Synthesis and biological activity of 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole derivatives, novel pro-apoptotic mitochondrial targeted agents.

This study reports the synthesis of a number of 1- and 2-phenyl derivatives of the 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole nucleus, which were obtained by the reaction of the versatile 7-substituted 2,3-dihydro-3-hydroxymethylene-4H-1-benzothiopyran-4-ones with hydrazine and substituted phenylhydrazines. The antiproliferative activity of the synthesized compounds was evaluated by an in vitro assay on human tumor cell lines (HL-60 and HeLa) and showed a significant capacity of the 7-methoxy-substituted benzothiopyrano[4,3-c]pyrazoles 3b-d, carrying the pendant phenyl group in the 1-position, to inhibit cell growth. Investigation of the mechanism of action indicated the induction of the mitochondrial permeability transition (MPT) as the molecular event responsible for the inhibition of cell growth. This phenomenon is related to the ability of the test compounds to cause a rapid Ca2+-dependent and cyclosporin A-sensitive collapse of the transmembrane potential (DeltaPsi) and matrix swelling. All this leads to the release of caspase activators, such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF), which trigger the pro-apoptotic pathway leading to DNA fragmentation.

Intratumoral vs systemic administration of meta-tetrahydroxyphenylchlorin for photodynamic therapy of malignant gliomas: assessment of uptake and spatial distribution in C6 rat glioma model.

Malignant gliomas, with an incidence of 5 cases per 100,000 population per year, represent the most common primary brain tumour. They have an overall survival length of less than 2 years. Many different adjuvant therapies have been developed. Among them, Photodynamic Therapy (PDT), that is based on photochemical reactions between light and tumoral tissue selectively labelled with exogenous photosensitizing agents. Among photosensitizers, m-THPC (Temoporfin), seems to be the most promising one for the treatment of brain tumors, but, unfortunately, it causes problems of high skin photosensitivity. To by-pass this problem, we devised an intratumoral route of administration of this photosensitizer. The aim of this study is to investigate and compare the uptake of m-THPC in brain tumor and normal tissue after systemic and intratumoral administration of the drug. 30 female Wistar rats received m-THPC 12 days after C6 tumor implantation. Temoporfin was administered intratumorally in 24 rats at two different concentrations. 6 rats constituted the control group and received m-THPC by means of an intraperitoneal injection. The brains were extracted at 4 h, 24 h and 96 h after Temoporfin injection. The samples were examined with a confocal laser scanning microscope. All samples showed high fluorescence emission exclusively in the tumour area, without appreciable differences between the samples taken at the different times of sacrifice and the two routes of administration. No fluorescence whatsoever was detected among normal brain tissue surrounding the tumour. The intratumoral route appears to give comparable results to the systemic one, regarding intracellular uptake efficiency and tumour--normal tissue ratio, with the advantage of a much shorter time needed to reach optimal intratumoural concentration--that is just four hours from m-THPC injection.

The physiological role of biogenic amines redox reactions in mitochondria. New perspectives in cancer therapy.

In tumours, polyamines and amine oxidases increase as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H2O2 and aldehydes produced by the reaction. Increasing the incubation temperature from 37 to 42 degrees C enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. Since the tumour cells release endogenous substrates of BSAO, the administration of spermine is not required. Combination with hyperthermia improves the cytocidal effect of polyamines oxidation products. Our findings show that multidrug resistant (MDR) cells are more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity which induces the generation of intracellular ROS prior to the onset of mitochondrial permeability transition (MPT). It makes this new approach attractive, since the development of MDR is one of the major problems of conventional cancer therapy.

Activation of rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells.

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.

Modulation of the cytotoxic effect of 5-fluorouracil by N-methylformamide on a human colon carcinoma cell line.

The cytotoxic effect of the combination of N-methylformamide (NMF) with 5-fluorouracil (5-FU) on cell survival of the human colon cancer line HT29 was assessed. The differentiating activity of NMF was evidenced by morphological maturation and conversion of cell culture characteristics to those consistent with a more benign phenotype. In combination experiments, the noncytotoxic concentration of 1% NMF was chosen and doses of 5-FU ranging from 5 to 25 micrograms/ml were employed. Two main schedules were tested either on exponentially or stationarily growing cells: (a) 1% NMF for 72 h followed by 12-h exposure to 5-FU; (b) 5-FU for 12 h followed by 72-h exposure to 1% NMF. The results obtained demonstrated that the 5-FU----NMF sequence determined a powerful reduction in the surviving fraction of HT29 cells, while the reverse sequence did not increase the killing effect of 5-FU given alone. Immunocytochemical and scanning electron microscopy studies seemed to confirm that the association in which the differentiating agent followed the 5-FU treatment strongly impaired cellular integrity and function and that cytoskeletal elements, particularly microfilaments, and surface structures could play an essential role in the mechanisms of cytotoxicity. Furthermore, the results of this work indicate that drug sequence is a critical factor for the optimal combination of 5-FU and NMF.

Enhancement of hyperthermic damage on M14 melanoma cells by liposome pretreatment.

Exponentially growing human melanoma cells (M14 cell line) were pretreated with various amounts of dipalmitoylphosphatidylcholine-containing multilamellar liposomes and then exposed to heat treatment (42.5 degrees C). Cell damage produced by the treatments, given separately or in combination, was evaluated in terms of cell survival. Our results demonstrate that the cell survival at 37 degrees C was not affected by liposome concentrations up to 1000 nmol of phospholipid/2.5 x 10(6) cells, while liposome treatment of cells before heat exposure determined a marked damaging effect even at 100 nmol of phospholipid/2.5 x 10(6) cells. The mechanisms of liposome-cell interaction have been investigated by electron microscopy or by electron spin resonance measurements of spin-labeled membranes of intact cells. Evidence has been obtained that liposomal lipids are either taken up by M14 cells or become incorporated in the cell membrane. The present data suggest the possibility that liposome treatments per se could be of potential value as a therapeutic approach, by increasing the effect of heat therapy.

The relationship between 1H-NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo).

The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated. In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared. The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts. However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells. In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types. The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts. From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals. The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts.

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells.

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.

Voacamine, an alkaloid extracted from Peschiera fuchsiaefolia, inhibits P-glycoprotein action in multidrug-resistant tumor cells.

Multidrug resistance (MDR) in tumor cells is generally associated with increased efflux of the cytotoxic compounds, due to the activation of mechanisms of intracellular transport and to the overexpression of surface proteins, such as P-glycoprotein (Pgp), which act as ATP-dependent molecular pumps. In a previous study, voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on resistant human osteosarcoma cells. The effects of voacamine on the cell survival and on accumulation of DOX were investigated on both the parental cell line, U-2 OS-WT, and its resistant counterpart, U-2 OS-R. A differential effect between sensitive and resistant cells on the intracellular DOX concentration and distribution was revealed. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. Moreover, the cell survival analysis and the electron microscopic observations revealed an enhancement of the cytotoxic effect of DOX induced by the plant extract. In the present study, a panel of monoclonal antibodies (MAbs), recognizing different and specific structural and functional state of Pgp, was used. By flow cytometry and immunofluorescence confocal microscopy, a dose-dependent increase of the reactivity of Pgp with MAb UIC2, which specifically recognizes an epitope of the drug transporter in its functional conformation, was detected in voacamine-treated U-2 OS-R cells. Conversely, the expression of the epitope recognized by MAb MC57 was downregulated while MAb MM4.17 did not change its binding level to treated and untreated MDR cells. These data suggest that the plant extract reacts with Pgp producing conformational changes with consequent epitope modulation. Taken together, our observations seem to demonstrate that voacamine is a substrate for Pgp and, therefore, interferes with the Pgp-mediated drug export, acting as a competitive antagonist of cytotoxic agents.

Glucan-associated protein modulations and ultrastructural changes of the cell wall in Candida albicans treated with micafungin, a water-soluble, lipopeptide antimycotic.

The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet beta-glucan synthesis inhibitory concentration (0.01 microg/ml) of FK463 (micafungin). Namely, a decrease in enzymes of glucose metabolism (mostly enolase and a novel 40 kDaltons component, here identified as the enzyme fructose-1,6-biphosphate aldolase) was observed, and this was coupled with an increase in two beta1-3 exo-glucanase isoforms (34 and 44 kDa, respectively). No GAP changes were detected in the same strain of the fungus made resistant to the drug, attesting to the specificity of the observed cell wall protein modulation. In addition, GAP changes were accompanied by marked ultrastructural alterations upon treatment with the sub-MIC dose of the drug, the majority of which was an aberrant cell surface morphology and a derangement of the normal layering of the cell wall. Our data demonstrate that sub-MIC doses of micafungin do critically affect not only the beta-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

Effects of daunomycin on the microtubular network: a cytochemical study on a human melanoma cell line.

The interaction of daunomycin (DAU), an anthracyclinic antibiotic employed as antitumoral agent, with microtubules, has been investigated by cytochemical and morphological methods on a human melanoma cell line (H14). Results obtained indicated that DAU was able to modulate the microtubule reassembly in cells treated with colcemid; such an effect proved to be dose-dependent. In particular, it has been observed that a low dose of DAU (0.05 microM) seemed to favor the microtubule reassembly whereas a higher dose (0.10 microM) impaired this process. In addition, when the anthracyclinic antibiotic was employed together with colcemid, both the cell detachment and the depolymerization of microtubules induced by the mitotic poison were hampered. These effects were dose-dependent and were better accomplished when DAU was used at an equimolar or at higher dose than that employed for the antimicrotubular agent. Moreover, the treatment of cells with DAU alone induced the stabilization of the microtubules, making them more resistant to the action of antimicrotubular agents. This effect could in part explain the antagonistic action exerted by DAU against colcemid. These observations seem to confirm that the microtubular network is an important target involved in the mechanism of action of the anthracyclinic antibiotics.

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes.

A freeze-fracture study has been carried out on human peripheral blood lymphocytes (hPBL) from healthy donors. Lymphocytes were frozen either from 37 degrees C or 4 degrees C. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed: a) a difference in size between IMPs on the external face (EF) and those on the protoplasmic face (PF); b) a remarkable influence of temperature either on size or density of the IMPs; c) the existence at 4 degrees C of two lymphocyte populations differing in IMP density.

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes. II. Ultrastructural differences between T and B cells.

Separated T and B lymphocytes from human peripheral blood were studied using the freeze-fracture technique. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed remarkable differences between cells belonging to the two main lymphocyte populations. In particular: (a) both fracture faces of the cytoplasmic membrane of B lymphocytes exhibit larger particles than T lymphocytes; (b) the mean densities, on both protoplasmic (PF) and external (EF) fracture faces, in B lymphocytes are lower than in T lymphocytes; (c) in B cells the partition ratio of particles between PF and EF is reversed with respect to T cells; (d) on both fracture faces of B lymphocytes, the IMP densities present a normal distribution while on T cells, density values show bimodal distributions indicating the existence of two cell subsets differing in particle density.

Influence of N-methylformamide on the intracellular transport of doxorubicin.

The polar solvent N-methylformamide proved to be capable of enhancing the cytotoxic potential of various antitumoral compounds, both in vitro and in vivo. In many cases, this ability depended on the sequence of treatment, and the enhancement of the cytotoxic effect occurred only when N-methylformamide administration succeeded anticancer drug treatment. The results obtained in the present study indicate that N-methylformamide interferes with the mechanisms of intracellular transport and efflux of the antitumoral drug doxorubicin. In particular, laser scanning confocal microscopy observations performed on melanoma cells (M14) after N-methylformamide administration revealed evident alterations of the microtubular network, including numerous interruptions of the microtubules. Moreover, when doxorubicin-treated cells were recovered in the presence of the polar solvent, the normal efflux of the anthracyclinic antibiotic appeared to be hampered, and the drug was localized mainly in well delimited perinuclear regions. Double staining experiments demonstrated the colocalization of the doxorubicin molecules and the WGA-stained regions as well as a close structural relationship between them and the microtubule system. These results indicate that N-methylformamide interferes with the doxorubicin transport inducing a damage in the microtubular network and the consequent persistence and entrapment of the drug in the regions likely occupied by the Golgi apparatus of tumor cells. This finding could account for the chemosensitizing properties exerted by N-methylformamide.

Adriamycin-plasma membrane interaction in human erythrocytes.

A great body of data increasingly point to the cell membrane as an important target for adriamycin (ADR). However, the exact mechanism by which ADR exerts its cytotoxic action through the interaction with the plasma membrane is still unknown. In this study, the interaction of ADR with red blood cells from healthy donors was investigated by freeze-fracturing (FF) and scanning electron microscopy (SEM). The results obtained can be summarized as follows: a) a dose-dependent modification in the intramembrane particle (IMP) distribution was revealed by FF on both fracture faces of the plasma membrane of erythrocytes treated with 50 or 100 microM ADR; b) SEM observations allowed to reveal a discocyte-stomatocyte transition induced by 50 microM ADR and the formation of mottled cells at the higher dose; c) these morphological and ultrastructural changes were not related to lipid peroxidation as demonstrated by experiments with radical scavengers or strong oxidant substances; d) the analysis of IMP density seemed to rule out a segregation process of membrane proteins suggesting that ADR interacts with the plasma membrane by becoming incorporated within the lipid bilayer.

Changes in cell volume and internal sodium concentration in HeLa cells during exponential growth and following lonidamine treatment.

Cell volumes decreased in HeLa cells as a function of time after seeding during exponential growth. Cell volume distributions revealed the presence of two cell populations in all stages of growth. When cells approached confluence, the ratio of the two populations abruptly shifted towards that characterised by the smallest volume. Percentages of G1-, S- and G2 + M-phase cells were also measured and it was found that G1 frequency increased as a function of cell density during exponential growth. Intracellular sodium concentration, [Na]i was monitored by 23Na NMR in the presence of 5 mM dysprosium (III) tripolyphosphate. [Na]i increased from 22.8 to 59.0 mM in cells from the second to the seventh day after seeding. Treatment with lonidamine, an antitumoral drug that it is known to slow down cell growth by affecting aerobic glycolysis, produced a complete block of cell progression after a few days of treatment. The progression of cell volume distributions towards smaller volumes and the increase in internal sodium concentration as a function of time after seeding were also affected by the drug. These phenomena were related to the existence of a subpopulation of mitotically inactive G1-phase cells during exponential growth, pointing out that a density-dependent cellular mechanism regulates the cell cycling in HeLa cells.

Differential cell surface expression of mannoprotein epitopes in yeast and mycelial forms of Candida albicans.

The ultrastructural localization of mannoprotein constituents (MP) of the cell wall of yeast and hyphal forms of Candida albicans was studied by immunoelectron microscopy. To this aim, two monoclonal antibodies (mAbs AF1 and 1D10), recognizing distinct oligomannoside epitopes of MP molecules, and a second antibody coupled to colloidal gold, were employed. Preembedding methods revealed the presence of both AF1- and, albeit to a lesser intensity, 1D10-epitopes within the fibrillar, capsular layer of yeast cells of the fungus, provided this capsule was preserved and stabilized by treatment of whole cells with Concanavalin A. These cell surface-associated MP were absent in hyphal cells, despite the presence in these cells of a capsular layer not different in form and thickness from that present in yeast cells. Postembedding methods showed that both yeast and hyphal forms of growth of C. albicans synthesized the relevant mannoproteins and similarly incorporated them into inner layers of the cell wall. Apparently, however, the "export" of these MP to the outermost, capsular layer occurred in yeast but not in hyphal cells. These ultrastructural data, coupled with previous biochemical ones, emphasize form-associated patterns of MP expression on Candida cell surface. Given the value of MP as main immunogenic components of Candida, this differential expression could be a means by which the fungus evades from, or attenuates host's response.

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

A study on the receptor for a mycobacteriophage : phage phlei.

From Mycobacterium phlei, glycolipid fractions have been isolated which inactivate phage Phlei. On the basis of the characteristics of the inactivation (specificity, kinetics, requirement for Ca++) typical of the phage-host cell system, it was concluded that these fractions contain the receptor sites for phage Phlei ; this conclusion was supported by electron microscopic studies. All the active fractions contain four kinds of components : fatty acids, glycerol, sugars (D-lyxose, 6-0-methyl-D-glucose, and low amounts of glucose and mannose), and water-soluble acids. These acids are isolated by degradation of the receptor fractions as oxalic and pyruvic acids. Variations of the ratio oxalic acid/pyruvic acid according to the mode of degradation and the absence of the peak characteristic of the protons of a pyruvic acid residue in the NMR spectrum, suggest that these acids might arise from the splitting of oxaloacetic acid. A tentative structure of the receptor is proposed, in many monoglycerides are linked through keto-acid to a polysaccharide core.