Structure and component dynamics in binary mixtures of poly(2-(dimethylamino)ethyl methacrylate) with water and tetrahydrofuran: A diffraction, calorimetric, and dielectric spectroscopy study.

We have combined X-ray diffraction, neutron diffraction with polarization analysis, small angle neutron scattering,differential scanning calorimetry, and broad band dielectric spectroscopy to investigate the structure and dynamics of binary mixtures of poly(2-(dimethylamino)ethyl methacrylate) with either water or tetrahydrofuran (THF) at different concentrations. Aqueous mixtures are characterized by a highly heterogeneous structure where water clusters coexist with an underlying nano-segregation of main chains and side groups of the polymeric matrix. THF molecules are homogeneously distributed among the polymeric nano-domains for concentrations of one THF molecule/monomer or lower. A more heterogeneous situation is found for higher THF amounts, but without evidences for solvent clusters. In THF-mixtures, we observe a remarkable reduction of the glass-transition temperature which is enhanced with increasing amount of solvent but seems to reach saturation at high THF concentrations. Adding THF markedly reduces the activation energy of the polymer ß-relaxation. The presence of THF molecules seemingly hinders a slow component of this process which is active in the dry state. The aqueous mixtures present a strikingly broad glass-transition feature, revealing a highly heterogeneous behavior in agreement with the structural study. Regarding the solvent dynamics, deep in the glassy state all data can be described by an Arrhenius temperature dependence with a rather similar activation energy. However, the values of the characteristic times are about three orders of magnitude smaller for THF than for water. Water dynamics display a crossover toward increasingly higher apparent activation energies in the region of the onset of the glass transition, supporting its interpretation as a consequence of the freezing of the structuralrelaxation of the surrounding matrix. The absence of such a crossover (at least in the wide dynamic window here accessed) in THF is attributed to the lack of cooperativity effects in the relaxation of these molecules within the polymeric matrix.

Dynamics of tetrahydrofuran as minority component in a mixture with poly(2-(dimethylamino)ethyl methacrylate): A neutron scattering and dielectric spectroscopy investigation.

We have investigated a mixture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and tetrahydrofuran (THF) (70 wt. % PDMAEMA/30 wt. % THF) by combining dielectric spectroscopy and quasielastic neutron scattering (QENS) on a labelled sample, focusing on the dynamics of the THF molecules. Two independent processes have been identified. The "fast" one has been qualified as due to an internal motion of the THF ring leading to hydrogen displacements of about 3 Å with rather broadly distributed activation energies. The "slow" process is characterized by an Arrhenius-like temperature dependence of the characteristic time which persists over more than 9 orders of magnitude in time. The QENS results evidence the confined nature of this process, determining a size of about 8 Å for the volume within which THF hydrogens' motions are restricted. In a complementary way, we have also investigated the structural features of the sample. This study suggests that THF molecules are well dispersed among side-groups nano-domains in the polymer matrix, ruling out a significant presence of clusters of solvent. Such a good dispersion, together with a rich mobility of the local environment, would prevent cooperativity effects to develop for the structural relaxation of solvent molecules, frustrating thereby the emergence of Vogel-Fulcher-like behavior, at least in the whole temperature interval investigated.

Effect of NGF on the subcellular localization of group IIA secretory phospholipase A(2) (GIIA) in PC12 cells: role in neuritogenesis.

Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.

Phosphatidylethanolamine of lung surfactant: proportion and fatty acid patterns of the alkylacyl, alkenylacyl and diacyl subclasses.

The phosphatidylethanolamine fraction isolated from lung surfactant consisted of 33% of the alkylacyl, 39% of the alkenylacyl and 25% of the diacyl subclass. Palmitic acid was the major fatty acid of the alkyl acyl and alkenylacyl subclasses. In diacylphosphatidylethanolamine, saturated and unsaturated fatty acids revealed a ratio of nearly 1.

The reverse reaction of cholinephosphotransferase in rat brain microsomes. A new pathway for degradation of phosphatidylcholine.

The synthesis of phosphatidylcholine is catalyzed by cholinephosphotransferase (EC 2.7.8.2) which is known to be reversible in liver. The reversibility of cholinephosphotransferase in rat brain in demonstrated in this paper. Labeled microsomes were prepared from young rats which had been given an intracerebral injection of labeled choline or oleate 2 h before killing. During incubation of choline-labeled microsomes with CMP, label was lost from ;choline glycerophospholipids and labeled CDPcholine was produced. The Km for CMP was 0.35 mM and V was 3.3 nmol/min per mg protein. Neither AMP nor UMP could substitute for CMP. Oleate-labeled microsomes were pretreated with e mM diisopropylfluorophosphate (lipase inhibitor). During incubation with CMP, label was lost from choline, and ethanolamine glycerophospholipid and labeled diacylglycerols were produced. When the lipase was not inhibited, labeled oleate was produced. We propose that a principal pathway for degradation of phosphatidylcholine, particularly during brain ischemia, is by reversal of cholinephosphotransferase, followed by hydrolysis of diacylglycerols by the lipase.

A comparison of the reversibility of phosphoethanolamine transferase and phosphocholine transferase in rat brain microsomes.

The reversibility of phosphoethanolamine transferase (EC 2.7.8.1) in rat brain is demonstrated in this paper. Microsomal ethanolamine glycerophospholipids were prelabeled with an intracerebral injection of [3H]ethanolamine 4 h before killing young rats. Labeled CDPethanolamine was produced by incubation of the microsomes with CMP, although to a lesser extent than for the previously observed release of CDPcholine. Ethanolamine and choline glycerophospholipids were labeled with [2-3H]glycerol by incubation with primary cultures of rat brain. Microsomes from rat brains, with diisopropyl phosphofluoridate for inhibition of lipases, were incubated with the labeled glycerophospholipids separately, and labeled diacylglycerols were produced. The kinetic parameters of phosphoethanolamine transferase and phosphocholine transferase (EC 2.7.8.2) were compared by incubating rat brain microsomes with [3H]CMP. Inclusion of AMP in the reaction mixture was necessary in order to inhibit the hydrolysis of CMP by an enzyme with the properties of 5'-nucleotidase (EC 3.1.3.5). For phosphoethanolamine transferase and phosphocholine transferase respectively, the Km values for CMP were 40 and 125 microM and the V values were 2.3 and 21.6 nmol/h per mg protein. The reversibility of both enzymes permits the interconversion of the diacylglycerol moieties of choline and ethanolamine glycerophospholipids. During brain ischemia, a principal pathway for degradation of ethanolamine glycerophospholipids may be by reversal of phosphoethanolamine transferase followed by hydrolysis of diacylglycerols by the lipase.

Activation of phospholipase A2 and beta-thromboglobulin release in human platelets: comparative effects of thrombin and fluoroaluminate stimulation.

Several reports have suggested that the activity of platelet phospholipase A2 is modulated by GTP-binding protein(s) whose nature and properties need to be defined. Fluoroaluminate is known to activate G-proteins and this leads to a number of cellular responses including the activation of phospholipases. This paper demonstrates that human platelets, prelabelled with [3H]arachidonic acid, produce free arachidonic acid when stimulated with fluoroaluminate and this effect is time- and dose-dependent. The production of arachidonic acid is not inhibited by neomycin, a PI-cycle inhibitor, but is completely abolished by mepacrine, an inhibitor of both phospholipase A2 and C. At low concentration of fluoroaluminate (10 mM NaF) phospholipase A2 but not phospholipase C is activated. In addition, fluoroaluminate treatment releases beta-thromboglobulin (beta-TG) and this effect is not inhibited by acetylsalicylic acid. Under identical conditions both neomycin and mepacrine suppress the release of arachidonic acid and beta-TG induced by thrombin. Sodium nitroprusside, which increases cGMP levels in platelets, inhibits arachidonic acid liberation and beta-TG release in thrombin-stimulated platelets but has no effect in fluoroaluminate-treated platelets; cGMP was reported to suppress phospholipase C activation. These results are consistent with the hypothesis that, in thrombin-stimulated platelets, the liberation of arachidonic acid and beta-TG are strictly dependent on the activation of phospholipase C. We have also provided evidence for the existence of a phospholipase A2 activated by a G-protein which is independent from the degradation of phosphoinositides and, contrary to phospholipase C, it is not down regulated by cGMP.

Evidence that cytosolic phospholipase A2 is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate.

We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF4-), an unspecific G-protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca2+ concentration and that AlF4(-)-induced release of AA is abolished by BAPTA, an intracellular Ca2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2). These observations indicate that cPLA2 is responsible for the AlF4(-)-induced release of arachidonic acid by a mechanism that is down-regulated by PKC.

Protein kinase C inhibitors enhance G-protein induced phospholipase A2 activation in intact human platelets.

Washed intact human platelets were prelabelled with [3H]arachidonic acid ([3H]AA) and stimulated with thrombin or with AlF4-, a known unspecific activator of G-proteins. Both stimuli induced the liberation of [3H]AA, the release of beta-thromboglobulin (beta-TG) and platelet aggregation. PMA did not induce liberation of [3H]AA although it induced beta-TG release and aggregation; preincubation with PMA did not modify significantly the amounts of [3H]AA and beta-TG released by thrombin or AlF4-. Different inhibitors of PKC (staurosporine, H-7 and calphostin C) increased the release of [3H]AA and inhibited beta-TG release and aggregation induced by AlF4- but they had no effect when platelets were stimulated with thrombin (0.5 U/ml). Calphostin C was able to release [3H]AA by itself without inducing aggregation of beta-TG release. Okadaic acid (a serine/threonine phosphoprotein phosphatase inhibitor) greatly inhibited the release of [3H]AA, beta-TG and aggregation in AlF4--stimulated platelets. These results indicate the presence of a G-protein mediated mechanism for the activation of a platelet phospholipase A2 which is negatively affected by a protein kinase, sensible to putative inhibitors of protein kinase C, and it is activated by a protein phosphatase, sensible to okadaic acid.

Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary for coupling postsynaptic and presynaptic phenomena, through the activation of group I metabotropic glutamate receptors, and its action lasts only for a short period. If this coupling does not occur, a full and long-lasting potentiation cannot develop.

Inhibition of PAF synthesis by stimulated human polymorphonuclear leucocytes with cloricromene, an inhibitor of phospholipase A2 activation.

1. A phospholipase A2 (PLA2) represents the key enzyme in the remodelling pathway of platelet-activating factor (PAF) synthesis in human polymorphonuclear (PMN) leucocytes. 2. PLA2 activation is also the rate-limiting step for the release of the arachidonic acid utilized for the synthesis of leukotrienes in stimulated leucocytes; however, it is unknown whether the PLA2s involved in the two biosynthetic pathways are identical. 3. Cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy- carbonylmethoxy coumarin) is an antithrombotic coumarin derivative which inhibits platelet and leucocyte function and suppresses arachidonic acid liberation by interfering with PLA2 activation. 4. The aim of the present study was to assess whether chloricromene inhibits PAF synthesis by stimulated human polymorphonuclear leucocytes (PMNs). 5. Cloricromene (50-500 microM) inhibited in a concentration-dependent manner the release of PAF, as measured by h.p.l.c. bioassay, from A23187-stimulated PMNs. Significant inhibition (45%) of PAF-release was obtained with 50 microM cloricromene and the IC50 was 85 microM. Mepacrine (500 microM), a non-specific PLA2 inhibitor, strikingly reduced PAF release. 6. The incorporation of [3H]-acetate into [3H]-PAF induced by serum-treated zymosan in human PMNs was also inhibited concentration-dependently by cloricromene, with an IC50 of 105 microM. Mepacrine also suppressed [3H]-acetate incorporation into [3H]-PAF. 7. Cloricromene did not affect the activities of the enzymes involved in PAF-synthesis acetyltransferase or phosphocholine transferase. 8. Our data demonstrate that cloricromene, an inhibitor of PLA2-activation in human leucocytes, reduces the synthesis of PAF by stimulated PMNs. This finding has a twofold implication: the PLA2s (or the mechanisms that regulate their activation) involved in PAF synthesis and arachidonate release in human leucocytes are either identical or else indistinguishable by their sensitivity to cloricromene; the inhibition of PAF release by activated leucocytes may contribute to the antithrombotic and anti-ischaemic activities exerted by cloricromene.

Cloricromene inhibits leukotriene formation by human polymorphonuclear leucocytes by suppressing arachidonate release from membrane phospholipids.

Cloricromene, an antithrombotic agent known to inhibit the release of arachidonic acid (AA) in stimulated human platelets, was tested for its effects on arachidonate release and metabolism in human polymorphonuclear leucocytes (PMNs). Cloricromene dose-dependently suppressed the release of leukotriene B4 (LTB4), as assessed by radioimmunoassay, from both isolated PMNs and human whole blood stimulated with the calcium ionophore A23187 or with serum-treated zymosan (STZ). The inhibitory effect was higher when the concentration of the stimulating agent was weaker. Cloricromene also inhibited dose-dependently the liberation of LTB4, LTC4, LTD4 and 5-hydroxy-6,8,11,14-eicosatraenoic acid as assessed by HPLC in the supernantant of A23187-stimulated PMNs. Finally, the drug was able to suppress the release of [3H]AA from purified human PMNs prelabeled with the radioactive fatty acid and stimulated with either A23187 or with STZ. The A23187-induced decrease in the radioactivity of phosphatidylinositol, the phospholipid class mainly involved in AA release in stimulated PMNs, was also inhibited by cloricromene. Cloricromene suppresses leukotriene formation in human PMNs by reducing AA release from membrane phospholipids, possibly through interference with phospholipase A2 activation; this activity may contribute to the leucocyte-inhibitory effects reported previously for cloricromene.

A double label retrograde tracing study of the olivocerebellar projection to the pyramis and uvula in the rat.

Microinjections of fluorescent tracers (Nuclear Yellow and Fast Blue) were used to study the organization of olivary cells in nucleus beta which give rise to climbing fibers to the medial zone of the uvula and the intermediate zone of the pyramis. It was determined that most cells in nucleus beta project to one or the other of these cortical sites. A third, smaller, population of cells were double labeled and, therefore, have climbing fiber axon collaterals which innervate these two cortical regions. There was no discernable segregation of these three populations of cells within nucleus beta.

Synaptic vesicle-bound phospholipase(s) acting on phosphatidylinositol exhibit(s) high susceptibility to brain ischemia.

The activity of phospholipase C acting against [3H]-inositol-phosphatidylinositol (PI) and the activity of arachidonic acid (AA) release from [1-14C]arachidonoyl-phosphatidylinositol by enzyme(s) located in synaptic vesicles (SV) isolated from normoxic and ischemic brains was investigated. Brain ischemia significantly activated phospholipase C (PhLC) by about 90% and AA release by about 50%. PhLC and AA release in SV isolated from brain submitted to ischemia were not further activated by 2 mM CaCl2 contrary to the enzymes from normoxic brain. The activation of PhLC and PhLA2 may produce conformational changes and rearrangement of the SV membranes leading to vesicle-membrane fusion and subsequently to massive neurotransmitter release known to occur during ischemia.

AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonyl-meth oxy coumarin) inhibits the release of arachidonic acid in human platelets stimulated by thrombin.

The coumarin derivative AD6 is known to inhibit platelet aggregation and release and it possesses vasodilatory properties on coronary arteries of laboratory animals. Furthermore, the inhibition of the production of TxB2 from endogenous substrates after stimulation of human platelets with collagen has been demonstrated. The present report demonstrates that AD6 inhibits the production of labeled arachidonic acid and diglycerides from phospholipids of platelets stimulated with thrombin. This effect is dose-dependent and is already evident at a concentration of the drug (25 microM) which is unable to prevent the aggregation. Apparently, AD6 inhibits the release of arachidonic acid from phosphatidylinositol and choline phosphoglycerides which are the main sources of the substrate for the synthesis of prostaglandins and thromboxanes.

Properties of PAF-synthesizing phosphocholinetransferase and evidence for lysoPAF acetyltransferase activity in rat brain.

Several reports have indicated that platelet-activating factor (PAF) may play a role in the physiopathology of nervous tissue. We previously have demonstrated the presence, in the microsomal fractions of rat brain, of a phosphocholinetransferase which is able to synthesize PAF by the de novo pathway. The presence of dithiothreitol in the medium increases the rate of PAF biosynthesis, whereas it inhibits the synthesis of long-chain alkylacyl- and diacyl-glycerophosphocholines (GPC), including dioctanoyl-GPC. This and other properties, such as pH dependence and thermal stability, indicate that rat brain may have two distinct enzymes for the synthesis of PAF and other choline phospholipids. The affinity of these enzymes for CDPcholine is similar to that reported for other tissues, the Km being 42 microns and 55 microns with alkylacetylglycerol and dioctanoylglycerol as lipid substrates, respectively. The Vmax values were 3.0 and 2.2 nmol/mg prot/min for PAF and dioctanoyl-GPC, respectively. In addition, it was shown that the microsomal fraction of rat brain contains an acetyltransferase which can convert lysoPAF to PAF. Since it has been reported previously that brain tissue possesses phospholipase A2 activity that can hydrolyze alkylacyl-GPC to lysoPAF, we conclude that brain tissue has all enzymic activities for the synthesis of PAF by the "remodeling pathway". The role of the two routes of PAF biosynthesis in nervous tissue remains to be established.

Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine through ethanolaminephosphotransferase activity in the neuronal and glial cells of rabbit in vitro.

The transfer of radioactivity from cytidine-5'-diphosphate ethanolamine into 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine of neuronal and glial cells from adult rabbit brain cortex has been investigated in vitro. The synthesis of 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine in both cell populations was stimulated 23-25-fold by the addition of 6 mM alkylacylglycerol. The neuronal cell-enriched fraction was found to possess/unit protein a 1.7-1.8-fold ethanolaminephosphotransferase activity (EC 2.7.8.1), as compared to the glial fraction, when saturating concentrations (6 mM) of alkylacylglycerols were added in the incubation system. The neuronal/glial ratio was 2.6-2.8 in the absence of lipid acceptor or with low concentrations of alkylacylglycerol. Under most favorable conditions, 6.4 and 3.3 nmoles 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine/mg protein/30 min was obtained for neurons and glia, respectively. Various kinetic properties of the 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine synthesizing phosphotransferase activity were found to be similar both in neurons and glia.

Cholinephosphotransferase activity in human platelets.

Disrupted human platelets possess a cholinephosphotransferase activity (EC 2.7.8.2) whose properties have been studied in this work. The labeling of choline glycerophospholipid (CGP) from radioactive cytidine-5'-diphosphate choline (CDP-choline) in vitro shows a maximum at pH 8.0 (using Hepes [4-(2-hydroxyethyl)-piperazine-1-ethane-2-sulfonic acid] as a buffer) and is stimulated by Mn2+, Mg2+ and diacylglycerol. The enzymic activity is inhibited by Ca2+. The dependence of human platelet cholinephosphotransferase upon CDP-choline concentration does not follow the Michaelis-Menten equation. CMP strongly inhibits the reaction. The functional implications of this newly discovered platelet activity are briefly considered.

Chronic effects of trichloroethylene upon S-100 protein content and lipid composition in gerbil cerebellum.

Gerbil rats were exposed to trichloroethylene (TCE) vapors intermittently (8 h/d) at 510 ppm or continuously at 170 ppm for five months. The cerebellar content of S-100 protein and the phospholipid fatty-acid profiles were determined. S-100 protein, a possible marker for astrocytic reactivity, indicated delayed astrocytic reactivity in the anterior cerebellar hemisphere and a decrease of S-100 protein in the posterior cerebellar vermis. Minor lipid changes were observed. The fatty-acid profiles of ethanolamine phosphoglycerides showed a tendency towards alterations among the 22-carbon fatty acids, with a decrease in 22:5 (N-3), similar to those shown earlier for cerebral cortex and hippocampus of the gerbil. Two monoenoic fatty acids were decreased, the 20:1 of phosphatidyl-ethanolamine and the 18:1 of the phosphatidyl-serine. This occurrence could indicate a decrease in myelin in areas where these two fatty acids were found to be enriched.

Hepatoid carcinoma of the stomach: is it still an unusual anatomo-clinical entity? Six cases-report.

Hepatoid carcinoma of the stomach is a rare neoplasm (especially in western countries) characterized by high levels of serum alpha-fetoprotein (AFP), the presence of "hepatoid foci" inside the gastric tumor and poor prognosis, due to the earlier onset of liver metastases. We treated six patients for hepatoid carcinoma of the stomach between 1990 and 1997. The female to male ratio was 1:1, the average age was 71 (54-81) and the average AFP-level was 1160 ng/ml (603-1531). We performed 2 total gastrectomies, 2 subtotal gastrectomies and 2 gastro-jejunostomies (due to presence of liver metastases): in one case, the patient underwent a splenectomy as well. All the tumors showed the presence of "hepatoid foci" (the morphological feature is close to the hepatocellular carcinoma) and a positive immunoreactivity to AFP. The mean survival was 3 months: only one patient is still alive and disease-free (with a 52 months follow-up). After radical surgery, she underwent a chemotherapic treatment with cisplatin, epirubicin, 5-fluorouracil and l-leucovorin. We conclude that our series (the widest in Italy and one of most impressive in Europe) confirm the poor prognosis of this neoplasm, but we also want to underline that this tumor is not so "unusual" any more and it requires new types of treatment, like postoperative chemotherapy, besides surgery, to be fighted properly.