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.     

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.     

Neuropeptide Y: the universal soldier

The peptidic neurotransmitter neuropeptide Y (NPY) has received great attention because it has been implicated in the regulation of several organ systems. In particular, NPY is involved in the regulatory loops that control food intake in the hypothalamus and appears also to be important for regulating the activity of neuroendocrine axes under poor metabolic conditions. Furthermore, NPY exerts vasoconstrictive action on the vasculature and potentiates the actions of many other vasoconstrictors. In addition, it was demonstrated to have trophic properties and could therefore contribute to cardiovascular remodeling. These various effects plus a number of others make NPY an attractive target for the potential treatment of human diseases, such as obesity, metabolic disorders, hypertension and heart failure. Received 17 July 2002; received after revision 7 November 2002; accepted 29 November 2002 RID="*" ID="*"Corresponding author.     

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.     

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.     

Glycoconjugates of the intestinal goblet cells of four cyprinids

The aim of this work was to show differences in the terminal and subterminal sugar composition of carbohydrate chains of glycoconjugates produced by the goblet cells of the intestines of four cyprinids. We analysed intestines of two herbivorous species – sneep and grass carp – and two omnivorous ones – chub and common carp. We compared four intestinal regions of every studied species. In every region, the presence of neutral and acidic glycoconjugates was confirmed. The smallest amount of acidic glycoconjugates was present in the second region of sneep intestine. Sulphated glycoconjugates were absent in the third and fourth region of chub intestine. Lectin histochemistry provided evidence for the presence of β-D-galactose, α-N-acetylgalactosamine, β-N-acetylglucosamine and sialic acids. Additionally, the occurrence of α-L-fucose in the goblet cells of chub, grass carp and sneep was confirmed. We tried to correlate the patern of glycoconjugate glycosylation with feeding habits of the studied fishes. Received 1 July 2002; received after revision 8 August 2002; accepted 19 August 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-α-tubulin monoclonal antibodies, polymorphic α-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of α-tubulin with relat ively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic α-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic a-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic α-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic α-tubulin isoform. The acidic α-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog. Received 1 October 2002; accepted 9 October 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.     

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.     

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.     

Role of glutamic acid decarboxylase in the pathogenesis of type 1 diabetes

Glutamic acid decarboxylase (GAD) is considered to be one of the strongest candidate autoantigens involved in triggering β-cell-specific autoimmunity. The majority of recent onset type 1 diabetes patients and prediabetic subjects have anti-GAD antibodies in their sera, as do nonobese diabetic (NOD) mice, one of the best animal models for human type 1 diabetes. Immunization of young NOD mice with GAD results in the prevention or delay of the disease as a result of tolerizing autoreactive T cells. Autoimmune diabetes can also be prevented by the suppression of GAD expression in antisense GAD trans genic mice backcrossed with NOD mice for seven generations. These results support the hypothesis that GAD plays an important role in the development of T-cell-mediated autoimmune diabetes. However, there is some controversy regarding the role of GAD in the pathogenesis of diabetes. Whether GAD truly plays a key role in the initiation of this disease remains to be determined. The examination of the development of insulitis and diabetes in β-cell-specific GAD knockout NOD mice will answer this remaining question. Received 12 April 2002; received after revision 24 May 2002; accepted 27 May 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.     

Increased demyelination and axonal damage in metallothionein I+II-deficient mice during experimental autoimmune encephalomyelitis

Metallothioneins I+II (MT-I+II) are antioxidant, neuroprotective factors. We previously showed that MT-I+II deficiency during experimental autoimmune encephalomyelitis (EAE) leads to increased disease incidence and clinical symptoms. Moreover, the inflammatory response of macrophages and T cells, oxidative stress, and apoptotic cell death during EAE were increased by MT-I+II deficiency. We now show for the first time that demyelination and axonal damage are significantly increased in MT-I+II deficient mice during EAE. Furthermore, oligodendroglial regeneration, growth cone formation, and tissue repair including expression of trophic factors were significantly reduced in MT-I+II-deficient mice during EAE. Accordingly, MT-I+II have protective and regenerative roles in the brain. Received 31 October 2002; received after revision 23 November 2002; accepted 26 November 2002 RID="*" ID="*"Corresponding author. M. Penkowa and C. Espejo contributed equally to this paper.