Antifungal activity of Euphorbia species against moulds responsible of cereal ear rots.

AIMS: This work aimed to identify secondary metabolites from aerial parts of Euphorbia species functional for control of toxigenic Fusarium species responsible of cereal grain rots. METHODS AND RESULTS: Aerial parts of Euphorbia serpens, Euphorbia schickendantzii and Euphorbia collina were sequentially extracted with hexane, ethyl acetate and methanol. The extracts were tested against strains of Fusarium verticillioides and Fusarium graminearum by microdilution tests. The hexane extract of E. collina provided the lowest IC50 s on both fungal species. Further fractionation showed that cycloartenol (CA) and 24-methylenecycloartanol are associated to the moderate inhibitory effect of the hexane extract on fungal growth.Sublethal concentrations of CA and 24MCA blocked deoxynivalenol (DON) and fumonisins production.CA and 24MCA co-applied with potassium sorbate, a food preservative used for Fusarium control, synergized the growth inhibition of fungi. The mixtures reduced mycotoxins accumulation when applied at sublethal concentrations. CONCLUSIONS: CA and 24MCA inhibited both fungal growth and mycotoxins production. This fact is an advantage respect to potassium sorbate which increased the mycotoxins accumulation at sublethal concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: CA and 24MCA synergized potassium sorbate and their mixtures offer a lower mycotoxigenic risk than potassium sorbate for control of the Fusarium species.

Nerve growth factor transcriptional control of c-fos promoter transfected in cultured spinal sensory neurons.

High efficiency gene transfer (greater than 90%) in chicken dorsal root ganglion neurons has been obtained by DNA calcium phosphate co-precipitation, hence providing an important tool to study control of gene expression in primary neurons. Transfection with c-fos promoter sequences linked to the chloramphenicol acetyltransferase reporter gene showed that the serum responsive element functions as a strong transcriptional enhancer. Transcription from this element is developmentally regulated, and mediates the genetic response to nerve growth factor (NGF) in developing avian sensory neurons. Furthermore, NGF exerts a negative effect on transcription from the cyclic AMP responsive element, thereby supporting the involvement of tyrosine kinase activation by NGF in primary sensory neurons.

Molecular mechanisms underlying wound healing and anti-inflammatory properties of naturally occurring biotechnologically produced phenylpropanoid glycosides.

Two phenylpropanoid glycosides, verbascoside (VB) and teupolioside (TP), produced biotechnologically by Syringa vulgaris and Ajuga reptans plant cell cultures, were studied in vitro and in vivo for their anti-inflammatory and wound healing activities. It was shown that TP- and VB-containing extracts significantly accelerated wound healing and possessed remarkable anti-inflammatory action in the excision wound model. These effects correlated with the inhibition of reactive oxygen species release from the whole blood leukocytes and with the ferrous ion chelating capacity. On the other hand, they don't correlate either with free radical scavenging or with the inhibition of lipid peroxidation in the cell-free systems. Furthermore, both VB- and TP-containing extracts were extremely effective inhibitors of chemokine and growth factor expression by cultured human keratinocytes treated with pro-inflammatory cytokines, TNF-alpha and interferon-gamma.

Nerve growth factor: from neurotrophin to neurokine.

Nerve growth factor (NGF) is largely known as a target-derived factor responsible for the survival and maintenance of the phenotype of specific subsets of peripheral neurones and basal forebrain cholinergic nuclei during development and maturation. However, NGF also exerts a modulatory role on sensory, nociceptive nerve physiology during adulthood that appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. Other NGF-responsive cells are now recognized as belonging to the haemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. The concentration of NGF is elevated in a number of inflammatory and autoimmune states in conjunction with an increased accumulation of mast cells. Mast cells and NGF appear to be involved in neuroimmune interactions and tissue inflammation, with NGF acting as a general 'alert' molecule capable of recruiting and priming tissue defence processes following insult as well as systemic defensive mechanisms. Moreover, mast cells themselves produce NGF, suggesting that alterations in normal mast cell behaviours can provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states. This review discusses recent discoveries involving novel and diverse biological activities of this fascinating molecule.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

Relationship of DNA topoisomerase II alpha and beta expression to cytotoxicity of antineoplastic agents in human acute lymphoblastic leukemia cell lines.

The levels of expression of topoisomerase II alpha and topoisomerase II beta were investigated in six established cell lines of human childhood acute lymphoblastic leukemia (ALL) as a function of doubling time, cell cycle distribution, and of sensitivity to the antineoplastic agents Adriamycin and etoposide. The slowest growing cell line, ALL-G, was most sensitive to both drugs, whereas the fastest growing cell line, ALL-C, was 15.3- and 6.4-fold more resistant than ALL-G to Adriamycin and etoposide, respectively. Furthermore, ALL-W, the second most rapidly dividing cell line, was most resistant to both Adriamycin (22.8-fold) and etoposide (14.1-fold). Expression of topoisomerase II alpha varied inversely with doubling time, whereas no correlation was found between topoisomerase II beta levels and doubling time. Expression of topoisomerase II beta varied inversely with that of topoisomerase II alpha. The level of topoisomerase II alpha correlated directly with the percentage of cells in S and G2-M phases, whereas topoisomerase II beta expression varied directly with the number of cells in G1. An inverse correlation was found between the level of expression of topoisomerase II beta and resistance to Adriamycin, whereas a direct correlation was observed between the level of expression of topoisomerase II alpha and resistance to Adriamycin. Studies with etoposide, although not statistically significant, were consistent with the pattern observed with Adriamycin. These findings suggest that in ALL cells, cytocidal activity of Adriamycin and etoposide may be mediated, at least in part, by topoisomerase II beta.

Factor V Leiden improves in vivo hemostasis in murine hemophilia models.

The role of factor V Leiden (FVL) as a modifier of the severe hemophilia phenotype is still unclear. We used mice with hemophilia A or B crossed with FVL to elucidate in vivo parameters of hemostasis. Real-time thrombus formation in the microcirculation was monitored by deposition of labeled platelets upon laser-induced endothelial injury using widefield microscopy in living animals. No thrombi formed in hemophilic A or B mice following vascular injuries. However, hemophilic mice, either heterozygous or homozygous for FVL, formed clots at all injured sites. Injection of purified activated FV into hemophilic A or B mice could mimic the in vivo effect of FVL. In contrast to these responses to a laser injury in a microvascular bed, FVL did not provide sustained hemostasis following damage of large vessels in a ferric chloride carotid artery injury model, despite of the improvement of clotting times and high circulating thrombin levels. Together these data provide evidence that FVL has the ability to improve the hemophilia A or B phenotype, but this effect is principally evident at the microcirculation level following a particular vascular injury. Our observations may partly explain the heterogeneous clinical evidence of the beneficial role of FVL in hemophilia.

Nerve growth factor directs differentiation of the bipotential cell line GH-3 into the mammotroph phenotype.

GH-3 is an established cell line which, for the production of both PRL and GH, may be related to the bipotential somatomammotroph from which both somatotroph and mammotroph cells derive. In the present study we first report that GH-3 cells express both the gp140trk and the gp75 components of the nerve growth factor (NGF) receptor and that NGF dictates a nonneuronal type of differentiation of this cell line of ectodermal origin. After exposure to NGF, GH-3 cells markedly decreased their proliferation rate. This effect, which was maximal (50% inhibition) 3 days after beginning the treatment and was maintained during the following days of exposure, was paralleled by a change in the hormone production. The secretion of PRL was increased 6-fold, but that of GH was remarkably inhibited. Moreover, GH-3 cells expressed the mammotroph-specific D-2 receptor protein in response to NGF, as shown by binding with the D-2 receptor ligand N-(p-aminophenetyl)spiperone coupled to fluorescein. The present data thus show that NGF induces the differentiation of GH-3 cells into one of their physiological counterparts, the mammotroph cell, and together with the finding that NGF receptors are expressed in the anterior pituitary suggest a physiological role for the neurotrophic factor in pituitary ontogenesis.

Beta-adrenergic receptor regulation of NGF-mRNA content in rat C6-2B glioma cells.

In C6-2B astrocytoma cells the Beta NGF content and secretion rate are increased by isoproterenol activation of beta-adrenergic receptors (Schwartz and Costa, 1977). Utilizing poly (A+) RNA hybridization analysis with a cRNA probe for mouse Beta NGF it was found that isoproterenol activation of C6-2B cells produces also a 4 fold increase of the content of messenger RNA encoding Beta NGF. This increase is specifically antagonized by 1-propanolol, but not by phentolamine. Furthermore, addition of dibutyryl-cAMP induces an increase of Beta NGF mRNA content similar to that obtained with isoproterenol. These results are consistent with the hypothesis that regulation of Beta NGF synthesis in neuroglial cells may be modulated by beta-adrenergic receptor activation.

Dopamine receptors on human T- and B-lymphocytes.

The presence of functional dopamine receptors on differentiated cells of the mammalian immune system is still under discussion. This study has utilized (-)-[3H]sulpiride as a ligand, to detect the presence of recognition sites of the dopamine D2 receptor family on human T- and B-lymphocytes. The (-)-[3H]sulpiride binding was of high affinity (Kd 0.9 nM +/- 0.2 nM), specific, saturable (Bmax 10.2 +/- 1.4 fmol/10(6) cells) and reversible. The pharmacological characterization of the recognition site suggests similarities mainly with the D2 and D4 rather than D3 subtype of dopamine receptor. Furthermore, dopamine treatment was able to reduce the intracellular cAMP levels of lymphocytes stimulated with forskolin, thus suggesting a potential functional significance of this dopamine receptor in mediating neural-immune interactions.

Characterization and growth-dependent regulation of the nerve growth factor receptor gp140trk in rat C6 glioma cells.

The glioma cell line C6 was used to study the expression and growth-dependent regulation of the nerve growth factor (NGF) tyrosine kinase receptor gp140trk, which is the mature protein product of the trk proto-oncogene. Chemical cross-linking of 125I-NGF to C6 cells, followed by immunoprecipitation with polyclonal anti-NGF antibodies and separation by polyacrylamide gel electrophoresis, revealed the presence of 90-95 and 150 kDa species. Immunocytochemical staining of C6 cells with antibodies directed against either the low-affinity NGF receptor gp75NGFR or trk proto-oncogene products demonstrated a heterogeneous cellular distribution of both antigens. Brief treatment of C6 cells with NGF led to the tyrosine phosphorylation of 80, 110 and 140 kDa protein species, as detected on anti-phosphotyrosine Western blots. Similar molecular weight species were found with anti-Trk antibodies in the NGF-treated cells. Intracellular localization of Trk-like immunoreactivity in C6 cells released from a growth-arrested state indicated an initial immunostaining of the nuclear periphery, progressing to cytoplasmic vesicles and finally to the plasma membrane. These observations at the light microscopic level were confirmed using immunoelectron microscopy with the same anti-Trk antibodies, and showed clearly the trafficking of Trk-like immunostained particles from the endoplasmic reticulum to the plasmalemma. The cellular localization of trk gene products also appeared to depend on their glycosylation state. Such growth-dependent expression of NGF receptors on glial cells may be important in controlling autocrine regulatory processes of glia to NGF, which these cells produce.

Choline acetyltransferase messenger RNA expression in developing and adult rat brain: regulation by nerve growth factor.

The polymerase chain reaction (PCR) was used to develop a method for detection and relative quantification of the choline acetyltransferase (ChAT) mRNA in neonatal and adult rat CNS. Oligonucleotide primers derived from a porcine ChAT cDNA sequence were used in coupled reverse transcriptase (RT)-PCR to amplify a cDNA sequence of 206 bp which arises in a cycle- and RNA-dependent manner and which hybridizes with both an internal oligonucleotide and a ChAT cDNA probe. ChAT mRNA was detected in spinal cord, septal area, striatum, cortex and hippocampus but not in cerebellum and cardiac or skeletal muscle. In the septal area, relative quantitative evaluation of ChAT mRNA levels by RT-PCR indicates that this transcript is developmentally regulated and increased following intracerebral administration of nerve growth factor (NGF) to both neonatal and young adult rats. This suggests that the increases of ChAT activity observed in basal forebrain during development or after NGF administration are, at least in part, associated with an increase in corresponding levels of mRNA.

Differential turnover rates of D1 dopamine receptors in the brain and retina of adult and senescent rats.

The present study was designed to investigate the turnover rates of D1 dopamine receptors in the brain and retina of adult and aged rats. To this aim, we monitored the increase in the density of 3H-SCH 23390 binding sites after the administration of the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2 dihydroquinoline (EEDQ). The results indicate that, in aged rats, the production rate of D1 receptors decreases by 41% to 61% in the striatum, nucleus accumbens and substantia nigra, whereas a smaller reduction in the degradation rate of these receptors is observed (-21% to -40%). In contrast, the production rate of D1 receptors in the retina of aged rats remains unchanged, whilst the degradation rate decreases by 25%. These alterations in the rates of receptor production and degradation may account for the age-related decrease in the density of D1 dopamine receptors in the striatum, nucleus accumbens and substantia nigra as well as for the increase in the density of these receptors in the retina of aged rats.

Age-related changes in the turnover rates of D1-dopamine receptors in the retina and in distinct areas of the rat brain.

The steady-state density and the turnover rates of D1-dopamine receptors were investigated in the striatum, nucleus accumbens, substantia nigra, and retina of adult (3-month-old) and aged (23-month-old) rats. The turnover rates were measured by monitoring the repopulation kinetics of D1-dopamine receptors labeled with [3H]-SCH 23390 after the irreversible inactivation induced by a single dose of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 10 mg/kg, s.c.). In all the neural tissues examined, the repopulation of D1 dopamine receptors could be adequately described by a theoretical model that assumes a constant rate of receptor production (i.e. zero order) and a rate of degradation that is dependent on the receptor density at any time (i.e. first order). The results obtained indicate that the reduction in the density of D1-dopamine receptors in the striatum, nucleus accumbens and substantia nigra of aged rats is the result of a larger decrease in the receptor production rate (-44 to -60%) than in the receptor degradation rate (-21 to -46%). By contrast, the production rate of D1-dopamine receptors in the retina of aged rats remains unchanged, whilst the degradation rate is reduced by 25%. This results in an age-related increase in the density of D1-dopamine receptors in the rat retina.

Parkinsonian serum carries complement-dependent toxicity for rat mesencephalic dopaminergic neurons in culture.

The presence of antibodies recognizing specific epitopes of dopaminergic neurons in serum of patients suffering of Parkinson's Disease (PD) as well as their capability to induce neuronal damage was investigated utilizing serum-free dissociated mesencephalic-striatal co-cultures. High affinity dopamine (DA) and GABA uptakes were assessed as specific, functional markers of dopaminergic and GABAergic cell viability, respectively. Heat-inactivated serum samples from 18 and 13 patients suffering from idiopathic and vascular parkinsonism, respectively and from 18 neurologic controls, were added to co-cultures on day 4 in vitro. Twenty four hours later, reconstituted rabbit complement was added for 60 min and uptake parameters as well as immunocytochemical staining for tyrosine hydroxylase (TH)-containing cells were subsequently assessed. DA, but not GABA, uptake was significantly decreased only when complement was added to cultures containing serum samples from 14 out of 18 patients with idiopathic parkinsonism and 3 out of 13 patients with vascular parkinsonism (Fisher test, P < 0.01). Complement addition to cultures containing serum samples from seropositive parkinsonian patients significantly reduced immunocytochemical staining of TH-containing cells. Seropositive and seronegative patients did not differ in demographic and clinical features. These results suggest that a complement-dependent humoral immune response occurs mainly in idiopathic parkinsonian patients, but its clinical relevance remains to be established.

Update of the NGF saga.

Nerve growth factor (NGF), initially characterized for its survival and differentiating actions on embryonic sensory and sympathetic neurons, is now known to display a greatly extended spectrum of biological functions. NGF exerts a profound modulatory role on sensory nociceptive nerve physiology during adulthood which appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. Other newly detected NGF-responsive cells belong to the hematopoietic-immune and neuroendocrine systems. In particular, mast cells and NGF both appear to be involved in neuroimmune interactions and tissue inflammation, with NGF acting as a general "alert" molecule capable of recruiting and priming both local tissue and systemic defense processes following stressful events. NGF can thus be viewed as a multifactorial mediator modulating neuroimmune-endocrine functions of vital importance to the regulation of homeostatic interactions, with potential involvement in pathological processes deriving from dysregulation of either local or systemic homeostatic balances.

Mechanisms of chromium toxicity in mammalian cell cultures.

Our observations about the cytotoxic and cytogenetic effects of hexavalent and trivalent chromium compounds in mammalian cells cultured in vitro are reviewed. Additional data concerning the induction of chromosomal aberrations and sister chromatid exchanges, the inhibition of nucleic acid and protein synthesis, the interference with nucleotide metabolism, and the modification of membrane-linked enzyme activity are reported. A possible mechanism of chromium action is proposed.

Effects of potassium dichromate on ATP content of mammalian cells cultured in vitro.

In order to elucidate the mechanism of the cytotoxic activity of hexavalent chromium (Cr(VI)), the alterations of intracellular ATP levels induced by potassium dichromate in cultured hamster fibroblasts (BHK line) have been studied. Two kinds of treatment procedures were adopted: (1) BHK cell suspensions were exposed to 0.05--1.00 mM K2Cr2O7 in Hanks' balanced salt solution (BSS) for up to 180 min and ATP concentrations were determined immediately after the exposure to Cr(VI). A decrease of ATP content was observed with 0.25--12.00 mM K2Cr2O7 but only in the case of the highest dose was it related in a linear fashion to the duration of the treatment. (2) Cells were preincubated in BSS for 30 min with 0.05--1.00 mM dichromate. They were then reincubated in Eagle's minimal essential medium (MEM) for up to 180 min and ATP was measured at different time points. Immediately after the exposure to chromium all the treated cultures showed a depletion of ATP content. However while the cells treated with 0.25--0.25 mM dichromate rapidly resumed ATP levels very similar to that of the control, no recovery was detected in cells treated with 0.50 and 1.0 mM K2Cr2O7, even after 180 min. The observed effects have been attributed to the oxidizing activity of Cr(VI), which subtracts electrons from electron donors involved in metabolic pathways producing ATP, and to the ability of Cr(III), deriving from Cr(VI) reduction, to form stable coordination complexes with ATP precursors and enzymes involved in ATP synthesis.

Effects of gangliosides on the functional recovery of damaged brain.

The effect of GM1 ganglioside on the recovery of dopaminergic nigro-striatal neurons was studied in rats after unilateral hemitransection. GM1 treatment favoured the collateral sprouting of dopaminergic axons in the striatum as indicated by the induced increase of tyrosine hydroxylase (TH) activity and immunofluorescence. Concomitantly GM1 partially prevented the decrease of TH activity caused by the hemitransection in the substantia nigra ipsilateral to the lesion. A significant increase of TH immunoreactivity was also detected in the substantia nigra: GM1 prevented the disappearance of TH-positive cell bodies and increased the formation of TH-positive collaterals and dendrites with respect to the saline treatment. The addition of GM1 to embryonic dissociated mesencephalic cell cultures stimulates the expression of dopaminergic characteristics as suggested by the increase of 3H-DA uptake.