FR901469, a novel antifungal antibiotic from an unidentified fungus No.11243. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological properties.

FR901469 is a novel antifungal antibiotic produced by an unidentified fungus No.11243. This compound was isolated from the culture broth by solvent extraction, HP-20 and YMC ODS gel column chromatography, and lyophilization. FR901469 is a white powder which melts at 182 approximately 187 degrees C and possesses the molecular formula C71H116N14O23. This compound has good water solubility. FR901469 inhibited the activity of 1,3-beta-glucan synthase from Candida albicans with an IC50 value of 0.05 microg/ml, and displayed greater inhibitory activity than other 1,3-beta-glucan synthase inhibitors such as, WF11899A, echinocandin B, aculeacin A, and papulacandin B.

FR901469, a novel antifungal antibiotic from an unidentified fungus No.11243. II. In vitro and in vivo activities.

FR901469 is a water-soluble macrocyclic lipopeptidolactone (C71H116N14O23) that has inhibitory activity against 1,3-beta-glucan synthase and exhibits in vitro and in vivo antifungal activity against both Candida albicans and Aspergillus fumigatus. The MICs of FR901469 against Candida albicans FP633 and Aspergillus fumigatus FP1305 in a micro-broth dilution test were 0.63 and 0.16 microg/ml, respectively. FR901469 showed excellent efficacy by subcutaneous injection against both Candida albicans and Aspergillus fumigatus in a murine systemic infection mode, with ED50s of 0.32 and 0.2 mg/kg, respectively. This compound also showed potent anti-Pneumocystis activity in the nude mice model with experimental Pneumocystis pneumonia. The hemolytic activity of FR901469 towards mouse red blood cells, is about 30-fold weaker than that of amphotericin B.

Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer.

We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method. These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C. At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells. Stress fibers, peripheral bands, and focal contacts were established in similar ways. After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating. After mild agitation cells floated free from the dish surface without trypsin treatment. Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces. An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment. An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment. These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton. While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive. The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment.

Release of neutrophil elastase and its role in tissue injury in acute inflammation: effect of the elastase inhibitor, FR134043.

Neutrophil elastase degrades extracellular matrix components and is involved in tissue destruction in several inflammatory states. We examined the inhibition of the elastase activity derived from activated neutrophils in vitro and in vivo by FR134043, disodium-(Z,1S,15S,18S,24S,27R,29S,34S,37R)-29-b enzyl-21-ethylidene-27-hydroxy-15-isobutyrylamino-34-isopropyl-31, 37-dimethyl-10,16,19,22,30,32,35,38-octaoxo-36-oxa-9,11,17,20,23,2 8,31,33-octaazatetracyclo[16.13.6.1(24,28).0(3,8)]octatriconta+ ++-3,5,7-trien-5,6-diyl disulfate, an elastase inhibitor with broad specificity, and elucidated the role of neutrophil elastase in pathogenesis of acute inflammation. In a culture of human neutrophils, phorbol myristate acetate (PMA) and calcium ionophore increased elastase activity in the supernatants, which was amplified by co-existing mononuclear leukocytes. Formyl-Met-Leu-Phe stimulated elastase release in the presence of, not without, mononuclear leukocytes. Intratracheal injection of lipopolysaccharide elevated the elastase activity in bronchoalveolar lavage fluid of rats. These elastase activities were significantly inhibited by FR134043. Intratracheal treatment with FR134043 in rats also inhibited the enzyme induced by lipopolysaccharide, though the maximum inhibition was 52%. Ear edema elicited by topical application of PMA in mice was significantly suppressed by pretreatment with FR134043 (38% inhibition at 1 mg/ear). In carrageenan-induced joint injury in rats, plasma extravasation into the synovial cavity was partially and significantly inhibited by FR134043 at 1 mg/knee, while an elastase-specific inhibitor showed no effect. These results suggest that neutrophil elastase is partially involved in tissue damage in acute inflammation provoked by irritants, but not in carrageenan-induced hyperpermeability.

Inhibition of elastase-induced acute inflammation and pulmonary emphysema in hamsters by a novel neutrophil elastase inhibitor FR901277.

OBJECTIVE AND DESIGN: A neutrophil elastase inhibitor FR901277 was examined for its inhibitory effect on degradation of natural substrate elastin in vitro, and on acute inflammatory states and pulmonary emphysema in vivo. MATERIAL AND TREATMENT: Elastin-congo red was used as a substrate for elastin degradation assay. Paw edema in male C57BL mice (6 weeks old) and pulmonary hemorrhage in female golden hamsters (5 weeks old) were induced by topical injection of human neutrophil elastase (HNE). Pulmonary emphysema in male golden Syrian hamsters (10 weeks old) was provoked by intratracheal instillation of porcine pancreatic elastase. In all in vivo experiments. FR901277 was administered prior to elastase treatment. METHODS: Elastin degradation by HNE was monitored spectrophotometrically with elastin-congo red. Foot swelling was measured by calipers. Pulmonary hemorrhage was assessed by hemoglobin concentration in bronchoalveolar lavage fluid. As emphysematous parameters, quasi-static lung compliance and vital capacity were measured. RESULTS: FR901277 inhibited HNE-induced elastin degradation. Systemic treatment with FR901277 significantly inhibited paw edema and pulmonary hemorrhage. Intratracheal treatment with FR901277 significantly ameliorated changes in pulmonary function. CONCLUSIONS: These results suggest that FR901277 inhibits the elastase activity potently both in vitro and in vivo, and that elastase may play a role at least in part in pathogenesis of pulmonary emphysema.

Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacrylamide)-grafted surfaces.

Temperature-responsive hydration/dehydration changes in surface-grafted poly(N-isopropylacrylamide) (PIPAAm) were utilized for hydrophilic/hydrophobic surface property alterations in cell culture. In this report, we utilized PIPAAm-grafted surfaces to recover confluently-cultured vascular endothelial cells as coherent monolayers from this cell culture substrate and to transfer to new cell culture substrates. For this purpose, we used two different methods to recover and transfer cell monolayer cultures: (1) chitin membranes used as an apical side cell support during cultured cell transfer, allowing cell basal side reattachment to new culture substrates after transfer; and (2) a cell culture insert (porous PET) used as both a support as well as new substrate, allowing basal surfaces of cultured cells to be exposed to the medium after transfer. In both cases, all cells grown on PIPAAm-grafted surfaces detach completely with maintenance of basement membrane-like structure. Recovered cells attach to the second culture surfaces, covering more than 60% of the new substrate, and retain approximately 90% viability and their original function as judged from tissue-type plasminogen activator secretion. This technique could be utilized to prepare novel bioartificial organs as well as cell co-culture systems by multi-layering different cell types to mimic tissue structures for tissue engineering.

Retinal pigmented epithelium cultures on thermally responsive polymer porous substrates.

A cross-linkable co-polymer of UV-sensitive 4-(N-cinnamoylcarbamide)methylstyrene (CCMS) and N-isopropylacrylamide (NIPAAm), was applied to porous tissue culture inserts. Surface chemical analyses of the inserts show an introduction of a thermally responsive polymer comparable to that on similarly incorporated non-porous polystyrene surfaces. Contact angle measurements as well as atomic force microscopy show a surface change in response to changing temperature in an aqueous environment, from hydrophilic, extended polymer chains below 32 degrees C to a dense hydrophobic film above 32 degrees C. Cell growth on porous inserts allowed measurement of cell expression, such as transepithelial resistance and fluid transport, which are not observable on cells from non-porous surfaces. Cultures of retinal pigmented epithelium (RPE) were able to restore an environment similar to in vivo by forming a tight junction barrier membrane upon confluence at 37 degrees C, as observed by changes in morphology, transepithelial resistance, and directionally-specific fluid transport. In addition, cells cultured on these surfaces detached as an oriented polarized sheet when the inserts were brought to 20 degrees C. This cell sheet was transplanted to other tissue culture surface without polymer detachment or dissolution, or cell damage caused by traditional detachment methods using proteolytic enzymes.

Biochemical and pharmacological characterization of FR134043, a novel elastase inhibitor.

FR134043, disodium(Z,1S,15S,8S,24S,27R,29S,34S,37R)-29-ben zyl-21-ethylidene-27-hydroxy-15-isobutyrylamino-34-isopropyl-31,37 -dimethyl-10,16,19,22,30,32,35,38-octaoxo-36-oxa-9,11,17,20,23,28, 31,33-octaazatetracyclo[16.13.6.1(24),(28).0(3),(8)]octatricont a-3,5,7-trien-5,6-diyl disulfate, is a water-soluble inhibitor of human neutrophil elastase with a molecular mass of 1166.15 Da. FR134043 demonstrated a characteristic competitive inhibition of human neutrophil elastase with a Ki of 8 nM. In studies using synthetic substrates, FR134043 inhibited both neutrophil elastase activity and porcine pancreatic elastase activity with IC50 values of 35 nM and 49 nM respectively. FR134043 also inhibited hydrolysis of bovine neck ligament elastin by human neutrophil elastase with an IC50 value of 210 nM. In in vivo experiments, FR134043 protected animals against human neutrophil elastase (50 microg/animal)-induced lung hemorrhage in hamsters with an ED50 value of 3.1 microg/animal for intratracheal administration and 5.0 mg/kg for intravenous administration. Subcutaneous treatment with FR134043 significantly suppressed human neutrophil elastase (20 microg/paw)-induced paw edema in mice with an ED50 value of 3.3 mg/kg when evaluated 4 h after elastase injection. The potency of FR134043 given intratracheally to protect against porcine pancreatic elastase (100 microg/animal)-induced emphysema in hamsters was relatively low (Quasi-static lung compliance; ED50 = 1590 microg/animal) compared to that in acute animal models. FR134043 (10 mg/kg per h i.v. infusion) significantly improved lipopolysaccharide (0.25 mg/kg per h i.v. infusion)-induced thrombocytopenia and some coagulation parameters in rats. These results suggest that systemic administration of FR134043 would be advantageous over intratracheal administration of FR134043 for the treatment of adult respiratory distress syndrome, septic shock and pulmonary emphysema and other pathophysiologic conditions in which elastases are thought to be involved.

Novel thermally reversible hydrogel as detachable cell culture substrate.

A novel UV crosslinkable co-polymer of 4-(N-cinnamoylcarbamide)methylstyrene (CCMS) and N-isopropylacrylamide (IPAAm) was partially entrapped in traditional tissue-culture-treated polystyrene and crosslinked by UV light irradiation. Dishes modified by this method showed a change in contact angle with respect to temperature as compared to tissue culture polystyrene controls. Surface chemical analysis indicated that the crosslinked hydrogel does not detach from the surface after successive rinsing in ethanol and water, keeping the cells or cell construct free of unwanted soluble polymer after detachment. Cultures of both bovine endothelium and human retinal pigmented epithelium were confirmed to be able to attach and grow on the polymer-modified surfaces morphologically identical to that on control tissue culture polystyrene surfaces. Corresponding to a change in temperature, these cultures would detach and could be transplanted to another culture surface without functional and structural changes. These results show that the new, photo-crosslinkable hydrogel system can utilize the hydrophobic/hydrophilic change of the surface for cell culture detachment while being permanently applicable to any tissue culture geometry.

WA8242A1, A2 and B, novel secretory phospholipase A2 inhibitors produced by Streptomyces violaceusniger. II. Biological properties.

WA8242A1, A2 and B, novel inhibitors of secretory phospholipase A2 (PLA2), were obtained from Streptomyces violaceusniger No. 8242. WA8242B inhibited secretory group I and II PLA2s in a dose-dependent manner. The mode of inhibition of group II PLA2 by WA8242B was in competitive fashion. WA8242B inhibited group II PLA2-induced Prostaglandin I2 (PGI2) production by human umbilical vein endothelial cells. Furthermore, WA8242B was effective in mouse zymosan writhing model.

FR167653, a dual inhibitor of interleukin-1 and tumor necrosis factor-alpha, ameliorates endotoxin-induced shock.

Increased production of interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) have been implicated in the pathophysiology of a variety of diseases including circulatory shock. The present study evaluated the efficacy of FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-pyridylpyrazolo[5,1-c] [1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate), a dual inhibitor of interleukin-1 and TNF-alpha production, to protect rabbits from the shock and lethality induced by lipopolysaccharide. In this sepsis model, FR167653 at a dose of 0.32 mg/kg per h ameliorated the 7-day mortality from 93% in the placebo group to 47% in the FR167653-treated group and, at doses of 0.10-0.32 mg/kg per h, attenuated the hypotensive response to lipopolysaccharide challenge and returned mean arterial blood pressure to almost normal levels. The increases in plasma interleukin-1 and TNF-alpha levels evoked by lipopolysaccharide administration were also inhibited by treatment with FR167653, which was efficacious at doses of 0.1-0.32 mg/kg per h. In addition, FR167653 treatment attenuated the increases in plasma creatinine concentrations consistent with renal damage in a dose-dependent manner. These findings suggested that FR 167653 has a beneficial potential as a drug for septic shock or multiple organ dysfunction syndrome.

The identification of an orally active, nonpeptide bradykinin B2 receptor antagonist, FR173657.

1. An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2-4-dichloro-3-[(2-methyl-8-quinolin yl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl] acrylamide) has been identified. 2. This compound displaced [3H]-BK binding to B2 receptors present in guinea-pig ileum membranes with an IC50 of 5.6 x 10(-10) M and in rat uterus with an IC50 of 1.5 x 10(-9) M. It did not inhibit different specific radio-ligand binding to other receptor sites. 3. In human lung fibroblast IMR-90 cells, FR173657 displaced [3H]-BK binding to B2 receptors with an IC50 of 2.9 x 10(-9) M and a Ki of 3.6 x 10(-10) M, but did not reduce [3H]-des]Arg10-kallidin binding to B1 receptors. 4. In guinea-pig isolated preparations, FR173657 antagonized BK-induced contractions with an IC50 of 7.9 x 10(-9) M, but did not antagonize acetylcholine or histamine-induced contractions even at a concentration of 10(-6) M. FR173657 caused parallel rightward shifts of the concentration-response curves to BK at concentrations of 10(-9) M and 3.2 x 10(-9) M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration-response curve at a concentration of 10(-8) M. Analysis of the data yield a pA2 of 9.2 +/- 0.2 (n = 5) and a slope of 1.5 +/- 0.2 (n = 5). 5. In vivo, the oral administration of FR173657 inhibited BK-induced bronchoconstriction dose-dependently in guinea-pigs with an ED50 of 0.075 mg kg-1, but did not inhibit histamine-induced bronchoconstriction even at 1 mg kg-1. FR173657 also inhibited carrageenin-induced paw oedema with an ED50 of 6.8 mg kg-1 2 h after the carrageenin injection in rats. 6. These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.

Effects of FR145237, an acyl-CoA:cholesterol acyltransferase inhibitor, on diet-induced hypercholesterolemia in diabetic rats.

Recent studies have shown that acyl-CoA:cholesterol acyltransferase (ACAT) plays an important role in the initiation of diabetes-associated hypercholesterolemia. To confirm this hypothesis, effects of a potent ACAT inhibitor, FR145237, on diet-induced hypercholesterolemia were examined in streptozotocin (STZ)-induced diabetic rats. One-week feeding of 1% cholesterol and 0.5% cholic acid to normal rats and STZ-induced diabetic rats increased plasma cholesterol levels in both groups, and the response was more remarkable in the STZ rats than in the normal ones (1266 +/- 476 mg/dl and 146 +/- 7 mg/dl, respectively). FR145237 dose-dependently reduced the rise in plasma cholesterol levels in the STZ rats and the levels were almost normalized by treatment with 1 mg/kg/day of the compound. These results suggest that hyperresponse to dietary cholesterol was induced in the STZ rats and that ACAT is involved in the hyperresponse. The effects of FR145237 on other plasma lipids such as high density lipoprotein (HDL) cholesterol and triglyceride (TG) levels were also examined.

New antitumor substances, FR901463, FR901464 and FR901465. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

New antitumor substances, FR901463, FR901464 and FR901465 were isolated from the culture broth of a bacterium of Pseudomonas sp. No.2663. FR901463, FR901464 and FR901465 remarkably enhanced the transcriptional activity of the promoter of SV40 DNA virus. Further, these compounds exhibited potent antitumor activities against murine and human tumor cell lines in vitro.

New antitumor substances, FR901463, FR901464 and FR901465. II. Activities against experimental tumors in mice and mechanism of action.

FR901463, FR901464 and FR901465, novel antitumor substances, were isolated from the fermentation broth of Pseudomonas sp. No. 2663. Their antitumor activities were examined in three mouse tumor systems and one human tumor system. The three FR compounds prolonged the life of mice bearing murine ascitic tumor P388 leukemia (T/C values were 160%, 145% and 127% for FR901463, FR901464 and FR901465, respectively), and inhibited the growth of a human solid tumor, A549 lung adenocarcinoma, with different effective dose ranges. FR901464 exhibited most prominent effects on these tumor systems among the three FR compounds. FR901464 also inhibited the growth of murine solid tumors, Colon 38 carcinoma and Meth A fibrosarcoma. To address the involvement of transcriptional activation ability of the three FR compounds in the antitumor effect, we selected FR901464 as a candidate compound and investigated cell cycle transition, chromatin status and endogenous gene expression in FR901464-treated tumor cells having elevated transcriptional activity. FR901464 induced characteristic G1 and G2/M phase arrest in the cell cycle and internucleosomal degradation of genomic DNA with the same kinetics as activation of SV40 promoter-dependent cellular transcription in M-8 tumor cells. In contrast to the potent activation of the viral promoter, FR901464 suppressed the transcription of some inducible endogenous genes but not house keeping genes in M-8 cells. These results suggest that FR901464 may induce a dynamic change of chromatin structure, giving rise to strong antitumor activity, and therefore may represent a new type of drug for cancer chemotherapy.

Effect of FR133605, a novel cytokine suppressive agent, on bone and cartilage destruction in adjuvant arthritic rats.

OBJECTIVE: We evaluated the effect of FR133605, a novel inhibitor of interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), on bone and cartilage destruction in adjuvant arthritic rats and compared it to corticosteroid, nonsteroidal antiinflammatory drugs (NSAID), and disease modifying antirheumatic drugs (DMARD). METHODS: The antiinflammatory responses were evaluated by measurement of hind paw swelling, body weight, femoral bone mineral density, and glycosaminoglycan content (GAG) in femoral condyles in adjuvant arthritic rats. RESULTS: FR133605 inhibited IL-1 and TNF-alpha production stimulated with lipopolysaccharide (LPS) in human monocytes. FR133605 also inhibited serum IL-1 and TNF-alpha concentrations in LPS treated mice. In contrast, among comparison antirheumatic drugs, only corticosteroid inhibited production at nontoxic concentrations. In adjuvant arthritis, FR133605 significantly inhibited paw swelling, bone and cartilage destruction, and increased body weight. On the other hand, indomethacin significantly inhibited paw swelling, but not bone and cartilage loss. Dexamethasone completely inhibited paw swelling and bone loss, but augmented cartilage breakdown. DMARD weakly restored the loss of GAG contents in articular cartilage. CONCLUSIONS: FR133605 improved bone loss and articular cartilage destruction in adjuvant arthritic rats and the inhibitory effect was closely correlated with the suppressive activity of IL-1 and TNF-alpha production.

Effect of FR167653, a cytokine suppressive agent, on endotoxin-induced disseminated intravascular coagulation.

FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5-1-c] [1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate) is a low molecular weight inflammatory cytokine inhibitor that inhibits the production of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha (TNF-alpha) in human monocytes stimulated with lipopolysaccharide, and in human lymphocytes stimulated with phytohemagglutinin-M. FR167653 inhibited these cytokines in a dose-dependent manner (IC50 values were 0.84, 0.088, 1.1 microM and 0.072, respectively). However, FR167653 did not inhibit even at 10 microM interleukin-6 production by human monocytes, and the production of interleukin-2 and interferon-gamma by human lymphocytes. We evaluated the effect of FR167653 on lipopolysaccharide-induced disseminated intravascular coagulation in rats. FR167653 (0.032-0.32 mg/kg/h for 4 h, intravenous infusion) markedly improved thrombocytopenia and plasma coagulation parameters in a dose-dependent manner, but not leukopenia in this mode. Plasma interleukin-1 and TNF-alpha levels were elevated by lipopolysaccharide administration and the treatment with FR167653 (0.31 mg/kg/h for 4 h) inhibited the increased plasma interleukin-1 (100.0%) and plasma TNF-alpha (89.2%) levels. These results suggest that interleukin-1 and TNF-alpha may play a pivotal role in the pathogenesis of DIC. FR167653 can act as a protective drug in lipopolysaccharide-induced DIC, and this protection is due to an inhibition of increased plasma interleukin-1 and TNF-alpha.

Flexibacter japonensis sp. nov., a new species that produces a novel inhibitor of human leukocyte elastase isolated from soil.

This strain 758 of a new member of Flexibacter species isolated from a soil is a Gram-negative, nonflagellated, gliding, long rod or filamentous. The GC contents of the deoxyribonucleic acid of this strain is 49.8 mol %GC. The isolate can be distinguished from other Flexibacter species on the basis of physiological and biochemical properties and DNA relatedness data. Therefore, we propose a new species, Flexibacter japonensis, for this strain. The strain 758 is deposited to the Japan Collection of Microorganisms as JCM 9735.

Effects of basic fibroblast growth factor and ganglioside GM1 on neuronal survival in primary cultures and on eight-arm radial maze task in adult rats following partial fimbria transections.

The effects of basic fibroblast growth factor (bFGF) and ganglioside GM1 (GM1) were evaluated alone and simultaneously in two types of experiments. First, the neuronal survival of primary culture neurons from fetal rat brain was measured. Then, performance on radial maze task in adult male rats following bilateral partial Fimbria-Fornix transections (F-F lesion) was tested. In primary culture neurons, bFGF (1-10 ng/ml) supported the neuronal survival from three regions (hippocampus, cortex and septum) of embryonic rat brain. However, GM1 (0.1-10 micrograms/ml) did not support the neuronal survival from any regions. Survival of cultured neurons was not supported by addition of 0.1 ng/ml bFGF, but when bFGF (0.1 ng/ml) and GM1 (0.1, 1 microgram/ml) were given to the cultured neurons simultaneously, the number of surviving neurons increased significantly. In the eight-arm radial maze task, where only the same four arms were baited, F-F lesion produced substantial memory impairment. In this task, administration of bFGF (10 micrograms/ml) or GM1 (1 mg/ml) alone did not produce any effects. However, when they were given simultaneously, the number of working memory errors decreased significantly, in spite of no amelioration for hippocampal choline acetyl transferase (ChAT) depletion. These findings indicate that actions of bFGF may be potentiated by the addition of GM1 in both primary neuronal cultures and radial maze task performance. These results suggest that the combination of bFGF and GM1 may synergistically improve spatial memory deficits.