National Jewish Health

About: National Jewish Health is a healthcare organization based out in Denver, Colorado, United States. It is known for research contribution in the topics: Asthma & T cell. The organization has 883 authors who have published 833 publications receiving 79201 citations. The organization is also known as: National Jewish Medical and Research Center.

Isolation and Characterization of Mouse Fibroblasts.

Abstract: The lung parenchyma is comprised of many cells including the structurally important stromal fibroblasts. Fibroblasts function to produce extracellular matrix and are important in the maintenance of alveolar epithelial cells. To understand the role of fibroblasts both in homeostasis and disease, we isolate fibroblasts and grow them in culture. Two methods are presented here for the isolation and maintenance of mouse primary lung fibroblasts.

Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay

Abstract: The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite Plasmodium falciparum, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells. We have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity in vitro. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wild-type cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme. The identification of amodiaquine as an inhibitor of PfPMT in vitro and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs.

Retrieval of missing data for meta-analysis: a practical example.

Abstract: The use of meta-analysis to review the scientific literature is now common across most fields of research. For example, a recent search of the Medline database for the year 2002 using “meta-analysis” as the keyword resulted in a total of 1,835 available citations (GAK, June 28, 2003). One of the primary goals of meta-analysis is to identify all studies, within the smallest margin of search error possible, that meet the meta-analyst's inclusion criteria on the topic of interest. However, a problem encountered by all meta-analysts is the absence of adequate data from eligible studies for the outcome of interest. For example, a recent meta-analysis on the effects of exercise on resting blood pressure in children and adolescents found that 34 percent of eligible studies in which resting systolic and diastolic blood pressure was assessed had necessary data that was not reported (i.e., missing). For example, the standard deviation of the outcome measure was neither reported nor could it be obtained (1). Because this may introduce a possible bias in the results (data retrieval bias), retrieving as much of this missing data as possible is important. While different statistical procedures for handling missing outcome data exist (replacement with the mean, multiple regression to predict missing values, etc.) all have weaknesses (5). Consequently, it would seem more appropriate to first try and retrieve the actual data from as many eligible studies as possible. Unfortunately, we are not aware of any research that has focused on both the methodology and success of the retrieval of outcome data for a summary meta-analysis. Given the importance of retrieving summary outcome data from study authors and the lack of research documenting methodology and success, the purpose of this study was to examine the feasibility of retrieving missing outcome data using a specific example of a meta-analysis dealing with the effects of aerobic exercise on lipids and lipoproteins in adults.

A novel miRNA produced during lytic HSV-1 infection is important for efficient replication in tissue culture

Abstract: The influence of miRNAs on the host-pathogen environment is largely unknown and under intensive investigation. Whether produced by the pathogen or by the host cell, these miRNAs will sculpt the intracellular landscape, as their activity will ultimately affect levels of target proteins. Using a high-throughput sequencing approach, we identified 19 novel small RNAs produced during the early hours of herpes simplex virus type 1 (HSV-1) infection in epithelial cells. Six of the novel RNAs had predicted folds characteristic of miRNAs. One of the six, miR-92944, which resides in the 5' UTR of the ul42 gene in the sense orientation, was confirmed as a bona fide miRNA by RT-PCR and stem-loop PCR analysis. Northern blot analysis was used to observe the precursor forms of miR-92944. Viral mutants that do not produce miR-92944 exhibited significant reductions in viral titers in both single and multi-step growth analysis and a fourfold reduction in plaque size. The miR-92944 mutants produce wild-type levels of ICP4, UL42, VP5, and gC proteins contain no additional changes in the DNA sequence surrounding the site of mutagenesis. The defective phenotype of miR-92944 mutants was complemented in V42.3 cells, which contain the 5'UTR of ul42. We also found that miR-H1 expression was diminished in cells infected with the miR-92944 mutant virus. This study provides new information on the miRNA landscape during the early stages of HSV-1 infection and reveals novel targets for antagonistic molecules that may curtail the establishment of lytic or latent virus infection.

Platelet-derived growth factor receptor is a novel modulator of type A gamma-aminobutyric acid-gated ion channels.

Abstract: Platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) are ubiquitously expressed in the mammalian central nervous system, where they exert trophic actions on both neuronal and glial cells. However, the acute actions of PDGF on synaptic transmission are unknown. We report a novel regulatory action of PDGF/PDGFR. Activation of PDGFRs inhibited the function of native type A gamma-aminobutyric acid (GABAA) receptors (GABAA-RS) in rat hippocampal CA1 pyramidal neurons and mouse brain membrane vesicles. The mechanism of this inhibition was studied with a panel of mutant PDGFRS-beta coexpressed with cloned human GABAA-Rs in Xenopus oocytes. These experiments revealed that phospholipase C-gamma is the protein that relays the inhibitory signal from PDGFRS to GABAA-Rs. Experiments with microinjected EGTA and inositol-1, 3, 4-triphosphate demonstrated that inhibition of GABAA-Rs depended on a phospholipase C-gamma-mediated increase in intracellular Ca(2+)-levels. The PDGFR-induced inhibitory effect was independent of the subunit composition of GABAA-RS. Moreover, GABAA-RS composed of alpha 1 beta 1 S409A subunits, which do not contain any known protein kinase C phosphorylation sites, were inhibited by PDGF to the same extent as wild-type GABAA-RS. Inhibitors of protein kinase C, CA2+/calmodulin-dependent protein kinase II, calcineurin, and tyrosine phosphatases did not affect the modulatory actions of PDGFR. In conclusion, our results suggest that PDGFRs exert potent modulatory actions on GABAA-R-dependent inhibitory synaptic transmission. These regulatory actions of PDGF could play important roles in the function of the mammalian central nervous system during physiological and pathophysiological conditions.