Response of resident murine peritoneal macrophages to in vivo administration of granulocyte-macrophage colony-stimulating factor

The effect of s.c. inoculation of purified recombinant derived granulocyte-macrophage (GM)-CSF on resident murine peritoneal macrophages was assessed in this study. From 18 to 24 h after s.c. administration of GM-CSF to normal mice, the resident peritoneal macrophages were harvested and the levels of membrane-bound IL-1, FcR, Mac-1 cell-surface Ag, and class II MHC expression were assessed. Peritoneal cells from GM-CSF-inoculated mice had significantly greater levels of membrane-bound IL-1 than did control mice. In addition when resident peritoneal macrophages from normal mice were purified by adherence and grown in the presence of GM-CSF, they produced greater levels of both membrane-bound and secreted IL-1. The peritoneal cells from GM-CSF-inoculated mice did not differ from controls in the expression of class II MHC-encoded Ag. This observation was confirmed by the finding that GM-CSF was unable to induce class II MHC expression on P388D1 cells, whereas a secondary mixed leukocyte culture supernatant was. Peritoneal cells from GM-CSF-inoculated mice also exhibited greater levels of expression of FcR and the Mac-1 cell-surface Ag. This resulted in an increase in their ability to phagocytose opsonized SRBC in vitro.     

Molecular determinants of phospholipid synergy in blood clotting

Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single "phospho-L-serine-specific" interaction and multiple "phosphate-specific" interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca(2+) in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphingomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-L-serine-specific and phosphate-specific interactions, respectively.     

Metabolomic profiling of regulatory lipid mediators in sputum from adult cystic fibrosis patients

Retained respiratory tract (RT) secretions, infection, and exuberant inflammatory responses are core abnormalities in cystic fibrosis (CF) lung disease. Factors contributing to the destructive CF airway inflammatory processes remain incompletely characterized. The pro-oxidative inflammatory CF RT milieu is known to contain enzymatically and nonenzymatically produced regulatory lipid mediators, a panel of structurally defined oxidized metabolites of polyunsaturated fatty acids known to play a role in pathology related to inflammation. Using an extraction protocol that maximizes recoveries of sputum-spiked deuterated standards, coupled with an LC/MS/MS detection system, this study presents a metabolomic method to assess a broad spectrum of regulatory lipid mediators in freshly obtained sputum from CF patients. A broad range of both proinflammatory and anti-inflammatory lipid mediators was detected, including PGE2, PGD2, TXB2, LTB4, 6-trans-LTB4, 20-OH-LTB4, 20-COOH-LTB4, 20-HETE, 15-HETE, 11-HETE, 12-HETE, 8-HETE, 9-HETE, 5-HETE, EpETrEs, diols, resolvin E1, 15-deoxy-PGJ2, and LXA4. The vast majority of these oxylipins have not been reported previously in CF RT secretions. Whereas direct associations of individual proinflammatory lipid mediators with compromised lung function (FEV-1) were observed, the relationships were not robust. However, multiple statistical analyses revealed that the regulatory lipid mediators profile taken in aggregate proved to have a stronger association with lung function in relatively stable outpatient adult CF patients. Our data reveal a relative paucity of the anti-inflammatory lipid mediator lipoxin A4 in CF sputum. Patients displaying detectable levels of the anti-inflammatory lipid mediator resolvin E1 demonstrated a better lung function compared to those patients with undetectable levels. Our data suggest that comprehensive metabolomic profiling of regulatory lipid mediators in CF sputum should contribute to a better understanding of the molecular mechanisms underlying CF RT inflammatory pathobiology. Further studies are required to determine the extent to which nutritional or pharmacological interventions alter the regulatory lipid mediators profile of the CF RT and the impact of potential modulations of RT regulatory lipid mediators on the clinical progression of CF lung disease.     

Sensitivity measurement and compensation in spectral imaging.

BACKGROUND: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. METHODS: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. RESULTS: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre- and post-compensation. CONCLUSIONS: Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi-automated manner.     

Nitric oxide and protein nitration in the cystic fibrosis airway

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.     

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.     

Establishment of DsRed.T3_S4T as an improved autofluorescent marker for microbial ecology applications

Autofluorescent proteins (AFPs), such as green fluorescent protein (GFP) and DsRed, are valuable tools for studying plant-microbe interactions. Nevertheless, because of some limitations, efforts are ongoing to generate improved AFP variants. Several groups have generated variants of GFP with altered spectral characteristics, and faster maturing and brighter variants of DsRed. In this study we used plasmid and chromosomal constructs to test the efficacy of a new variant of DsRed, DsRed.T3_S4T, in Pseudomonas fluorescens F113rif. In addition, we compared the ecological fitness of strains carrying chromosomal copies of EGFP, DsRed or DsRed.T3_S4T. Strains expressing DsRed.T3_S4T fluoresced significantly brighter than strains expressing DsRed. Furthermore, it was found that although all strains grew equally well in vitro, only strains carrying DsRed.T3_S4T functioned as well as wild type in a competitive rhizosphere colonization assay. In particular, it was observed that DsRed.T3_S4T is an improved marker over DsRed for microbial ecology studies in this strain.     

Intracellular Accumulation of High Levels of γ-Aminobutyrate by Listeria monocytogenes 10403S in Response to Low pH: Uncoupling of γ-Aminobutyrate Synthesis from Efflux in a Chemically Defined Medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular γ-aminobutyrate (GABA_i). The glutamate is then decarboxylated to GABA_i, a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA_e) in response to acidification. In addition, high levels of GABA_i (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA_i in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA_i. These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.The capacity to produce γ-aminobutyric acid (GABA) through glutamate decarboxylation is commonly found in both Gram-negative and Gram-positive bacterial genera (10, 12). In several cases, this reaction has been shown to be critical for bacteria to survive potentially lethal acidic environments (15, 18, 20). It is generally held that the hydrogen ion consumed during the decarboxylation reaction helps to prevent excessive acidification of the cytoplasm, thereby protecting the cells against acidic environments. The GABA produced in the reaction is removed from the cell through the activity of an antiporter that exchanges a GABA molecule for an extracellular glutamate (Glu) molecule (6, 12).In Listeria monocytogenes, the Gram-positive food-borne pathogen that was the focus of the present study, the glutamate decarboxylase (GAD) system has been shown to play an essential role in acid tolerance (8, 9). Mutants compromised in their ability to catalyze this decarboxylation reaction survive poorly both in acidic foods (8) and gastric juice (9). The GAD system in most L. monocytogenes strains is encoded by a total of five genes. There are three genes, designated gadD1, gadD2, and gadD3, that encode distinct glutamate decarboxylase enzymes and two genes, designated gadT1 and gadT2, that encode two Glu-GABA antiporters. These genes are organized at three separate genetic loci: gadD1T1, gadT2D2, and gadD3 (11). The decarboxylase/antiporter system encoded by gadT2D2 plays a central role in allowing survival under extreme acidic conditions; mutants lacking either the GadT2 antiporter or the GadD2 decarboxylase are highly sensitive to low pH (9, 11). In contrast, the GadD1/GadT1 decarboxylase/antiporter system appears to be more important for growth under moderately acidic conditions (11). The genes encoding this system are absent from most serotype 4 strains, and this generally correlates with a reduced ability of these strains to grow well at low pH (11). The role of GadD3 is less clear since it has not been possible to generate a deletion mutant lacking the corresponding gene (9).Although the activity of the decarboxylase is generally thought to be coupled directly to the antiporter activity (i.e., the efflux of GABA is coupled to the supply of Glu) there is little direct evidence for this, even in bacteria where the system has been very well characterized. Most studies of the bacterial GAD system have used complex growth media when studying acid tolerance and GABA production (7, 8, 15). In the present study, we sought to determine whether extracellular Glu is a requirement for the production of GABA in L. monocytogenes. To do this, we have used a chemically defined growth medium (DM) that supports the growth of L. monocytogenes but does not include Glu. The results indicate that cells cultured in this medium do not produce extracellular GABA (GABA_e) in response to low pH but are capable of accumulating substantial pools of intracellular GABA (GABA_i). We establish that some component of complex medium is indispensable for efficient efflux of GABA. Surprisingly, supplementation of the DM with Glu failed to stimulate the extracellular release of GABA. We show that the GadD2/GadT2 decarboxylase/antiporter system is not transcribed when cells are grown in DM and suggest that this accounts for much of the difference in GABA production between cells cultured in DM and complex growth medium.     

Internal work and physiological responses during concentric and eccentric cycle ergometry

Internal mechanical work during cycling, required to raise and lower the legs and change their velocities, is shown to be an important factor when interpreting physiological responses to cycle ergometer exercise. The internal work required to move the legs during concentric and eccentric cycle ergometry at different speeds and workloads was calculated from segmental energy changes determined using cinematography and directly using an eccentric ergometer. The mean internal work rates obtained at pedal frequencies of 30, 60 and 90 min-1 were 11.5, 20 and 62 W respectively. When these estimates were added to the external work rates, they increased concentric and decreased eccentric work rates. The largest differences were seen at low work rates and high pedal frequencies during which concentric work rates increased by 51% and eccentric decreased 60% by the inclusion of internal work. When comparisons of concentric and eccentric cycling at equal uncorrected work rates were made, neglecting to include internal work introduced errors ranging from 12 to 97%. The calculated estimates of internal work agreed well with the power supplied by the eccentric ergometer to move the legs passively. The investigations show that the inclusion of internal work is important when comparing physiological responses during concentric and eccentric ergometry, especially when pedal frequencies exceed 60 min-1 and when work rates are small.