Creation of genome-wide protein expression libraries using random activation of gene expression

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.     

Orbital response indicates nasal pungency: analysis of biomechanical strain on the skin

Stimulation of the human nasal passage with pungent vapor elicits motor responses in a zone around the eye. This investigation addressed whether quantification of such responses, particularly activity of the orbicularis oculi muscle, could yield a sensitive index of nasal pungency. We placed an array of small, high-contrast targets just beneath the lower eyelid and videotaped their movement to capture deformation of the skin atop the orbicularis oculi during 3 s stimulation with pungent concentrations of ethyl acetate. Eleven subjects participated. Analysis of the movements served to determine mechanical strain, which yielded a single index that we termed 'maximum strain'. This increased with concentration of the vapor and with time during and just after stimulation. Comparison with psychophysical data showed that the strain became evident at concentrations just detectable as pungent. Maximum strain measured on the skin shows promise as an objective index of pungency.     

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.     

Acute inflammation increases selective uptake of HDL cholesteryl esters into adrenals of mice overexpressing human sPLA2

The acute-phase protein secretory phospholipase A2 (sPLA2) influences the metabolism of high-density lipoproteins (HDL). The adrenals are known to utilize HDL cholesterol as a source of sterols. The aim of the present study was to test the hypothesis that sPLA2 enhances the selective uptake of HDL into the adrenals in response to acute inflammation as a possible physiological role for the sPLA2-HDL interaction. Human sPLA2-transgenic mice, in which sPLA2 expression is upregulated by inflammatory stimuli, were used. Ten hours after induction of the acute-phase response (APR) by injection of bacterial lipopolysaccharide (LPS), plasma levels of HDL cholesterol decreased significantly in sPLA2-transgenic mice (-18%, P < 0.05) but remained unchanged in wild-type mice. The fractional catabolic rates of both 125I-labeled tyraminecellobiose (TC)-HDL and [3H]cholesteryl ether increased significantly in the sPLA2-transgenic mice after induction of the APR (0.18 +/- 0.01 vs. 0.21 +/- 0.01 pool/h, P < 0.05, and 0.31 +/- 0.02 vs. 0.42 +/- 0.05 pool/h, P < 0.05, respectively) but remained unchanged in the wild-type mice (0.10 +/- 0.01 vs. 0.22 +/- 0.02 pool/h, respectively). After induction of the APR, in both groups HDL holoparticle uptake by the liver was increased (P < 0.001). sPLA2-transgenic mice had 2.4-fold higher selective uptake into the adrenals after induction of the APR than wild-type mice (156 +/- 6 vs. 65 +/- 5%/ micro g tissue protein, P < 0.001). In summary, upregulation of sPLA2 expression during the APR specifically increases the selective uptake of HDL cholesteryl ester into the adrenals. These data suggest a novel metabolic role for sPLA2: modification of HDL during the APR to promote increased adrenal uptake of HDL cholesteryl ester to serve as source for steroid hormone synthesis.     

Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity

A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa. Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.     

Overexpression of secretory phospholipase A(2) causes rapid catabolism and altered tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I

Plasma levels of high density lipoprotein (HDL) cholesterol and its major protein component apolipoprotein (apo) A-I are significantly reduced in both acute and chronic inflammatory conditions, but the basis for this phenomenon is not well understood. We hypothesized that secretory phospholipase A(2) (sPLA(2)), an acute phase protein that has been found in association with HDL, promotes HDL catabolism. A series of HDL metabolic studies were performed in transgenic mice that specifically overexpress human sPLA(2) but have no evidence of local or systemic inflammation. We found that HDL isolated from these mice have a significantly lower phospholipid and cholesteryl ester and significantly greater triglyceride content. The fractional catabolic rate (FCR) of (125)I-HDL was significantly faster in sPLA(2) transgenic mice (4.08 +/- 0.01 pools/day) compared with control wild-type littermates (2.16 +/- 0.48 pools/day). (125)I-HDL isolated from sPLA(2) transgenic mice was catabolized significantly faster than (131)I-HDL isolated from wild-type mice after injection in wild-type mice (p < 0.001). Injection of (125)I-tyramine-cellobiose-HDL demonstrated significantly greater degradation of HDL apolipoproteins in the kidneys of sPLA(2) transgenic mice compared with control mice (p < 0.05). The fractional catabolic rate of [(3)H]cholesteryl ether HDL was significantly faster in sPLA(2)-overexpressing mice (6.48 +/- 0.24 pools/day) compared with controls (4.80 +/- 0.72 pools/day). Uptake of [(3)H] cholesteryl ether into the livers and adrenals of sPLA(2) transgenic mice was significantly enhanced compared with control mice. In summary, these data demonstrate that overexpression of sPLA(2) alone in the absence of inflammation causes profound alterations of HDL metabolism in vivo and are consistent with the hypothesis that sPLA(2) may promote HDL catabolism in acute and chronic inflammatory conditions.     

Comparison of two stimulus-delivery systems for measurement of nasal pungency thresholds

Using representative members of each of three homologous series of chemicals-ketones, acetates and alcohols-we measured nasal pungency thresholds in anosmics via two stimulus-delivery systems. The first system consists of the fairly commonly used 270 ml, plastic 'squeeze bottles'. The second system consists of 1900 ml, glass vessels with Teflon tubing and nose-pieces. Although bulkier and more susceptible to mechanical breakage, the glass vessels possess advantages that can allow them to provide 'environmentally realistic' chemosensory thresholds, i.e. thresholds closer in absolute values to those that might be obtained under whole-body exposures. Such advantages include a larger volume of the vapor-source to accommodate whole sniffs, and a tight nose-nose-piece connection to avoid stimulus dilution. The outcome revealed that, for every chemical, the glass vessels provided nasal pungency thresholds significantly lower than those provided by the squeeze bottles. The difference amounted, on average, to a factor of 4.6, though the relative potency of the compounds remained the same under both systems. Additionally, when tested with the highest homologues used here, namely, octyl acetate and 1-octanol, anosmics using the glass vessels had little or no difficulty achieving the criterion for threshold whereas they did have difficulty when using the squeeze bottles.