Expression and Characterization of a Novel Antimicrobial Protein from Loblolly Pine ('Pinus taeda' L.)

Department of Natural Resource Ecology and Managemen

Gene silencing in mammalian cells by PCR-based short hairpin RNA

AbstractRNA interference (RNAi) provides a powerful tool to silence genes in a sequence-specific manner in a variety of systems. However, not all sequences are effective in the RNAi-mediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)-based RNAi strategy for a quick screening of small interfering RNA (siRNA) efficiency. This method utilized a two-step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in specific depressions of exogenous (luciferase, green fluorescent protein and β-galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning

Characterization and Functional Study of Ionotropic Gaba Receptors in Alveolar Epithelial Cells

We began this project from a target gene GABRP, identified from DNA microarray. We firstly examined the expression patterns of GABRP in various culture conditions by using real-time PCR, western blot and immunostaining. Then we isolated lung tissue or type II cells from fetal, neonate, and adult rats, as well as hyperoxia-exposed rats. By using real-time PCR, we demonstrated the differential expression of all the ionotropic GABA receptor subunits during fetal lung development and after hyperoxia exposure. We further performed the functional study on fresh or cultured type II cells and rats, by using real-time PCR, western blot, immunostaining, biotinylation, immunproecipitation, isotope, RNA intereference, and lung fluid clearance assays. (1)The GABRP expression was regulated by culture conditions and closely associated with the type II cell-phenotype. (2)Among 19 known GABA receptor subunits, 17 were expressed in rat lungs and type II cells throughout the fetal and adult stages. Those subunits were dVeterinary Pathobiolog

Identification of rat lung – prominent genes by a parallel DNA microarray hybridization

BACKGROUND: The comparison of organ transcriptomes is an important strategy for understanding gene functions. In the present study, we attempted to identify lung-prominent genes by comparing the normal transcriptomes of rat lung, heart, kidney, liver, spleen, and brain. To increase the efficiency and reproducibility, we first developed a novel parallel hybridization system, in which 6 samples could be hybridized onto a single slide at the same time. RESULTS: We identified the genes prominently expressed in the lung (147) or co-expressed in lung-heart (23), lung-liver (37), lung-spleen (203), and lung-kidney (98). The known functions of the lung-prominent genes mainly fell into 5 categories: ligand binding, signal transducer, cell communication, development, and metabolism. Real-time PCR confirmed 13 lung-prominent genes, including 5 genes that have not been investigated in the lung, vitamin D-dependent calcium binding protein (Calb3), mitogen activated protein kinase 13 (Mapk13), solute carrier family 29 transporters, member 1 (Slc29a1), corticotropin releasing hormone receptor (Crhr1), and lipocalin 2 (Lcn2). CONCLUSION: The lung-prominent genes identified in this study may provide an important clue for further investigation of pulmonary functions

Correlations and the Cross Section of Exclusive (e,e′pe,e'p) Reactions for 16^{16}O

The reduced cross section for exclusive ($e,e'p$) reactions has been studied in DWIA for the example of the nucleus $^{16}$O using a spectral function containing effects of correlations. The spectral function is evaluated directly for the finite nucleus starting from a realistic nucleon-nucleon interaction within the framework of the Green's function approach. The emphasis is focused on the correlations induced by excitation modes at low energies described within a model-space of shell-model configurations including states up to the $sdg$ shell. Cross sections for the $p$-wave quasi-hole transitions at low missing energies are presented and compared with the most recent experimental data. In the case of the so-called perpendicular kinematics the reduced cross section derived in DWIA shows an enhancement at high missing momenta as compared to the PWIA result. Furthermore the cross sections for the $s$- and $d$-wave quasi-hole transitions are presented and compared to available data at low missing momenta. Also in these cases, which cannot be described in a model without correlations, a good agreement with the experiment is obtained.Comment: 12 pages, LaTeX, 4 figures include

Role of GABA Receptors in Fetal Lung Development in Rats

Fluid accumulation is critical for lung distension and normal development. The multi-subunit γ-amino butyric acid type A receptors (GABAA) mainly act by mediating chloride ion (Cl−) fluxes. Since fetal lung actively secretes Cl−-rich fluid, we investigated the role of GABAA receptors in fetal lung development. The physiological ligand, GABA, and its synthesizing enzyme, glutamic acid decarboxylase, were predominantly localized to saccular epithelium. To examine the effect of activating GABAA receptors in fetal lung development in vivo, timed-pregnant rats of day 18 gestation underwent an in utero surgery for the administration of GABAA receptor modulators into the fetuses. The fetal lungs were isolated on day 21 of gestation and analyzed for changes in fetal lung development. Fetuses injected with GABA had a significantly higher body weight and lung weight when compared to phosphate-buffered saline (control)-injected fetuses. GABA-injected fetal lungs had a higher number of saccules than the control. GABA increased the number of alveolar epithelial type II cells as indicated by surfactant protein C-positive cells. However, GABA decreased the number of α-smooth muscle actin-positive myofibroblasts, but did not affect the number of Clara cells or alveolar type I cells. GABA-mediated effects were blocked by the GABAA receptor antagonist, bicuculline. GABA also increased cell proliferation and Cl− efflux in fetal distal lung epithelial cells. In conclusion, our results indicate that GABAA receptors accelerate fetal lung development, likely through an enhanced cell proliferation and/or fluid secretion

Arsenic-induced changes in the gene expression of lung epithelial L2 cells: implications in carcinogenesis

Background: Arsenic is a carcinogen that is known to induce cell transformation and tumor formation. Although studies have been performed to examine the modulation of signaling molecules caused by arsenic exposure, the molecular mechanisms by which arsenic causes cancer are still unclear. We hypothesized that arsenic alters gene expression leading to carcinogenesis in the lung.Results: In this study, we examined global gene expression in response to 0.75 uM arsenic treatment for 1 - 7 days in a rat lung epithelial cell line (L2) using an in-house 10 k rat DNA microarray. One hundred thirty one genes were identified using the one-class statistical analysis of microarray (SAM) test. Of them, 33 genes had a fold change of >/= 2 between at least two time points. These genes were then clustered into 5 groups using K-means cluster analysis based on their expression patterns. Seven selected genes, all associated with cancer, were confirmed by real-time PCR. These genes have functions directly or indirectly related to metabolism, glycolysis, cell proliferation and differentiation, and regulation of transcription.Conclusion: Our findings provide important insight for the future studies of arsenic-mediated lung cancer.Peer reviewedPhysiological Science

Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-211The nucleotide sequence reported herein has been deposited in the DDBJ, EMBL and GenBank databanks under the accession number AB034697.

AbstractA mouse cDNA that encodes a DNA-binding protein was identified by yeast two-hybrid screening, using activating transcription factor-2 (ATF-2) as the bait. The protein contained a bZIP (basic amino acid-leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2). Mouse JDP2 interacted with ATF-2 both in vitro and in vivo via its bZIP domain. It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues. Furthermore, mouse JDP2 bound to the cAMP-response element (CRE) as a homodimer or as a heterodimer with ATF-2, and repressed CRE-dependent transcription that was mediated by ATF-2. JDP2 was identified as a novel repressor protein that affects ATF-2-mediated transcription

Meson exchange currents in electromagnetic one-nucleon emission

The role of meson exchange currents (MEC) in electron- and photon-induced one-nucleon emission processes is studied in a nonrelativistic model including correlations and final state interactions. The nuclear current is the sum of a one-body and of a two-body part. The two-body current includes pion seagull, pion-in-flight and the isobar current contributions. Numerical results are presented for the exclusive 16O(e,e'p)15N and 16O(\gamma,p)15N reactions. MEC effects are in general rather small in (e,e'p), while in (\gamma,p) they are always large and important to obtain a consistent description of (e,e'p) and (\gamma,p) data, with the same spectroscopic factors. The calculated (\gamma,p) cross sections are sensitive to short-range correlations at high values of the recoil momentum, where MEC effects are larger and overwhelm the contribution of correlations.Comment: 9 pages, 6 figure

Overlap functions in correlation methods and quasifree nucleon knockout from 16^{16}O

The cross sections of the ($e,e'N$) and ($\gamma,p$) reactions on $^{16}$O are calculated, for the transitions to the $1/2^{-}$ ground state and the first $3/2^{-}$ excited state of the residual nucleus, using single-particle overlap functions obtained on the basis of one-body density matrices within different correlation methods. The electron-induced one-nucleon knockout reaction is treated within a nonrelativistic DWIA framework. The theoretical treatment of the ($\gamma,p$) reaction includes both contributions of the direct knockout mechanism and of meson-exchange currents. The results are sensitive to details of the different overlap functions. The consistent analysis of the reaction cross sections and the comparison with the experimental data make it possible to study the nucleon--nucleon correlation effects.Comment: 26 pages, LaTeX, 5 Postscript figures, submitted to PR