AT(1) receptor ligands: virtual-screening-based design with TOPP descriptors, synthesis, and biological evaluation of pyrrolidine derivatives

As a continuing effort to establish the structure-activity relationships (SARs) within the series of the angiotensin II antagonists (sartans), a pharmacophoric model was built by using novel TOPP 3D descriptors. Statistical values were satisfactory (PC4: r(2)=0.96, q(2) ((5) (random) (groups))=0.84; SDEP=0.26) and encouraged the synthesis and consequent biological evaluation of a series of new pyrrolidine derivatives. SAR together with a combined 3D quantitative SAR and high-throughput virtual screening showed that the newly synthesized 1-acyl-N-(biphenyl-4-ylmethyl)pyrrolidine-2-carboxamides may represent an interesting starting point for the design of new antihypertensive agents. In particular, biological tests performed on CHO-hAT(1) cells stably expressing the human AT(1) receptor showed that the length of the acyl chain is crucial for the receptor interaction and that the valeric chain is the optimal one.

Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) A1 from human macrophage foam cells

High circulating levels of triglyceride-rich lipoproteins (TGRL) represent an independent risk factor for coronary artery disease. Here, we show that TGRL inhibit the efflux of cholesterol from 'foam cell' macrophages to lipid-poor apolipoprotein (apo) A1, and may thereby inhibit arterial reverse cholesterol transport and promote the formation of atherosclerotic lesions. Human (THP-1) monocyte-derived macrophages were pre-incubated (48h) with acetylated low-density lipoprotein (AcLDL) to provide a foam cell model of cholesterol efflux to apoA1. Pre-incubation of macrophage 'foam cells' with TGRL (0-200 mug/ml, 0-24 h) inhibited the efflux of exogenously radiolabelled ([H-3]), endogenously synthesised ([C-14]) and cellular cholesterol mass to lipid-poor apoA1, but not control medium, during a (subsequent) efflux period. This inhibition is dependent upon the length of prior exposure to, and concentration of, TGRL employed, but is independent of changes in intracellular triglyceride accumulation or turnover of the cholesteryl ester pool. Despite the negative impact of TGRL on cholesterol efflux, major proteins involved in this process-namely apoE, ABCA1, SR-B1 and caveolin-1-were unaffected by TGRL pre-incubation, suggesting that exposure to these lipoproteins inhibits an alternate, and possibly novel, anti-atherogenic pathway. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2 to induce inflammation and hyperalgesia

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased [Ca(2+)](i) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca(2+)](i) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.

Modulation of hTREK-1 by carbon monoxide

TREK-1 is a background K channel important in the regulation of neuronal excitability. Here, we demonstrate that recombinant human TREK-1 is activated by low concentrations of carbon monoxide (CO) and nitric oxide (NO), applied via their respective donor molecules. Related channels hTASK-1 and hTASK-3 were unaffected by CO. Effects of both CO and NO were prevented by preincubation of cells with the protein kinase G inhibitor, Rp-8-Br-PET-cGMPS. The effects of CO were independent of NO formation. At higher concentrations, both NO and CO were inhibitory. As both NO and CO are important neuronal gasotransmitters and TREK is crucial in regulating neuronal excitability, our results provide a novel means by which these gases may modulate neuronal activity.

Hemokinins and endokinins

The mammalian tachykinins are a family of peptides that, until recently, has included substance P (SP), neurokinin A and neurokinin B. Since, the discovery of a third preprotachykinin gene (TAC4), the number of tachykinins has more than doubled to reveal several species-divergent peptides. This group includes hemokinin-1 (HK-1) in mouse and rat, endokinin-1 (EK-1) in rabbit, and EKA, EKB, human HK-1 (hHK-1) and hHK(4-11) in humans. Each exhibits a remarkable selectivity and potency for the tachykinin NK1 receptor similar to SP. Their peripheral expression has led to the proposal that they are the endogenous peripheral SP-like endocrine/paracrine agonists where SP is not expressed. Moreover, their strong cross-reactivity with a specific SP antibody leads us to question many of the proposed locations and roles of SP in the periphery. Additionally, three orphan tachykinin gene-related peptides are identified on TAC4, in rabbit, EK-2 and in humans, EKC and EKD.

Invertebrate food supply and breeding success of Mallards Anas platyrhynchos at flooded gravel quarries in southern Britain

Reproductive parameters of Mallard Anas platyrhynchos were investigated in relation to vegetative development, physical characteristics and invertebrate food abundance of a complex of flooded gravel quarries in southern Britain. Breeding pair density over the study area was particularly high with 2.2 pairs km(-1) of shoreline. Across all lakes breeding success was limited to an average of 0.9 ducklings fledged pair(-1), yet an associated study showed that very little of the mortality could be attributed to predation. The results of stepwise multiple regressions indicated that the availability of emerging insects was important in determining the breeding density and breeding success of Mallards rather than habitat structure. We suggest that the overall great availability of suitable nest-sites combined with the variable but unmeasured stocking densities of fish may explain the lack of the latter correlations.

Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Effect of low extracellular pH on NF-κB activation in macrophages

Objective: Many diseases, including atherosclerosis, involve chronic inflammation. The master transcription factor for inflammation is NF-κB. Inflammatory sites have a low extracellular pH. Our objective was to demonstrate the effect of pH on NF-κB activation and cytokine secretion. Methods: Mouse J774 macrophages or human THP-1 or monocyte-derived macrophages were incubated at pH 7.0–7.4 and inflammatory cytokine secretion and NF-κB activity were measured. Results: A pH of 7.0 greatly decreased pro-inflammatory cytokine secretion (TNF or IL-6) by J774 macrophages, but not THP-1 or human monocyte-derived macrophages. Upon stimulation of mouse macrophages, the levels of IκBα, which inhibits NF-κB, fell but low pH prevented its later increase, which normally restores the baseline activity of NF-κB, even though the levels of mRNA for IκBα were increased. pH 7.0 greatly increased and prolonged NF-κB binding to its consensus promoter sequence, especially the anti-inflammatory p50:p50 homodimers. Human p50 was overexpressed using adenovirus in THP-1 macrophages and monocyte-derived macrophages to see if it would confer pH sensitivity to NF-κB activity in human cells. Overexpression of p50 increased p50:p50 DNA-binding and in THP-1 macrophages inhibited considerably TNF and IL-6 secretion, but there was still no effect of pH on p50:p50 DNA binding or cytokine secretion. Conclusion: A modest decrease in pH can sometimes have marked effects on NF-κB activation and cytokine secretion and might be one reason to explain why mice normally develop less atherosclerosis than do humans.

A Linguistic Analysis of Spanglish: Relating Language to Identity.

According to the 2000 census, 35.3 million Hispanics live in the United States. This number comprises 12.5% of the overall population rendering the Latino community the largest minority in the United States. The Mexican community is not only the largest Hispanic group but also the fastest growing: from 1990 to 2000, the Mexican population grew 52.9% increasing from 13.5 million to 20.6 million (U.S. Department of Commerce News, 2001). The influx of Mexican immigrants coupled with the expansion of their community within the United States has created an unparalleled situation of language contact. Language is synonymous with identity (cf. Granger, 2004, and works cited within). To the extent that this is true, Spanish is synonymous with being Mexican and by extension, Chicano. With the advent of amnesty programs such as Immigration Reform and Control Act (IRCA), which naturalized millions of Mexican migrants, what was once a temporal migratory population has become increasingly permanent (Durand et al., 1999). In an effort to conserve Mexican traditions and identity, the struggle to preserve the mother tongue while at the same time acculturate to mainstream Americana has resulted in a variant of Spanglish that has received little attention. This paper will examine the variant of Spanglish seen in the greater Los Angeles area and liken it to the bi-national identity under which these Mexican Americans thrive.

Mapping the immune response to the outer domain of a human immunodeficiency virus-1 clade C gp120

The outer domain (OD) of human immunodeficiency virus (HIV)-1 gp120 represents an attractive, if difficult, target for a beneficial immune response to HIV infection. Unlike the entire gp120, the OD is structurally stable and contains the surfaces that interact with both the primary and secondary cellular receptors. The primary strain-specific neutralizing target, the V3 loop, lies within the OD, as do epitopes for two cross-reactive neutralizing monoclonal antibodies (mAbs), b12 and 2G12, and the contact sites for a number of inhibitory lectins. The OD is poorly immunogenic, at least in the context of complete gp120, but purposeful OD immunization can lead to a substantial antibody response. Here, we map the antibody generated following immunization with a clade C OD. In contrast to published data for the clade B OD, the majority of the polyclonal response to the complete clade C OD is to the V3 loop; deletion of the loop substantially reduces immunogenicity. When the loop sequence was substituted for the epitope for 2F5, a well-characterized human cross-neutralizing mAb, a polyclonal response to the epitope was generated. A panel of mAbs against the clade C OD identified two mAbs that reacted with the loop and were neutralizing for clade C but not B isolates. Other mAbs recognized both linear and conformational epitopes in the OD. We conclude that, as for complete gp120, V3 immunodominance is a property of OD immunogens, that the responses can be neutralizing and that it could be exploited for the presentation of other epitopes.

Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2

Oxidized low-density lipoproteins (LDL) play a central role in atherogenesis and induce expression of the antioxidant stress protein heme oxygenase 1 (HO-1). In the present study we investigated induction of HO-1 and adaptive increases in reduced glutathione (GSH) in human aortic smooth muscle cells (SMC) in response to moderately oxidized LDL (moxLDL, 100 mu g protein/ml, 24 h), a species containing high levels of lipid hydroperoxides. Expression and activity of HO-1 and GSH levels were elevated to a greater extent by moxLDL than highly oxidized LDL but unaffected by native or acetylated LDL. Inhibitors of protein kinase C (PKC) or mitogen-activated protein kinases (MAPK) p38(MAPK) and MEK or c-jun-NH2-terminal kinase (JNK) significantly attenuated induction of HO-1. Phosphorylation of p38(MAPK), extracellular signal-regulated kinase (ERK1/2), or JNK and nuclear translocation of the transcription factor Nrf2 were enhanced following acute exposure of SMC to rnoxLDL (100 mu g proteiri/ml, 1-2 h). Pretreatment of SMC with the antioxidant vitamin C (100 mu M, 24 h) attenuated the induction of HO-1 by moxLDL. Native and oxidized LDL did not alter basal levels of intracellular ATP, mitochondrial dehydrogenase activity, or expression of the lectin-like oxidized LDL receptor (LOX-1) in SMC. These findings demonstrate for the first time that activation of PKC, p38(MAPK), JNK, ERK1/2, and Nrf2 by oxidized LDL in human SMC leads to HO-1 induction, constituting an adaptive response against oxidative injury that can be ameliorated by vitamin C. (C) 2005 Elsevier Inc. All rights reserved.

Domain swapping in the human histamine H-1 receptor

G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H-1 receptor. We also show the presence of domain-swapped H-1 receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate(107) in transmembrane (TM) 3 or phenylalanine(432) in TM6 to alanine results in two radioligand-binding-deficient mutant H-1 receptors. Coexpression of H-1 D(107)A and H-1 F(432)A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wildtype H-1 receptor. Interestingly, the H-1 receptor radioligands [H-3] mepyramine and [H-3]-(-)- trans-1-phenyl-3-N, N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B-max) for wild-type H-1 receptors but not for the radioligand binding site that is formed upon coexpression of H-1 D(107)A and H-1 F(432)A receptors, suggesting the presence of different H-1 receptor populations.

Regulation of SHP-1 tyrosine phosphatase in human platelets by serine phosphorylation at its C terminus

SHP-1 is a Src homology 2 (SH2) domain-containing tyrosine phosphatase that plays an essential role in negative regulation of immune cell activity. We describe here a new model for regulation of SHP-1 involving phosphorylation of its C-terminal Ser(591) by associated protein kinase Calpha. In human platelets, SHP-1 was found to constitutively associate with its substrate Vav1 and, through its SH2 domains, with protein kinase Calpha. Upon activation of either PAR1 or PAR4 thrombin receptors, the association between the three proteins was retained, and Vav1 became phosphorylated on tyrosine and SHP-1 became phosphorylated on Ser(591). Phosphorylation of SHP-1 was mediated by protein kinase C and negatively regulated the activity of SHP-1 as demonstrated by a decrease in the in vitro ability of SHP-1 to dephosphorylate Vav1 on tyrosine. Protein kinase Calpha therefore critically and negatively regulates SHP-1 function, forming part of a mechanism to retain SHP-1 in a basal active state through interaction with its SH2 domains, and phosphorylating its C-terminal Ser(591) upon cellular activation leading to inhibition of SHP-1 activity and an increase in the tyrosine phosphorylation status of its substrates.

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha(2)beta(1)

Background: Activation of the platelet integrin alpha(2)beta(1) is closely regulated due to the high thrombogenicity of its ligand. As a beta(1) interacting kinase, ILK represents a candidate intracellular regulator of alpha(2)beta(1) in human platelets. Objectives We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha(2)beta(1) activation in HEL cells, a megakaryocytic cell line. Methods: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta(1)-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha(2)beta(1) function assessed by overexpression studies in HEL cells. Results: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta(1)-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha(2)beta(1)-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha(2)beta(1). Conclusions: Our findings that ILK regulates alpha(2)beta(1) in HEL cells, is activated in platelets and associates with beta(1)-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.