Evaluating the detection efficacy of advanced bimetallic plasmonic nanoparticles for heavy metals, hazardous materials and pesticides of leachate in contaminated groundwater.

During the decomposition of trashes, leachate is created and leaching is gradually pollutes the surface and groundwater. Thus, the most severe ecological impact is the risk of ground water pollution because of collection of leachate from unlined insecure landfills. Due to the low biodegradable organic strength, irregular productivity and composition, the environmentally neglected landfill leachate treatment is challenging. This work was conducted on a synthetically effective bimetallic surface enhanced Raman spectroscopic (SERS) nanosensor by gold/silver-bimetallic nanoparticles (Au/Ag-NPs), and used for the specific detection of municipal solid waste (MSW) landfill leachate in groundwater. The optical study of Au/Ag-NPs led to reflections from Ag cores and small Au shells. The structural studies represent the FCC structure of Au/Ag-NPs. The core-shell nanocrevice NPs with particle size of 23 nm played an important role with plasmonic behaviour enhances the electromagnetic excitation to achieve SERS detection and plasmonic photocatalysis. Thus, obtained results clearly show that Au was successfully added to Ag-NPs, and its existence can also be confirmed by energy dispersive spectroscopy (EDAX). The prepared SERS based sensors have the potential to detect aromatic hydrocarbon, pesticides and heavy metals from environmentally ignored MSW landfill leachate. In general, the application of this new synergetic strategy of the photocatalytic degradation of leachate was irradiated by visible wavelength with the rate constant of 0.0036/min, 0.0047/min and 0.005/min by Ag-NPs, Au-NPs and Au/Ag-NPs respectively. Overall, this is the only study achieved efficiently with photocatalytic degradation and SERS detection of environmentally ignored real sample (leachate) to make pollutant free homeland aquifers.

Surface Enhanced Raman Spectroscopic investigations of 2-bromo-3-methylamino-1,4-naphthoquinone on silver nanoparticles.

Surface Enhanced Raman Spectroscopic technique has been employed to investigate the orientation of 2-bromo-3-methylamino-1,4-naphthoquinone (BMANQ) on silver nanoparticles. Silver nanoparticles have been prepared by solution combustion method with citric acid as fuel. Silver nanoparticles were characterized by X-ray Diffraction (XRD), High Resolution Transmission Electron Microscopy (HRTEM) and Scanning Electron Microscopy (SEM). XRD and morphological results confirmed the nanocrystalline nature of the prepared silver nanoparticles. The observed intense CO stretching, CBr stretching and NH2 vibration suggests that the BMANQ molecule may be adsorbed in a 'stand-on' orientation to the silver surface. The calculated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy show that charge transfer occurs within the molecule.

Surface enhanced Raman spectral studies of 2-bromo-1,4-naphthoquinone.

Silver nanoparticles have been synthesized by a simple and inexpensive solution combustion method with urea as fuel. The structural and morphology of the silver nanoparticles were investigated through X-ray powder diffraction (XRD), Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersion Spectra (EDS) techniques. Structural and morphological results confirmed the nanocrystalline nature of the silver nanoparticles. Density Functional Theory (DFT) calculations were also performed to study the ground and excited state behavior of 2-bromo-1,4-naphthoquinone (2-BrNQ) and 2-BrNQ on silver nanoparticles. Surface-Enhanced Raman Scattering (SERS) spectra of 2-BrNQ adsorbed on silver nanoparticles were investigated. The CO, CH in-plane bending and CBr stretching modes were enhanced in SERS spectrum with respect to normal Raman spectrum. The spectral analysis reveals that the 2-BrNQ adsorbed 'stand-on' orientation on the silver surface. Density Functional Theory (DFT) calculations are also performed to study the vibrational features of 2-BrNQ molecule and 2-BrNQ molecule on silver surface.

Spectroscopic investigations on the orientation of 1,4-dibromonaphthalene on silver nanoparticles.

Silver nanoparticles (Ag NPs) have been prepared by solution combustion method with glycine as fuel. Silver nanoparticles were characterized by X-Ray Diffraction (XRD), High Resolution Transmission Electron Microscopy (HRTEM) and UV-visible spectroscopy. The prepared silver nanoparticles exhibit cubic crystalline structure with grain size of 59 nm. HRTEM image shows that the silver nanoparticles have strain and four-fold symmetry formed by twinning in the crystal structure. The optical adsorption spectrum shows that the surface plasmon resonance peak of silver is observed at 380 nm. The orientation of 1,4-dibromonaphthlaene (1,4-DBrN) on silver nanoparticles has been inferred from nRs and SERS spectral features. The absence of a C-H stretching vibrations, the observed high intense C-H out-of-plane bending modes and high intense C-Br stretching vibration suggest that the 1,4-DBrN molecule may be adsorbed in a 'stand-on' orientation to the surface.

Identification and initial characterization of 5000 expressed sequenced tags (ESTs) each from adult human normal and osteoarthritic cartilage cDNA libraries.

OBJECTIVE: To prepare, sequence and analyse adult human cartilage cDNA libraries to study the gene expression pattern between normal and osteoarthritic cartilage. METHODS: Poly A(+)RNA from adult human normal and osteoarthritic articular cartilage was isolated and used to prepare cDNA libraries. Approximately 5000 ESTs from each library were sequenced and analysed using bioinformatic tools. The expression of select genes was confirmed by Northern blot and in situ hybridization analysis. RESULTS: Multiple gene families including several classical cartilage matrix protein encoding genes were identified. Approximately 28-40% of the genes sequenced from these libraries were novel, while half of the genes encoded known proteins and 4-6% of the genes encoded novel homologs of known proteins. Several known genes, whose expression has not been reported previously in cartilage, were also identified. We have confirmed the cartilage expression of three known (CTGF, CTGF-L and clusterin) and two novel homologs of known genes (PCPE-2 and Gal-Nac transferase) by Northern blot and in situ hybridization analysis. CONCLUSION: This is the first report of the preparation and sequencing of cDNA libraries from adult human normal and osteoarthritic articular cartilage. Further analysis of genes identified from these libraries may provide molecular targets for diagnosis and/or treatment of osteoarthritis (OA).

Oligonucleotide-mediated, PCR-independent cloning by homologous recombination.

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).

Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir.

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv(r) clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv(r) mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.

Rapid isolation of tissue-specific genes from rat kidney.

A systematic effort to isolate kidney-specific genes was performed using recently described PCR-select methodology. Using this technique, a kidney-specific mini-gene library was generated and a number of kidney-specific genes that share significant homology to previously characterized kidney genes from rats and other species were isolated. These included three renal-specific transporters (an ADH water channel, the anion transporters RST and ROAT1), a cell adhesion molecule (K-cadherin) and a kidney-specific protein upregulated in renal carcinoma (DD96). In addition, we isolated two novel genes from a rat kidney. One of the genes shares limited homology to rat profilin-1 while the other did not share any similarity to genes in the Genbank. Northern blot analysis revealed that the mRNA for each of these genes is expressed in a highly kidney-restricted fashion. Our results suggested that tissue-specific genes can be rapidly isolated and characterized using PCR-select techniques and this methodology may be generally applicable to isolate specific genes from a variety of tissues.

Clusterin expression in adult human normal and osteoarthritic articular cartilage.

OBJECTIVE: To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage. METHODS: Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization. RESULTS: Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. CONCLUSION: The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.

Identification of a novel kidney-specific gene downregulated in acute ischemic renal failure.

To gain further insights into the molecular mechanisms involved in acute renal failure, we have isolated a new gene from rat and human, named KSP32 (kidney-specific protein with a molecular mass of 32 kDa). KSP32 encodes a novel gene that shows little homology to other mammalian proteins. It, however, shares extensive homology with several proteins found in the nematode Caenorhabditis elegans and plants. The expression of KSP32 mRNA is highly restricted to kidney. In situ hybidization analysis revealed that the expression of KSP32 mRNA was prominent in the boundary of kidney cortex and outer medulla, exhibiting a raylike formation extending from the medulla into the cortex. Finally, KSP32 mRNA was dramatically downregulated in rat following induction of acute ischemic renal failure. Rapid loss of KSP32 mRNA expression was observed beginning at approximately 5 h following renal injury and mRNA levels remained depressed for at least 96 h. Both KSP32 mRNA levels as well as renal function recovered 14 days after injury. Administration of an endothelin receptor antagonist (SB-209670), known to restore renal function, significantly increased KSP32 expression.

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.

Identification, molecular cloning, expression, and characterization of a cysteinyl leukotriene receptor.

The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.

hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome.

Like other members of the Flaviviridae family, the 3' non-translated region (NTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication by interacting with components of the viral replicase complex. To inves-tigate the possibility that host components may also participate in this process, we used UV cross-linking assays to determine if any cellular proteins could bind specifically to the 3'NTR RNA. We demonstrate the specific interaction of two host proteins with the extensive pyrimidine-rich region within the HCV 3'NTR. One host protein migrates as a doublet with a molecular weight of 57 kDa and is immunoreactive with antisera specific for polypyrimidine tract-binding protein (PTB), and the other protein (35 kDa) is recognized by a monoclonal antibody specific for heterogeneous nuclear ribonucleoprotein C (hnRNP C). These results suggest that recognition of the large pyrimidine-rich region by PTB and hnRNP C may play a role in the initiation and/or regulation of HCV RNA replication.

The coiled coil stem loop miniprotein as a presentation scaffold.

The coiled coil helical dimer found in naturally occurring proteins is conformationally stable and can tolerate significant sequence variation on the solvent-exposed surfaces of the helices. We are interested in exploring the use of a de novo designed coiled coil stem loop miniprotein (CCSL) as a template for presenting (1) helical and loop sequences from heterologous proteins and (2) constrained libraries of peptides. Towards this end, we synthesized a 56 residue prototype CCSL and verified its structure by extensive biophysical characterization. CCSL variants with altered sequences in the solvent-exposed helical positions were found to fold similarly to the prototype design. Based on the results with the CCSL produced by peptide synthesis, we assembled a synthetic cDNA for the CCSL prototype and expressed the CCSL miniprotein on filamentous phage. This genetic construction can be used to introduce random peptide libraries into regions within the scaffold loop and helices in order to identify key side chains of native proteins involved in binding, to establish structural models for their pharmacophores and to identify novel peptide recognition mimics of macromolecular ligands and their receptors.

Type I IL-1 receptor: ligand specific confirmation differences and the role of glycosylation in ligand binding.

The extracellular domain of the type I Interleukin-1 receptor (sIL-1R) was expressed in Drosophila S2 cells as a secreted 43 kDa glycoprotein, as evidenced by its binding to Concanavalin A and enzymatic deglycosylation. sIL-1R bound IL-1 beta with a K(D) of 2 nM as determined by competition ELISA. N-Glycanase treated sIL-1R had a C. 100 fold lower affinity than glycosylated sIL-1R for IL-1 beta, suggesting that glycosylation is a key component of the IL-1 beta/IL-1 receptor interaction. Crosslinking of sIL-1R to (125)I-IL-1 beta could be competed with unlabelled IL-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), and a mutant of IL-1 (Th9Gly) which has reduced bioactivity but wild type receptor binding affinity. Limited proteolysis of sIL-1R in the presence of IL-1 alpha, IL-1 beta, IL-1ra, and Thr9Gly IL-1 beta with several different proteases followed by analysis of sIL-1R by Western blot was used to assess the effect of binding on sIL-1R conformation. While some proteases showed no differences in cleavage patterns or sensitivity between free and bound sIL-1R, others showed differences in either cleavage sites or sensitivity with different ligands. This implies that upon ligand binding there is a conformational change in the receptor which is sensitive to the particular ligand bound, and hence has implications for the ability of different ligands to trigger responses after binding to receptor.

Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.

Cloning of the galactokinase cDNA and identification of mutations in two families with cataracts.

Galactokinase is an essential enzyme for the metabolism of galactose and its deficiency causes congenital cataracts during infancy and presenile cataracts in the adult population. We have cloned the human galactokinase cDNA, which maps to chromosome 17q24, and show that the isolated cDNA expresses galactokinase activity in bacteria and mammalian cells. We also describe two different mutations in this gene in unrelated families with galactokinase deficiency and cataracts. The availability of the cloned galactokinase gene provides an important reference to identify mutations in patients with galactokinase deficiency and cataracts.

In vitro antibody maturation. Improvement of a high affinity, neutralizing antibody against IL-1 beta.

Abs as therapeutic antagonists should be of relatively high affinity to effectively neutralize their target Ag. Typically, Abs with nanomolar affinities to protein Ags can be obtained in vivo, however, this may not be the upper limit of affinity because the biologic process attempts to optimize Ab function, of which affinity is only one component. SK48, a high affinity neutralizing murine Fab against human IL-1 beta was used to explore the nature of Ab-Ag interactions and the potential for further affinity improvement in vitro using mutagenesis and selection via phage display. The codons of six amino acids in the third complementarity-determining region of the heavy chain (CDR3-H) were both individually and combinatorialy randomized and the resultant libraries were screened for IL-1 binding phenotype. Mutations that reduced affinity suggested that both the backbone conformation of the CDR3 loop and certain side chains are essential for binding, yet alterations to the canonical salt bridge residues at the base of the loop had minimal effect on affinity. Four rounds of selection of the phage Ab libraries on immobilized IL-1 beta gave predominantly the wild-type sequence, indicating efficient affinity maturation of this CDR in vivo. However, a twofold improvement in affinity was observed for both single and double amino acid changes at two positions in the middle of the CDR. Moreover, a 10-fold increase in affinity for IL-1 beta was achieved by combining two of the phage-selected single amino acid substitutions in CDR3-H, thereby demonstrating that a significant improvement in affinity can be achieved through CDR mutagenesis, even in a matured Ab. The increased affinity of this Fab did not, however, enhance its neutralizing activity in vitro.

Suppression of monocyte 85-kDa phospholipase A2 by antisense and effects on endotoxin-induced prostaglandin biosynthesis.

Studies were conducted to characterize a human monocyte model where the role of the 85-kDa phospholipase A2 (PLA2) in prostanoid formation could be evaluated. The presence of an immunologically related 85-kDa PLA2 and type II 14-kDa PLA2 was demonstrated in human monocytes and their roles examined in lipopolysaccharide (LPS)-induced monocyte prostaglandin E2 (PGE2) formation. Exposure of human monocytes to LPS over 18 h resulted in the up-regulation of the mitogen-inducible cyclooxygenase-2 and was accompanied by production and release of prostaglandin E2 but not leukotriene C4. This coincided with a 2-fold increase in the 85-kDa PLA2 protein and activity levels. In contrast, there was no effect on the type II 14-kDa-like PLA2 activity measured in the 100,000 x g particulate fraction nor did LPS induce the release of type II 14-kDa PLA2 into the medium. Treatment with cycloheximide over 18 h resulted in a time-dependent decrease in cytosolic 85-kDa PLA2 protein and activity (half-life = 4 h), but there was no change in the particulate type II 14-kDa-like PLA2 activity. Monocytes were therefore exposed to an 85-kDa PLA2 initiation site-directed antisense oligonucleotide which specifically decreased the cytosolic 85-kDa PLA2 protein levels and activity in a concentration-dependent manner. This had no effect on the cyclooxygenase-2 (protein mass or the ability to convert arachidonic acid to PGE2) or the particulate fraction sn-2 acylhydrolytic activity but was associated with a decrease in LPS-induced PGE2 production. Taken together, these data support a role for the cytosolic 85-kDa PLA2 in LPS-induced monocyte PGE2 formation.

Differentiation between binding sites for angiotensin II and nonpeptide antagonists on the angiotensin II type 1 receptors.

To characterize binding sites for nonpeptide angiotensin antagonists on the human angiotensin II receptor type 1 (AT1 receptor) we have systematically exchanged segments of the human receptor with corresponding segments from a homologous Xenopus laevis receptor, which does not bind the nonpeptide compounds. Substitution of transmembrane segment VII of the human AT1 receptor dramatically reduced the binding affinity of all of the 11 nonpeptide antagonists tested (55- to > 2000-fold) with no effect on the binding of angiotensin. The affinity for the nonpeptide compounds decreased additionally one order of magnitude when transmembrane segment VI and the connecting extracellular loop 3 from the Xenopus receptor were also introduced into the human AT1 receptor. Exchanges of smaller segments and single residues in transmembrane segments VI and VII and extracellular loop 3 revealed that the binding of nonpeptide antagonists was dependent on nonconserved residues located deep within the transmembrane segments VI and VII, in particular Asn295 in transmembrane segment VII. Surprisingly, all exchanges in transmembrane segment VII, including the Asn295 to Ser substitution, had a more pronounced effect on the binding of the competitive antagonists relative to the insurmountable antagonists. It is concluded that the binding mode for peptide and nonpeptide ligands on the AT1 receptor is rather different and that competitive and insurmountable antagonists presumably bind to overlapping but distinct sites located in transmembrane segments VI and VII.