic abnormalities must be time independent. Analysis of the core gene set revealed a “stromal gene signature” consisting of 16 matricellular and collagen genes, which, when subjected to unsupervised hierarchical clustering, not only led to the same group distinctions but resolved the genetic heterogeneity of the larger group. Remarkably, the stromal genes were down regulated in the more aggressive group and up regulated in the more indolent one. To determine the overall genetic differences between these two clinically distinct groups, we used Gene Set Enrichment Analysis to identify the top 50 up or down regulated genes in each. Among the notable genes down regulated in the indolent group were BAALC, DACH1, CD44, DDX17 and VIM, and in the aggressive group, FOXO1, IRF8, ABCD1, HOXA5, SOX4 and CRIM1. In the indolent group, LSM1, FADS2, CDKN1A, E2F1, MAZ and FHL2 were up regulated. FHL2 was also the only top 50 up regulated gene shared in common with the aggressive group, and one of the top two most up regulated genes in the patient population as a whole, LSM1 being the other, emphasizing the importance of these genes in disease pathogenesis. Equally important, in the aggressive group, there was an up regulated 17 gene signature involving DNA replication unique to it alone (p = 0.032). These data solidify the contention that despite the commonality of JAK2 V617F, PV is not a monolithic illness, that prognosis is independent of disease duration, provide insight into the pathogenesis of PV and offer the possibility of genetically identifying those patients most at risk of disease-associated morbidity and mortality when they can still benefit from therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Abstract 4892: Epigenetic reprogramming associated with melanocyte malignant transformation induced by sustained stress condition

Abstract Tissue-specific epigenetic patterns are fundamental for an appropriate gene expression control and aberrant epigenetic patterns are invariably seen in cancer, resulting both in oncogene activation and loss of tumor suppressor genes. These genes may have their expression altered by modifications in the promoter epigenetic status and also by epigenetic regulation of miRNAs which may control the expression of the same genes. It has been strongly suggested that epigenetic aberrant patterns could be established early during the carcinogenesis. In this way, epigenetic alterations observed during specific steps of malignant transformation and progression may be useful as tumor detection, diagnosis and prognosis markers. Melanoma is one of the most deadly human cancers, thus urgency needs to exist for the better understand of the regulatory pathways involved in melanocyte malignant transformation and melanoma progression. Results from our laboratory showed that transformed phenotype may be achieved by submitting non-tumorigenic melanocytes (murine melan-a lineage) to sequential cycles of anchorage blockade, which suggests that sustained cell adhesion changes may lead to intracellular alterations with important gene expression impact. This model represents distinct phases of melanoma genesis and seems to be suitable for studying early phases of melanocyte malignant transformation. Both DNA methyltransferases levels and histone marks were found changed throughout this process. In order to gain an improved understanding of the molecular targets of epigenetic machinery along melanoma genesis, we have compared gene expression profiles through oligonucleotide microarray technology (GeneChip® Mouse Genome 430 2.0 Array, Affymetrix Inc.) and miRNA expression profiles through Taqman Low Density Array (Applied Biosystems) of non-tumorigenic melanocytes (melan-a), melanocytes submitted to 4 deadhesion cycles (4C cells), non-metastatic (4C11-) and metastatic melanoma (4C11+), treated or not with DNA demethylating agent (5-Aza-2′-deaxycitidine). The expression of the selected genes was validated by qPCR and ONCOMINE Cancer Microarray Database analysis identified several of these genes differentially expressed in our murine model by altered epigenetic status as aberrantly expressed in human melanomas. This combined analysis reveals epigenetic signatures associated with early phases of melanocyte malignant transformation as well as melanoma progression and may be useful to define new biomarkers associated with different phases of melanoma genesis. Supported by FAPESP (06/61293-1) and CNPq (473139/2009). 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 4892.

Oligonucleotide Microarray and QRT-PCR Study of Adhesion Protein Gene Expression in Acute Coronary Syndrome Patients

Cardiovascular diseases, including acute coronary syndrome (ACS), are the leading cause of death among humans. Adhesion proteins, owing to their involvement in the initiation and progression of atherosclerotic lesions, contribute to the progression of coronary disease and ACS occurrence. Considering ambiguosity of results reported to date, we decided to conduct a preliminary investigation of adhesion protein gene expression in ACS patients as well as in healthy subjects by making use of oligonucleotide microarray technology. Analysis of eight microarrays revealed ten upregulated genes differentiating between the two groups: intercellular adhesion molecule-2, platelet/endothelial cell adhesion molecule-1, zyxin, integrin-linked kinase, calcium and integrin binding protein-1 (calmyrin), integrin beta 2, integrin beta 3 (ITGB3), integrin beta 7, integrin alpha 2b, and selectin P ligand. The expression of ITGB3 was found to have been downregulated.

An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

BackgroundAlthough oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches.ResultsIn this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA) will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay.ConclusionsBy integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.

Design of 16 S rRNA-based Oligonucleotide Array Using Group-specific Non-unique Probes in Large Scale Bacteria Detection*

With thousands of sequenced 16 S rRNA genes available, and advancements in oligonucleotide microarray technology, the detection of microorganisms in microbial communities consisting of hundreds of species may be possible. The existing algorithms developed for sequence-specific probe design are not suitable for applications in large-scale bacteria detection due to the lack of coverage, flexibility and efficiency. Many other strategies developed for group-specific probe design focus on how to find a unique group-specific probe that can specifically detect all target sequences of a group. Unique group-specific probe for each group can not always be found. Hence, it is necessary to design non-unique probes. Each probe can specifically detect target sequences of a different subgroup. Combination of multiple probes can achieve higher coverage. However, it is a time-consuming task to evaluate all possible combinations. A feasible algorithm using relative entropy and genetic algorithm (GA) to design group-specific non-unique probes was presented.

Analysis of Gene Expression in Mouse Spinal Cord-derived Neural Precursor Cells During Neuronal Differentiation

The differentiation of neural precursor cells (NPCs) into neurons and astrocytes is a process that is tightly controlled by complicated and ill-defined gene networks. To extend our knowledge to gene networks, we performed a temporal analysis of gene expression during the differentiation (2, 4, and 8 days) of spinal cord-derived NPCs using oligonucleotide microarray technology. Out of 32,996 genes analyzed, 1878 exhibited significant changes in expression level (fold change>2, p

Oligonucleotide Microarray Technology and its Application to Mycobacterium avium subsp. paratuberculosis Research: A Review

Microarrays represent a modern powerful technology, which have potential applications in many areas of biological research and provide new insights into the genomics and transcriptomics of living systems. The aim of this review is to describe the application of microarray technology for Mycobacterium avium subsp. paratuberculosis (MAP) research. The main focus points include a summary of results obtained for MAP using microarrays, examination of the fields of MAP research which are currently being investigated and possible areas of future research. This article is divided into two parts according to the type of nucleic acid used for array hybridisation. Articles related to MAP research using microarray technology are then divided according to the field of study, such as comparative genome analysis, diagnostics, expression or environmental studies.

Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human epithelial cancers and is a determinant of malignant cellular behavior in pancreatic adenocarcinoma cells. Moreover, it is expressed in tumor endothelium and its activation promotes angiogenesis. To better clarify the therapeutic potential of monoclonal antibodies (mAbs) directed to the EphA2 receptor, we generated a large number of mAbs by differential screening of phage-Ab libraries by oligonucleotide microarray technology and implemented a strategy for the rapid identification of antibodies with the desired properties. We selected two high-affinity and highly specific EphA2 monoclonal antibodies with different in vitro properties on the human pancreatic tumor cell line MiaPaCa2. One is a potent EphA2-agonistic antibody, IgG25, that promotes receptor endocytosis and subsequent degradation, and the second is a ligand antagonist, IgG28, that blocks the binding to ephrin A1 and is cross-reactive with the mouse EphA2 receptor. We measured the effect of antibody treatment on the growth of MiaPaCa2 cells orthotopically transplanted in nude mice. Both IgG25 and IgG28 had strong antitumor and antimetastatic efficacy. In vivo treatment with IgG25 determined the reduction of the EphA2 protein levels in the tumor and the phosphorylation of FAK on Tyr576 while administration of IgG28 caused a decrease in tumor vascularization as measured by immunohistochemical analysis of CD31 in tumor sections. These data show that in a pancreatic cancer model comparable therapeutic efficacy is obtained either by promoting receptor degradation or by blocking receptor activation.

A MCMC strategy for group-specific 16S rRNA probe design

Revealing biodiversity in microbial communities is essential in metagenomics researches. With thousands of sequenced 16S rRNA gene available, and advancements in oligonucleotide microarray technology, the detection of microor-ganisms in microbial communities consisting of hundreds of species may be possible. Many of the existing strategies developed for oligonucleotide probe design are dependent on the result of global multiple sequences alignment, which is a time-consuming task. We present a novel program named OligoSampling that uses MCMC method to design group-specific oli-gonucleotide probes. The probes generated by OligoSampling are group specific with weak crosshybridization potentials. Furthermore a high coverage of target sequences can be obtained. Our method does not need to globally align target sequences. Locally aligning target sequences iteratively based on a Gibbs sampling strategy has the same effect as globally aligning sequences in the process of seeking group-specific probes. OligoSampling provides more flexibility and speed than other software programs based on global multiple sequences alignment.

Differential Screening of Phage-Ab Libraries by Oligonucleotide Microarray Technology

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.

Computational analysis of small RNA cloning data

Cloning and sequencing is the method of choice for small regulatory RNA identification. Using deep sequencing technologies one can now obtain up to a billion nucleotides—and tens of millions of small RNAs—from a single library. Careful computational analyses of such libraries enabled the discovery of miRNAs, rasiRNAs, piRNAs, and 21U RNAs. Given the large number of sequences that can be obtained from each individual sample, deep sequencing may soon become an alternative to oligonucleotide microarray technology for mRNA expression profiling. In this report we present the methods that we developed for the annotation and expression profiling of small RNAs obtained through large-scale sequencing. These include a fast algorithm for finding nearly perfect matches of small RNAs in sequence databases, a web-accessible software system for the annotation of small RNA libraries, and a Bayesian method for comparing small RNA expression across samples.

Gene profiling of growth factor independence 1B gene (Gfi-1B) in leukemic cells

To investigate the molecular effects of growth factor independence 1B (Gfi-1B), a transcription factor essential for the development of hematopoietic cells and differentiation of erythroid and megakaryocytic lineages, the naturally Gfi-1B overexpressing cell line K562 was cultured in the presence of Gfi-1B target-specific small interfering RNA (siRNA). SiRNA treatment significantly knocked down Gfi-1B expression with an efficiency of nearly 90%. Analysis of the siRNA silencing protocol by colony-forming units ensured that it was not cytotoxic. Samples from Gfi-1B overexpressing cells and cells with knocked-down Gfi-1B were analyzed by oligonucleotide microarray technology and based upon rigorous statistical analysis of the data; relevant genes were chosen for confirmation by reserve transcriptase-polymerase chain reaction, including MYC/MYCBP and CDKN1A. Interestingly, transcripts within components of the signalling cascade of immune cells (PLD1, LAMP1, HSP90, IL6ST), of the tyrosine kinase pathway (TPR, RAC3) and of the transcription factors (RAC3, CEP290, JEM-1, ATR, MYC, SMC3, RARA, RBBP6) were found to be differentially expressed in Gfi-1B overexpressing cells compared to controls. Individual genes such as ZDHHC17, DMXL1, ZNF292 were found to be upregulated in Gfi-1B overexpressing cells. In addition, down-regulated transcripts showed cell signaling transcripts for several chemokine gene members including GNAL, CXCL5, GNL3L, GPR65, TMEM30, BCL11B and transcription factors (GTF2H3, ATXN3). In conclusion, several essential cell signalling factors, as well as transcriptional and post-translational regulation genes were differentially expressed in cells that overexpressed Gfi-1B compared to control cells with knocked-down Gfi-1B. Our data indicate that Gfi-1B signalling is important for commitment and maturation of hematopoietic cell populations.

Oligonucleotide microarrays: widely applied poorly understood

Microarray technology, which has been around for almost two decades, now provides an indispensable service to the biomedical research community. Soaring demand for high-throughput screening of genes potentially associated with cancer and other diseases, as well as the increased need for identifying microorganisms, have substantially opened up the application of this technology to many fields of science, including new ones such as array-based comparisons of whole genomes. Yet, despite this significant progress, the fundamental understanding of the pillars of this technology, have been largely unexplored, in particular for oligonucleotide-based microarrays. In fact, most of the current approaches for the design of microarrays are based on 'common-sense' parameters, such as guanine-cytosine content, secondary structure, melting temperature or possibility of minimizing the effects of nonspecific hybridization. However, recent experiments suggest that these are inadequate. Here we discuss these results, which challenge the basic principles and assumptions of oligonucleotide microarray technology. It is clear that more systematic physicochemical studies will be required to better understand the hybridization and dissociation behaviour of oligonucleotides.

Transcriptome profiling of the small intestinal epithelium in germfree versus conventional piglets

BackgroundTo gain insight into host-microbe interactions in a piglet model, a functional genomics approach was used to address the working hypothesis that transcriptionally regulated genes associated with promoting epithelial barrier function are activated as a defensive response to the intestinal microbiota. Cesarean-derived germfree (GF) newborn piglets were colonized with adult swine feces, and villus and crypt epithelial cell transcriptomes from colonized and GF neonatal piglets were compared using laser-capture microdissection and high-density porcine oligonucleotide microarray technology.ResultsConsistent with our hypothesis, resident microbiota induced the expression of genes contributing to intestinal epithelial cell turnover, mucus biosynthesis, and priming of the immune system. Furthermore, differential expression of genes associated with antigen presentation (pan SLA class I, B2M, TAP1 and TAPBP) demonstrated that microbiota induced immune responses using a distinct regulatory mechanism common for these genes. Specifically, gene network analysis revealed that microbial colonization activated both type I (IFNAR) and type II (IFNGR) interferon receptor mediated signaling cascades leading to enhanced expression of signal transducer and activator of transcription 1 (STAT1), STAT2 and IFN regulatory factor 7 (IRF7) transcription factors and the induction of IFN-inducible genes as a reflection of intestinal epithelial inflammation. In addition, activated RNA expression of NF-kappa-B inhibitor alpha (NFκBIA; a.k.a I-kappa-B-alpha, IKBα) and toll interacting protein (TOLLIP), both inhibitors of inflammation, along with downregulated expression of the immunoregulatory transcription factor GATA binding protein-1 (GATA1) is consistent with the maintenance of intestinal homeostasis.ConclusionThis study supports the concept that the intestinal epithelium has evolved to maintain a physiological state of inflammation with respect to continuous microbial exposure, which serves to sustain a tight intestinal barrier while preventing overt inflammatory responses that would compromise barrier function.

Gene expression profiling reveals two separate mechanisms regulating apoptosis in rectal carcinomas in vivo

The level of apoptosis in rectal carcinomas of patients treated by surgery only predicts local failure; patients with intrinsically high-apoptotic tumors develop less local recurrences than patients with low levels of apoptosis. To identify genes involved in this intrinsic apoptotic process in vivo, 47 rectal tumors with known apoptotic phenotype (24 low- and 23 high-apoptotic) were analyzed by oligonucleotide microarray technology. We identified several genes differentially expressed between low- and high-apoptotic tumors. Unsupervised clustering of the tumors based on expression levels of these genes separated the low-apoptotic from the high-apoptotic tumors, indicating a gene expression-dependent regulation. In addition, this clustering revealed two subgroups of high-apoptotic tumors. One high-apoptotic subgroup showed subtle differences in mRNA and protein expression of the known apoptotic regulators BAX, cIAP2 and ARC compared to the low-apoptotic tumors. The other subgroup of high-apoptotic tumors showed high expression of immune-related genes; predominantly HLA class II and chemokines, but also HLA class I and interferon-inducible genes were highly expressed. Immunohistochemistry revealed HLA-DR expression in epithelial tumor cells in 70% of these high-apoptotic tumors. The expression data suggest that high levels of apoptosis in rectal carcinoma patients can be the result of either slightly altered expression of known pro- and anti-apoptotic genes or high expression of immune-related genes.Electronic supplementary materialThe online version of this article (doi: 10.1007/s10495-007-0088-2) contains supplementary material, which is available to authorized users.

Investigation of the Molecular Mechanisms Preceding PDE4 Inhibitor-Induced Vasculopathy in Rats: Tissue Inhibitor of Metalloproteinase 1, a Potential Predictive Biomarker

Phosphodiesterase (PDE) 4 inhibitors are a class of drugs that can provide novel therapies for asthma and chronic obstructive pulmonary disease. Their development is frequently hampered by the induction of vascular toxicity in rat mesenteric tissue during preclinical studies. Whereas these vascular lesions in rats have been well characterized histologically, little is known about their pathogenesis and in turn, sensitive and specific biomarkers for preclinical and clinical monitoring do not exist. In order to investigate the early molecular mechanisms underlying vascular injury, time-course studies were performed by treating rats for 2-24 h with high doses of the PDE4 inhibitor CI-1044. Transcriptomics analyses in mesenteric tissue were performed using oligonucleotide microarray and real-time RT-PCR technologies and compared to histopathological observations. In addition, protein measurements were performed in serum samples to identify soluble biomarkers of vascular injury. Our results indicate that molecular alterations preceded the histological observations of inflammatory and necrotic lesions in mesenteric arteries. Some gene expression changes suggest that the development of the lesions could follow a primary modulation of the vascular tone in response to the pharmacological effect of the compound. Activation of genes coding for pro- and antioxidant enzymes, cytokines, adhesion molecules, and tissue inhibitor of metalloproteinase 1 (TIMP-1) indicates that biomechanical stimuli may contribute to vascular oxidant stress, inflammation, and tissue remodeling. TIMP-1 appeared to be an early and sensitive predictive biomarker of the inflammatory and the tissue remodeling components of PDE4 inhibitor-induced vascular injury.

Microarray analysis to reveal genes involved in colon carcinogenesis

Sporadic colon cancer is a major cause of death throughout the world. Multistage development of the disease has been associated with remarkable genetic events, mainly at the level of oncogenes and oncosuppressor genes, most notably APC (adenomatous polyposis coli), ras and p53. Despite all of these efforts, the development of a sensitive and convenient diagnostic system for detecting colorectal cancers at the early stage is still in progress. In recent years, cDNA and oligonucleotide microarray technologies have made the analysis of gene expression profiles of colorectal tumours at the genomic level possible and have identified signatures of gene expression associated with pre-cancerous phenotypes, cancers of the early stage and/or metastatic cancer. The contribution of this powerful technology in identification of novel important genes for prognosis, diagnosis and therapy of sporadic colorectal will be discussed.

Molecular Factors and Biochemical Pathways Induced by Febrile Temperature in Intraerythrocytic Plasmodium falciparum Parasites

Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37 degrees C and 41 degrees C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as "hypothetical." We find that cultivation of P. falciparum parasites at 41 degrees C leads to parasite death in a time-dependent manner. The presence of the "crisis forms" and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.

Gene Array Profiles of Alcohol and Aldehyde Metabolizing Enzymes in Brains of C57BL/6 and DBA/2 Mice

Differences in ethanol metabolizing enzymes expressed in brain have been suggested to contribute to the significant differences in ethanol (alcohol) preference between inbred C57BL/6 and DBA/2 mouse strains. We have utilized 2 different platforms of oligonucleotide microarray technology (CodeLink UniSet I BioArray from G.E. Healthcare and MG U74A v2.0 from Affymetrix) to simultaneously assess expression of alcohol and acetaldehyde metabolizing enzymes in the whole brain of naïve (no exposure to alcohol) C57BL/6 and DBA/2 mice. There were no significant differences between the 2 strains of mice in gene expression intensity for alcohol dehydrogenases (ADH), catalase, and a number of the cytochrome P450 family of genes, which can be involved in ethanol catabolism. However, significantly higher expression of mRNA for aldehyde dehydrogenase 2 (ALDH2), an isoform mainly responsible for the catabolism of acetaldehyde, was observed in whole brains of DBA/2 mice with both platforms. Aldehyde dehydrogenase 2 protein was also higher in DBA/2 brain. Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA was found to be higher in brains of DBA/2 mice, when measured with the CodeLink platform, but not when measured with Affymetrix arrays or quantitative reverse transcriptase-real-time polymerase chain reaction (qRT-PCR). The ALDH1A1 protein, however, reflected the results obtained with the CodeLink arrays and was higher in DBA/2 brain, compared with brains of C57BL/6 mice. In contrast, the expression intensity for the aldehyde dehydrogenase 7A1 (ALDH7A1) mRNA and protein was significantly higher in C57BL/6 mice than DBA/2 mice. These expression differences are consistent with more rapid metabolism of acetaldehyde in brains of DBA/2 mice. The use of 2 different microarray platforms provides important cross-validation of many results, and some discrepancies can be resolved with qRT-PCR and immunoblotting. The expression differences that were validated may affect alcohol/aldehyde metabolism in brain and/or alcohol preference in the 2 strains of mice.

Global Gene Expression Patterns in Mouse Wolffian Duct Development

We identified genes responsible for terminal differentiation of the mouse ureter. We isolated the wolffian ducts of Black Swiss mice from embryonic days 12.5 to 14.5. These ducts were processed for total RNA extraction. RNA was amplified and converted to biotinylated cDNA, which was hybridized to Affymetrix(R) mouse 430 microarray GeneChips. Data were analyzed using Affymetrix software provided by the core facility at our institution. Data were confirmed by semiquantitative polymerase chain reaction and immunohistochemistry. Of 42,000 evaluable transcripts 412 were up-regulated and 133 were down-regulated more than 1.5-fold between embryonic days 12.5 and 14.5. Of up-regulated genes 17 were up-regulated more than 3-fold. Of these genes 5 were selected for further study and they were identified as having roles in cell growth/regulation in general. Increased expression of Foxa1 was seen at embryonic day 14.5 in the distal ureter and urogenital sinus. To our knowledge this is the first report of differential gene expression patterns in the developing mouse wolffian duct. Our results diverge from expression patterns reported in the kidney. This information will enable future comparisons between WT and transgenic mice with an abnormal phenotype.