Biomedical subjects

Zhongming Chen

Publications and source records attributed to Zhongming Chen.

9 recordsLinked to original sources

PubMed2006

Gene expression profiling identifies regulatory pathways involved in the late stage of rat fetal lung development.

Fetal lung development is a complex biological process that involves temporal and spatial regulations of many genes. To understand the molecular mechanisms of this process, we investigated gene expression profiles of fetal lungs on gestational days 18, 19, 20, and 21, as well as newborn and adult rat lungs. For this analysis, we used an in-house rat DNA microarray containing 6,000 known genes and 4,000 expressed sequence tags (ESTs). Of these, 1,512 genes passed the statistical significance analysis of microarray (SAM) test; an at least twofold change was shown for 583 genes (402 known genes and 181 ESTs) between at least two time points. K-means cluster analysis revealed seven major expression patterns. In one of the clusters, gene expression increased from day 18 to day 20 and then decreased. In this cluster, which contained 10 known genes and 5 ESTs, 8 genes are associated with development. These genes can be integrated into regulatory pathways, including growth factors, plasma membrane receptors, adhesion molecules, intracellular signaling molecules, and transcription factors. Real-time PCR analysis of these 10 genes showed an 88% consistency with the microarray data. The mRNA of LIM homeodomain protein 3a (Lhx3), a transcription factor, was enriched in fetal type II cells. In contrast, pleiotrophin, a growth factor, had a much higher expression in fetal lung tissues than in fetal type II cells. Immunohistochemistry revealed that Lhx3 was localized in fetal lung epithelial cells and pleiotrophin in the mesenchymal cells adjacent to the developing epithelium and blood vessel. Using GenMAPP, we identified four regulatory pathways: transforming growth factor-beta signaling, inflammatory response, cell cycle, and G protein signaling. We also identified two metabolic pathways: glycolysis-gluconeogenesis and proteasome degradation. Our results may provide new insights into the complex regulatory pathways that control fetal lung development.

Animals↗

PubMed2006

Alveolar type I cells protect rat lung epithelium from oxidative injury.

The lung alveolar surface is covered by two morphologically and functionally distinct cells: alveolar epithelial cell types I and II (AEC I and II). The functions of AEC II, including surfactant release, cell differentiation and ion transport, have been extensively studied. However, relatively little is known regarding the physiological functions of AEC I. Global gene expression profiling of freshly isolated AEC I and II revealed that many genes were differentially expressed in AEC I. These genes have a diversity of functions, including cell defence. Nine out of 10 selected genes were verified by quantitative real-time PCR. Two genes, apolipoprotein E (Apo E) and transferrin, were further characterized and functionally studied. Immunohistochemistry indicated that both proteins were specifically localized in AEC I. Up-regulation of Apo E and transferrin was observed in hyperoxic lungs. Functionally, Apo E and transferrin play a protective role against oxidative stress in an animal model. Our studies suggest that AEC I is not just a simple barrier for gas exchange, but a functional cell that protects alveolar epithelium from injury.

Animals↗

PubMed2006

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation.

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.

Animals↗

PubMed2006

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.

Animals↗

PubMed2005

RealSpot: software validating results from DNA microarray data analysis with spot images.

The spot images from DNA microarray highly affect the discovery of biological knowledge from gene expression data. However, results from quality analysis, normalization, differential expression, and cluster analysis are rarely validated with spot images in current data analysis methods or software packages. We designed RealSpot, a software package, to validate the results by directly associating spot quality and data with spot images in a spreadsheet table. RealSpot splits hybridization images into individual spots stored in a spreadsheet table. It subsequently associates microarray data with spot images and performs data validation through the standard table operation such as sorting, searching, and editing. RealSpot has several built-in functions to facilitate data validation, including spot quality analysis, data organization, one-way ANOVA, gene ontology association, verification, import, and export. We used RealSpot to evaluate 77 slides (30,000 features each) from real hybridization experiments and to validate results from each step of data analysis. It took approximately 10 min to validate results of spot quality after initial evaluation and correct approximately 0.3% of falsely assigned qualities of 10,000 spots. We validated 1,641 of 2,110 differentially expressed genes identified by SAM analysis in approximately 1/2 h by comparing each gene with its respective spot image. Furthermore, we found that 6 of 48 genes in one cluster from k-mean clustering method showed inconsistent trends of spot images. RealSpot is efficient for validating microarray results and thus helpful for improving the reliability of the whole microarray experiment for experimentalists.

Algorithms↗

PubMed2005

[Determination of peroxides in environmental samples by high performance liquid chromatography with fluorescence detection].

A high performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of hydrogen peroxide and organic peroxides in environmental samples, and the method has been applied to peroxides detection in urban air and rain samples. The analytical sensitivity was improved. Post-column derivatization involved the oxidation of peroxides to a fluorescent dimer using p-hydroxyphenylacetic acid, a reaction catalyzed by hemin. The optimal excitation wavelength was 315 nm, while the emission wavelength was 400 nm. The temperature of the reaction coil was controlled at about 30 degrees C. Based on the ratio of signal to noise of 3, the detection limits were 4.0 x 10(-9) mol/L for hydroperoxide (H2O2), 4.1 x 10(-8) mol/L for methylhydroperoxide (MHP) and 6.7 x 10(-8) mol/L for ethylhydroperoxide (EHP) for aqueous samples. While the corresponding detection limits were 2.4 ng/m3 for H2O2, 35.2 ng/m3 for MHP, and 74.4 ng/m3 for EHP for air samples. Air samples were collected by Horibe cold trap at the temperature of about -90 degrees C. The results show that H2O2, hydroxymethyl hydroperoxide (HMHP), MHP, and EHP were the major peroxides in air, and that peroxyacetic acid (PAA) was occasionally detected. In rain samples, the two major peroxides were H2O2 and HMHP.

Chromatography, High Pressure Liquid↗

PubMed2004

Identification of two novel markers for alveolar epithelial type I and II cells.

Alveolar epithelial type I and type II cells (AEC I and II) are closely aligned in alveolar surface. There is much interest in the precise identification of AEC I and II in order to separate and evaluate functional and other properties of these two cells. This study aims to identify specific AEC I and AEC II cell markers by DNA microarray using the in vitro trans-differentiation of AEC II into AEC I-like cells as a model. Quantitative real-time PCR confirmed five AEC I genes: fibroblast growth factor receptor-activating protein 1, aquaporin 5, purinergic receptor P2X 7 (P2X7), interferon-induced protein, and Bcl2-associated protein, and one AEC II gene: gamma-aminobutyric acid receptor pi subunit (GABRP). Immunostaining on cultured cells and rat lung tissue indicated that GABRP and P2X7 proteins were specifically expressed in AEC II and AEC I, respectively. In situ hybridization of rat lung tissue confirmed the localization of GABRP mRNA in type II cells. P2X7 and GABRP identified in this study could be used as potential AEC I and AEC II markers for studying lung epithelial cell biology and monitoring lung injury.

Animals↗

PubMed2004

Isolation of highly pure alveolar epithelial type I and type II cells from rat lungs.

There are no ideal cell lines available for alveolar epithelial type I and II cells (AEC I and II) at the present time. The current methods for isolating AEC I and II give limited purities. Here, we reported improved and reproducible methods for the isolation of highly pure AEC I and II from rat lungs. AEC I and II were released from lung tissues using different concentrations of elastase digestion. Macrophages and leukocytes were removed by rat IgG 'panning' and anti-rat leukocyte common antigen antibodies. For AEC II isolation, polyclonal rabbit anti-T1alpha (an AEC I apical membrane protein) antibodies were used to remove AEC I contamination. For AEC I isolation, positive immunomagnetic selection by polyclonal anti-T1alpha antibodies was used. The purities of AEC I and II were 91 +/- 4 and 97 +/- 1%, respectively. The yield per rat was approximately 2 x 10(6) for AEC I and approximately 33 x 10(6) for AEC II. The viabilities of these cell preparations were more than 96%. The protocol for AEC II isolation is also suitable to obtain pure AEC II (93-95%) from hyperoxia-injured and recovering lungs. The purified AEC I and II can be used for gene expression profiling and functional studies. It also offers an important tool to the field of lung biology.

Animals↗

PubMed2003

Protein nitration in rat lungs during hyperoxia exposure: a possible role of myeloperoxidase.

Several studies have suggested that exposure to hyperoxia causes lung injury through increased generation of reactive oxygen and nitrogen species. The present study was aimed to investigate the effects of hyperoxia exposure on protein nitration in lungs. Rats were exposed to hyperoxia (>95%) for 48, 60, and 72 h. Histopathological analysis showed a dramatic change in the severity of lung injury in terms of edema and hemorrhage between 48- and 60-h exposure times. Western blot for nitrotyrosine showed that several proteins with molecular masses of 29-66 kDa were nitrated in hyperoxic lung tissues. Immunohistochemical analyses indicate nitrotyrosine staining of alveolar epithelial and interstitial regions. Furthermore, immunoprecipitation followed by Western blot revealed the nitration of surfactant protein A and t1alpha, proteins specific for alveolar epithelial type II and type I cells, respectively. The increased myeloperoxidase (MPO) activity and total nitrite levels in bronchoalveolar lavage and lung tissue homogenates were observed in hyperoxic lungs. Neutrophils and macrophages isolated from the hyperoxia-exposed rats, when cocultured with a rat lung epithelial L2 cell line, caused a significant protein nitration in L2 cells. Inclusion of nitrite further increased the protein nitration. These studies suggest that protein nitration during hyperoxia may be mediated in part by MPO generated from activated phagocytic cells, and such protein modifications may contribute to hyperoxia-mediated lung injury.

Amino Acid Sequence↗