Relative Reverse Transcriptase-polymerase Chain Reaction Research Articles

Extracellular matrix changes in the umbilical arteries of growth-restricted fetuses

This study was undertaken to determine whether the expression of extracellular matrix components (ECM) is altered in the umbilical arteries from preterm fetal growth-restricted (FGR) pregnancies. Preterm pregnancies with FGR were compared with appropriately grown preterm pregnancies. Umbilical artery messenger RNA (mRNA) levels for fibrillar collagens I and III, nonfibrillar collagen XIV, and decorin were determined by using relative reverse transcriptase polymerase chain reaction (RT-PCR). The mRNA expression was compared between cases and controls by using the Student t test. P < or = .05 was considered significant. Eight FGR cases and 5 control pregnancies were analyzed. Mean counts per minute (cpm) +/- SEM for collagen I and collagen III were increased in FGR pregnancies. There were no differences in mRNA expression of collagen XIV or decorin. Increased mRNA expression of collagen I and III, but not collagen XIV or decorin, is found in the umbilical arteries of preterm FGR pregnancies.

Increased diacylglycerol acyltransferase activity is associated with triglyceride accumulation in tissues of diet-induced insulin-resistant hyperlipidemic hamsters

Over-accumulation of triglyceride (TG) in insulin-sensitive tissues is associated with the development of insulin resistance. We investigated whether enhanced de novo lipogenesis via diacylglycerol acyltransferase (DGAT) may contribute to the over-accumulation of TG in various tissues (liver, adipose, muscle, and intestine) using 2 well-characterized hyperlipidemic, insulin-resistant hamster models. In general, a marked increase in TG accumulation was noted in most tissues. Interestingly, the increase in TG accumulation corresponded to an increase in microsomal DGAT activity which ranged from 114% to 575% in all of the examined tissues (n = 7 per group). To delineate the mechanism for the increase in DGAT activity, we measured the expression of DGAT-1 and DGAT-2 messenger RNA by relative reverse transcriptase polymerase chain reaction (RT-PCR). In general, DGAT gene expression changed with DGAT-1 changing the most in the liver and adipose tissue, whereas DGAT-2 showed responses mainly in muscle and intestine. The increases in messenger RNA expression were not remarkable (averaging 35%; n = 4 per group) indicating that posttranscriptional mechanism(s) may play a larger role in regulating DGAT activity. In summary, the data suggest that elevated DGAT activity/expression and the subsequent increase in de novo lipogenesis could in part induce the insulin-resistant state.

Expression of the ?-carotene desaturase gene in sunflower

A relative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) approach was performed to assess ζ-carotene desaturase (HaZds) gene expression in sunflower (Helianthus annuus L.). HaZds was highly expressed in cotyledons and in young and mature leaves, but transcript levels were comparatively lower in both stems and roots. The HaZds transcript levels were influenced by leaf expansion, which suggests that HaZds expression is regulated during leaf development. The key role of HaZds in controlling carotenoid biosynthesis is indicated by the concurrent increase of HaZds transcript levels with the light-dependent enhanced carotenoid production in cotyledons of sunflower.

α lipoic acid inhibits human T‐cell migration: Implications for multiple sclerosis

We have demonstrated previously the ability of the antioxidant alpha lipoic acid (ALA) to suppress and treat a model of multiple sclerosis (MS), relapsing experimental autoimmune encephalomyelitis (EAE). We describe the effects of ALA and its reduced form, dihydrolipoic acid (DHLA), on the transmigration of human Jurkat T cells across a fibronectin barrier in a transwell system. ALA and DHLA inhibited migration of Jurkat cells in a dose-dependent fashion by 16-75%. ALA and DHLA reduced matrix metalloproteinase-9 (MMP-9) activity by 18-90% in Jurkat cell supernatants. GM6001, a synthetic inhibitor of MMP, reduced Jurkat cell migration, but not as effectively as ALA and DHLA did. Both ALA and DHLA downmodulated the surface expression of the alpha4beta1 integrin (very late activation-4 antigen; VLA-4), which binds fibronectin and its endothelial cell ligand vascular cell adhesion molecule-1 (VCAM-1). Moreover, ALA, but not DHLA, reduced MMP-9-specific mRNA and extracellular MMP-9 from Jurkat cells and their culture supernatants as detected by relative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. ALA and DHLA inhibited Jurkat cell migration and have different mechanisms for inhibiting MMP-9 activity. These data, coupled with its ability to treat relapsing EAE, suggest that ALA warrants investigation as a therapy for MS.

Stimulation of intestinal glutamine absorption in chronic metabolic acidosis

BackgroundAmino acid glutamine plays a central role in inter-organ nitrogen transfer in acidosis, a compensatory mechanism that is essential in maintaining acidbase balance. Intestinal glutamine absorption is a key exogenous glutamine source in maintaining glutamine homeostasis. The purpose of this in vivo study was to investigate the regulation of intestinal glutamine absorption during chronic metabolic acidosis. MethodsMetabolic acidosis in adult male Sprague-Dawley rats was induced by adding 1.5% NH4Cl to drinking water. [3H]-L-glutamine transport activity across brush border membrane vesicles and glutamine transporter ATB0 messenger RNA levels by relative reverse transcriptase-polymerase chain reaction were measured in rat jejunum. Data were analyzed by t test (P < .05). ResultsAcidosis occurred as early as 1 day and was partially compensated by 7 days. Glutamine transport in brush border membrane vesicles was increased after 2 days of acidosis. Chronic acidosis (7 days) resulted in an 8-fold increase of glutamine transport activity. The glutamine transport maximal capacity (Vmax) was stimulated 5-fold, while the transport affinity (Km) was not affected by acidosis. Relative reverse transcriptase-polymerase chain reaction showed a 2.5-fold increase of glutamine transporter ATB0 messenger RNA levels. ConclusionsChronic metabolic acidosis stimulates intestinal glutamine absorption via a mechanism that involves an increase of functional membrane glutamine transporter units.

Identification of 9 genes differentially expressed in head and neck squamous cell carcinoma.

Current treatment modalities in squamous cell carcinoma of the head and neck have failed to improve survival. Advances in the discovery of novel biomarkers and targets for therapy are necessary. Differential display and microarray analysis were used to identify differences in gene expression between squamous carcinoma and matched nonmalignant biopsy specimens. Differences in gene expression found in vivo were also tested in vitro by comparing primary cultured normal oral epithelium with head and neck squamous cell carcinoma (HNSCC) cell lines. Results were confirmed by relative reverse transcriptase-polymerase chain reaction and immunohistochemical analysis. In tumors, microarray analysis showed down-regulation of calgranulin B (CAGB), CD24, lymphoepithelial Kazal-type-related inhibitor (LEKTI), zinc finger protein (ZNF-185), transglutaminase-3 (TGM3), and the ETS homologous factor (EHF). In addition, differential display revealed down-regulation of headpin. In contrast, periostin and the human homologue of the Drosophila white gene (ABCG1) were found to be up-regulated by microarray analysis and differential display, respectively. In HNSCC cell lines, LEKTI, ZNF-185, TGM3, headpin, and ABCG1 showed an expression pattern similar to that observed in tumor specimens. Periostin showed an opposite expression pattern in cell lines compared with that of tumor specimens. No consistent pattern of expression was found for CAGB, CD24, and EHF in cell lines. Immunohistochemical analysis revealed that the expression of headpin in nonmalignant mucosa was undetectable in tumors. Using differential display and microarray analysis, we have identified and confirmed the differential expression of 9 genes in HNSCC. Work is in progress to determine the biological significance of these genes and their potential as biomarkers or targets for therapy.

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Lopinavir

Selenium regulates expression in rat liver of genes for proteins involved in iron metabolism.

Suppression subtractive hybridization analysis in our laboratory recently revealed that transferrin mRNA may be elevated in Sedeficient rat liver. In this work, we compared expression in rat liver of genes for transferrin, transferrin receptor, ferritin light and heavy chains, and iron-regulatory proteins 1 and 2 in Se adequacy and deficiency. Weanling male Sprague-Dawley rats were fed Torula yeast diets supplemented with 0 or 0.15 microg Se/kg diet as sodium selenite for 15 wk. Activity of cellular glutathione peroxidase was virtually abolished in Se-deficient rat liver, whereas activity of glutathione S-transferase was 43% higher than in Se-adequate liver. There were no differences in hematocrit, hemoglobin, or liver iron content. To examine differential gene expression, we used a multiplex relative reverse transcriptase-polymerase chain reaction method. Three of the six genes examined showed modest but consistent upregulation in Se deficiency. Transferrin mRNA was 30% more abundant in Se-deficient than in Se-adequate liver. For the transferrin receptor, the difference was 32%, and for iron regulatory protein 1, it was 63%. No consistent differences were observed for iron regulatory protein 2 or for ferritin light or heavy chain. These findings suggest a possible role for dietary Se in moderating iron metabolism.