Biomedical subjects

Grazyna Kwapiszewska

Publications and source records attributed to Grazyna Kwapiszewska.

8 recordsLinked to original sources

PubMed2006

Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia.

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O(2) between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

Animals↗

PubMed2006

Systematic comparison of the T7-IVT and SMART-based RNA preamplification techniques for DNA microarray experiments.

BACKGROUND: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification. METHODS: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. RESULTS: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. CONCLUSIONS: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.

3' Untranslated Regions↗

PubMed2006

Laser-microdissection for cell type- and compartment-specific analyses on genomic and proteomic level.

Morphological study and identification of cell differentiation within tissues are the basis for assessment of physiological and pathological processes. Applying molecular techniques, the analysis of tissue homogenates may lead to masking of genetic deviations or expression changes of an individual cell type by the bulk of surrounding cells. To overcome the tissue heterogeneity, cells have to be isolated selectively. Therefore, microdissection techniques were developed making retrieval of target cells simple, rapid and precise. Presenting an overview of our approaches, it is demonstrated that single cell isolation is often preconditional for the investigation of splicing isoform expression. To reveal gene regulation combining microdissection of few cells and real-time RT-PCR allows to determine relative mRNA expression in a cell type-specific manner, even after immunofluorescence staining of target cells. Combination with RNA preamplification techniques and micro arrays results in cell type- or compartment-specific expression profiles that especially differ from those of tissue homogenates when minor represented cell types are investigated. For proteomic biomarker screening, the application of mass spectrometry techniques (SELDI/MALDI TOF) turned out to be feasible in combination with microdissected minute amounts of tissues. However, further identification of marker peaks still needs a remarkable effort. Strategies to deal with this problem are presented. In consequence, the isolation of cells or cell types allows a more accurate investigation of complex tissues and gives deeper insight to regulation processes and crosstalk of the respective cells.

Animals↗

PubMed2006

Impact of TASK-1 in human pulmonary artery smooth muscle cells.

The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K+) conductances. Although studies suggest that background K+ currents carried by 2-pore domain K+ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K+ channels contribute to the K+ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K+ current performed by TASK-1 K+ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K+ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo.

Cells, Cultured↗

PubMed2005

Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension.

BACKGROUND: Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling. METHODS AND RESULTS: Analysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO2 = 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the growth mediators PDGF-B, TGF-beta, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription factors. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media. CONCLUSION: Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ.

Animals↗

PubMed2004

Identification of proteins in laser-microdissected small cell numbers by SELDI-TOF and Tandem MS.

BACKGROUND: Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques. RESULTS: Hemalaun stained mouse lung sections were used to isolate 500-2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChip Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks. CONCLUSION: Laser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization.

Animals↗

PubMed2003

Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection.

Circulating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detect and quantify platelet mRNA that excludes contamination with leukocyte RNA is mandatory. Applying laser-assisted microdissection and manipulation (LMM) we could isolate platelets from hemalaun-stained cytospins under permanent visual control and after laser-photolysis of nucleated blood cells. For mRNA quantification, the platelet-specific mRNAs were subsequently measured by real time RT-PCR. High-copy beta3 integrin and low-copy a alpha2 integrin as well as tissue factor (TF) transcripts were analyzed in LMM-harvested platelets. In 91.2% (83/91) beta3 integrin was detectable with a mean threshold cycle (CT) value of 32.5+/-3.2 (< or =50,000 cells). The low-copy a 2 integrin mRNA was positive in 84.4% (38/45) with CT mean value of 36.9+/-1.3, indicating that the relative expression of alpha2 integrin mRNA in platelets was about 130 times lower than beta3. The TF transcript was undetectable in all samples. Comparing platelet mRNA from LMM isolation to that from limiting dilution series resulted in a high accordance for beta3 integrin transcript in both, recovery (91.2% vs. 95.2%) and CT value (32.5 vs. 32.8). These results demonstrate that the combination of LMM and real-time RT-PCR is a valuable tool for precise, platelet-specific mRNA analysis without contamination of other cells.

Blood Platelets↗

PubMed2002

RFLP analysis of 1-aminocyclopropane-1-carboxylate synthase ACC2 and ACC4 genes from Polish cultivars of tomato.

An important trait of tomato is the rate of fruit ripening, strongly dependent on ethylene production. The ripening-related ethylene synthesis in tomato is controlled mainly by 1-aminocyclopropane-1-carboxylate synthase LE-ACS2 and LE-ACS4 isoenzymes (Rottmann et al., 1991, J. Mol. Biol. 222: 937; Lincoln et al., 1993, J. Biol. Chem. 268: 19422; Barry et al., 2000, Plant Physiol. 123: 979). In spite of numerous reports on the LE-ACS2 and LE-ACS4 gene expression, only ones considered the genomic organisation each of these genes (Rottmann et al., 1991; Lincoln et al., 1993) reported one copy of each of these genes in tomato cv VF36. In this article we suggest that the genomic organisation of LE-ACS2 and LE-ACSS4 genes may depend on tomato cultivars and may differ from that described by the above authors. The results of Southern analyses of genomic DNAs from 17-day old seedlings (cultivars Jaga, Halicz, Betalux, New Yorker) imply that the genomic organisation of LE-ACS2 and LE-ACS4 genes in Polish cultivars differs from that reported for cv VF36.

Blotting, Southern↗