Stephen Tsoi

Identification of Immune-Relevant Genes from Atlantic Salmon Using Suppression Subtractive Hybridization

In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314–BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription–polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.

Use of human cDNA microarrays for identification of differentially expressed genes in Atlantic salmon liver during Aeromonas salmonicida infection.

Commercially available human complementary DNA microarrays were used to compare differential expression in the livers of Atlantic salmon (Salmo salar) infected with Aeromonas salmonicida and of healthy fish. Complementary DNA probes were prepared from total RNA isolated from livers of control salmon and infected salmon by reverse transcription in the presence of 33P-dCTP and independently hybridized to human GENEFILTERS GF211 microarrays. Of the 4131 known genes on the microarray, 241 spots gave clearly detectable signals using labeled RNA from the control salmon liver. Of these, 4 spots were consistently found to have a greater than 2-fold increase in infected salmon compared with controls when using the same pair of filters to generate hybridization data from triplicates. These up-regulated genes were ADP/ATP translocase (AAT2), Na+/K+ ATPase, acyloxyacyl hydrolase (AOAH), and platelet-derived growth factor (PDFG-A). A BlastN search revealed an AAT2 homolog from Atlantic salmon, and a reverse transcriptase polymerase chain reaction assay using primers based on this sequence confirmed its up-regulation (approx. 1.8-fold) during early infection. This work demonstrates the feasibility of using human microarrays to facilitate the discovery of differentially expressed genes in Atlantic salmon, for which no homologous microarrays are available.

cDNA microarray analysis of gene expression profiles in human placenta: up-regulation of the transcript encoding muscle subunit of glycogen phosphorylase in preeclampsia.

Objective: Third-trimester human placentas from normal and preeclamptic pregnancies were evaluated for possible changes in gene expression patterns by microarray analysis. Methods: Placentas from four normal pregnancies and four pregnancies complicated by preeclampsia were studied. In a preliminary effort to identify possible differences between the two groups, complementary DNA (cDNA) probes were prepared from pooled total RNA by reverse transcription in the presence of 33-P-dCTP. After hybridization to human GeneFilter cDNA microarrays (GF211; Research Genetics, Huntsville, AL), 319 positive signals were detected above background out of a possible 4131 human cDNAs spotted on the filters. Results: Ten most highly expressed mRNA species, ten most up-regulated, and ten most down-regulated genes in placentas from both groups of patients were identified for future studies. Of the 319 positive hybridizations, one transcript was clearly evaluated in preeclamptic pregnancy. This cDNA encodes the muscle subtype of glycogen phosphorylase (GP-M) and was increased more than 2.8-fold (P < .05) in the preeclamptic placentas. In contrast, cDNA for glycogen synthase (muscle and liver isoforms) was not significantly increased, being near the limits of detection. The preeclampsia-induced increase of placental GP-M mRNA expression (approximately 3.5-fold) was confirmed by northern blot analysis. Conclusion: We conclude that microarray analysis can detect trends in mRNA and gene expression in placentas from normal and preeclamptic pregnancies and may be further studied in a more targeted fashion. We found that placental GP-M mRNA level is up-regulated in preeclampsia, which is consistent with previous reports of increased glycogen phosphorylase activity, and we suggest that it may be largely regulated at the level of transcription. Further studies may determine whether such up-regulation might be a response to hypoxia.

Markers of oxidative stress in placental villi exposed to ethanol.

Objective: Ethanol exposure during pregnancy may result in fetal alcohol syndrome (FAS). The mechanism by which this occurs is unknown. Recent studies in several organ systems, including the placenta, suggest that oxidative stress is involved. In this study we investigated the presence and levels of three oxidative stress markers in placental villous tissue exposed to ethanol. Methods: Villous tissues from normal placentas were perfused with Dulbecos modified Eagles medium (DMEM) with HEPES buffer, sodium bicarbonate, and glucose at pH 7.4. After stabilization, 100 mM ethanol was added to the perfusate. After 2 hours of perfusion, the tissue was removed, fixed and stained for nitrotyrosine, 4-hydroxy-2-nonenal (4HNE) and 8-hydroxyguanosine (8-OHDG). Staining within the trophoblasts was quantified with densitometry. Results: Nitrotyrosine and 4HNE immunostaining was seen in the trophoblasts. 4HNE was also seen in the stroma. In contrast, 8-OHDG was seen only in the stroma and endothelial cells in the fetal circulation. Ethanol exposure significantly increased nitrotyrosine levels in the trophoblasts beyond levels in the control tissue. Nitrotyrosine and 8-OHDG levels were also increased in stroma. Conclusion: Within the placental villi, markers of oxidative stress are present in the trophoblasts and stroma after a short period of ethanol exposure. There is an increase in oxidative stress, primarily involving the nitric oxide pathway, in the trophoblasts as well as DNA damage in the stroma. Lipid peroxidation is not acutely changed in our 2-hour exposure window.

Expressed Sequence Tags — A Snapshot of the Fish Genome

Expressed Sequence Tags (ESTs) represent the transcribed portion of an organism’s genome and as such provide a snapshot of the expression of that genome at a particular time. Since non-coding DNA such as introns, intergenic spacers, repeat elements, etc. which comprise the bulk of the genome, is not sequenced, ESTs provide an information-rich source of data that is relatively easily interpreted and managed. EST surveys of non-normalized libraries can also provide an estimate of the abundance of particular transcripts. At the Institute for Marine Biosciences, we have optimized inexpensive template preparation and automated sequencing methodologies to produce large numbers of ESTs in an efficient and cost-effective manner. Tissue-specific EST libraries for winter flounder, our model system for studying flatfish biology, have been generated both for gene discovery purposes and for comparative purposes with other organisms. We have also produced an EST database from liver, head kidney and spleen of Atlantic salmon that will aid in genome mapping, identification of duplicated genes and DNA microarray construction, as well as providing information on genes critical to the fish immune system. An EST approach is also being used with subtractive libraries constructed from salmon infected with the pathogen Aeromonas salmonicida in order to focus on transcripts that are differentially regulated in response to disease.

Molecular Cloning of Ovine Endothelial Nitric Oxide Synthase and Expression in COS-7 Cells:

While studies of human and bovine endothelial nitric oxide synthase (eNOS) demonstrate activation by Ca2+ /calmodulin, recent progress demonstrates that eNOS phosphorylation can alter sensitivity to intracellular free calcium ([Ca2+]). The sheep, however, is widely used as a model for cardiovascular adaptation to pregnancy and ovine uterine artery endothelial cell (UAEC) eNOS undergoes pregnancy-specific (P) enhancement of activity associated with increased Ca2+ and protein kinase signaling in response to a number of agonists, including adenosine triphosphate (ATP). The degree of homology between the ovine and human full-length cDNAs was not previously known and yet is necessary to determine the validity in using an ovine model to study human physiology. The objectives of this study were to isolate and validate the clone of ovine eNOS cDNA and investigate ovine eNOS activation when expressed in COS-7 cells. The ovine eNOS cDNA has high homology to published human and bovine sequences and shares identity with the bovine amino acid sequence. When ovine eNOS was transiently expressed in COS-7 cells (COS-7/oeNOS), A23187 increased specfic catalytic activity in a dose- and time-dependent manner. A23187-stimulated activation of eNOS was, however, also accompanied by phosphorylation of eNOS S1179 and dephosphorylation of T497, demonstrating that an increase in [Ca2+]i may not be the sole mechanism of activation. The physiologic relevance of this was further underscored by the finding that ATP dose dependently increased peak [Ca2+]i and eNOS activity in COS-7/oeNOS, but also increased eNOS p-S1179 and decreased p-T497. This finding was similar to those in ovine P-UAEC treated with the Ca2+-mobilizing agonist ATP, wherein activation of eNOS was again concomitant with a rise p-S1179 as well as a slight decrease in p-T497. In conclusion, we describe the full-length ovine eNOS cDNA sequence and show that both physiologic and nonphysiologic calcium-mobilizing agents, which activate ovine eNOS in COS7 and PUAEC, do so in association with changes in eNOS phosphorylation. Given this information we can now begin to dissect the relationship between Ca2+ elevation and specflc phosphorylation events in eNOS activation in the ovine model, and thereby gain insight into the possible basis for pregnancy-related dysfunction.

Differential gene expression profiling in HELLP syndrome placentas.

The aim of this study was to identify differentially expressed genes by suppression subtractive hybridization (SSH) in HELLP placentas. Two cDNA libraries were constructed; HSI (HELLP subtracted induced or upregulated) and HSS (HELLP subtracted suppressed or downregulated). Two hundred eighty-eight cDNA clones were sequenced; 37 were matched to GenBank entries and included genes in cell communication and organization, cellular processes, genetic information processing, and metabolic processes. A subgroup of 11 genes of interest was further selected for real-time quantitative polymerase chain reaction confirmation. Results showed no differences in expression of chosen upregulated genes between HELLP and non-HELLP placentas; 6 HELLP downregulated genes were significantly suppressed. Two genes related to production of secreted proteins, CTHRC1 and SERPINE2. SERPINE2 (PAI-1) is a soluble protease inhibitor and is a potential biomarker by Western blot analysis, and the protein is significantly decreased in HELLP placentas. SERPINE2 might be tested clinically in patients for early diagnosis of HELLP syndrome.