William A. Hanlon

Integrated Genomic and Proteomic Analyses of Gene Expression in Mammalian Cells

Using DNA microarrays together with quantitative proteomic techniques (ICAT reagents, two-dimensional DIGE, and MS), we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation, as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists. We observe that the differential expression of mRNA (up or down) can capture at most 40% of the variation of protein expression. Although the overall pattern of protein expression is similar to that of mRNA expression, the incongruent expression between mRNAs and proteins emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both proteins and mRNAs.

Pyrroles and other heterocycles as inhibitors of P38 kinase

Investigation of furans, pyrroles and pyrazolones identified 3-pyridyl-2,5-diaryl-pyrroles as potent, orally bioavailable inhibitors of p38 kinase. 3-(4-pyridyl-2-(4-fluoro-phenyl)-5-(4-methylsulfinylphenyl)-pyrrol e (L-167307) reduces secondary paw swelling in the rat adjuvant arthritis model: ID50 = 7.4 mg/kg/b.i.d.

rTNFα facilitates human polymorphonuclear leukocyte adherence to fibrinogen matrices with mobilization of specific and tertiary but not azurophilic granule markers

rTNFα facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen‐coated surfaces in a concentration‐ and time‐dependent manner. The adhesion was maximal with 1.0 nM rTNFα within 40–50 min at 37°C. A monoclonal antibody (1B4) directed toward the β2‐chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNFα induced adhesion. TNFα caused a time‐dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of Aα(1–21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNFα could be further stimulated with cytochalasin B and N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and Aα(1–21) production over time. Thus the rTNFα‐facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.

Serum-induced monocyte differentiation and monocyte chemotaxis are regulated by the p38 MAP kinase signal transduction pathway.

Regulation by the p38 mitogen‐activated protein (MAP) kinase signaling pathway of monocytic inflammatory functions was evaluated using L‐790,070, a potent and selective inhibitor of p38 MAP kinase. Three major functions of monocytes were investigated: differentiation, chemotaxis, and phagocytosis. L‐790,070 inhibited serum‐induced monocyte differentiation with an IC50 of 0.5 nM. Monocyte chemotaxis induced by RANTES, macrophage inflammatory protein‐1α (MIP‐1α), monocyte chemotactic protein‐1 (MCP‐1), and fMLP were all sensitive to L‐790,070. When titrated, L‐790,070 inhibited MCP‐1‐induced chemotaxis in a concentration‐dependent manner with an IC50 of 0.3 nM. However, the ability of serum‐derived macrophages to phagocytose apoptotic neutrophils was unaffected by L‐790,070. The concentration with which L‐790,070 inhibited both differentiation and chemotaxis was similar to that necessary to inhibit p38 MAP kinase activation of MAPKAP kinase (0.3 nM) in response to stimulation by lipopolysaccharide. Therefore, the data in this report suggest that the mechanism by which L‐790,070 blocked monocyte differentiation and prevented chemotaxis was by inhibiting p38 MAP kinase activity. J. Leukoc. Biol. 67: 869–875; 2000.

Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38

Early events in the response of cells to lipopolysaccharide (LPS) include activation of NF‐κB and stress‐activated MAP kinase p38. Recent studies have shown that the human Toll‐like receptor 2 (TLR2) mediates activation of NF‐κB in response to commercial preparations of LPS (comLPS), membrane lipoproteins, and Gram‐positive bacterial products. Here, we show that expression of TLR2 in human embryonic kidney 293 cells enabled p38 phosphorylation in response to comLPS, a synthetic bacterial lipoprotein, and B. subtilis. Activation of p38 was confirmed by an in vitro kinase assay using ATF2 as substrate and by an assay measuring activation of the downstream effector of p38, MAP kinase‐activated protein kinase in cells. Thus, TLR2 initiated the signaling pathway for p38 in response to bacterial products.

Hypophosphatemic Effect of Niacin in Patients without Renal Failure: A Randomized Trial

BACKGROUND AND OBJECTIVES Niacin administration lowers the marked hyperphosphatemia that is characteristic of renal failure. We examined whether niacin administration also reduces serum phosphorus concentrations in patients who have dyslipidemia and are free of advanced renal disease. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We performed a post hoc data analysis of serum phosphorus concentrations that had been determined serially (at baseline and weeks 4, 8, 12, 18, and 24) among 1547 patients who had dyslipidemia and were randomly assigned in a 3:2:1 ratio to treatment with extended release niacin (ERN; 1 g/d for 4 weeks and dose advanced to 2 g/d for 20 weeks) combined with the selective prostaglandin D2 receptor subtype 1 inhibitor laropiprant (L; n = 761), ERN alone (n = 518), or placebo (n = 268). RESULTS Repeated measures analysis revealed that ERN-L treatment resulted in a net mean (95% confidence interval) serum phosphorus change comparing ERN-L with placebo treatment of -0.13 mmol/L (-0.15 to -0.13 mmol/L; -0.41 mg/dl [-0.46 to -0.37 mg/dl]). These results were consistent across the subgroups defined by estimated GFR of <60 or > or =60 ml/min per 1.73 m(2), a serum phosphorus of >1.13 mmol/L (3.5 mg/dl) versus < or =1.13 mmol/L (3.5 mg/dl), the presence of clinical diabetes, or concomitant statin use. CONCLUSIONS We have provided definitive evidence that once-daily ERN-L treatment causes a sustained 0.13-mmol/L (0.4-mg/dl) reduction in serum phosphorus concentrations, approximately 10% from baseline, which is unaffected by estimated GFR ranging from 30 to > or =90 ml/min per 1.73 m(2) (i.e., stages 1 through 3 chronic kidney disease).

Formation of polymorphonuclear leukocyte elastase: α1 proteinase inhibitor complex and Aα(1–21) fibrinopeptide in human blood stimulated with the calcium ionophore A23187: A model to characterize inhibitors of polymorphonuclear leukocyte elastase

Incubation of human blood with the secretagogue A23187 resulted in the formation of increased plasma concentrations of polymorphonuclear leukocyte (PMN) elastase: alpha 1 proteinase inhibitor (PMNE:alpha 1 PI) complex as well as A alpha(1-21) fibrinopeptide [A alpha(1-21)]. The formation of these species was both time and A23187 concentration dependent. Using a sandwich ELISA and a radioimmunoassay, we determined the comparative potencies of several compounds to inhibit the formation of PMNE: alpha 1 PI complexes and A alpha(1-21), respectively. L-658,758, a substituted cephalosporin, essentially irreversible elastase inhibitor, inhibited the formation of PMNE: alpha 1 PI and A alpha(1-21) with IC50 values of 38 and 15 microM, respectively. L-683,845, a monocyclic beta-lactam, was much more potent against isolated PMNE than L-658,758. However in this system it was approximately equivalent to L-658,758 with an IC50 of 15 microM against both species. ICI-200,880, a competitive slow-binding elastase inhibitor, was significantly less potent to inhibit A alpha(1-21), having an IC50 of 75 microM, while Declaben, a reversible noncompetitive inhibitor, was inactive at concentrations as great as 200 microM. We propose that evaluating inhibitors in the complex milieu of blood will provide a useful method to predict their therapeutic potential in vivo.

Expression and characterization of the chemokine receptor CCR2B from rhesus monkey

Species selectivity of chemokine receptor antagonists is a potential deterrent to making preclinical assessments in vivo. To determine if rhesus monkey disease models could support these assessments, we pharmacologically and functionally characterized recombinant rhesus CCR2B receptor. For these studies we obtained the CCR2B coding region by PCR from genomic rhesus DNA and expressed the receptor as stable transfectants in Chinese Hamster Ovary cells. The surface expression of recombinant rhesus CCR2B was detected by flow cytometry using a commercially available monoclonal anti-hCCR2B antibody. This antibody was used to detect rhCCR2B on monocytes in peripheral blood mononuclear cell preparations from rhesus whole blood. The recombinantly expressed CCR2B exhibited similar high affinity binding to the CCR2 chemokine ligands from rhesus and human 125I-rhMCP-1 (K(d)=433+/-14 pM) and 125I-hMCP-1 (K(d)=550+/-256 pM). In competition binding, the receptor exhibited selective high affinity binding to the monocyte chemoattractant protein (MCP) family chemokines with little affinity for most other members of the CC family of chemokines. One exception was eotaxin, a high affinity ligand for CCR3, which bound to rhesus CCR2B receptor (K(i)=1467+/-205 pM). Chemokines which exhibited binding affinity for the receptor were tested for their ability to induce intracellular calcium release. In these experiments the relative potencies of the MCP family of chemokines for rhCCR2B were similar to the observed binding affinities. In contrast, eotaxin was functionally inactive as an antagonist or agonist to this receptor. TAK-799 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride), a dual CCR2/CCR5 antagonist, demonstrated high affinity for the rhesus CCR2B in competition with 125I-hMCP-1 binding to the receptor (K(i)=0.5 nM) and also potently blocked the MCP-1 induced calcium mobilization mediated through the receptor.

Phosphorous acid analogs of L-680,833, a potent monocyclic β-lactam inhibitor of human leukocyte elastase

Abstract Analogs of the monocyclic β-lactam human leukocyte elastase (HLE) inhibitor L-680,833 in which the carboxyl group is replaced by phosphorous acid moieties were synthesized and found to be potent inhibitors of the enzyme ( k inact / K i in the range 217,000–1,326,000 M −1 s −1 ). Cellular activity was demonstrated by inhibition of the generation of the N-terminal cleavage product Aα-(1–21) from the Aα chain of fibrinogen.

Protein Profiling using Two-Dimensional Gel Electrophoresis with Multiple Fluorescent Tags

Publisher Summary This chapter describes and characterizes a new enabling technology from Amersham Biosciences called Difference In Gel Electrophoresis (DIGE), which leverages the use of fluorescent cyanine dyes that are used to label proteins prior to electrophoresis. This helps reduce the overall background during scanning, and provides more reproducible labeling. The existence of three different cyanine dyes with separate excitation and emission wavelengths also enables analysis of up to three samples in a single gel. Multiple samples per gel reduce the number of gels necessary for each study, thereby reducing the need to match as many gels to the master gel. The use of an internal pooled standard run on every gel aids in reducing false positives and false negatives. Reproducibility of protein separation between gels using 2D electrophoresis (2DE) is hindered by many factors such as inhomogeneities in acrylamide polymerization along with temperature inequities and power fluctuations. These variables and others prevent 2DE gel images from being directly superimposable and require warping capability to overlay and compare them. Therefore, detection of differences between samples by 2DE requires a high level of skill and sophisticated spot-detection and matching software along with appropriately powered experimental design to compensate the technical and biological variation in order to increase the statistical significance of the data. Robotic spot cutters can be utilized for excision and identification is made via traditional mass spectrometry approaches after in-gel trypsin digestion.