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This review article that complements the previous review article on 'The discovery of antiviral agents: ten different compounds, ten different stories' presents ten more compounds and ten more stories in which I have been closely involved at one or another point of my scientific career: (i) interferon (IFN) (in particular, IFN-β); (ii) poly(I).poly(C); (iii) suramin; (iv) novel acyclic nucleoside phosphonates; (v) the double prodrug of [9-(2-phosphonomethoxyethyl)guanine]; (vi) cyclic nucleoside phosphonates; (vii) picornavirus inhibitors; (viii) human immunodeficiency virus (HIV) co-receptor inhibitors; (ix) nonimmunosuppressive cyclosporin A analogues; and (x) bicyclic (furanopyrimidine) nucleoside analogues. With the exception of the HIV co-receptor CCR5 inhibitor none of the compounds described here have already been marketed (for the indication they were initially developed). Successful antiviral drug development depends on the interplay of three disciplines, chemistry, biology/medicine, and industry, crucial factors being open mindedness for the unexpected, preparedness to explore serendipitous observations, and perseverance (in trying) to overcome the hurdles or setbacks inevitably compounding any drug development. © 2009 Wiley Periodicals, Inc. Med Res Rev [ABSTRACT FROM AUTHOR]

ErbB4 isoforms mediate different cellular activities depending on their susceptibility to proteolytic cleavage. The biological significance of ErbB4 cleavage in tumorigenesis, however, remains poorly understood. Here, we describe characterization of a monoclonal antibody (mAb 1479) that selectively recognizes the ectodomain of cleavable ErbB4 JM-a isoforms both in vitro and in vivo. mAb 1479 was used to analyse ErbB4 JM-a expression and ectodomain shedding in a series of 17 matched breast cancer/histologically normal peripheral breast tissue pairs. ErbB4 ectodomain was observed in 75% of tumors expressing ErbB4 but only in 18% of normal breast tissue samples expressing ErbB4. Difference in the relative quantity of ErbB4 ectodomain between normal and tumor tissue pairs was statistically significant (P=0.015). Treatment with mAb 1479 suppressed ErbB4 function by inhibiting ErbB4 tyrosine phosphorylation and ectodomain shedding, and by stimulating ErbB4 downregulation and ubiquitination. mAb 1479 suppressed both anchorage-dependent and -independent growth of human breast cancer cell lines that naturally express cleavable ErbB4 JM-a. These findings indicate that ErbB4 ectodomain shedding is enhanced in breast cancer tissue in vivo, and that mAb 1479 represents a potential drug candidate that suppresses breast cancer cell growth by selectively binding cleavable ErbB4 isoforms.Oncogene (2009) 28, 1309–1319; doi:10.1038/onc.2008.481; published online 19 January 2009 [ABSTRACT FROM AUTHOR]

Aims: The association between tissue factor (TF) expression and increased rate of tumour metastasis is well established. In this study, we have examined the hypothesis that the expression of TF by disseminated tumour cells confers protection against immune recognition and cytotoxicity. Materials and methods: A hybrid EGFP-TF protein was expressed in HT29 colon carcinoma and K562 lymphoblast cell lines. To assess the cytotoxic activity against tumour cells over-expressing TF, a novel method was used, based on the direct measurement of fluorescently labelled HT29 or K562 target cells. Results: Upon challenge with peripheral blood mononuclear cells (PBMC), tumour cells expressing TF partially evaded cellular cytotoxicity (Δ=15–40% reduction in cytotoxicity). Moreover, the influence of TF was not primarily dependent on its procoagulant function, although the inclusion of 20% (v/v) plasma did lower the rate of cytotoxicity against untransfected cells. However, expression of a truncated form of TF, devoid of the cytoplasmic domain, did not mediate any degree of inhibition of cytotoxicity, suggesting that the protective function of TF is principally due to this domain. Conclusions: We conclude that TF can promote immune evasion in tumour cells expressing this protein leading to increased survival and therefore metastatic rate in such cells. [ABSTRACT FROM AUTHOR]

The article presents a study on the role of sodium (Na) and potassium (K) in enzyme function. It examines the diversity of monovalent cation-activated enzymes and discusses its theoretical and practical aspects. A derivation of the kinetic expressions for the analysis of cofactor (type I) and allosteric effector (type II) activation is offered. The article also discusses the implications on the molecular evolution of monovalent cation-activated enzymes in biochemistry and protein engineering.

The enzymatic activity of acetylcholinesterase (AChE) has been shown to be altered by environmental contaminants such as metals. However, the available literature illustrates a background of contradictory results regarding these effects. Therefore, the main purpose of this study was to investigate the potential of five metal ions (nickel, copper, zinc, cadmium and mercury) to inhibit AChE activity in vitro . First, to accomplish this objective, the possible interference of metals as test toxicants in the Ellman's assay, which is widely used to assess AChE activity, was studied. The potential influence of two different reaction buffers (phosphate and Tris) was also determined. The results suggest that the selected metals react with the products of this photometric technique. It is impossible to ascertain the artefactual contribution of the interaction of the metals with the technique when measuring AChE inhibition. This constitutes a major obstacle in obtaining accurate data. The presence of phosphate ions also makes enzymatic inhibition difficult to analyse. Attending to this evidence, an assay using the substrate o-nitrophenyl acetate and Tris buffer was used to investigate the effects of metals on AChE activity. O-nitrophenyl acetate is also a substrate for esterases other than cholinesterases. It is therefore only possible to use it for the measurement of cholinesterase activity with purified enzymes or after a previous verification of the absence of other esterases in the sample tissue. Under these conditions, the results indicate that with the exception of nickel, all tested metals significantly inhibit AChE activity. [ABSTRACT FROM AUTHOR]

Tissue factor (TF) is the most important trigger of blood coagulation in vascular pathology. Rabbit TF, with or without (ΔC) its COOH-terminal intracellular tail, has been conjugated to green fluorescent protein (GFP) to study subcellular localization and other functions of TF. TF-GFP and TFΔC-GFP are associated with Na2CO3-resistant buoyant fractions in KEK-293 cells (lipid rafts); there is no morphological difference in the surface distribution of these or other GFP-labeled membrane proteins present in or excluded from rafts (confocal microscopy, HEK-293 cells). Endogenous TF expressed by rabbit aortic smooth muscle cells (SMCs) is also raft associated. Membranes from HEK293 cells expressing recombinant TF-GFP or wild-type TF were equipotent to clot human plasma; however, TRΔC-GFP was ∼20-fold more active (per membrane weight). Immunoblot confirmed that the deletion mutant is more abundantly expressed, and confocal microscopy showed that it has preferential membrane localization, whereas TF-GFP is mainly intracellular (nuclear lining and multiple granules). With a similar half-life (lt;4 h), the two constructions differ by their intracellular retention, lower for TFΔC-GFP. In serum-starved SMCs, the expression of endogenous TF was upregulated by interleukin-1β and/or PBS treatment (immunoblot, immunofluorescence, clotting assay). However, TF secretion or surface expression was not regulated by stimuli of physiological intensity (such as stimulation of the coexpressed kinin B1 receptors), although a calcium ionophore was highly active in this respect. TF is a raft-associated molecule whose surface expression (secretion) is apparently retarded or impaired by structural determinant(s) located in its COOK-terminal tail. [ABSTRACT FROM AUTHOR]

Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa (FVIIa) and the formation of TF-VIIa complexes on cell surfaces triggers the coagulation cascade. Further, TF-VIIa, either directly or indirectly, influences various biological processes, such as development, inflammation and tumor metastasis. Therefore, a proper regulation of TF-VIIa expression is critical for the maintenance of hemostatic balance and health in general. TF-VIIa functional expression on cell surfaces is regulated primarily by transcriptional regulation of TF gene or by specific plasma inhibitors, particularly tissue factor pathway inhibitor (TFPI). However, a number of other mechanisms that are yet to be well defined also regulate TF-VIIa functional expression. One such mechanism is the endocytosis of TF-VIIa. In this article, we provide a comprehensive review on the regulation of TF-VIIa functional expression by these other mechanisms, with a particular emphasis on TF-VIIa endocytosis, and our perspective of these studies. [ABSTRACT FROM AUTHOR]

Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 μM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 ± 120.0 s, mean ± SE, n = 20), had a peak density of 11.3 ± 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 ± 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC. [ABSTRACT FROM AUTHOR]

Recently, we have shown that a widely used antagonist of the human bradykinin B[sub 2] receptor (B2R) HOE 140 acts as a full agonist of the chicken ornithokinin receptor (B[sub o]R). To understand the molecular mechanisms underlying differential efficacy of HOE 140 for the various kinin receptors, we have constructed chimeric kinin receptors (CKR) in which the amino-terminal portion including the first two transmembrane regions and the first extracellular loop (CKR-2) or only the second transmembrane region and the first extracellular loop (CKR-1) of B[sub 2]R were substituted with the corresponding segments of B[sub o]R. Ligand efficacy of synthetic ligand HOE 140 decreased in the order B[sub o]R > CKR-2 > CKR1 > B[sub 2]R, whereas the efficacy of the endogenous kinin ligand was unchanged. Enhanced HOE 140 efficacy was not due to a structural change in the ligand binding site or to an enhanced receptor expression level. Rather, heterologous binding competition studies indicated that structural change(s) introduced into the engineered receptors caused a selective reduction in apparent affinity of HOE 140 for the uncoupled inactive receptor state R but not for the active G protein-coupled state R[sup *], thereby increasing the ratio of R[sup *] over R for a given ligand concentration. Our results may help explain the unusually broad efficacy spectrum of HOE 140, which varies from inverse to full agonism, depending on kinin receptor subtype, tissue origin, or species. [ABSTRACT FROM AUTHOR]

Objective: Fibrin accumulation in the joint cavity is a common feature of chronic arthritides, such as rheumatoid arthritis (RA). Complex formation between tissue factor (TF) and factor VII (FVII) is the initial step in such a fibrin formation.¶ Methods: To assess the role of the TF/FVII complex in the pathogenesis of joint inflammation, 1) the levels of TF/FVII complex were measured in synovial fluid of RA patients; 2) the complex was injected to healthy mice intra-articularly.¶ Results: Morphological analysis of the joints 4 days after TF/FVII injection revealed influx of CD4-Mac1+ mononuclear leukocytes into synovial tissue followed by cartilage and bone destruction. Inflammation induced by TF/FVII complex was more profound than that caused by each of the proteins separately, both with respect to frequency, severity and duration of arthritis. Interaction between macrophages and lymphocytes in sustaining joint inflammation was proved by the requirement of the combined lymphocyte/ monocyte depletion to abolish TF/FVII induced arthritis. Induction of monocyte attracting chemokines (MIP-1α and RANTES) was shown to be one of the potential mechanisms for TF/FVII complex triggered inflammatory cell influx. Interestingly, TF/FVII complexes were detected in synovial fluid of 20/40 patients with RA.¶ Conclusions: Altogether these findings indicate that TF/FVII complexes, frequently found intra-articularly in joints of RA patients, may be an important component in both induction and progression of chronic destructive arthritis. [ABSTRACT FROM AUTHOR]

DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases. Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. Most DNA primases can be divided into two classes. The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases. These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase. The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit. The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle. [ABSTRACT FROM AUTHOR]

Aim of this study was to identify optimal conditions for suspension transfection in the absence of serum. Transfection parameters for suspension culture can be very different to ones in adherent cells. Most transfection protocols have been developed and optimizedfor adherent culture. Using green fluorescent protein (GFP) as reporter, FCS was eliminated from the transfection process by altering critical parameters and by substituting serum with albumin. Using standard phosphate and calcium concentrations for transfection in the absence of serum resulted in titers of only 1% of those observed in the presence of serum. A reduction of the calcium concentration from 250 mM to 100 mM, yielded a 25-fold increase in the expression of the recombinant protein compared to the serum-free standard conditions. Altering the phosphate concentration, 1.4 mM in the transfection buffer, did not improve the protein expression. Interestingly, reduction of DNA quantity by half to a concentration of 0.5 μg per milliliter of culture volume resulted in a two-fold increase of protein production. Addition of albumin to serum-free medium protected the cells against the toxicity of the calcium phosphate transfection particles (CaPi) yielding higher protein expression. All the experiments were executed in a shaken multi-well system, allowing high multiplicity parameter screening to speed up optimizations. The culture system is inexpensive, simple and efficient, minimizing costs for labor and consumables. [ABSTRACT FROM AUTHOR]

The HER2/ neu oncogene product, p185HER2/ neu, is overexpressed on the surface of many human breast cancers. Strains of transgenic mice have been developed that express the rat neu oncogene in mammary epithelial cells and develop spontaneous mammary tumors that overexpress p185 neu. This model provides an ideal system for testing interventions to prevent tumor development. In this study, we immunized neu-transgenic mice with a vaccine consisting of the extracellular domain of p185 neu (NeuECD). Immunized mice developed Neu-specific humoral immune responses, as measured by circulating anti-Neu antibodies in their sera, and cellular immune responses, as measured by lymphocyte proliferation to NeuECD in vitro. In addition, the subsequent development of mammary tumors was significantly lower in immunized mice than in controls and vaccine treatment was associated with a significant increase in median survival. [ABSTRACT FROM AUTHOR]

This is the first report of transient transfection of suspended cells with purified plasmid DNA in bioreactors or spinner flasks. DNA/calcium phosphate complexes were pumped or injected directly into stirred cultures of the immortalized human embryo kidney cell line 293 (HEK-293) which had been adapted to growth in suspension. We identified culture conditions suitable for this approach and modified the protocol for the generation of precipitate complexes, based on our earlier work. In order to stabilize the ‘DNA-vehicle’-complex in the culture medium, we identified pH ranges and ion-concentrations which prevent dissolution or aggregation of the precipitate particles. Such conditions maintained suspended fine particles in spinners or bioreactors for up to 6 hr. During that period, cells and precipitate complexes interacted sufficiently to allow DNA transfer and subsequent expression of recombinant protein. In a simple 5 day batch process, with a starting density of 0.3 × 106 cells mL-1, about 0.5 mg L-1 of a recombinant tissue plasminogen activator variant was observed. [ABSTRACT FROM AUTHOR]

The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at PI, P2, and P3 reveals a strong preference for Arg over Lys at PI, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+was discovered. The divalent cation presumably binds to H188 near Dl89 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 μM. In an attempt to engineer Na+binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-I and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+binding site of allosteric serine proteases. [ABSTRACT FROM AUTHOR]

A variant of human interferon-gamma (IFN-γ) has been created in which the two chains of the homodimeric cytokine were linked N- to C-terminus by an eight residue polypeptide linker. The sequence of this linker was derived from a loop in bira bifunctional protein, and was determined from a structural database search. This 'single-chain' variant was used to create an IFN-γ molecule that binds only a single copy of the α-chain receptor, rather than the 2 α-chain receptor: 1 IFN-γ binding stoichiometry observed for the native hormone. Crystals have been grown of a 1:1 complex between this single-chain molecule and the extracellular domain of its α-chain receptor. These crystals diffract beyond 2.0 Å, significantly better than the 2.9 Å observed for the native 2:1 complex. Density calculations suggest these crystals contain two complexes in the asymmetric unit; a self-rotation function confirms this conclusion. [ABSTRACT FROM AUTHOR]

Antithrombin agents of recombinant and synthetic origin arc now validated in experimental models as useful anticoagulant and antithrombic drugs. Several clinical trials including surgical anticoagulation, management of coronary syndromes, adjunct to thrombolytic agents and treatment of thromboembolism have also shown the comparative efficacy of these agents in reference to heparin. Argatroban and hirudin arc now available for specific clinical indications such as thrombotic and ischemic stroke and alternate anticoagulants for heparin-induced thrombocytopenia (HIT) patients in Japan and European countries, respectively. While these agents produce strong anticoagulant effects, their mechanism of action is distinct from that of heparins, thus these agents should be used carefully using specific guidelines provided for each product. Thrombin inhibitors are effective anticoagulants however, their therapeutic index is narrower than heparin and as such their nonoptimized use is potentially associated with hemorrhagic complications. [ABSTRACT FROM AUTHOR]

The expression of a range of surface molecules/receptors that arc important in the host response lo infection and foreign antigens was examined using peritoneal macrophages isolated from patients on continuous ambulatory peritoneal dialysis (CAPD) with peritonitis. The macrophage phenotypic profile was compared with that of normal peripheral blood monocytes. Consistently there was increased expression by macrophages of CDI4, ICAM-I (CD54), FcγRI (CD64), FcγRII (CDw32), FcγRlIl (CD16), transferrin receptors (CD7I)and tissue factor. Increased expression of MHC class II was marginally significant. There was no detectable expression of either the p55 (CD25) or p70 chains of the IL-2 receptor. The expression of the complement receptors. CRI (CD35) and CR3 (CDllb, CDI8). was reduced. The activity of well-known inflammatory cytokines, rather than uraemic molecules, can account for the phenotypic profile of these extravasated peritoneal macrophages. The results of this study indicate that peritoneal macrophages from CAPD patients with peritonitis display a phenotype consistent with them being in vivo-derived inflammatory macrophages. and that they are appropriate for use in studies of anti-inflammatory agents. [ABSTRACT FROM AUTHOR]

Mer/Nyk/Eyk is an orphan receptor tyrosine kinase expressed at high levels in monocytes and cells derived from epithelial and reproductive tissues. Overexpression of Mer has been associated with lymphoid malignancies. Here we identify Gas6, the product of a growth arrest specific gene, as a ligand for Mer. Gas6 has previously been shown to activate both Axl and Rse/Tyro3, two other receptor tyrosine kinases in the same family as Mer. The apparent relative association and dissociation rate constants of Gas6 for soluble Axl, Rse/Tyro3 and Mer were compared using surface plasmon resonance. Gas6 was shown to induce rapid phosphorylation of Mer expressed in several different types of cells. We also observed a transient activation of p42 MAP kinase following activation of Mer by Gas6. Thus, Gas6 exerts its biological effects through multiple receptor tyrosine kinases. [ABSTRACT FROM AUTHOR]

Antithrombin drugs represent a wide group of natural agents, recombinant agents equivalent to some of the naturally occurring proteins, and synthetic agents. This group of drugs is characterized by marked structural and functional heterogeneity. Several of these drugs are currently in various phases of development. Argatroban represents the first clini cally approved antithrombin agent, which was made available in Japan several years ago. Two recombinant hirudin prepara tions, Revasc (Novartis) and Refludan (Aventis), are available for postsurgical DVT prophylaxis and alternate anticoagulant use in patients with heparin-induced thrombocytopenia. A syn thetic antithrombin agent based on the combined structures of hirudin and antithrombin peptides, hirulog (Bivalirudin), is un dergoing clinical trials in cardiovascular indications. Additional studies on the hirudins are being carried out to test their effi cacy as surgical and interventional anticoagulants as replace ments for heparin. However, the need for a proper antagonist is one of the limiting factors for the optimal development of hi rudin in this indication. Several of the synthetic thrombin in hibitors are also being developed for oral use for the prophy laxis of DVT in surgical patients. Since the therapeutic index of thrombin inhibitors is narrower than that of heparin, this route may not be an optimal approach for the development of these agents. Despite several unresolved developmental issues, the thrombin inhibitors provide a useful alternative to heparin an ticoagulation and may prove to be useful in validated clinical use. Key Words: Antithrombin—Anticoagulants—Hirudin— Heparin. [ABSTRACT FROM PUBLISHER]

Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.Ser117HisfsTer10) in F3, the gene encoding TF, causing premature termination of TF (TFshort) in a woman with unexplained bleeding. Routine hematological laboratory evaluation of the proposita was normal. CRISPR-edited human induced pluripotent stem cells recapitulating the variant were differentiated into vascular smooth muscle and endothelial cells that demonstrated haploinsufficiency of TF. The variant F3 transcript is eliminated by nonsense-mediated decay. Neither overexpression nor addition of exogenous recombinant TFshort inhibited factor Xa or thrombin generation, excluding a dominant-negative mechanism. [ [F3.sup.+/-] mice provide an animal model of TF haploinsufficiency and exhibited prolonged bleeding times, impaired thrombus formation, and reduced survival following major injury. Heterozygous TF deficiency is present in at least 1 in 25,000 individuals and could limit coagulation initiation in undiagnosed individuals with abnormal bleeding but a normal routine laboratory evaluation., Introduction Tissue factor (TF) is an integral membrane protein of the cytokine receptor superfamily that binds factor VII/VIIa to catalyze proteolytic activation of factors IX and X to trigger the [...]