Peptide fragment of thymosin ß4 increases hippocampal neurogenesis and facilitates spatial memory.

Although several studies have suggested the neuroprotective effect of thymosin ß4 (TB4), a major actin-sequestering protein, on the central nervous system, little is understood regarding the action of N-acetyl-serylaspartyl-lysyl-proline (Ac-SDKP), a peptide fragment of TB4 on brain function. Here, we examined neurogenesis-stimulative effect of Ac-SDKP. Intrahippocampal infusion of Ac-SDKP facilitated the generation of new neurons in the hippocampus. Ac-SDKP-treated mouse hippocampus showed an increase in ß-catenin stability with reduction of glycogen synthase kinase-3ß (GSK-3ß) activity. Moreover, inhibition of vascular endothelial growth factor (VEGF) signaling blocked Ac-SDKP-facilitated neural proliferation. Subchronic intrahippocampal infusion of Ac-SDKP also increased spatial memory. Taken together, these data demonstrate that Ac-SDKP functions as a regulator of neural proliferation and indicate that Ac-SDKP may be a therapeutic candidate for diseases characterized by neuronal loss.

Gi-protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), induces senescence-associated beta-galactosidase positive cell formation through CREB phosphorylation.

AIMS: We evaluated Gi-protein inhibitor, guanosine 5'-O-(2-thiodiphosphate)(GOT)-induced senescence-associated(SA)-beta-galactosidase(Gal) positive cell formation to determine if it occurred through phosphorylation of cyclic AMP-dependent response element binding protein (CREB). MAIN METHODS: IMR-90 human lung fibroblast cells were used. SA-beta-Gal positive cells and senescence-associated heterochromatic foci (SAHF) were determined by assessing blue color formation of substrate, X-gal inside cells and DAPI staining, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis. CREB phosphorylation and molecular changes were analyzed by western blot. KEY FINDINGS: GOT treatment led to SA-beta-Gal positive cell formation and SAHF. CREB phosphorylation increased in response to GOT treatment but then decreased over 24h. SA-beta-Gal positive cell formation increased in response to transient transfection of pS6-RSV-CREB and no changes were detected following CREB knockdown with CREB-siRNA. In addition, CREB phosphorylation was delayed by treatment with the anti-cellular senescence agents, clitocybins which also reduced the number of SA-beta-Gal positive cells. Collectively, our data showed that GOT-induced CREB phosphorylation initiated SA-beta-Gal positive cell formation after which decreased in SA-beta-Gal positive cells. SIGNIFICANCE: These findings suggest for the first time that CREB phosphorylation by GOT could induce cellular senescence as judged by SA-beta-Gal positive cell formation.

Asarone inhibits adipogenesis and stimulates lipolysis in 3T3-L1 adipocytes.

Asarone is a molecule found in certain plants such as Acorus calamus, the root of which is used in traditional medicine to treat diabetes. We determined the molecular mechanism underlying the anti-diabetic activity of asarone. Treatment of asarone significantly inhibited the differentiation of 3T3-L1 preadipocytes through suppression of expression of the transcription factors, CCAAT/enhancer binding protein-alpha and peroxisome proliferator activated receptor-gamma, which activate adipogenesis. Intracellular triglyceride levels were reduced by asarone in a dose-dependent manner and asarone treatment stimulated the phosphorylation of hormone-sensitive lipase. Together, the present findings indicate that asarone inhibits adipogenesis by down-regulation of PPARgamma and C/EBPalpha and reduces lipid accumulation by stimulation of lipolysis through an increase in hormone-sensitive lipase activity.

Stimulation of various functions in murine peritoneal macrophages by high mannuronic acid-containing alginate (HMA) exposure in vivo.

High mannuronic acid-containing alginate (HMA) was tested to affect murine peritoneal macrophages. In the present study, we measured various functions of murine peritoneal macrophages that were isolated 20 h after intraperitoneal injection with HMA (25 and 100 mg/kg). HMA increased the number of peritoneal macrophages and phagocytosis. Macrophages from HMA-treated mice significantly inhibited growth of tumor cells compared to macrophages from control mice. In addition, supernatants from macrophages of HMA-treated mice contained nitric oxide (NO), hydrogen peroxide (H2O2) and TNF-alpha. The increased production of these cytotoxic molecules induced by HMA is consistent with tumoricidal activity of activated macrophages. Furthermore, HMA-induced tumoricidal activity was partially abrogated by anti-TNF-alpha, inhibitors of NO and the scavenger of reactive oxygen. Thus, the tumoricidal activity induced by HMA appeared to be mediated by the production of TNF-alpha, NO and H2O2. Taken together, these results suggest that HMA has the immunostimulating effect on macrophages after in vivo exposure of it.

Hahella chejuensis gen. nov., sp. nov., an extracellular-polysaccharide-producing marine bacterium.

A bacterial strain, designated 96CJ10356T, which produced abundant extracellular polysaccharides and red pigment was isolated from marine sediment collected from Marado, Cheju Island, Republic of Korea. The organism is Gram-negative, aerobic, rod-shaped and motile. Growth was not observed in the absence of NaCl, and was optimal at an NaCl concentration of 2%. The strain contained oxidase and catalase, and was able to hydrolyse aesculin and gelatin. The major cellular fatty acids were saturated or monounsaturated straight-chain fatty acids. An almost complete 16S rDNA sequence of the test strain was determined. Phylogenetic analysis based on the neighbour-joining and Fitch-Margoliash methods indicated that the organism formed a distinct phyletic line within the gamma Proteobacteria. This relationship was also supported by sequence comparison, as no valid bacterial species showed more than 90% sequence homology with the isolate. It is clear from polyphasic evidence that the isolate merits the status of genus in the gamma subclass of the Proteobacteria, and the name Hahella chejuensis gen. nov., sp. nov. is proposed for the marine isolate 96CJ10356T (= KCTC 2396T = IMSNU 11157T).

Nocardiopsis kunsanensis sp. nov., a moderately halophilic actinomycete isolated from a saltern.

A moderately halophilic actinomycete, designated HA-9T, was isolated from a saltern in Kunsan, Republic of Korea, and was the subject of polyphasic identification. Analysis of 16S rDNA indicated that the isolate belonged to the genus Nocardiopsis, but differed genetically from other Nocardiopsis species. Strain HA-9T contained meso-diaminopimelic acid, no diagnostic sugars, hexa- or octa-hydrogenated menaquinones with 10 isoprene units, straight-chain saturated or monounsaturated, iso-, anteiso-, 10-methyl branched fatty acids with 13-18 carbons and type III phospholipids. All of these characters consistently assign the isolate to the genus Nocardiopsis. All of the validly described Nocardiopsis species, including moderately halophilic Nocardiopsis halophila, can be differentiated from the saltern isolate using morphological and physiological traits. On the basis of polyphasic evidence, the name Nocardiopsis kunsanensis sp. nov. is proposed for strain HA-9T (= KCTC 9831T), which is designated the type strain.

Activation of murine peritoneal macrophages by Streptococcus pneumoniae type II capsular polysaccharide: involvement of CD14-dependent pathway.

In this study we examined the ability of capsular polysaccharide type 2 (PS) from Streptococcus pnemoniae to induce secretory and cellular responses in peritoneal macrophages. Tumour cytotoxicity induced by preincubation with PS was demonstrated to be concentration-dependent. PS-induced tumouricidal activity was partially abrogated by anti-tumour necrosis factor (TNF)-alpha and inhibitor of nitric oxide, whereas anti-interferon (IFN)-alpha/beta antibody and the scavengers of reactive oxygen intermediates had no effect. In addition, supernatants from macrophages treated with PS contained TNF-alpha, and their iNOS-enzymatic activity was significantly increased. Thus, the tumouricidal activity induced by PS appeared to be mediated by the production of TNF-alpha and nitrite. Treatment of macrophages with PS increased the expression of CD14, the receptor for lipolysaccharide (LPS)/LPS-binding protein. Moreover, blocking antibody to CD14 abrogated partially TNF-alpha and nitrite induction by PS, suggesting that the PS-induced CD14 upregulation was correlated with secretion of TNF-alpha and nitrite. Taken together, these results demonstrate that PS may induce macrophage-secretory and cellular activities, in part, possibly via CD14-dependent pathway.

Alteration of kinase-mediated signalings in murine peritoneal macrophages by aflatoxin B(1).

Aflatoxin B(1) (AFB(1)) is a potent hepatocarcinogen which is thought to exhibit an impairment of specific and non-specific immunity. Macrophages are responsible for non-specific immunity in host defense against tumors and microorganisms, and release a number of cytotoxic compounds, including nitric oxide (NO). We investigate whether the effect of AFB(1) on signal transduction is related to the decrease of NO production in murine peritoneal macrophages. When macrophages were stimulated with lipopolysaccharide (LPS) after AFB(1)-pretreatment, AFB1 decreased the NO production. The percentage of NO production in AFB(1)-pretreated macrophages was inversely increased by the addition of cholera toxin, phorbol 12-myrisate 13-acetate (PMA) and ionomycin. This suggests that AFB(1) affects the function of signaling constituents, including guanine nucleotide-binding protein (G protein), protein kinase C (PKC) and the calcium ion. AFB(1)-pretreatment significantly decreased PKC activity and tyrosine phosphorylation after LPS-stimulation. Taken together, these data propose that the inhibition of LPS-stimulated NO production by AFB(1) is related to the suppression of kinase-mediated intracellular signal transduction in murine peritoneal macrophages.

Aflatoxin B(1) inhibits CD14-mediated nitric oxide production in murine peritoneal macrophages.

Aflatoxin B(1) (AFB(1)), a potent hepatocarcinogen, has been known to impair non-specific and specific immunity. Macrophages play an important role in host defense against tumors and microorganisms and a number of compounds are implicated in macrophage cytotoxicity. Since activated by the reaction of LPS with CD14, macrophages produce nitric oxide (NO) that is a cytotoxic effector molecule in cell killing. In the present study, we investigated whether the alteration of CD14 level on macrophages by AFB(1) affects NO production in murine peritoneal macrophages. When macrophages were stimulated with LPS after AFB(1)-pretreatment, or they were co-treated with LPS and AFB(1), the NO production decreased in a dose-dependent manner. In contrast, when macrophages were post-treated with AFB(1) after LPS-stimulation, NO production was unchanged. DNA, RNA, and protein synthesis were reduced by AFB(1)-pretreatment of macrophages. The addition of anti-CD14 antibodies to the cultures decreased NO production further. FACS analysis showed that the binding of anti-CD14 antibodies to the macrophages was suppressed by AFB(1)-pretreatment followed by LPS-stimulation. However, AFB(1) does not alter the binding anti-CD14 antibodies to the macrophages without LPS-stimulation. In contrast, AFB(1) pretreatment increased an amount of CD14 released in culture medium. Taken together, these data indicate that the reduced NO production in murine peritoneal macrophages by AFB(1)-pretreatment is related to the suppressed expression of CD14 on macrophage membrane and to the increased secretion of it to culture medium after LPS-stimulation.

Inhibition of various functions in murine peritoneal macrophages by aflatoxin B1 exposure in vivo.

Aflatoxin B1 (AFB1) has been known to impair specific and nonspecific immunity. In the present study, we tested various functions of murine peritoneal macrophages that were isolated and stimulated with LPS after AFB1 (400 microg/5 ml/kg) was administered every other day for 2 weeks. AFB1 decreased phagocytosis and the production of superoxide anion (O2-) and hydrogen peroxide (H2O2), compared to those of corn oil-treated control group. In addition, the production of NO and TNF-alpha was decreased in macrophages of AFB1-treated mice. In vitro antitumor activity of in vivo AFB1-treated macrophages was reduced against target cell, L929. Taken together, these results suggested that AFBI might have the immunosuppressive effect on macrophages after in vivo exposure, which was related to the antitumor activity reduction.

Antitumor activity of 4-phenyl-1-arylsulfonylimidazolidinone, DW2143.

We examined the ability of sulfonylurea derivative, DW2143 (4-phenyl-1-[1-(4-aminobenzoyl)-indoline-5-sulfonyl]-4,5-dihydro-2-imida zolone hydrochloride), to inhibit the growth of tumor cells in vitro and in vivo. When its anti-proliferative activities were tested on five murine tumor (B 16, Colon26, E1-4, 3LL and P388) and nine human tumor (BxPC-3, HepG2, Lovo, MCF-7, NCI-H69, SW480, WiDR, KB and KBV20C) cells of diverse tissue origins, the in vitro antitumor activities of DW2143 were comparable to those of doxorubicin against all tumor cell lines. In addition, the anti-proliferative activities of DW2143 against KBV20C, a vincristine-resistant cell line, are similar or superior to those of doxorubicin. When the in vivo antitumor activities using three murine tumor cells were tested after oral administration of DW2143, a wide range of tumor growth inhibition was observed. Tumor growth inhibition against 3LL at doses of 50 and 100 mg/kg DW2143 was 84.3% and 47.2%, respectively, which was comparable or superior to those of doxorubicin (5 mg/kg). Tumor growth inhibition of B16 at a dose of 100 mg/kg in the DW2143-treated group was 42% as compared to 54% for doxorubicin (5 mg/kg). When mice implanted with Colon26 were tested, tumor growth inhibition at a dose of 80 mg/kg DW2143 was 36% as compared with 37% for doxorubicin (5 mg/kg). Taken together, these results indicate that the novel sulfonylurea derivative, DW2143, is an attractive candidate for further development as a useful oral anticancer drug.

Involvement of NO, H2O2 and TNF-alpha in the reduced antitumor activity of murine peritoneal macrophages by aflatoxin B1.

Aflatoxin B, (AFB1), a potent hepatocarcinogen, has been known to impair non-specific and specific immune responses. Nitric oxide (NO), hydrogen peroxide (H2O2), superoxide anion (O2-) and tumor necrosis factor-alpha (TNF-alpha) produced by macrophages play an important role in host defense against tumors and microorganisms. In the present studies, we investigated the involvement of those products in the reduced antitumor activities by AFB1. When macrophages are stimulated with LPS after AFB1-pretreatment, the cytolytic activities decrease in a dose-dependent manner. The addition of N(G)-monomethyl arginine (NMMA), anti-TNF-alpha antibodies, catalase and peroxidase decreases antitumor activities further. In contrast, superoxide dismutase (SOD) does not change the antitumor activities. NO and TNF-alpha production was reduced by the addition of NMMA and anti-TNF-alpha antibodies, respectively. Taken together, these data indicate that the reduced antitumor activities in murine peritoneal macrophages are mediated by the suppressed production of NO, TNF-alpha and H2O2 by AFB1 pretreatment, suggesting that the inhibitory effect of AFB1 on those materials may provide the tumors with readily growing condition in vivo.

In vitro suppressive effect of aflatoxin B1 on murine peritoneal macrophage functions.

We examined the immunosuppressive effects of aflatoxin B1 (AFB1), a toxic compound produced by the Aspergillus flavus, on murine peritoneal macrophages after in vitro pre-exposure. When thioglycollate-elicited macrophages pre-exposed to AFB1 were stimulated with lipopolysaccharide (LPS), antitumor activity induced by LPS was suppressed by 10 and 50 microM AFB1. In addition, the production of reactive intermediates including nitric oxide (NO), superoxide anion and hydrogen peroxide which have been known to be implicated in macrophage-mediated cytotoxicity, was decreased by AFB1 pretreatment in a dose-dependent manner. We also determined whether the macrophage-mediated cytokine production was altered by AFB1 in vitro pretreatment. AFB1 markedly inhibited TNF-alpha interleukin-1 (IL-1) and IL-6 production by LPS-stimulated macrophages. Taken together, these data indicate that AFB1 inhibits the killing ability of murine macrophages, decreases various secretory molecules in those cells and the macrophages would be one of many systems affected by AFB1.

Cytotoxic constituents from the roots of Anthriscus sylvestris.

Activity-guided fractionation of the roots of Anthriscus sylvestris resulted in the isolation and characterization of five cytotoxic compounds, deoxypodophyllotoxin (1), falcarindiol (2), and angeloyl podophyllotoxin (5) from the hexane soluble fraction and morelensin (3), bursehernin (4) from the chloroform soluble fraction. It is the first report of the occurrence of compound 5 in nature.

Effect of DW2282 on the induction of methemoglobinemia, hypoglycemia or WBC count and hematological changes.

DW2282,(S)-(+)-4-phenyl-1-[1-(4-aminobenzoyl)-indoline-5-sulfonyl] -4,5-dihydro-2-imidazolone hydrochloride, is a new anticancer agent which is thought to exhibit a characteristic mechanism of action in the inhibition of tumor growth. In this study, we estimated the toxicities of DW2282 in mice. When mice were orally dosed for five consecutive days at the dosages of 50, 100 and 150 mg/kg, DW2282 did not induce methemoglobinemia and hypoglycemia at any of these doses. However, increased ALT and AST values were observed in the 150 mg/kg dosing group, and white blood cells (WBC) were significantly decreased at all doses. However, the changes in WBC count, ALT and AST immediately reversed after the cessation of drug administration. In addition, we found that DW2282 did not cause an increase in hemolysis in human blood. Taken together, these data suggested that DW2282 may have a relatively low level of toxicity, and that there may be a quick recovery from any toxicity it does produce.

Characterization of the anticancer activity of DW2282, a new anticancer agent.

DW2282 [(S)-(+)-4-phenyl-1-[N-(4-aminobenzoyl) indoline-5-sulfonyl]-4,5-dihydro-2-imidazolone].hydrochloride] was derived from diarylsulfonylurea and was identified as a prominent new anticancer agent. We examined the characteristics of DW2282 activity on the proliferation of human lung carcinoma cells, A549 and human leukemic cells, K562. DW2282 effectively inhibited cancer cell proliferation in vitro. Colony forming assay and viability tests demonstrated that DW2282 is a cytotoxic agent rather than a cytostatic agent. The isotope uptake test exhibited that DW2282 inhibited or inactivated protein synthesis. Also, under conditions which cause RNA or protein synthesis inhibition, by co-treatment with actinomycin D or cycloheximide, reduced the anticancer effects of DW2282. This means that the cytotoxicity of DW2282 depends partially on RNA or protein synthesis and proteins affected by DW2282 may inactivate or alter the process of the synthesis of another protein. DW2282 activity was highly diminished in the presence of colcemid, a metaphase spindle blocker. This result suggests that DW2282 may be related to the cell cycle. After exposure to DW2282, morphologically apoptotic cells appeared in A549 cells and fragmented DNA was detected in K562 cells. It demonstrated that apoptosis is one of the mechanisms by which DW2282 inhibits the proliferation of A549 and K562 cells.

Aflatoxin B1-induced suppression of nitric oxide production in murine peritoneal macrophages.

Aflatoxin B1 (AFB1), a potent hepatocarcinogen, is known to impair specific and non-specific immune responses. AFB1 mainly decreases lymphocyte functions and may also affect macrophages assisting lymphocyte functions. Macrophages play an important role in a host defense against tumors and bacteria. Furthermore, some macrophage products, including nitric oxide (NO), may be involved in cytotoxicity. The effect of aflatoxin B1 (AFB1) was investigated on NO production from murine peritoneal macrophages. Macrophages were pretreated with AFB1 for 24 h and then stimulated with lipopolysaccharide (LPS) for 24 h. AFB1 at 10 or 50 microM reduced the production of NO. Compared to vehicle control, there was a greater reduction of NO production with increased AFB1 pretreatment and LPS stimulation. AFB1 at 10 or 50 microM decreased inducible nitric oxide synthase (iNOS) activity about 24% and 28%, respectively, after stimulation with 1 microg/ml LPS and about 12% and 24%, respectively, after stimulation with 10 microg/ml LPS. AFB1 pretreatment also decreased the synthesis of iNOS protein and the mRNA of macrophages. Taken together, these results suggest that AFB1 pretreatment reduces NO production from murine peritoneal macrophages stimulated by LPS, which is mediated by the reduction of iNOS activity, mRNA, and protein.

Momordins inhibit both AP-1 function and cell proliferation.

The activation of Jun/Fos is a crucial factor in transmitting the tumor promoting signal from the extracellular environment to nuclear transcription machinery. One of the final steps in signal transduction is the binding of Jun/Fos to the AP-1 site in order to express gene transcription. Utilizing this concept, we screened about 100 extracts of natural plants to search for a Jun-Fos function inhibitor. The methanol extract of Ampelopsis radix reduced Jun/Foc retardation remarkably. The active principles of the extract were isolated and purified by repeated column chromatography and their structures were identified as oleanolic acid glycosides known as momordin I, Id, and Ie. These compounds reduced the Jun/Fos-DNA interaction and their activities were quantitated with liquid scintillation counting of corresponding bands. Among them, momordin I had the strongest inhibitory activity, with an IC50 value of 22.8 micrograms/ml. The methanol extract and momordin I, Id and Ie also showed cell cytotoxicity against human cancer cell lines. As expected from a gel shift assay, momordin I showed the strongest cytotoxicity and its IC50 value was from 7.280 micrograms/ml to 16.05 micrograms/ml depending on the cell line. With these data, it may be concluded that the mechanism of anticancer activity of momordin I comes from its inhibitory effect on the protein-DNA interaction. The in vivo test was done only with the methanol extract. The extract showed measurable anticancer activity against murine colon cancer. The wet tumor weight reduction rate was 17.73% at 90 mg/kg dose. We suggest that the Jun/Fos-DNA interaction results in cell cytotoxicity.

Toxicities of 166Holmium-chitosan in mice.

166Holmium (166Ho) is a radionuclide of rare earth chemical and is known to have antitumor activity. Several chemicals were complexed with 166Ho to facilitate the transport of this radionuclide to the site of action. In this study, 166Ho was complexed to chitosan (Chit) which decreases the distribution of Ho into other tissues when applied intrahepatically. To investigate the single dose toxicity, mice were administered intravenously with 1 mCi/kg body weight of 166Ho-Chit (DW-166HC), Chit or nothing. Organ weights, hematological and histopathological studies were performed in 6 animals per group at 1, 3 and 14 days after administration. In 166Ho-Chit treated animals, a slight decrease of erythrocyte number was observed at day 14 and increases of relative liver and lung weights were found at day 3. Although marked multiple necrotic foci in the white pulp and depletion of marginal zone in the spleen were noted at day 1, these findings were decreased in severity and fully recovered at day 3 and day 14, respectively. Slightly decreased kidney weights were observed both in Chit and in 166Ho-Chit treated groups without histological alterations. Thus it is suggested that most effects of 166Ho-Chit observed at an early stage after administration are limited to rapidly dividing cells and reversible within 14 days.

Synthesis and antitumor activity of 4-phenyl-1-arylsulfonyl imidazolidinones.

Novel 1-(1-benzoylindoline-5-sulfonyl)-4-phenyl-4,5-dihydroimidazolon es 3 synthesized show highly potent and broad cytotoxicities. Among them compound 3b (DW2143) exhibits much more potent cytotoxicities than doxorubicin and highly effective antitumor activities against murine (3LL, Colon 26) and human xenograft (NCI-H23, SW620) tumor models.