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High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)'[sub.2]-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB[sub.2]) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)'[sub.2]-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)'[sub.2]. This binding could interfere with the role of C3a and C5a in inflammation., Author(s): Milan Basta (corresponding author) [1]; Fredric Van Goor [2]; Stefano Luccioli [3]; Eric M. Billings [4]; Alexander O. Vortmeyer [5]; Lajos Baranyi [6]; Janos Szebeni [6]; Carl R. Alving [...]

Transition-state analog complex crystal structure of the phosphonate derivative of Enterobacter cloacae P99 cephalosporinase is homologous to the tetrahedral intermediate structure in beta-lactamase catalysis. The Tyr 150 hydroxyl group is preferred as an acid/base catalyst in the beta-lactamase active site. The sensitivity of serine beta-lactamases is opposite to that of serine proteases in phosphonate monoanion inhibition and indicates a positive potential in the catalytic sites.

Acylated activating elements, precatalytic structures and acyl-enzyme intermediates for sulbactam are produced from the crystal structure of class A-beta lactamases of Bacillus licheniformis. Sulbactam functions as a mechanism-based lactamase inactivator of the Lys-234, Lys-73 and Ser-130 residues. The leaving group penicillanic acid does not affect inactivation rate.

Energy-minimized constructs for active-binding of the precatalytic ('Michaelis') complex with the clinically used inactivator, clavulanate acid, for the acyl enzyme intermediate, and for the ultimate acylated species that inactivates class A beta-lactamases by clavulanate, were developed from the refined 2 angstrom crystal structure of the Bacillus licheniformis 749/C beta-lactamase. The constructs were used to reexamine the chemistry of inactivation of clavulanate. The capture of the beta-hydroxyl of concerned Ser-130 by the iminium moiety of the acylated acyclic intermediate and the subsequent deprotonation at C6 of clavulanate are most probably the essence of irrevocable inactivation.

Abstract Background The vascular disease in-stent restenosis (ISR) is characterized by formation of neointima and adverse inward remodeling of the artery after injury by coronary stent implantation. We hypothesized that the analysis of gene expression in peripheral blood mononuclear cells (PBMCs) would demonstrate differences in transcript expression between individuals who develop ISR and those who do not. Methods and Results We determined and investigated PBMC gene expression of 358 patients undergoing an index procedure to treat in de novo coronary artery lesions with bare metallic stents, using a novel time-varying intercept model to optimally assess the time course of gene expression across a time course of blood samples. Validation analyses were conducted in an independent sample of 97 patients with similar time-course blood sampling and gene expression data. We identified 47 probesets with differential expression, of which 36 were validated upon independent replication testing. The genes identified have varied functions, including some related to cellular growth and metabolism, such as the NAB2 and LAMP genes. Conclusions In a study of patients undergoing bare metallic stent implantation, we have identified and replicated differential gene expression in peripheral blood mononuclear cells, studied across a time series of blood samples. The genes identified suggest alterations in cellular growth and metabolism pathways, and these results provide the basis for further specific functional hypothesis generation and testing of the mechanisms of ISR.

Two new techniques for modeling chemical processes in condensed phases with combined quantum mechanical and molecular mechanical (QM/MM) potentials are introduced and tested on small, model compounds. The first technique, the double link atom (DLA) method, is an extension of the traditional, single link atom (SLA) method to avoid some of the problems with the latter method. These problems are primarily electrostatic, as the SLA method can produce an unphysical overall charge or dipole. The second technique, the delocalized Gaussian MM charge (DGMM) method, is an empirical way to include the delocalized character of the electron density of atoms in the MM region. This can be important for the electrostatic interaction of the QM region with nearby atoms in the MM region, and it can simplify the rules governing which classical interactions are included in the energies and forces. Even for very short distances, the DGMM method does not require the neglect of the MM host in the QM calculation. The DGMM method can be used for modeling reactions in solution, and it can be combined with methods such as the link atom, frozen orbital, or pseudopotential methods for terminating the QM region at a covalent bond. The DLA and the DGMM methods have been combined effectively. Presented here are tests on small, model systems that mimic properties important for reactions in proteins, in particular rotational barriers, proton affinities, and deprotonation energies. The new methods yield improved energetics for model compounds, vis-à-vis a point-MM-charge and SLA treatment. [ABSTRACT FROM AUTHOR]

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)2 activates ADP-ribosylation factors, ∼20-kDa GTPase proteins critical for continuity of intracellular vesicular trafficking by accelerating the replacement of ADP-ribosylation factor-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. Here, the participation of BIG2 in integrin β1 cycling through actin dynamics during cell migration was identified using small interfering RNA (siRNA) and difference gel electrophoresis analyses. After a 72-h incubation with BIG2 siRNA, levels of cytosolic Arp2, Arp3, cofilin-1, phosphocofilin, vinculin, and Grb2, known to be involved in the effects of integrin β1-extracellular matrix interactions on actin function and cell translocation, were increased. Treatment of HeLa cells with BIG2 siRNA resulted in perinuclear accumulation of integrin β1 and its delayed return to the cell surface. Motility of BIG2-depleted cells was simultaneously decreased, as were actin-based membrane protrusions and accumulations of Arp2, Arp3, cof ¡lin, and phosphocofilin at the leading edges of migrating cells, in wound-healing assays. Taken together, these data reveal a mechanism(s) through which BIG2 may coordinate actin cytoskel-eton mechanics and membrane traffic in cell migration via integrin β1 action and actin functions. [ABSTRACT FROM AUTHOR]

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations ("two-hit" cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Two-hit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal-epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin. [ABSTRACT FROM AUTHOR]

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)'[SUB2]-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB[SUB2]) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)'[SUB2]-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)'[SUB2]. This binding could interfere with the role of C3a and C5a in inflammation. [ABSTRACT FROM AUTHOR]

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg × LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of 125I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7α1, CYP27α1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions. [ABSTRACT FROM AUTHOR]

High throughput microarray analysis has great potential in scientific research, disease diagnosis, and drug discovery. A major hurdle toward high throughput microarray analysis is the time and effort needed to accurately locate gene spots in microarray images. An automatic microarray image processor will allow accurate and efficient determination of spot locations and sizes so that gene expression information can be reliably extracted in a high throughput manner. Current microarray image processing tools require intensive manual operations in addition to the input of grid parameters to correctly and accurately identify gene spots. This work developed a method, herein called auto-spot, to automate the spot identification process. Through a series of correlation and convolution operations, as well as pixel manipulations, this method makes spot identification an automatic and accurate process. Testing with real microarray images has demonstrated that this method is capable of automatically extracting subgrids from microarray images and determining spot locations and sizes within each subgrid, regardless of variations in array patterns and background noises. With this method, we are one step closer to the goal of high throughput microarray analysis.

Mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) regulate cell growth, protein synthesis, and apoptosis in response to nutrients and mitogens. As an important source of nitric oxide during inflammation, human inducible nitric oxide synthase also plays a role in the regulation of cytokine-driven cell proliferation and apoptosis. The role of mTOR and PI3K in the activation of human inducible nitric oxide synthase transcription by cytokines and lipopolysaccharide (LPS) was investigated in lung epithelial adenocarcinoma (A549) cells. LY294002, a dual mTOR and PI3K inhibitor, blocked human inducible nitric oxide synthase (hiNOS) promoter activation and mRNA induction by cytokines and LPS in a PI3K-independent fashion. On gene expression analysis, LY294002 selectively blocked the induction of a subset of 14 LPS/interferon-gamma (IFN-gamma)-induced genes, previously characterized as signal transducer and activator of transcription-1 (STAT1)-dependent. LY294002, but not wortmannin, inhibited LPS/IFN-gamma-dependent STAT1 phosphorylation at Ser-727 and STAT1 activity. Consistent with dual inhibition of mTOR and PI3K by LY294002, dominant-negative mTOR, anti-mTOR small interfering RNA, or rapamycin each inhibited phosphorylation of STAT1 only in the presence of wortmannin. LPS/IFN-gamma led to the formation of a macromolecular complex containing mTOR, STAT1, as well as protein kinase C delta, a known STAT1alpha kinase. Thus, LPS and IFN-gamma activate the PI3K and mTOR pathways, which converge to regulate STAT1-dependent transcription of pro-apoptotic and pro-inflammatory genes in a rapamycin-insensitive manner.