ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Gαs-mediated stimulation of adenylylcyclase (AC), Gαq-mediated activation of phospholipase Cβ (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast prolifera-tion. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways – an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulate d by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects. Received 2 August 2002; received after revision 10 September 2002; accepted 18 October 2002 RID="*" ID="*"Corresponding author.     

Aptamers against prion proteins and prions

Prion diseases are fatal neurodegenerative and infectious disorders of humans and animals, characterized by structural transition of the host-encoded cellular prion protein (PrP^c) into the aberrantly folded pathologic isoform PrP^Sc. RNA, DNA or peptide aptamers are classes of molecules which can be selected from complex combinatorial libraries for high affinity and specific binding to prion proteins and which might therefore be useful in diagnosis and therapy of prion diseases. Nucleic acid aptamers, which can be chemically synthesized, stabilized and immobilized, appear more suitable for diagnostic purposes, allowing use of PrP^Sc as selection target. Peptide aptamers facilitate appropriate intracellular expression, targeting and re-routing without losing their binding properties to PrP, a requirement for potential therapeutic gene transfer experiments in vivo. Elucidation of structural properties of peptide aptamers might be used as basis for rational drug design, providing another attractive application of peptide aptamers in the search for effective anti-prion strategies.     

Microtubule associated protein tau binds to double-stranded but not single-stranded DNA

Tau, a major microtubule-associated protein of the neuron, which is known to promote the assembly of and to stabilize microtubules, has also been seen associated with chromatin in neuronal cell lines, but its role in this subcellular compartment is still unknown. In this study, the binding of tau to DNA was investigated using the electrophoretic mobility shift assay. Using polynucleotide as probe, we found that tau bound to double-stranded but not to single-stranded DNA. Formation of tau-polynucleotide complex was disrupted by alkaline pH and a high concentration of NaCl, but was not affected by dithiothreitol. Electron microscopy revealed that the protein associated with the nucleic acid in a necklacelike manner. DNA-cellulose chromatography and radioimmunodot-blot analyses showed that calf thymus histones VI-S, VII-S and VIII-S could replace both recombinant human brain tau₃₅₂ (tau-23) and tau₄₄₁ (tau-40) from DNA. Thus, tau appears to bind to DNA reversibly in the presence of histones. Received 24 November 2002; received after revision 28 December 2002; accepted 30 December 2002 RID="*" ID="*"Corresponding author.     

New endo-beta-1,4-glucanases from the parabasalian symbionts,Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites

An endo-β-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus. The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself. The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed. This showed high homology to EGs from glycoside hydrolase family (GHF) 7. PCR-based cloning methods were applied to the hindgut contents of C. lacteus and individual protozoan symbionts from C. formosanus. cDNAs encoding putative EGs homologous to GHF7 members were then identified. The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli. Received 18 September 2002; accepted 20 September 2002 RID="*" ID="*"Corresponding author.     

HLA-G protein processing and transport to the cell surface

Data are presented on the intracellular trafficking of HLA-G protein, taking the unique features of this non-classical molecule into consideration: the existence of seven isoforms resulting from alternative splicing (HLA-G1 to G7), and reduced tail length compared with HLA class I antigens. Biochemical studies and analysis of viral strategies for escaping the host immune system led to the demonstration that (i) both the membrane-bound (HLA-G1) and the soluble (HLA-G5) forms of the molecule require peptide association for cell surface expression, using TAP-dependent or TAP-independent pathways; (ii) peptide loading onto the HLA-G protein plays a critical role in controlling the quality of the molecule reaching the cell surface; (iii) surface expression of truncated HLA-G molecules is possible, and (iv) HLA-G expression may be restricted to soluble HLA-G5. These data reveal that HLA-G presents specific cell trafficking pathways and strongly support the contention that the primary function of HLA-G is as of an inhibitor ligand for immune-competent cells. Received 4 June 2002; accepted 2 July 2002 RID="*" ID="*"Corresponding author.     

SecB,one small chaperone in the complex milieu of the cell

SecB is only one of a plethora of cytosolic chaperones in E. coli whose common property is that they bind nonnative proteins. It plays a crucial role during protein export via the general secretory pathway by modulating the partitioning of precursors between folding or aggregation and delivery to the membrane-bound translocation apparatus. In this latter role SecB demonstrates specific binding to a unique partner, SecA. SecB has the potential to participate in functions outside of export acting as a general nonspecific chaperone to provide buffering capacity of the nonnative state of proteins in the cytosolic pool. We discuss the interactions of SecB with its many binding partners in light of its recently determined structure, emphasizing both kinetic and thermodynamic parameters. RID="*" ID="*"Corresponding author.     

Structure to function relationships in ceruloplasmin: a 'moonlighting' protein

Specialised copper sites have been recruited during evolution to provide long-range electron transfer reactivity and oxygen binding and activation in proteins destined to cope with oxygen reactivity in different organisms. Ceruloplasmin is an ancient multicopper oxidase evolved to insure a safe handling of oxygen in some metabolic pathways of vertebrates. The presently available knowledge of its structure provides a glimpse of its plasticity, revealing a multitude of binding sites that point to an elaborate mechanism of multifunctional activity. Ceruloplasmin represents an example of a 'moonlighting' protein that overcomes the one gene-one structure-one function concept to follow the changes of the organism in its physiological and pathological conditions. Received 19 February 2002; received after revision 29 March 2002; accepted 2 April 2002 RID="*" ID="*"Corresponding author.     

Structural features of prions explored by sequence analysis. II. A PrP<Superscript>Sc</Superscript> model

Prion diseases are neurodegenerative disorders associated with a conformational conversion of the prion PrP protein, in which the β-strand content increases and that of the α helix decreases. However, the structure of the pathogenous form PrP^Sc, occurring after conformational conversion of the normal cellular form PrP^C, is not yet known. From sequence analysis, we have previously proposed that helix H2 of the prion PrP^C structure might be a key region for this structural conversion. More recently, we identified the TATA box-binding protein fold as a putative scaffold that may locally satisfy the predicted secondary-structure organisation of PrP^Sc. In the present analysis, we detail the schematic construction of PrP^Sc monomeric and dimeric models, based on this hypothesis. These models are globally compatible with available data and therefore may provide further insights into the structurally and functionally elusive PrP protein. Some comments are also devoted to a comparison of the yeast Ure2p prion and animal prions. Received 29 July 2002; received after revision 24 October 2002; accepted 24 October 2002 RID="*" ID="*"Corresponding author.     

Isolation of peptides blocking the function of anti-apoptotic Livin protein

Livin (ML-IAP) is a cancer-associated member of the inhibitor of apoptosis protein (IAP) family. By yeast two-hybrid screening of a randomized peptide expression library, we isolated short linear peptides that specifically bind to Livin, but not to other IAPs. Intracellular expression of the peptides sensitized livin-expressing cancer cells toward different pro-apoptotic stimuli. The bioactive peptides neither showed sequence homologies to Smac-derived IAP inhibitors, nor did they interfere with the binding of Livin to Smac. Intracellular expression of the peptides did not affect the levels or the subcellular distribution of Livin. Growth of livin-expressing tumor cells was inhibited in colony formation assays by the Livin-targeting peptides. These findings provide evidence that the targeted inhibition of Livin by peptides represents a viable approach for the apoptotic sensitization and growth inhibition of tumor cells. The inhibitory peptides isolated here could form a novel basis for the development of therapeutically useful Livin inhibitors.     

Protein misfolding and disease: the case of prion disorders

Recent findings strongly support the hypothesis that diverse human disorders, including the most common neurodegenerative diseases, arise from misfolding and aggregation of an underlying protein. Despite the good evidence for the involvement of protein misfolding in disease pathogenesis, the mechanism by which protein conformational changes participate in the disease is still unclear. Among the best-studied diseases of this group are the transmissible spongiform encephalopathies or prion-related disorders, in which misfolding of the normal prion protein plays a key role in the disease. In this article we review recent data on the link between prion protein misfolding and the pathogensis of spongiform encephalopathies. Received 15 July 2002; received after revision 19 August 2002; accepted 23 August 2002 RID="*" ID="*"Corresponding author.     

Ectoprotein kinase-mediated phosphorylation of FAT/CD36 regulates palmitate uptake by human platelets

Glycoprotein IV (FAT/CD36) has been shown to be phosphorylated by a cAMP-dependent, platelet membrane-bound ectokinase. In this study, we demonstrate that ectophosphorylation of FAT/CD36 regulates initial palmitate uptake. This is the first time that short-term regulation of the activity of a long-chain fatty acid carrier could be shown. Phosphorylation of FAT/CD36 was paralleled by a significant decrease in initial palmitate uptake by morphologically and functionally intact platelets. Maximum inhibition of palmitate uptake was achieved at 0.5 nM extracellular ATP, being significantly decreased to 72% compared to the control. Inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI or with β,γ-methylene-ATP, and was reversible upon addition of alkaline phosphatase. An extracellular ATP concentration above 5 μM completely prevented the ectophosphorylation-mediated inhibition of palmitate uptake. We conclude that FAT/CD36-mediated palmitate uptake by human platelets is short-term regulated via cAMP-dependent ectophosphorylation of FAT/CD36. Received 18 July 2002; received after revision 29 August 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Cyanovirin-N: a sugar-binding antiviral protein with a new twist

Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium Nostoc ellipsosporum, is a highly potent virucidal agent that has generated interest as a lead natural product for the prevention and chemotherapy of human immunodeficiency virus infection. The antiviral activity of CV-N is mediated through specific, high-affinity interactions with the viral surface envelope glycoproteins. A number of structures of wild-type, mutant and sequence-shuffled CV-N have been solved by nuclear magnetic resonance and crystallography, showing that the protein exists as either a quasi-symmetric two-domain monomer or a domain-swapped dimer. Structures of several complexes of CV-N with oligosaccharides help in explaining the unique mode of high-affinity binding of these molecules to both forms of CV-N. RID="*" ID="*"Corresponding author.     

Oligo-1,6-glucosidase and neopullulanase enzyme subfamilies from the alpha-amylase family defined by the fifth conserved sequence region

The α-amylase enzyme family is the largest family of glycoside hydrolases. It contains almost 30 different enzyme specificities covering hydrolases, transferases and isomerases. Some of the enzyme specificities from the family are closely related, others less so. This study, based on the analysis of 79 amino acid sequences, postulates two subfamilies in the framework of the α-amylase family: the oligo-1,6-glucosidase subfamily and the neopullulanase subfamily. The specific sequence in the fifth conserved sequence region of the family served as the basis for defining the subfamilies: QpDln for the oligo-1,6-glucosidase subfamily and MPKln for the neopullulanase subfamily. This conserved sequence region is proposed to be the selection marker that enables one to distinguish between the two subfamilies. The 'intermediary' sequence MPDLN can be characteristic of the so-called intermediary group with a mixed enzyme specificity of α-amylase, cyclomaltodextrinase and neopullulanase. The evolutionary trees clearly supported the proposed definition of the two subfamilies. Received 12 July 2002; received after revision 28 August 2002; accepted 24 September 2002 RID="*" ID="*"Corresponding author.     

Small,novel proteins from the mistletoe <Emphasis Type="Italic">Phoradendron tomentosum</Emphasis> exhibit highly selective cytotoxicity to human breast cancer cells

Four novel proteins (phoratoxins C–F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC₅₀ values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples. Received 30 September 2002; received after revision 28 October 2002; accepted 7 November 2002 RID="*" ID="*"Corresponding author.     

Hsp90 binds CpG oligonucleotides directly: implications for hsp90 as a missing link in CpG signaling and recognition

CpG motifs originating from bacterial DNA (CpG DNA) can act as danger signals for the mammalian immune system. These CpG DNA motifs like many other pathogen-associated molecular patterns are believed to be recognized by a member of the toll-like receptor family, TLR-9. Here we show results suggesting that heat shock protein 90 (hsp90) is also implicated in the recognition of CpG DNA. Hsp90 was characterized as a binder to oligodeoxynucleotides (ODNs) containing CpG motifs (CpG ODNs) after several purification steps from crude protein extracts of peripheral blood mononuclear cells. This finding was further supported by direct binding of CpG ODNs to commercially available human hsp90. Additionally, immunohistochemistry studies showed redistribution of hsp90 upon CpG ODN uptake. Thus, we propose that hsp90 can act as a ligand transfer molecule and/or play a central role in the signaling cascade induced by CpG DNA. Received 18 December 2002; accepted 6 January 2002 RID="*" ID="*"Corresponding author. B. Agerberth and G. H. Gudmundsson contributed equally to this work.     

Anti-DNA antibodies: aspects of structure and pathogenicity

Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus. Received 4 July 2002; accepted 23 July 2002 RID="*" ID="*"Corresponding author.     

Morphofunctional changes in the mitochondrial subpopulations of conceptus tissues during the placentation process

To establish the role of mitochondrial subpopulations in the mitochondrial maturation process, we studied morphological and functional changes in the mitochondria of different mammalian conceptus tissues during the organogenic and the placentation processes. Mitochondrial subpopulations of three different conceptus tissues, embryo and visceral yolk sac placenta on gestational days 11, 12 and 13 and placenta on days 12 and 13, were examined morphologically by transmission electron microscopy. Cytochrome oxidase activity and protein levels were also measured in each mitochondrial subpopulation. The results indicate two different mitochondrial subpopulation profiles: a homogeneous one, which corresponds to immature mitochondria, and a heterogeneous one, which represents the mature mitochondria. The three tissues studied show different morphologic and metabolic patterns of mitochondrial maturation during the placentation process, rendering them suitable as experimental models to establish the p ossible relationship between mitochondrial maturation and the mitochondrial subpopulations. Received 5 August 2002; received after revision 23 September 2002; accepted 8 October 2002 RID="*" ID="*"Corresponding author.     

Statins: the new aspirin?

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, have been described as the principal and the most effective class of drug to reduce serum cholesterol levels. Statin therapies have been shown to reduce cardiovascular events, including myocardial infarction, stroke, and death, significantly, by altering vascular atherosclerosis development in patients with or without coronary artery disease symptoms. Extensive use of statins has led to the increase of some undesirable effects that are heavily counterbalanced by the benefits. Indeed, pleiotropic effects extend far beyond cholesterol reduction and involve non-lipid-related mechanisms that modify endothelial functions, immunoinflammatory responses, smooth muscle cell activation, proliferation and migration, atherosclerotic plaque stability, and thrombus formation. In this review, we describe in detail the targets and mechanisms of action of statins. Received 6 June 2002; received after revision 6 September 2002; accepted 6 September 2002 RID="*" ID="*"Corresponding author.     

Identification of the bioactive peptide PEC-60 in brain

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60may play a role in the central nervous system disorders involving dopamine dysregulation. Received 6 December 2002; received after revision 10 December 2002; accepted 11 December 2002 RID="*" ID="*"Corresponding author.     

Physicochemical profiling in drug research: a brief survey of the state-of-the-art of experimental techniques

This review begins with a general presentation of the new paradigm of drug discovery, with its emphasis on the rapid identification and elimination of compounds with unsuitable physicochemical and pharmacokinetic properties. The focus of the paper is on the various experimental methods used to determine such key physicochemical properties as ionization, lipophilicity and distribution in isotropic and anisotropic systems, solubility, and permeability across artificial membranes. Both traditional and high-throughput methods are presented and their limits highlighted. The text concludes with the trade-off between quantity/speed in high-throughput screening techniques versus greater data quality in the more labor-intensive methods. Received 23 April 2002; received after revision 25 June 2002; accepted 11 July 2002 RID="*" ID="*"Corresponding author.