The processing and presentation of endogenous and exogenous antigen by Schwann cells in vitro

The expression of major histocomatibility complex class II in vitro and in vivo by Schwann cells indicates a potential facultative role of Schwann cells in the presentation of antigen to neuritogenic T cells during inflammatory demyelinating neuropathies. Using a T cell proliferation assay, this study demonstrated that processing and presentation of endogenous and exogenous antigen by Schwann cells influences T cell proliferation. Statistical analysis of proliferation and its relation to processing and presentation of antigen by Schwann cells had not been previously addressed. Different combinations of factors including treatment of cultures (untreated, irradiated or fixed), concentration of exogenous antigen (0 or 40 μmg/ml), the presence of interferon-γ and the timing of exogenous antigen addition influence the proliferation P₂-specific, non-mammalian protein ovalbumin-specific T cell lines and naive T cells. Received 25 July 2002; received after revision 9 September 2002; accepted 7 October 2002     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Genes involved in breast cancer metastasis to bone

Metastasis to bone occurs frequently in advanced breast cancer and is accompanied by debilitating skeletal complications. Current treatments are palliative and new therapies that specifically prevent the spread of breast cancer to bone are urgently required. While our understanding of interactions between breast cancer cells and bone cells has greatly improved, we still know little about the molecular determinants that regulate specific homing of breast cancer cells to the bone. In this review, we focus on genes that have been implicated in migration and adhesion of breast cancer cells to bone, as well as genes that promote tumor cell proliferation in the bone microenvironment. In addition, the review discusses new technologies, including better animal models, that will further assist with the identification of the molecular determinants of bone metastasis and will guide the development of new therapies. Received 25 January 2002; received after revision 27 March 2002; accepted 5 April 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.     

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.     

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC₅₀ values of 4.35 and 6.47 μM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 μM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N¹-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H₂O₂ and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis. Received 17 January 2002; received after revision 22 February 2002; accepted 22 February 2002     

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.     

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.     

Specific localization of the catalytic subunits of protein kinase CK2 at the centrosomes

The protein kinase CK2 holoenzyme is composed of two regulatory β subunits and two catalytic α or α' subunits. Although experimental evidence for involvement of the enzyme in the regulation of cell proliferation is accumulating, the exact mechanism of its action is still unclear. The subcellular localization of the enzyme may be a key to its function. We have recently shown that the CK2 holoenzyme is tightly associated with the Golgi complex and the endoplasmic reticulum. Centrosomes, which organize spindle formation during the cell cycle and microtubule cytoskeleton formation and, thereby, the location and orientation of different organelles in the cell, are in close vicinity to the Golgi complex. Because several kinases and phosphatases have been described to regulate the functions of the centrosome, we analysed the association of CK2 with these organelles. Using biochemical cell fractionation and coimmunoprecipitation, we never found the holoenzyme but only the catalytic asubunits associated with the centrosome. These data were confirmed by immunoelectron microscopy. Thus, the present data point to a particular role of the catalytic α and α' subunit of protein kinase CK2, which may be different from their roles in the holoenzyme. Received 2 August 2002; received after revision 2 October 2002; accepted 22 October 2002 RID="*" ID="*"Corresponding author.     

Transgenic and knock-out mice for deciphering the roles of EGFR ligands

Generation of genetically engineered mice with either gain-of-function or loss-of-function mutations is the most popular technique for determining gene functions and the interrelationship between molecules in vivo. These models have provided a wealth of information about the developmental and physiological roles of oncogenes and growth factors. To date, transgenic techniques have been used extensively to study the functions of the epidermal growth factor (EGF) family. This review highlights some of the major recent findings pertinent to the EGF receptor (EGFR) and its ligands with special reference to elucidating how EGF and its related growth factors work together to regulate reproduction, growth and development. Finally, future investigations on ligand-ligand communications, EGFR and its ligands in neural stem cell research, and the mechanisms of EGFR signaling and trafficking in cells are also suggested. Received 24 May 2002; received after revision 15 July 2002; accepted 16 July 2002     

Colloidal drug carriers: achievements and perspectives

Colloidal drug carriers such as liposomes and nanoparticles are able to modify the distribution of an associated substance. They can therefore be used to improve the therapeutic index of drugs by increasing their efficacy and/or reducing their toxicity. If these delivery systems are carefully designed with respect to the target and route of administration, they may provide one solution to some of the delivery problems posed by new classes of active molecules such as peptides, proteins, genes, and oligonucleotides. They may also extend the therapeutic potential of established drugs such as doxorubicin and amphotericin B. This article discusses the use of colloidal, particulate carrier systems (25 nm to 1 μm in diameter) in such applications. In particular, systems which show diminished uptake by mononuclear phagocytes are described. Specific targeting of carriers to particular tissues or cells is also considered. Received 8 April 2002; received after revision 25 June 2002; accepted 26 June 2002     

Effect of maternal under-nutrition on pup body weight and hypothalamic endocannabinoid levels

Dietary long-chain polyunsaturated fatty acids are known to influence brain levels of the endocannabinoid anandamide in newborn pigs and mice. Furthermore, endocannabinoids were shown to control pup suckling and body weight in mice, and food intake in adult rodents. Here we determined the effect of maternal under-nutrition during gestation, lactation, or both, on body weight, and on the levels of endocannabinoids and expression of cannabinoid CB₁ receptors and fatty acid amide hydrolase in the hypothalamus of rat pups at weaning (21 days old) or adult rats (4 months old). Maternal under-nutrition resulted in a striking decrease in body weight of weaning rats, paralleled by a decrease in the hypothalamic levels of the endocannabinoid anandamide, but not of 2-arachidonoylglycerol. No significant change in the hypothalamic expression of either cannabinoid CB₁ receptors or fatty acid amide hydrolase mRNA was detected in any of the three groups of weaned pups. The decrease in pup body weight and hypothalamic anandamide levels was not observable in 4-month-old rats from any of the three groups. These data suggest that maternal under-nutrition causes a decrease in hypothalamic anandamide levels and loss of body weight, and confirm a crucial role for endocannabinoid signalling in neonatal development. Received 4 November 2002; received after revision 29 November 2002; accepted 16 December 2002 RID="*" ID="*"Corresponding author.     

Carboxypeptidase E and thrombospondin-1 are differently expressed in subcutaneous and visceral fat of obese subjects

The aim of this study was to identify candidate genes for visceral obesity by screening for genes strongly differentially expressed between human subcutaneous and visceral adipose depots. A cDNA microarray with human adipose-derived cDNAs was used as an initial screening to identify genes that are potentially differentially expressed between human subcutaneous and visceral abdominal fat tissues. For the two best candidates, carboxypeptidase E (CPE) and thrombospondin-1 (THBS1) (EST N72406), real-time RT-PCR was performed to confirm their depot specific expression in extremely obese individuals. Both genes appeared to be strongly differentially expressed, having a higher expression in the visceral depot than in the subcutaneous one. For THBS1, the difference in expression between the depots was greater in women than in men. The involvement of CPE and THBS1 in obesity allows us to suggest that the physiological processes controlled by these genes contribute to depot and gender-related differences in the metabolic complications of obesity. Received 7 August 2002; received after revision 19 Septemer 2002; accepted 24 September 2002