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Chemical investigation of Indonesian marine sponges Agelas linnaei and A. nakamurai afforded 24 alkaloid derivatives representing either bromopyrrole or diterpene alkaloids. A. linnaei yielded 16 bromopyrrole alkaloids including 11 new natural products with the latter exhibiting unusual functionalities. The new compounds include the first iodinated tyramine-unit bearing pyrrole alkaloids, agelanesins A-D. These compounds exhibited cytotoxic activity against L5178Y mouse lymphoma cells with IC(50) values between 9.25 and 16.76 muM. Further new compounds include taurine acid substituted bromopyrrole alkaloids and a new dibromophakellin derivative. A. nakamurai yielded eight alkaloids among them are three new natural products. The latter include the diterpene alkaloids (-)-agelasine D and its oxime derivative and the new bromopyrrole alkaloid longamide C. (-)-Agelasine D and its oxime derivative exhibited cytotoxicity against L5178Y mouse lymphoma cells (IC(50) 4.03 and 12.5 microM, respectively). Furthermore, both agelasine derivatives inhibited settling of larvae of Balanus improvisus in an anti-fouling bioassay and proved to be toxic to the larvae. (-)-Agelasine D inhibited the growth of planktonic forms of biofilm forming bacteria S. epidermidis (MIC<0.0877 microM) but did not inhibit biofilm formation whereas the oxime derivative showed the opposite activity profile and inhibited only biofilm formation but not bacterial growth. The structures of the isolated secondary metabolites were elucidated based on extensive spectroscopic analysis involving one- and two-dimensional NMR as well as mass spectrometry and comparison with literature data., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

The antifouling (AF) activity of 2-hydroxy-4-(3-hydroxy-5-methylphenoxy)- 6-methylbenozoic acid methyl ester (1), 3,5-dibromo-2-(2',4'-dibromophenoxy)phenol (2); 3,4,5-tribromo-2-(2',4'-dibromophenoxy)phenol (3), 3,4,5-tribromo-2-(2'-bromophenoxy)phenol (4), 3,5-dibromo-2(2',4'-dibromophenoxy)phenol (5), 3,4,5,6-tetrabromo-2-(2'-bromophenoxy)phenol (6); 4-phenoxyphenol (7), 4-phenoxyaniline (9), 1-chloro-4-phenoxybenzene (10); 1-bromo-4-phenoxybenzene (13) was investigated against marine bacteria, a diatom, barnacle larvae and mussel juveniles. The naturally occurring compound 2 showed the strongest AF activity in all bioassays but lacked toxicity. It inhibited the growth of all tested bacterial strains (MIC = 0.02 - 1.52 microM) and its 50% effective concentrations (EC(50)) were 0.24 microM (diatom test), 0.66 microM (mussel test) and 1.26 microM (barnacle test). Among the commercially available derivates, compound 7 was the most active in bacterial and diatom bioassays but its activity was lower than that of compound 2. Overall, the naturally occurring compounds showed stronger activity than the commercially available analogues and could be possible future non-toxic AF candidates.

Eighteen brominated sponge-derived metabolites and synthetic analogues were analyzed for antilarval settlement of Balanus improvisus. Only compounds exhibiting oxime substituents including bastadin-3 (4), -4 (1), -9 (2), and -16 (3), hemibastadin-1 (6), aplysamine-2 (5), and psammaplin A (10) turned out to inhibit larval settling at 1 to 10 microM. Analogues of hemibastadin-1 (6) were synthesized and tested for structure activity studies. Debromohemibastadin-1 (8) inhibited settling of B. improvisus, albeit at lower concentrations than hemibastadin-1 (6). Both 6 and 8 also induced cyprid mortality. 5,5'-dibromohemibastadin-1 (7) proved to be nontoxic, but settlement inhibition was observed at 10 microM. Tyrosinyltyramine (9), lacking the oxime function, was not antifouling active and was non-toxic at 100 microM. Hemibastadin-1 (6) and the synthetic products showed no general toxicity when tested against brine shrimp larvae. In contrast to the lipophilic psammaplin A (10), the hydrophilic sulfated psammaplin A derivative (11) showed no antifouling activity even though it contains an oxime group. We therefore hypothesize that the compound needs to cross membranes (probably by diffusion) and that the target for psammaplin A lies intracellularly.

Chromatographic separation of a crude extract obtained from the aerial parts of the Mongolian medicinal plant Scorzonera divaricata yielded the two new quinic acid derivatives feruloylpodospermic acids A and B. Both compounds feature a feruloyl group and two dihydrocaffeoyl substituents. For feruloylpodospermic acid A, the dihydrocaffeic acid substituents were found esterified at positions 1 and 5 of the quinic acid moiety, while the feruloyl group was attached at position 3. For feruloylpodospermic acid B, the substituents were linked at positions 1, 3, and 4. The aerial parts of S. pseudodivaricata that are likewise used in Mongolian traditional medicine yielded two further new natural products, for which the names scorzoneric acid and scorzonerin are proposed. Scorzoneric acid is an unusual phenolic compound featuring a central tetrasubstituted phenyl ring to which a glucose unit is bound, which in turn is substituted by an esterified acyl side chain. Further substituents of the central phenyl ring system include a butan-2-one group, which is linked to a second para-substituted phenyl ring system. Scorzonerin is a matricarin-based sesquiterpene lactone that carries an esterified dihydrocoumaric acid moiety, which in turn is glycosidically bound to glucose. The structures of all new compounds were unambiguously established from NMR (1H, 13C, COSY, HMBC) spectroscopic and mass spectrometric data. The new quinic acid derivatives feruloylpodospermic acids A and B exhibited strong antioxidative activity when analyzed in the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay.

E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells. A polymorphism, in which serine is substituted for arginine at position 128 (S128R) in the EGF domain, has been associated with both early-onset atherosclerosis and SLE. We investigated whether the substitution alters the ligand-binding properties of E-selectin under shear flow by studying the capacity of Chinese hamster ovary cell transfectants expressing wild type (WT) or S128R E-selectin to support interactions of neutrophils, K562 cells or HL60 cells. We initially chose to study non-fucosylated K562 cells. No interactions were observed on WT E-selectin, whereas S128R supported a transient tethering interaction of K562 cells, which was resistant to digestion with either neuraminidase or O-sialoglycoprotein endopeptidase, and, in turn, could result in firm adhesion in the presence of a beta2-integrin. HL60 cells exhibited increased rolling on S128R E-selectin. Although neuraminidase treatment inhibited all HL60 interactions with WT E-selectin, it unmasked transient tethers on S128R. We further observed that S128R recruited significantly more neutrophils than WT E-selectin, without affecting neutrophil rolling velocity. This polymorphism may therefore amplify leukocyte-endothelial cell interactions and may be a factor linking the S128R polymorphism to vascular disease.

The LW blood group glycoprotein, ICAM-4, is a member of the intercellular adhesion molecule (ICAM) family expressed in erythroid cells. To begin to address the function of this molecule, ligands for ICAM-4 on hemopoietic and nonhemopoietic cell lines were identified. Peptide inhibition studies suggest that adhesion of cell lines to an ICAM-4-Fc construct is mediated by an LDV-inhibitable integrin on hemopoietic cells and an RGD-inhibitable integrin on nonhemopoietic cells. Antibody inhibition studies identified the hemopoietic integrin as alpha(4)beta(1.) Antibody inhibition studies on alpha(4)beta(1)-negative, nonhemopoietic cell lines suggested that adhesion of these cells is mediated by alpha(V) integrins (notably alpha(V)beta(1) and alpha(V)beta(5)). The structure of ICAM-4 modeled on the crystal structure of ICAM-2 was used to identify surface-exposed amino acid residues for site-directed mutagenesis. Neither an unusual LETS nor an LDV motif in the first domain of ICAM-4 was critical for integrin binding. ICAM-4 is the first ICAM family member shown to be a ligand for integrins other than those of the beta(2) family, and the data suggest that ICAM-4 has a novel integrin-binding site(s). These findings suggest a role for ICAM-4 in normal erythropoiesis and may also be relevant to the adhesive interactions of sickle cells.

Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coli alpha-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the beta2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins.

Recent clinical trials suggest that blockade of integrins is a promising strategy for the treatment of acute coronary syndromes. Administration of 7E3 monoclonal antibody (mAb) Fab fragment (c7E3 Fab) directed against platelet integrin IIb/IIIa (alpha IIb beta 3, CD41/CD61) reduces acute ischemic complications of coronary angioplasty and clinical restenosis at 6 months. However, 7E3 mAb is not selective for platelet IIb/IIIa but also cross-reacts with the leukocyte integrin Mac-1 (alpha M beta 2, CD11b/CD18) and the vitronectin receptor (alpha v beta 3, CD51/CD61). Information regarding how this mAb may affect other cells important in vascular repair is scant. Potential interactions of c7E3 Fab with inflammatory (i.e., monocytes and neutrophils), vascular smooth muscle, and endothelial cells may contribute to the in vivo actions of c7E3 Fab. In this study we explored the binding of 7E3 to monocytic cells and the functional effect of 7E3 and c7E3 Fab on Mac-1-mediated adhesion to fibrinogen (FGN) and intercellular adhesion molecule-1 (ICAM-1), ligands abundant in the injured vessel wall. Flow cytometry demonstrated that 7E3 bound to THP-1 monocytic cells and identified a subpopulation (approximately 10%) of Mac-1 that was qualitatively similar to that recognized by CBRM1/5, a mAb directed to an activation-specific neoepitope present on a subset of Mac-1 molecules. mAb 7E3 bound to K562 cells transfected with just the alpha subunit (CD11b) of Mac-1 but not to nontransfected cells, confirming a direct interaction between 7E3 and Mac-1. mAb 7E3 and c7E3 Fab blocked the adhesion of Mac-1-bearing cells to FGN (80 +/- 11% and 78 +/- 9% inhibition, respectively) and ICAM-1 (62 +/- 14% and 62 +/- 17%). Both 7E3 and c7E3 Fab significantly inhibited (70 +/- 6% and 62 +/- 26%) soluble FGN binding to human peripheral blood monocytes. Thus, c7E3 Fab cross-reacts with the CD11b subunit of Mac-1 and interrupts cell-extracellular matrix and cell-cell adhesive interactions and may thereby influence the recruitment of circulating monocytes to sites of vessel injury. Given the recent evidence that adherent and infiltrating monocyte number directly correlates with the extent of neointimal hyperplasia, inhibition of Mac-1-dependent adhesion and IIb/IIIa-dependent function by c7E3 Fab may jointly contribute to the regulation of vascular repair and to the sustained clinical benefits observed with c7E3 Fab after angioplasty.

In this study we have compared the ligand binding activity of the two closely related beta 2 integrins, Mac-1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac-1 has previously been shown to associate with Fc gamma R, particularly Fc gamma RIII, but K562 cells express only endogenous Fc gamma RIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between Fc gamma RIIA with Mac-1 and p150,95 in the absence of other Fc gamma R. The main finding is that anti-Fc gamma RII mAb have a profound inhibitory effect on cell adhesion mediated by Mac-1, but not on the adhesion mediated by p150,95. Thus, in spite of the fact that Mac-1 and p150,95 bind to the same or at least a very similar selection of ligands, their association with other receptors on the cellular membrane, and therefore their mode of regulation may be different.

A human erythroleukemic cell line (K562) that does not normally express beta 2 integrins has been transfected with the genes encoding these integrins. The resulting cell lines show minimal background adhesion but can be stimulated to bind to appropriate substrates when activated with either of two different antibodies to CD18. The two antibodies appear to generate different ligand binding states in LFA-1 such that different members of the ICAM family are recognized. Antibody-activated complement receptor type 3 and p150,95-transfected cells bind protein-coated surfaces, although they require slightly different activation conditions for optimal binding.

Unactivated peripheral blood leukocytes show little tendency to bind to other cells or matrix components, whilst, in the presence of inflammatory mediators, adhesive interactions can rapidly increase. The Leu-CAM (beta 2 integrin) family of adhesion molecules have been shown to mediate a variety of these induced adhesion events. Here we describe a monoclonal antibody against CD18, KIM185, which stimulates JY cell homotypic aggregation by a CD11 a pathway as well as inducing the adherence of neutrophils to protein-coated plastic by a CD11b-dependent mechanism. The antibody recognizes an epitope distinct from the previously described KIM127 antibody and evidence is presented that the binding of KIM185 can cause a change in the conformation of the CD18 molecule.

It has been suggested that the V3 domain of human immunodeficiency virus type 1 (HIV-1) isolates has to interact with a cell-surface-associated or endosomal proteinase during virus entry into susceptible cells. To investigate this hypothesis, we examined the effect of several mutations in the V3 loop on its susceptibility to proteolytic cleavage by thrombin and cathepsin E and compared it with the effect of these mutations on viral infectivity. The data obtained indicate that, if an interaction between the V3 loop and a proteinase is indeed crucial for viral entry, the substrate requirements for such a proteinase(s) would have to be very complex. In particular, it seems unlikely that a single enzyme with a unique specificity would be able to interact with all of the different HIV-1 and HIV-2/SIV strains isolated so far. Therefore, one would have to postulate the involvement of several cellular proteinases, or proteases with multiple specificities, in V3-based viral tropism.

The Leukocytic cell-adhesion molecule (beta 2 integrin) family of adhesion molecules play a key role in the intercellular adhesive interactions necessary for normal immune cell function. In this study, we report an antibody that recognizes an epitope on the Leukocytic cell-adhesion molecule common beta-chain (CD18) and promotes both lymphocyte function-associated Ag-1- and CR3-dependent adhesion events. The antibody recognizes a temperature-sensitive epitope that is not dependent on the presence of divalent cations. It is proposed that antibody binding promotes a conformational change in both lymphocyte function-associated Ag-1 and CR3, which may mimic a natural activation mechanism, resulting in increased cellular adhesion.