Expression of nerve growth factor-like polypeptides and immunoreactivity related to the two types of neurotrophin receptors in earthworm tissues

Nerve growth factor (NGF) belongs by sequence homology to the neurotrophins, a family of proteins binding the same p75 receptor and closely related members of the Trk family of receptor tyrosine kinases. Fundamental in the vertebrate nervous system, neurotrophin signals have also been suggested as essential for relatively complex nervous systems occurring in invertebrate species that live longer than Caenorhabditis elegans and Drosophila melanogaster. Mammalian neurotrophins have been found to influence invertebrate neuronal growth. However, there are only a few data on the presence of molecules related to neurotrophin signalling components in invertebrates. Our studies provide evidence that analogues of neurotrophins and neurotrophin receptors are expressed in Eisenia foetida earthworms. In particular, NGF-like and Trk-like immunoreactive proteins are both expressed in the nervous system, whereas p75-like positivity identifies tubular structures associated with dorsal pores that are involved in the earthworm response to mechanical irritation or stress. Received 12 November 2001; received after revision 8 January 2002; accepted 8 January 2002     

Receptor binding, internalization, and retrograde transport of neurotrophic factors

This review deals with the receptor interactions of neurotrophic factors, focusing on the neurotrophins of the nerve growth factor (NGF) family, the glial cell derived neurotrophic factor (GDNF) family, and the ciliary neurotrophic factor (CNTF) family. The finding that two proteins, p75^NTR and Trk, act as receptors for NGF in neurons generated the discovery of other neurotrophic factors/receptor families and has enhanced our understanding of the development, survival, regeneration, and degeneration of the nervous system. The kinetics of binding, the structure of the ligand-receptor complex, and the mechanism of retrograde transport of the neurotrophins are discussed in detail and compared to information available on the GDNF and CNTF families. Each neurotrophic factor family, i.e., NGF, GDNF, and CNTF, has a set of receptors with specificity for individual members of the family and a common receptor without member specificity that, in some families, generates the cellular signal and retrograde transport.     

The neurotrophic factors in non-neuronal tissues

Although neurotrophic factors are defined as molecules that maintain neuronal cells, they possess a range of functions outside the nervous system. For example, glial cell line-derived neurotrophic factor is essential for ureteric branching in kidney morphogenesis and for regulating the fate of stem cells during spermatogenesis. Leukemia inhibitory factor, a member of the interleukin-6 (IL-6) ciliary neurotrophic factor family, inhibits differentiation of embryonic stem cells, induces tubulogenesis in the embryonic kidney, and regulates sperm differentiation. Other IL-6 family members are important in cardiac differentiation and they have pleiotropic functions in the hematopoietic and immune systems. Although neurotrophin receptors have been found on a number of non-neuronal tissues, they represent mostly truncated receptor isoforms that are incapable of signal transduction and may have scavenger or dominant negative functions. However, several examples can be presented of essential non-neuronal functions played by neurotrophins in e.g., cardiac, hair follicle, and vascular differentiation, and the maintenance of immune cells.     

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.     

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.     

Neurotrophins and neuronal differentiation in the central nervous system

The central nervous system requires the proper formation of exquisitely precise circuits to function properly. These neuronal circuits are assembled during development by the formation of synaptic connections between hundreds of thousands of differentiating neurons. For these circuits to form correctly, neurons must elaborate precisely patterned axonal and dendritic arbors. Although the cellular and molecular mechanisms that guide neuronal differentiation and formation of connections remain mostly unknown, the neurotrophins have emerged recently as attractive candidates for regulating neuronal differentiation in the developing brain. The experiments reviewed here provide strong support for a bifunctional role for the neurotrophins in axonal and dendritic growth and are consistent with the exciting possibility that the neurotrophins might mediate activity-dependent synaptic plasticity.     

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.     

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.     

Chronic gestational exposure to ethanol impairs insulin-stimulated survival and mitochondrial function in cerebellar neurons

Chronic gestational exposure to ethanol has profound adverse effects on brain development. In this regard, studies using in vitro models of ethanol exposure demonstrated impaired insulin signaling mechanisms associated with increased apoptosis and reduced mitochondrial function in neuronal cells. To determine the relevance of these findings to fetal alcohol syndrome, we examined mechanisms of insulin-stimulated neuronal survival and mitochondrial function using a rat model of chronic gestational exposure to ethanol. In ethanol-exposed pups, the cerebellar hemispheres were hypoplastic and exhibited increased apoptosis. Isolated cerebellar neurons were cultured to selectively evaluate insulin responsiveness. Gestational exposure to ethanol inhibited insulin-stimulated neuronal viability, mitochondrial function, Calcein AM retention (membrane integrity), and GAPDH expression, and increased dihydrorosamine fluorescence (oxidative stress) and pro-apoptosis gene expression (p53, Fas-receptor, and Fas-ligand). In addition, neuronal cultures generated from ethanol-exposed pups had reduced levels of insulin-stimulated Akt, GSK-3β, and BAD phosphorylation, and increased levels of non-phosphorylated (activated) GSK-3β and BAD protein expression. The aggregate results suggest that insulin-stimulated central nervous system neuronal survival mechanisms are significantly impaired by chronic gestational exposure to ethanol, and that the abnormalities in insulin signaling mechanisms persist in the early postnatal period, which is critical for brain development. Received 21 January 2002; received after revision 28 February 2002; accepted 25 March 2002     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Local and target-derived actions of neurotrophins during peripheral nervous system development

Neurotrophic factors are present in limiting quantities, and neurons that obtain an adequate supply of the required neurotrophic factor survive whereas those that compete unsuccessfully die. Analysis of null mutant mice for neurotrophins and Trk receptors as well as in vivo experiments in ovo in the chick applying exogenous neurotrophins or neutralising antisera have significantly increased knowledge of the roles they play during development. This review focuses on recent advances in understanding the various roles of neurotrophins in dorsal root ganglion sensory neuron development at different times in embryonic development - an early local role for differentiation of the sensory precursor cells and a later survival-promoting target-derived role for the mature neurons. Neurotrophic factors are present in limiting quantities, and neurons that obtain an adequate supply of the required neurotrophic factor survive whereas those that compete unsuccessfully die. Analysis of null mutant mice for neurotrophins and Trk receptors as well as in vivo experiments in ovo in the chick applying exogenous neurotrophins or neutralising antisera have significantly increased knowledge of the roles they play during development. This review focuses on recent advances in understanding the various roles of neurotrophins in dorsal root ganglion sensory neuron development at different times in embryonic development - an early local role for differentiation of the sensory precursor cells and a later survival-promoting target-derived role for the mature neurons.     

Cerivastatin: a cellular and molecular drug for the future?

The 'statin story' began in 1987 when the first-generation, fungal HMG-CoA reductase inhibitor lovastatin received FDA approval in the USA. Ten years later, the sixth compound of this class came onto the world market - the fully synthetic statin cerivastatin. A number of clinical studies had confirmed its high pharmacological efficacy, its excellent pharmacokinetic properties with fast and nearly complete absorption after oral uptake, a linear kinetic over a broad concentration range, and its favorable safety profile. The greatest advantages, of cerivastatin, however, are its lipophilicity, its high bioavailability of about 60% after oral application and its potency at 100-fold lower doses compared to other lipophilic statins. Nevertheless, the most exciting findings are certainly its non-lipid-related, pleiotropic effects at the cellular and molecular level. Statin therapy was also found to reduce mortality in cases where cholesterol levels or atherosclerotic plaque formation remained unaltered. However, cerivastatin improves endothelial dysfunction, possesses anti-inflammatory, antioxidant, anticoagulant, antithrombotic, antiproliferative, plaque-stabilizing, immunmodulatory, and angiogenic effects, and may even prevent tumor growth, Alzheimer's disease, and osteoporosis. Most of these effects seem to be based on the inhibition of isoprenoid synthesis. Although cerivastatin is no longer on the market because of some problematic side effects, it could be one of the most potent cellular and molecular drugs for the future. Received 29 May 2002; received after revision 23 August 2002; accepted 26 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.