AIM: To study the chemical constituents of the solid culture of the endophyte Phomopsis sp. IFB-E060 in Vatica mangachapoi. METHOD: Isolation and purification were performed through silica gel column chromatography, gel filtration over Sephadex LH-20, ODS column chromatography, and HPLC. Structures of the isolated compounds were elucidated by a combination of spectroscopic analyses (UV, CD, IR, MS, 1D, and 2D NMR). The cytotoxicity of the isolates was evaluated in vitro by the MTT method against the human hepatocarcinoma cell line SMMC-7721. RESULTS: Five compounds were isolated from the solid culture of the endophyte Phomopsis sp. IFB-E060 and their structures were identified as 18-methoxy cytochalasin J (1), cytochalasin H (2), (22E, 24S)-cerevisterol (3), ergosterol (4), and nicotinic acid (5). Compound 1 had an inhibition rate of 24.4% at 10 μg·mL(-1) and 2 had an IC50 value of 15.0 μg·mL(-1), while a positive control 5-fluorouracil had an inhibition rate of 28.7% at 10 μg·mL(-1). CONCLUSION 18-Methoxy cytochalasin J (1), produced by endophytic Phomopsis sp. IFB-E060, is a new cytochalasin with weak cytotoxicity to the human hepatocarcinoma cell line SMMC-7721.
Ascomycota ; chemistry ; isolation & purification ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cytochalasins ; chemistry ; isolation & purification ; toxicity ; Endophytes ; chemistry ; isolation & purification ; Humans ; Magnoliopsida ; microbiology ; Molecular Structure ; Plant Bark ; microbiology

The dynamics of the actin cytoskeleton plays a pivotal role in the process of cell division, the transportation of organelles, vesicle trafficking and cell movement. Human immunodeficiency virus type 1 (HIV-1) hijacks the actin dynamics network during the viral entry and migration of the pre-integration complex (PIC) into the nucleus. Actin dynamics linked to HIV-1 has emerged as a potent therapeutic target against HIV infection. Although some inhibitors have been intensely analyzed with regard to HIV-1 infection, their effects are sometimes disputed and the exact mechanisms for actin dynamics in HIV infection have not been well elucidated. In this study, the small molecules regulating HIV-1 infection from diverse inhibitors of the actin dynamic network were screened. Two compounds, including Chaetoglobosin A and CK-548, were observed to specifically bar the viral infection, while the cytochalasin family, 187-1, N-WASP inhibitor, Rho GTPase family inhibitors (EHop-016, CID44216842, and ML-141) and LIMK inhibitor (LIM domain kinase inhibitor) increased the viral infection without cytotoxicity within a range of ~ µM. However, previously known inhibitory compounds of HIV-1 infection, such as Latrunculin A, Jasplakinolide, Wiskostatin and Swinholide A, exhibited either an inhibitory effect on HIV-1 infection combined with severe cytotoxicity or showed no effects. Our data indicate that Chaetoglobosin A and CK-548 have considerable potential for development as new therapeutic drugs for the treatment of HIV infection. In addition, the newly identified roles of Cytochalasins and some inhibitors of Rho GTPase and LIMK may provide fundamental knowledge for understanding the complicated actin dynamic pathway when infected by HIV-1. Remarkably, the newly defined action modes of the inhibitors may be helpful in developing potent anti-HIV drugs that target the actin network, which are required for HIV infection.
Actin Cytoskeleton ; Actins ; Anti-HIV Agents ; Cell Division ; Cell Movement ; Cytochalasins ; GTP Phosphohydrolases ; HIV Infections ; HIV-1 ; Humans ; Organelles ; Phosphotransferases ; Transportation

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-1beta and TNF-alpha was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-kappaB activation and cytokine expression. Treatment with M. leprae significantly increased NF-kappaB activation and expression of TNF-alpha and IL-1beta in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.
Cytochalasin D ; Cytokines ; Humans ; Leprosy ; Macrophage Activation ; Macrophages ; Mycobacterium ; Mycobacterium leprae ; NF-kappa B ; Phagocytosis ; Proteins ; Receptors, Cytoplasmic and Nuclear ; Tumor Necrosis Factor-alpha

Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-alpha. The present study investigated the mechanisms involved in TNF-alpha production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-alpha. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF-alpha by E. faecalis. In addition, antioxidant treatment reduced TNF-alpha production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF-alpha expression by RAW 264.7 cells. Furthermore, activation of NF-kappaB and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF-alpha in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-kappaB, and AP-1.
Bacteria ; Cell Line ; Cytochalasin D ; Cytokines ; Endocytosis ; Enterococcus ; Enterococcus faecalis ; Gene Expression ; Macrophages ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Periapical Periodontitis ; Phosphotransferases ; Reactive Oxygen Species ; RNA, Messenger ; Transcription Factor AP-1 ; Tumor Necrosis Factor-alpha ; Up-Regulation

Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-alpha. The present study investigated the mechanisms involved in TNF-alpha production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-alpha. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF-alpha by E. faecalis. In addition, antioxidant treatment reduced TNF-alpha production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF-alpha expression by RAW 264.7 cells. Furthermore, activation of NF-kappaB and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF-alpha in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-kappaB, and AP-1.
Bacteria ; Cell Line ; Cytochalasin D ; Cytokines ; Endocytosis ; Enterococcus ; Enterococcus faecalis ; Gene Expression ; Macrophages ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Periapical Periodontitis ; Phosphotransferases ; Reactive Oxygen Species ; RNA, Messenger ; Transcription Factor AP-1 ; Tumor Necrosis Factor-alpha ; Up-Regulation

Aggregatibacter actinomycetemcomitans is the most important etiologic agent of aggressive periodontitis and can interact with endothelial cells. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are chemokines, playing important roles in periodontal pathogenesis. In our current study, the effects of A. actinomycetemcomitans on the production of MCP-1 and IL-8 by human umbilical vein endothelial cells (HUVEC) were investigated. A. actinomycetemcomitans strongly induced the gene expression and protein release of both MCP-1 and IL-8 in a dose- and time-dependent manner. Dead A. actinomycetemcomitans cells were as effective as live bacteria in this induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, did not affect the mRNA up-regulation of MCP-1 and IL-8 by A. actinomycetemcomitans. However, genistein, an inhibitor of protein tyrosine kinases, substantially inhibited the MCP-1 and IL-8 production by A. actinomycetemcomitans, whereas pharmacological inhibition of each of three members of mitogen-activated protein (MAP) kinase family had little effect. Furthermore, gel shift assays showed that A. actinomycetemcomitans induces a biphasic activation (early at 1-2 h and late at 8-16 h) of nuclear factor-kappaB (NF-kappaB) and an early brief activation (0.5-2 h) of activator protein-1 (AP-1). Activation of canonical NF-kappaB pathway (IkappaB kinase activation and IkappaB-alpha degradation) was also demonstrated in these experiments. Although lipopolysaccharide from A. actinomycetemcomitans also induced NF-kappaB activation, this activation profile over time differed from that of live A. actinomycetemcomitans. These results suggest that the expression of MCP-1 and IL-8 is potently increased by A. actinomycetemcomitans in endothelial cells, and that the viability of A. actinomycetemcomitans and bacterial internalization are not required for this effect, whereas the activation of protein tyrosine kinase(s), NF-kappaB, and AP-1 appears to play important roles. The secretion of high levels of MCP-1 and IL-8 resulting from interactions of A. actinomycetemcomitans with endothelial cells may thus contribute to the pathogenesis of aggressive periodontitis.
Aggressive Periodontitis ; Bacteria ; Chemokine CCL2 ; Chemokines ; Cytochalasin D ; Endocytosis ; Endothelial Cells ; Gene Expression ; Genistein ; Human Umbilical Vein Endothelial Cells ; Humans ; I-kappa B Proteins ; Interleukin-8 ; Monocytes ; NF-kappa B ; Phosphotransferases ; Protein-Tyrosine Kinases ; RNA, Messenger ; Transcription Factor AP-1 ; Tyrosine ; Up-Regulation

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.
Actin Cytoskeleton ; Animals ; Cytochalasin B* ; Cytoskeleton ; Formaldehyde ; Immunoglobulin G ; Mice* ; Microtubules ; Nitrogen ; Oocytes* ; Propidium ; Sucrose ; Vitrification*

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.
Actin Cytoskeleton ; Animals ; Cytochalasin B* ; Cytoskeleton ; Formaldehyde ; Immunoglobulin G ; Mice* ; Microtubules ; Nitrogen ; Oocytes* ; Propidium ; Sucrose ; Vitrification*

OBJECTIVE: The purpose of this study was to evaluate the effect of CCB treatment on the survivability and in vitro development of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then exposed to EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes on EM grid was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M sucrose at 37degrees C for 3 minutes. This was followed by 0.5 M and 0.25 M sucrose for 3 minutes, each. We compared the survivability, cleavage and developmental rate of mouse frozen by vitrification between CCB treated and non-treated groups. Chi-square was used to determine statistical significance. statistical significance was defined as p<0.05. RESULTS: Survivability (79.3%) and developmental rate into blastocyst (52.3%) of mouse oocyte were markedly decreased after vitrification. There were no significant differences between CCB treated and non- treated groups regarding survivability of oocyte frozen by vitrification (80.3% vs 78.5%). The developmental rate into 2-cell in CCB treated group was significantly higher than that in non-treated group (69.7% vs 61.9%, p<0.05). The developmental rate into blastocyst in CCB treated group was higher than that in non-treated group (54.9% vs 51.5%), but the difference was not significant. CONCLUSION: Survivability of mouse oocyte could not be affected by CCB treatment and developmental rate into 2-cell was improved in CCB treated group. It is suggested that CCB treatment prior vitrification improve stability of cytoskeleton and then improve fertilization and early stage embryo development.
Animals ; Blastocyst ; Cytochalasin B* ; Cytoskeleton ; Embryonic Development ; Female ; Fertilization ; Mice* ; Nitrogen ; Oocytes* ; Pregnancy ; Sucrose ; Vitrification*

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [3H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [3H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [3H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [3H]3-OMG uptake was increased at 48 h. However, [3H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [3H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [3H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.
Alendronate ; Animals ; Blood-Retinal Barrier ; Capillaries* ; Cytochalasin B ; Diphosphonates* ; Endothelial Cells* ; Glucose* ; Hand ; Histamine ; Mevalonic Acid ; Rats* ; Retinaldehyde*