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.     

Dictyostelium mobile elements: strategies to amplify in a compact genome

Dictyostelium discoideum is a eukaryotic microorganism that is attractive for the study of fundamental biological phenomena such as cell-cell communication, formation of multicellularity, cell differentiation and morphogenesis. Large-scale sequencing of the D. discoideum genome has provided new insights into evolutionary strategies evolved by transposable elements (TEs) to settle in compact microbial genomes and to maintain active populations over evolutionary time. The high gene density (about 1 gene/2.6 kb) of the D. discoideum genome leaves limited space for selfish molecular invaders to move and amplify without causing deleterious mutations that eradicate their host. Targeting of transfer RNA (tRNA) gene loci appears to be a generally successful strategy for TEs residing in compact genomes to insert away from coding regions. In D. discoideum, tRNA gene-targeted retrotransposition has evolved independently at least three times by both non-long termina l repeat (LTR) retrotransposons and retrovirus-like LTR retrotransposons. Unlike the nonspecifically inserting D. discoideum TEs, which have a strong tendency to insert into preexisting TE copies and form large and complex clusters near the ends of chromosomes, the tRNA gene-targeted retrotransposons have managed to occupy 75% of the tRNA gene loci spread on chromosome 2 and represent 80% of the TEs recognized on the assembled central 6.5-Mb part of chromosome 2. In this review we update the available information about D. discoideum TEs which emerges both from previous work and current large-scale genome sequencing, with special emphasis on the fact that tRNA genes are principal determinants of retrotransposon insertions into the D. discoideum genome. Received 10 May 2002; received after revision 10 June 2002; accepted 12 June 2002 RID="*" ID="*"Corresponding author.     

The functions of mucosal T cells in containing the indigenous commensal flora of the intestine

There is an immense load of non-pathogenic commensal bacteria in the distal small intestine and the colon of mammals. The physical barrier that prevents penetration (translocation) of these organisms into the body is a simple epithelium comprised of the single enterocyte/colonocyte cell layer with its overlying mucus. In this review, we discuss the roles of intestinal T cells in initiating and regulating innate and adaptive mucosal immune responses of the mucosal immune system that avoid or limit penetration of the commensal intestinal bacteria. Received 9 August 2002; accepted 9 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.     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Circular proteoglycans from sponges: first members of the spongican family

Species-specific cell adhesion in marine sponges is mediated by a new family of modular proteoglycans whose general supramolecular structure resembles that of hyalectans. However, neither their protein nor their glycan moieties have significant sequence homology to other proteoglycans, despite having protein subunits equivalent to link proteins and to proteoglycan monomer core proteins, and glycan subunits equivalent to hyaluronan and to the glycosaminoglycans of hyalectans. In some species, these molecular components are assembled into a structure with a circular core formed by the link protein- and hyaluronan-like subunits. Besides their involvement in cell adhesion, these sponge proteoglycans, for which we propose the term spongicans, participate in signal transduction processes and are suspected to play a role in sponge self-nonself recognition. Their in vivo roles and the mild methods used to purify large amounts of functionally active spongicans make them ideal models to study the functions and possible new applications of proteoglycans in biomedical research. Received 21 May 2002; received after revision 5 July 2002; accepted 10 July 2002 RID="*" ID="*"Corresponding author.     

Cellular and molecular aspects of drugs of the future: oxaliplatin

Oxaliplatin (Eloxatine) is a third-generation platinum compound which has shown a wide antitumour effect both in vitro and in vivo, a better safety profile than cisplatin and a lack of cross-resistance with cisplatin and carboplatin. In this scenario, oxaliplatin may represent an innovative and challenging drug extending the antitumour activity in diseases such as gastrointestinal cancer that are not usually sensitive to these coordination complexes. Oxaliplatin has a non-hydrolysable diaminocyclohexane (DACH) carrier ligand which is maintained in the final cytotoxic metabolites of the drug. Like cisplatin, oxaliplatin targets DNA producing mainly 1,2-GG intrastrand cross-links. The cellular and molecular aspects of the mechanism of action of oxaliplatin have not yet been fully elucidated. However, the intrinsic chemical and steric characteristics of the DACH-platinum adducts appear to contribute to the lack of cross-resistance with cisplatin. To date, mismatch repair and replicative bypass appear to be the processes most likely involved in differentiating the molecular responses to these agents. Received 15 March 2002; received after revision 13 May 2002; accepted 21 May 2002 RID="*" ID="*"Corresponding author.     

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.     

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.     

Molecular aspects of membrane fission in the secretory pathway

Membrane fission is essential in various intracellular dissociative transport steps. The molecular mechanisms by which endocytic vesicles detach from the plasma membrane are being rapidly elucidated. Much less is known about the fission mechanisms operating at Golgi tubular networks; these include the Golgi transport and sorting stations, the trans-Golgi and cis-Golgi networks, where the geometry and physical properties of the membranes differ from those at the cell surface. Here we discuss the lipid and protein machineries that have so far been related to the fission process, with emphasis on those acting in the Golgi complex. Received 10 May 2002; received after revision 20 June 2002; accepted 26 June 2002 RID="*" ID="*"Corresponding author.     

Retinoic acid modulates gap junctional intercellular communication in hepatocytes and hepatoma cells

Gap junctional communication permits the direct exchange of small molecules and ions and has been implicated in tissue homeostasis/metabolite exchange. The lack of gap junctional intercellular communication (GJIC) plays important roles in the promotion and progression of carcinogenesis. In the present study, we demonstrate that treatment of human hepatoma Hep G2 cells with retinoic acid (RA) results in increased amounts and phosphorylation of connexins, their stabilisation in plasma membrane plaques and enhanced GJIC. In cultured fetal hepatocytes, which represent a non-transformed, proliferating and incompletely differentiated liver system, the effects of RA are limited to the establishment of connexin in areas of cell-cell contact and the improvement of GJIC. This suggests that modulation of cell-cell channel communication by RA occurs differently in these two experimental models: while RA is able to revert cell transformation in Hep G2 cells, in fetal hepatocytes it may induce the expression of a more differentiated phenotype. Received 19 June 2002; received after revision 29 July 2002; accepted 8 August 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.     

Sensing life: regulation of sensory neuron survival by neurotrophins

Neurotrophins are a family of structurally and functionally related neurotrophic factors which, in mammals, include: nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and NT-4/5. In addition to their canonical role in promoting neuronal survival, these molecules appear to regulate multiple aspects of the development of the nervous system in vertebrates, including neuronal differentiation, axon elongation and target innervation, among others. Actions of neurotrophins and of their receptors in vivo are being analyzed by loss-of-function or gain-of-function experiments in mice. Here, we review the phenotypes of the primary sensory system in these mutant mouse strains and the different strategies specifically involved in the regulation of neuronal survival by neurotrophins in this portion of the nervous system. Received 10 December 2001; received after revision 11 May 2002; accepted 13 May 2002 RID="*" ID="*"Corresponding author.     

Immunomodulatory properties of cystatins

Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. Because these cysteine proteases exist in all living organisms and because they are involved in various biological and pathological processes, the control of these protease functions by cystatins is of cardinal importance. Cystatins are found in mammals but cystatin-like molecules are also present in mammals and parasites. In the immune system, cystatins modulate cathepsin activities and antigen presentation. They also induce tumor necrosis factor α and interleukin 10 synthesis, and they stimulate nitric oxide production by interferon γ-activated murine macrophages. In turn, nitric oxide has inhibitory activity on cysteine proteases, especially those from parasitic protozoa. Cystatins isolated from parasitic nematodes also have immunomodulatory activities that are distinguishable from those induced by lipopolysacharide-like molecules from endosymbiotic bacteria. On the whole, cystatins and cystatin-like molecules belong to a new category of immunomodulatory molecules. Doubtless increasing data will improve our knowledge of this property, leading to practical applications in immunotherapy. Received 11 April 2002; accepted 18 April 2002 RID="*" ID="*"Corresponding author.     

Melatonin regulation of antioxidant enzyme gene expression

Antioxidant enzymes (AOEs) are part of the primary cellular defense against free radicals induced by toxins and/or spontaneously formed in cells. Melatonin (MLT) has received much attention in recent years due to its direct free radical scavenging and antioxidant properties. In the present work we report that MLT, at physiological serum concentrations (≈ 1 nM), increases the mRNA of both superoxide dismutases (SODs) and glutathione peroxidase (GPx) in two neuronal cell lines. The MLT effect on both SODs and GPx mRNA was mediated by a de novo synthesized protein. MLT alters mRNA stability for Cu-Zn SOD and GPx. Experiments with a short time treatment (pulse action) of MLT suggest that the regulation of AOE gene expression is likely to be receptor mediated, because 1-h treatment with MLT results in the same response as a 24-h treatment. Received 18 June 2002; received after revision 5 August 2002; accepted 27 August 2002 RID="*" ID="*"Corresponding author.     

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.     

Functions and malfunctions of the tau proteins

The tau proteins belong to the family of microtubule-associated proteins. They are mainly expressed in neurons where they play major regulatory roles in the organization and integrity of the cytoskeleton network. Neurofibrillary changes of abnormally hyperphosphorylated tau are a key lesion in Alzheimer's disease and a number of other tauopathies. However, despite an ever-increasing body of data on the changes which tau undergoes in disease, its role regarding the fundamental disease process is still unclear. Moreover, conceptions of tau functions continue to evolve, which complicates an understanding of its role in the disease process. This review attempts to summarize data on the role of tau proteins in the context of both normal cellular function and dysfunction. Furthermore, we try to develop a mechanistic framework for the involvement of tau during the disease process. The review closes with a look towards various approaches to elucidate the functions and malfunctions of tau. Received 21 June 2002; received after revision 24 July 2002; accepted 29 July 2002 RID="*" ID="*"Corresponding author.     

Common Molecular Mechanisms of Mammary Gland Development and Breast Cancer

Evidence is accumulating that breast cancer is not one disease but many separate diseases. DNA microarray-based gene expression profiling has demonstrated subtypes with distinct phenotypic features and clinical responses. Prominent among the new subtypes is 'basal-like' breast cancer, one of the 'intrinsic' subtypes defined by negativity for the estrogen, progesterone, and HER2/neu receptors and positivity for cytokeratins-5/6. Focusing on basal-like breast cancer, we discuss how molecular technologies provide new chemotherapy targets, optimising treatment whilst sparing patients from unnecessary toxicity. Clinical trials are needed that incorporate long-term follow-up of patients with well-characterised tumour markers. Whilst the absence of an obvious dominant oncogene driving basal-like breast cancer and the lack of specific therapeutic agents are serious stumbling blocks, this review will highlight several promising therapeutic candidates currently under evaluation. Thus, new molecular technologies should provide a fundamental foundation for better understanding breast and other cancers which may be exploited to save lives. (Part of a Multi-author Review).     

Epithelial supporting cells can differentiate into outer hair cells and Deiters' cells in the cultured organ of Corti

The organ of Corti is a complex structure containing a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs), supported respectively by one row of inner phalangeal cells and three rows of Deiters' cells. When fetal rat organ of Corti explants are cultured, supernumerary OHCs and supernumerary Deiters' cells are produced, without any additional cell proliferation. Analysis of semi- and ultrathin sections revealed that supernumerary OHCs are produced at the distal edge of the organ of Corti. Quantitative analysis of cell types present in the organ of Corti demonstrates that when the number of OHCs increases: (i) the total number of cells remains constant; (ii) the number of Deiters' cells increases; (iii) the number of tectal cells decreases and of Hensen's cells decreases. Using specific HC markers, i.e. jagged2 (Jag2) and Math1, we showed that in addition to existing OHCs, supernumerary OHCs, tectal cells and Hensen's cells expressed these markers in embryonic day 19 organ of Corti explants after 5 days in vitro. The results of this study suggest that Hensen's cells retain the capacity to differentiate into either tectal cells, which differentiate into OHCs, or into undertectal cells which differentiate into Deiters' cells. Received 15 May 2002; received after revision 18 July 2002; accepted 7 August 2002 RID="*" ID="*"Corresponding author.     

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.