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.

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.

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.

Modulation of factor VII levels by intron 7 polymorphisms: population and in vitro studies.

Previous studies have established that factor VII gene (F7) polymorphisms (5'F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype-phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with the IVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7 variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7G allele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.

Molecular mechanisms of FVII deficiency: expression of mutations clustered in the IVS7 donor splice site of factor VII gene.

In three Italian patients, two point mutations and a short deletion were found in the intron 7 of factor VII gene, clustered in the donor splice site and located in the first of several repeats. The mutation 9726+5G-->A, the most frequent cause of symptomatic factor VII deficiency in Italy, as well as the deletion (9729del4) gave rise in expression studies to abnormally spliced transcripts, which were exclusively produced from the cryptic site in the second repeat. The insertion in the mature mRNA of the first intronic repeat caused (9726+5G-->A) a reading frameshift, abolishing most of the factor VII catalytic domain, or produced (9729del4), an altered factor with 11 additional residues, the activity of which was not detectable in the cell medium after mutagenesis and expression studies. Studies of factor VII ectopic mRNA from leukocytes and expression studies indicated that the deleted gene produced 30% of normally spliced transcript. Differently, the 9726+5G-->A mutation permitted a very low level (0.2% to 1%) of correct splicing to occur, which could be of great importance to prevent the onset, in the homozygous patients, of most of the life-threatening bleeding symptoms. The 9726+7A-->G mutation was found to be a rare and functionally silent polymorphism. These findings, which provide further evidence of the interplay of sequence and position in the 5' splice site selection, throw light on the heterogeneous molecular bases and clinical phenotypes of FVII deficiency.

Interactions between brain-derived neurotrophic factor and the TRKB receptor. Identification of two ligand binding domains in soluble TRKB by affinity separation and chemical cross-linking.

The extracellular domain of the human neurotrophin TRKB receptor expressed in Chinese hamster ovary cells is a highly glycosylated protein, possessing binding ability for brain-derived neurotrophic factor (BDNF). Two distinct ligand binding domains of TRKB were isolated from proteolytic digests of the receptor by affinity separation on immobilized BDNF. One of these domains consists of amino acid residues 103-181 and contains both the third leucine-rich motif and the second cysteine cluster domain. The second domain is close to the second immunoglobulin-like domain (amino acid residues 342-394). Each of these two domains can bind BDNF independently. Disulfide linkages present in the first domain are necessary for BDNF binding, probably because of preservation of the native conformation. To study the second domain in greater detail, a truncated form of TRKB containing the second immunoglobulin-like domain (residues 248-398) was expressed in Escherichia coli. This domain was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling reaction. Several synthetic peptides corresponding to amino acid residues 343-379 were able to bind immobilized BDNF. Amino acid substitution and cross-linking analysis indicated that amino acids Phe347, Asp354, and Tyr361 are intimately involved in BDNF binding. These results, obtained from a variety of experimental techniques, highlight the importance of two distinct regions of the extracellular domain of the TRKB receptor in binding BDNF.

The majority of stem cell factor exists as monomer under physiological conditions. Implications for dimerization mediating biological activity.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

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.

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism.

Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-)-sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control.

B61, a ligand for the Eck receptor protein-tyrosine kinase, exhibits neurotrophic activity in cultures of rat spinal cord neurons.

Although the Eph subfamily represents the largest group of receptor protein-tyrosine kinases, the biological roles of the Eph-related receptors and their ligands are not well understood. B61 has been identified recently by receptor affinity chromatography as a ligand for the Eph-related receptor Eck (Bartley et al.: Nature 368:558-560, 1994). Here we show that Eck immunoreactivity is localized in areas of the embryonic rat spinal cord that are rich in axons, suggesting that Eck plays a role in this region of the developing nervous system. To examine the biological function of Eck, monolayer cultures of dissociated cells from embryonic rat spinal cord were treated with soluble B61. With an ED50 of approximately 10 ng/ml, B61 treatment improved the survival of the overall neuronal population. Furthermore, in the presence of B61 neurites were longer and more elaborated. B61 similarly affected survival and neurite length in cultures enriched in motor neurons. These neurotrophic effects of B61 were not observed in the presence of anti-Eck antibodies, indicating that these effects are likely to be mediated by the Eck receptor.

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.

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.

Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide.

Mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play important roles in tissue inflammation and in neuroimmune interactions. Very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. Palmitoylethanolamide, found in peripheral tissues, has been proposed to behave as a local autacoid capable of downregulating mast cell activation and inflammation. A cognate N-acylamide, anandamide, the ethanolamide of arachidonic acid, occurs in brain and is a candidate endogenous agonist for the central cannabinoid receptor (CB1). As a second cannabinoid receptor (CB2) has been found in peripheral tissues, the possible presence of CB2 receptors on mast cells and their interaction with N-acylamides was investigated. Here we report that mast cells express both the gene and a functional CB2 receptor protein with negative regulatory effects on mast cell activation. Although both palmitoylethanolamide and anandamide bind to the CB2 receptor, only the former downmodulates mast cell activation in vitro. Further, the functional effect of palmitoylethanolamide, as well as that of the active cannabinoids, was efficiently antagonized by anandamide. The results suggest that (i) peripheral cannabinoid CB2 receptors control, upon agonist binding, mast cell activation and therefore inflammation; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for the CB2 receptor on mast cells; (iii) modulatory activities on mast cells exerted by the naturally occurring molecule strengthen a proposed autacoid local inflammation antagonism (ALIA) mechanism; and (iv) palmitoylethanolamide and its derivatives may provide antiinflammatory therapeutic strategies specifically targeted to mast cells ("ALIAmides").

Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6.

The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.

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.

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.

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.