PubMed Health⌕ Search

Biomedical subjects

Paul B Savage

Publications and source records attributed to Paul B Savage.

13 recordsLinked to original sources
PubMed2003

The paradox of immune molecular recognition of alpha-galactosylceramide: low affinity, low specificity for CD1d, high affinity for alpha beta TCRs.

CD1 resembles both class I and class II MHC but differs by the important aspect of presenting lipid/glycolipids, instead of peptides, to T cells. Biophysical studies of lipid/CD1 interactions have been limited, and kinetics of binding are in contradiction with functional studies. We have revisited this issue by designing new assays to examine the loading of CD1 with lipids. As expected for hydrophobic interactions, binding affinity was not high and had limited specificity. Lipid critical micelle concentration set the limitation to these studies. Once loaded onto CD1d, the recognition of glycolipids by alphabeta T cell receptor was studied by surface plasmon resonance using soluble Valpha14-Vbeta8.2 T cell receptors. The Valpha14 Jalpha18 chain could be paired with NK1.1 cell-derived Vbeta chain, or any Vbeta8 chain, to achieve high affinity recognition of alpha-galactosylceramide. Biophysical analysis indicated little effect of temperature or ionic strength on the binding interaction, in contrast to what has been seen in peptide/MHC-TCR studies. This suggests that there is less accommodation made by this TCR in recognizing alpha-galactosylceramide, and it can be assumed that the most rigid part of the Ag, the sugar moiety, is critical in the interaction.

Animals↗
PubMed2003

Detection of VX contamination in soil through solid-phase microextraction sampling and gas chromatography/mass spectrometry of the VX degradation product bis(diisopropylaminoethyl)disulfide.

A solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) sampling and analysis method was developed for bis(diisopropylaminoethyl)disulfide (a degradation product of the nerve agent VX) in soil. A 30-min sampling time with a polydimethylsiloxane-coated fiber and high temperature alkaline hydrolysis allowed detection with 1.0 microg of VX spiked per g of agricultural soil. The method was successfully used in the field with portable GC-MS instrumentation. This method is relatively rapid (less than 1 h), avoids the use of complex preparation steps, and enhances analyst safety through limited use of solvents and decontamination of the soil before sampling.

Chemical Warfare Agents↗
PubMed2003

Synthesis of lipid A derivatives and their interactions with polymyxin B and polymyxin B nonapeptide.

Lipid A is the causative agent of Gram-negative sepsis, a leading cause of mortality among hospitalized patients. Compounds that bind lipid A can limit its detrimental effects. Polymyxin B, a cationic peptide antibiotic, is one of the simplest molecules capable of selectively binding lipid A and may serve as a model for further development of lipid A binding agents. However, association of polymyxin B with lipid A is not fully understood, primarily due to the low solubility of lipid A in water and inhomogeneity of lipid A preparations. To better understand lipid A-polymyxin B interaction, pure lipid A derivatives were prepared with incrementally varied lipid chain lengths. These compounds proved to be more soluble in water than lipid A, with higher aggregation concentrations. Isothermal titration calorimetric studies of these lipid A derivatives with polymyxin B and polymyxin B nonapeptide indicate that binding stoichiometries (peptide to lipid A derivative) are less than 1 and that affinities of these binding partners correlate with the aggregation states of the lipid A derivatives. These studies also suggest that cooperative ionic interactions dominate association of polymyxin B and polymyxin B nonapeptide with lipid A.

2-Naphthylamine↗
PubMed2003

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts.

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.

Adult↗
PubMed2002

Antibacterial properties of cationic steroid antibiotics.

Cationic steroid antibiotics have been developed that display broad-spectrum antibacterial activity. These compounds are comprised of steroids appended with amine groups arranged to yield facially amphiphilic morphology. Examples of these antibiotics are highly bactericidal, while related compounds effectively permeabilize the outer membranes of Gram-negative bacteria sensitizing these organisms to hydrophobic antibiotics. Cationic steroid antibiotics exhibit various levels of eukaryote vs. prokaryote cell selectivity, and cell selectivity can be increased via charge recognition of prokaryotic cells. Studies of the mechanism of action of these antibiotics suggest that they share mechanistic aspects with cationic peptide antibiotics.

Anti-Bacterial Agents↗
PubMed2002

Synthesis and NKT cell stimulating properties of fluorophore- and biotin-appended 6"-amino-6"-deoxy-galactosylceramides.

Alpha-galactosylceramides are potent stimulators of human T cells. Stimulation occurs through binding of the glycolipids by CD1d, presentation to T cells, and formation of a CD1d-glycolipid-T cell receptor complex. To facilitate the elucidation of the structural features of glycolipids necessary for T cell stimulation, alpha-galactosylceramides have been prepared with small molecules appended at the C6 position of the sugar. The appended molecules do not significantly influence the abilities of the glycolipids to stimulate T cells. [reaction: see text]

Antigens, CD1↗
PubMed2002

Formation of 2-chlorobenzylidenemalononitrile (CS riot control agent) thermal degradation products at elevated temperatures.

2-Chlorobenzylidenemalononitrile (CS riot control agent) has been shown to produce a number of thermal degradation products when dispersed at high temperature. We hypothesized that these CS-derived compounds are formed by energy input from heating during the dispersion process. Here we identified organic CS-derived compounds formed from purified CS subjected to temperatures ranging from 300 to 900 degrees C in an inert atmosphere with analysis of tube furnace effluent by gas chromatography and mass spectrometry. We conclude that the production of many CS-derived compounds previously observed during high-temperature dispersion is likely to be heat related.

Gas Chromatography-Mass Spectrometry↗
PubMed2002

Correlation of the antibacterial activities of cationic peptide antibiotics and cationic steroid antibiotics.

The antibacterial activities of cationic steroid antibiotics and cationic peptide antibiotics have been compared. Depolarization of bacterial membranes, activation of bacterial stress-related gene promoters, and changes in bacterial morphologies caused by these antibiotics suggest that cationic steroid and peptide antibiotics share mechanistic aspects. Modified cationic steroid antibiotics display improved selectivity for prokaryotic cells over eukaryotic cells presumably due to increased charge recognition.

Amino Acid Sequence↗
PubMed2002

A quantitative approach for studying IgE-FcepsilonRI aggregation.

Aggregation of cell surface receptors is a ubiquitous means of initiating signal transduction in many cellular systems. In this manuscript, we describe a combined theoretical and experimental approach based on multiparameter flow cytometry for measuring the time course of ligand induced aggregation of IgE-FcepsilonRI on RBL cells. By fluorescently labeling both the ligand and surface IgE (sIgE), we have developed an assay that permits us to simultaneously measure both occupancy of sIgE combining sites and association of antigen with the cell surface. This allows for a direct calculation of the degree of receptor aggregation present on the cell. By employing new mixing technologies developed for flow cytometry, we are able to look at aggregation in the sub second time domain. To extend our work, we have synthesized a new set of chemically well defined ligands (of valences 1-3) to use as probes in our studies. We show that the magnitude of the cellular response is dramatically increased as the valence of our ligand is raised from two to three.

Animals↗
PubMed2001

Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail-truncated CD1d.

For members of the CD1 family of beta(2)-microglobulin-associated lipid-presenting molecules, tyrosine-based motifs in the cytoplasmic tail and invariant chain (Ii) govern glycoprotein trafficking through endosomal compartments. Little is known about the intracellular pathways of CD1 trafficking and antigen presentation. However, in vitro studies with cells transfected with mutant CD1 that had a truncated cytoplasmic tail have suggested a role for these tyrosine motifs in some, but not all, antigenic systems. By introducing a deletion of the tyrosine motif into the germ line, and through homologous recombination in embryonic stem cells, we now describe knock-in mice with the CD1d cytoplasmic tail deleted. Despite adequate surface CD1d expression and the presence of Ii, these mutant mice showed multiple and selective abnormalities in intracellular trafficking, antigen presentation and T cell development, demonstrating the critical functions of the CD1d cytoplasmic tail motif in vivo.

Amino Acid Motifs↗
PubMedDate not supplied

Traditional sampling with laboratory analysis and solid phase microextraction sampling with field gas chromatography/mass spectrometry by military industrial hygienists.

The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the United States Department of Defense or the Uniformed Services University of the Health Sciences. Rapid on-site detection and identification of environmental contaminants to which personnel may be exposed is often needed during military deployment situations. The availability of military industrial hygienists with capabilities for "complete" on-site exposure assessment of chemical species should allow detection and identification of a number of important stressors almost immediately following sample collection. Portable gas chromatography/mass spectrometry (GC/MS) provides a rapid and efficient separation of volatile and semivolatile organic analytes, accompanied by sensitive electron impact ionization-mass spectrometry (EI-MS) detection. The use of GC/MS in the field is limited, however, by equipment cost, complexity of the equipment, and the analytical process. Additionally, a skilled operator is needed to obtain useful separations and to interpret mass spectral data. To demonstrate benefits and limitations of "complete" exposure assessment capabilities, a previously unidentified complex mixture, produced by thermal dispersion of riot control agents, was examined. Established active sampling methods were used with laboratory analyses. Solid phase microextraction, a passive sampling method that simplifies preparation for GC/MS analysis, also was used with a field-portable GC/MS system. Both sampling/analysis methods were used to detect CS riot control agent-derived air contaminants dispersed from riot control type canisters through oxidizer-supported combustion of a chemical fuel.

Chemistry Techniques, Analytical↗
PubMedDate not supplied

Liberation of hydrogen cyanide and hydrogen chloride during high-temperature dispersion of CS riot control agent.

High temperature dispersion (greater than 700 degrees C) of the riot control agent orthochlorobenzylidenemalononitrile (CS) has previously been shown to produce a number of organic thermal degradation products through rearrangements and loss of cyano and chlorine substituents present on the parent CS compound. Until now the possibility that HCN and HCl might also be air contaminants produced during high temperature CS dispersion has not been examined. Air samples were collected to detect HCN and HCl as air contaminants released during high-temperature CS dispersion indoors. Sampling and analysis based on National Institute of Occupational Safety and Health methods 7904 and 6010 for HCN, and 7903 for HCl, showed evidence that both compounds were present in air samples collected. A reassessment of human health risks associated with exposure to CS riot control agent dispersed at high temperature should be conducted, and should consider the full range of contaminants produced during the dispersion process.

Environmental Exposure↗