Stimulation of the farnesoid X receptor promotes M2 macrophage polarization.

This skewed polarization towards M2 macrophages by an FXR agonist was accompanied by increased expression of signaling molecules related to the retinoic acid receptor. Inhibition of the retinoic acid receptor suppressed FXR agonist-mediated M2 macrophage polarization, indicating that this polarization was, at least, partly dependent on the retinoic acid receptor pathway. These data demonstrate that FXR has a role in polarization toward M2 macrophages and suggest a possible therapeutic potential of FXR agonists in M2 macrophage-related conditions.


Blockade of the CXCR3/CXCL10 axis ameliorates inflammation caused by immunoproteasome dysfunction.

Immunoproteasomes regulate the degradation of ubiquitin-coupled proteins and generate peptides that are preferentially presented by MHC class I. Mutations in immunoproteasome subunits lead to immunoproteasome dysfunction, which causes proteasome-associated autoinflammatory syndromes (PRAAS) characterized by nodular erythema and partial lipodystrophy. It remains unclear, however, how immunoproteasome dysfunction leads to inflammatory symptoms. Here, we established mice harboring a mutation in Psmb8 (Psmb8-KI mice) and addressed this question. Psmb8-KI mice showed higher susceptibility to imiquimod-induced skin inflammation (IMS). Blockade of IL-6 or TNF-α partially suppressed IMS in both control and Psmb8-KI mice, but there was still more residual inflammation in the Psmb8-KI mice than in the control mice. DNA microarray analysis showed that treatment of J774 cells with proteasome inhibitors increased the expression of the Cxcl9 and Cxcl10 genes. Deficiency in Cxcr3, the gene encoding the receptor of CXCL9 and CXCL10, in control mice did not change IMS susceptibility, while deficiency in Cxcr3 in Psmb8-KI mice ameliorated IMS. Taken together, these findings demonstrate that this mutation in Psmb8 leads to hyperactivation of the CXCR3 pathway, which is responsible for the increased susceptibility of Psmb8-KI mice to IMS. These data suggest the CXCR3/CXCL10 axis as a new molecular target for treating PRAAS.


Necroptosis protects against exacerbation of acute pancreatitis.

mice exhibit weight loss, edematous pancreatitis, necrotizing pancreatitis, and acinar cell dedifferentiation in response to tissue damage. Genetic deletion of Mlkl resulted in downregulation of the antiapoptotic genes Bclxl and Cflar in association with increases in the numbers of apoptotic cells, as detected by TUNEL assay. These findings suggest that RIPK3 and MLKL-mediated necroptosis exerts protective effects in AP and caution against the use of necroptosis inhibitors for AP treatment.


Quantitative evaluation of SARS-CoV-2 inactivation using a deep ultraviolet light-emitting diode.

for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.


Distinct Roles of IL-1 and IL-18 in NLRC4-Induced Autoinflammation.

The NLRC4 inflammasome assembles in response to detection of bacterial invasion, and NLRC4 activation leads to the production of IL-1 and IL-18 together with pyroptosis-mediated cell death. Missense activating mutations in cause autoinflammatory disorders whose symptoms are distinctly dependent on the site of mutation and other aspects of the genetic background. To determine the involvement of IL-1 and IL-18 in the inflammation induced by mutation, we depleted IL-1 , IL-18, or both cytokines in Nlrc4-transgenic mice in which mutant is expressed under the MHC class II promoter (Nlrc4-H443P-Tg mice). The deletion of the or gene in Nlrc4-H443P-Tg mice reduced the neutrophil numbers in the spleen, and mice with deletion of both genes had an equivalent number of neutrophils compared to wild-type mice. Deletion of ameliorated but did not eliminate bone marrow hyperplasia, while mice deficient in showed no bone marrow hyperplasia. In contrast, tail bone deformity remained in the presence of deficiency, but deficiency completely abolished bone deformity. The decreased bone density in Nlrc4-H443P-Tg mice was counteracted by but not deficiency. Our results demonstrate the distinct effects of IL-1 and IL-18 on NLRC4-induced inflammation among tissues, which suggests that blockers for each cytokine should be utilized depending on the site of inflammation.


Notch Balances Th17 and Induced Regulatory T Cell Functions in Dendritic Cells by Regulating Aldh1a2 Expression.

Dendritic cells (DCs) are important for adaptive immune responses through the activation of T cells. The molecular interplay between DCs and T cells determines the magnitude of T cell responses or outcomes of functional differentiation of T cells. In this study, we demonstrated that DCs in mice that are Rbpj deficient in CD11c(+) cells (Rbpj(-/-) mice) promoted the differentiation of IL-17A-producing Th17 cells. Rbpj-deficient DCs expressed little Aldh1a2 protein that is required for generating retinoic acid. Those DCs exhibited a reduced ability for differentiating regulatory T cells induced by TGF-. Rbpj protein directly regulated Aldh1a2 transcription by binding to its promoter region. The overexpression of Aldh1a2 in Rbpj-deficient DCs negated their Th17-promoting ability. Transfer of naive CD4(+) T cells into Rag1-deficient Rbpj(-/-) mice enhanced colitis with increased Th17 and reduced induced regulatory T cells (iTreg) compared with control Rag1-deficient mice. The cotransfer of iTreg and naive CD4(+) T cells into Rag1-deficient Rbpj(-/-) mice improved colitis compared with transfer of naive CD4(+) T cell alone. Furthermore, cotransfer of DCs from Rbpj(-/-) mice that overexpressed Aldh1a2 or Notch-stimulated DCs together with naive CD4(+) T cells into Rbpj(-/-)Rag1-deficient mice led to reduced colitis with increased iTreg numbers. Therefore, our studies identify Notch signaling in DCs as a crucial balancer of Th17/iTreg, which depends on the direct regulation of Aldh1a2 transcription in DCs.


Differentiation of preadipocytes and mature adipocytes requires PSMB8.

The differentiation of adipocytes is tightly regulated by a variety of intrinsic molecules and also by extrinsic molecules produced by adjacent cells. Dysfunction of adipocyte differentiation causes lipodystrophy, which impairs glucose and lipid homeostasis. Although dysfunction of immunoproteasomes causes partial lipodystrophy, the detailed molecular mechanisms remain to be determined. Here, we demonstrate that Psmb8, a catalytic subunit for immunoproteasomes, directly regulates the differentiation of preadipocytes and additionally the differentiation of preadipocytes to mature adipocytes. Psmb8(-/-) mice exhibited slower weight gain than wild-type mice, and this was accompanied by reduced adipose tissue volume and smaller size of mature adipocytes compared with controls. Blockade of Psmb8 activity in 3T3-L1 cells disturbed the differentiation to mature adipocytes. Psmb8(-/-) mice had fewer preadipocyte precursors, fewer preadipocytes and a reduced ability to differentiate preadipocytes toward mature adipocytes. Our data demonstrate that Psmb8-mediated immunoproteasome activity is a direct regulator of the differentiation of preadipocytes and their ultimate maturation.


CD98 Heavy Chain Is a Potent Positive Regulator of CD4+ T Cell Proliferation and Interferon- Production In Vivo

Upon their recognition of antigens presented by the MHC, T cell proliferation is vital for clonal expansion and the acquisition of effector functions, which are essential for mounting adaptive immune responses. The CD98 heavy chain (CD98hc, Slc3a2) plays a crucial role in the proliferation of both CD4+ and CD8+ T cells, although it is unclear if CD98hc directly regulates the T cell effector functions that are not linked with T cell proliferation in vivo. Here, we demonstrate that CD98hc is required for both CD4+ T cell proliferation and Th1 functional differentiation. T cell-specific deletion of CD98hc did not affect T cell development in the thymus. CD98hc-deficient CD4+ T cells proliferated in vivo more slowly as compared with control T cells. C57BL/6 mice lacking CD98hc in their CD4+ T cells could not control Leishmania major infections due to lowered IFN- production, even with massive CD4+ T cell proliferation. CD98hc-deficient CD4+ T cells exhibited lower IFN- production compared with wild-type T cells, even when comparing IFN- expression in cells that underwent the same number of cell divisions. Therefore, these data indicate that CD98hc is required for CD4+ T cell expansion and functional Th1 differentiation in vivo, and suggest that CD98hc might be a good target for treating Th1-mediated immune disorders.


CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4(+) T cells.

CD4(+) T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4(+) T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4(+) T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4(+)CD25(-) T cells into Rag2(-/-) mice did not cause colitis accompanied by increasing Foxp3(+) inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4(+)CD25(-) T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4(+) T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.

Biochemical and Biophysical Research Communications


The ARNT-STAT3 axis regulates the differentiation of intestinal intraepithelial TCRαβ+CD8αα+cells.

Intestinal intraepithelial T cells contribute to the regulation of inflammatory responses in the intestine; however, the molecular basis for their development and maintenance is unknown. The aryl hydrocarbon receptor complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and senses environmental factors, including gut microbiota. Here, we identify ARNT as a critical regulator of the differentiation of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. Mice deficient in either ARNT or aryl hydrocarbon receptor show a greater than- eight-fold reduction in the number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. The number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells is increased by treatment with an aryl hydrocarbon receptor agonist in germ-free mice and is decreased by antibiotic treatment. The Arnt-deficient precursors of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells express low amounts of STAT3 and fail to differentiate towards the TCRαβ(+)CD8αα(+) cell fate after IL-15 stimulation, a deficiency that is overcome by overexpression of Stat3. These data demonstrate that the ARNT-STAT3 axis is a critical regulator of TCRαβ(+)CD8αα(+) intestinal intraepithelial T-cell development and differentiation.


Reduced Diversity and Imbalance of Fecal Microbiota in Patients with Ulcerative Colitis

Clinical observations and experimental colitis models have indicated the importance of intestinal bacteria in the etiology of ulcerative colitis (UC), but a causative bacterial agent has not been identified. To determine how intestinal bacteria are associated with UC, fecal microbiota and other components were compared for UC patients and healthy adults. Fresh feces were collected from 48 UC patients. Fecal microbiota were analyzed by use of terminal-restriction fragment length polymorphism (T-RFLP), real-time PCR, and culture. The concentrations of organic acids, indole, and ammonia, and pH and moisture, which are indicators of the intestinal environment, were measured and compared with healthy control data. T-RFLP data divided the UC patients into four clusters; one cluster was obtained for healthy subjects. The diversity of fecal microbiota was significantly lower in UC patients. There were significantly fewer Bacteroides and Clostridium subcluster XIVab, and the amount of Enterococcus was higher in UC patients than in healthy subjects. The fecal concentration of organic acids was significantly lower in UC patients who were in remission. UC patients have imbalances in the intestinal environment-less diversity of fecal microbiota, lower levels of major anaerobic bacteria (Bacteroides and Clostridium subcluster XIVab), and a lower concentration of organic acids.


In vitro cytotoxic effect of ethanol extract prepared from sporophyll of Undaria pinnatifida on human colorectal cancer cells.

Brown seaweed Undaria pinnatifida (Harvey) Suringar is popular as a foodstuff, and used for medical care in East Asian countries. The major components of this seaweed are shown to benefit hypertension and hyperlipidemia, and considered to reduce the risks of infarction and ischemic diseases. Furthermore, the intake of dietary fiber of seaweeds is considered to prevent the production and proliferation of cancer in the gastrointestinal tract. The direct effect of an ethanol extract prepared from Undaria pinnatifida sporophyll (mekabu) on HCT116 human colorectal cancer cells was examined, and the mekabu extract was shown to induce the non-oxidative apoptotic damage to the cells, thus resulting in the reduction of their viabilities in a concentration-dependent manner. Moreover, the cytotoxic effects of carcinostatic drugs, such as 5-fluorouracil (5-FU) and irinotecan (CPT-11), were observed only in the medium containing sera, while the mekabu extract could effectively reduce the cell viabilities even in the serum-free medium. These findings suggest that the mekabu extract may contain a potential active substance inducing the non-oxidative apoptotic cell death probably through a mechanism different from those of 5-FU and CPT-11, and hence mekabu is possibly useful as an auxiliary drug to the chemotherapy of colorectal cancer.


Manipulation of CD98 resolves type 1 diabetes in nonobese diabetic mice.

The interplay of CD4(+) and CD8(+) T cells targeting autoantigens is responsible for the progression of a number of autoimmune diseases, including type 1 diabetes mellitus (T1D). Understanding the molecular mechanisms that regulate T cell activation is crucial for designing effective therapies for autoimmune diseases. We probed a panel of Abs with T cell-modulating activity and identified a mAb specific for the H chain of CD98 (CD98hc) that was able to suppress T cell proliferation. The anti-CD98hc mAb also inhibited Ag-specific proliferation and the acquisition of effector function by CD4(+) and CD8(+) T cells in vitro and in vivo. Injection of the anti-CD98hc mAb completely prevented the onset of cyclophosphamide-induced diabetes in NOD mice. Treatment of diabetic NOD mice with anti-CD98hc reversed the diabetic state to normal levels, coincident with decreased proliferation of CD4(+) T cells. Furthermore, treatment of diabetic NOD mice with CD98hc small interfering RNA resolved T1D. These data indicate that strategies targeting CD98hc might have clinical application for treating T1D and other T cell-mediated autoimmune diseases.


Notch2 regulates the development of marginal zone B cells through Fos.

B cells are classified into several subsets depending on their functions, marker expression pattern and localization. Marginal zone B (MZB) cells are a distinct lineage from follicular B cells, and regulate host defenses against blood-borne pathogens. Notch2/RBP-J signaling regulates the development of MZB cells by interacting with delta-like 1 ligand, although the target genes for Notch2 signaling remain unclear. We identified Fos as an upregulated gene in LPS-stimulated B cells that received Notch2 signaling. Fos is expressed in CD21(high)CD23(low) MZB cells at a higher level compared to CD21(Int)CD23(high) follicular B cells. Deleting the Notch2 gene in CD19(+) B cells decreased Fos expression in B cells. Overexpression of Fos in Notch2-deficient B cells or bone marrow cells partially restored MZB development. Fos promoter activity was upregulated by Notch2 signaling, indicating that Notch2 directly controls Fos transcription associated with MZB development. These data identify Fos as one of the target genes for Notch2 signaling that is crucial for MZB development.


Histone deacetylase inhibitors promote neurosteroid-mediated cell differentiation and enhance serotonin-stimulated brain-derived neurotrophic factor gene expression in rat C6 glioma cells

Progesterone treatment has previously been reported to promote the differentiation of glial cells probably through the production of 5alpha-reduced neurosteroids, resulting in the enhancement of serotonin-stimulated brain-derived neurotrophic factor (BDNF) gene expression, which is considered to contribute to the survival, regeneration, and plasticity of neuronal cells in the brain and hence has been suggested to improve mood disorders and other symptoms in depressive patients. Based on these previous observations, the effects on glial cells of histone deacetylase (HDAC) inhibitors, which are known as agents promoting cell differentiation, were examined using rat C6 glioma cells as a model for in vitro studies. Consequently, trichostatin A (TSA), sodium butyrate (NaB), and valproic acid (VPA) stimulated glial fibrillary acidic protein (GFAP) gene expression, and their stimulatory effects on GFAP gene expression were inhibited by treatment of these cells with finasteride, an inhibitor of the enzyme producing 5alpha-reduced neurosteroids. In addition, HDAC inhibitors enhanced serotonin-stimulated BDNF gene expression, the enhancement of which could be abolished by the inhibition of 5alpha-reduced neurosteroid production in the glioma cells. These results suggest that HDAC inhibitors may be able to promote the differentiation of rat C6 glioma cells through the production of 5alpha-reduced neurosteroids, resulting in the enhancement of serotonin-stimulated BDNF gene expression as a consequence of promoting their differentiation, indicating the possibility that differentiated glial cells may be implicated in preserving the integrity of neural networks as well as improving the function of neuronal cells in the brain.


Production of apoptosis-inducing substances from soybean protein by Clostridium butyricum: Characterization of their toxic effects on human colon carcinoma cells

Microbial metabolism of soybean constituents is known to produce novel active substances as a chemopreventive agent during the fermentation, and enterobacteria are expected to produce chemopreventive agents as a consequence of metabolizing soybean constituents in the intestinal tract. Then, the conditioned medium was prepared by culturing an enterobacterium Clostridium butyricum (C. butyricum) with soybean protein, and its direct effect on human colon carcinoma HCT116 cells was examined. The conditioned medium was shown to induce the cell death, and suggested to contain novel apoptosis-inducing substances. Thus, enterobacteria are predicted to produce anti-tumor substances from food-derived proteins within the intestinal tract.


Inhibitory effects of fermented brown rice on induction of acute colitis by dextran sulfate sodium in rats.

Although the pathogenic mechanisms of inflammatory bowel diseases are not fully understood, colonic microbiota may affect the induction of colonic inflammation, and some probiotics and prebiotics have been reported to suppress colitis. The inhibitory effects of brown rice fermented by Aspergillus oryzae (FBRA), a fiber-rich food, on the induction of acute colitis by dextran sulfate sodium (DSS) were examined. Feeding a 5% and 10% FBRA-containing diet significantly decreased the ulcer and erosion area in the rat colon stained with Alcian blue. In another experiment, 10% FBRA feeding decreased the ulcer index (percentage of the total length of ulcers in the full length of the colon) and colitis score, which were determined by macroscopic observation. It also decreased myeloperoxidase activity in the colonic mucosa. Viable cell numbers of Lactobacillus in the feces decreased after DSS administration and was reversely correlated with severity of colitis, while the cell number of Enterobacteriaceae increased after DSS treatment and was positively correlated with colitis severity. These results indicate that FBRA has a suppressive effect on the induction of colitis by DSS and suggest FBRA-mediated modification of colonic microbiota.


Desipramine induces apoptotic cell death through nonmitochondrial and mitochondrial pathways in different types of human colon carcinoma cells.

Cytotoxic effects of desipramine on human colon carcinoma HT29 and HCT116 cells were examined. Desipramine reduced the viability of HT29 cells in a concentration-dependent manner, but failed to cause any significant change in the viability of HCT116 cells by the concentration up to 50 mumol/l, at which an approximately 60% reduction of the viability of HT29 cells was observed. Despite their different sensitivities, desipramine caused the nonoxidative apoptotic damage to both of them. In contrast to HT29 cells, desipramine might cause the apoptotic death of HCT116 cells through the disturbance of mitochondrial function. These results suggest that desipramine may cause the nonoxidative apoptotic damage to different types of human colon carcinoma cells through either a nonmitochondrial or a mitochondrial pathway, which may confer the different sensitivities to this drug on these tumor cells.


Modifying effects of fermented brown rice on fecal microbiota in rats

Brown rice fermented by Aspergillus oryzae (FBRA) is a fiber-rich food. Effects of dietary administration of FBRA on rat fecal microbiota composition were examined. Male Wistar rats were fed a basal diet or a 5% FBRA- or 10% FBRA-containing diet, and fecal microbiota was analyzed by culture and terminal-restriction fragment length polymorphism (T-RFLP) analysis. The viable cell number of lactobacilli significantly increased after feeding 10% FBRA diet for 3 weeks compared with that in the basal diet group and that in the same group at the beginning of the experiment (day 0). An increase in the viable cell number of lactobacilli was also observed after feeding 10% FBRA for 12 weeks compared with the effect of a basal diet. T-RFLP analysis showed an increase in the percentage of lactobacilli cells in feces of rats fed 10% FBRA for 14 weeks. Lactobacilli strains isolated from rat feces were divided into six types based on their randomly amplified polymorphic DNA (RAPD) patterns, and they were identified as Lactobacillus reuteri, L. intestinalis and lactobacilli species based on homology of the partial sequence of 16S rDNA. FBRA contains lactic acid bacteria, but their RAPD patterns and identified species were different from those in rat feces. These results indicated that dietary FBRA increases the number of lactobacilli species already resident in the rat intestine.


Suppressive effect of Clostridium perfringens-produced heat-stable substance(s) on proliferation of human colon adenocarcinoma HT29 cells in culture

Clostridium perfringens has been regarded as one of the intestinal bacteria increasing colon cancer risk. In previous studies, we have shown that the oral administration of C. perfringens culture medium can inhibit the mutagen-induced formation of pre-neoplastic lesions in rat colon, thus proposing the existence of factor(s) preventing colon tumorigenesis in this culture medium. However, the properties of effective factor(s) and the mechanism of this inhibitory action still remain to be investigated. Then, the effect of C. perfringens culture medium on human colon adenocarcinoma HT29 cells was examined. The exposure of HT29 cells to C. perfringens culture medium was found to suppress the proliferation of these cells probably through the reduction of immediate early gene egr-1 expression. These observations suggest that C. perfringens culture medium has a cytostatic action on colon tumor cells, which may be responsible for the prevention of pre-neoplastic formation in rat colon.


Characterization of cytotoxic actions of tricyclic antidepressants on human HT29 colon

Preclinical studies have suggested that the long-term use of antidepressants may result in the initiation and/or promotion of tumor in the gastrointestinal tract. However, a possible relationship between the use of antidepressants and the production of colon cancer has not yet been confirmed, and hence requires to be further investigated. To address this issue, the effects of antidepressants on the proliferation of colorectal tumor cells were examined using human HT29 colon carcinoma cells, and tricyclic antidepressant, such as imipramine, desipramine and amitriptyline, were shown to reduce the cell viability in a manner dependent on the time exposing to these drugs. In addition to these drugs, a selective serotonin reuptake inhibitor fluoxetine, but not a monoamine oxidase inhibitor tranylcypromine, caused the reduction of cell viability, similar in extent to that caused by imipramine. Further studies showed that desipramine caused the apoptotic cell death, which could be prevented by neither catalase, reduced-form glutathione (GSH), nor N-acetylcysteine (NAC), without accompanying the disruption of mitochondrial membrane potential within the cells and the release of cytochrome c into the cell cytoplasm. Moreover, desipramine caused the arrest of cell-cycle progression at either G0/G1-phase or G2/M-phase, which might be depending upon the drug concentration. Thus, these results suggest that tricyclic antidepressants may be cytotoxic, and induce the non-oxidative apoptotic death of human HT29 colon carcinoma cells probably through a non-mitochondrial pathway associated with the cell-cycle progression.


Possible involvement of 5a-reduced neurosteroids in adrenergic and serotonergic stimulation of GFAP gene expression in rat C6 glioma cells.

Influence of adrenergic and serotonergic stimulation on glial fibrillary acidic protein (GFAP) gene expression in rat C6 glioma cells was first examined as an in vitro model experiment for investigating the neuronal regulation of glial cell differentiation. Stimulation of these cells with isoproterenol and serotonin elevated GFAP mRNA levels followed by an increase in its protein contents, thus suggesting that both adrenergic and serotonergic stimulation might induce the differentiation of the glioma cells. In addition, progesterone and its 5alpha-reduced metabolite dihydroprogesterone also elevated GFAP mRNA levels in rat C6 glioma cells, consistent with their stimulatory actions on GFAP gene expression observed in rat astrocytes. Further studies showed that the elevation of GFAP mRNA levels induced by isoproterenol and serotonin as well as progesterone was abolished by pretreatment of the glioma cells with finasteride, an inhibitor of 5alpha-reduced steroid production. Moreover, the stimulatory actions of isoproterenol and serotonin on GFAP gene expression were inhibited by pretreatment with a GABA(A) receptor antagonist bicuculline and a progesterone receptor antagonist RU486. These findings suggest that both adrenergic and serotonergic stimulation may indirectly activate GFAP gene expression probably through the production of 5alpha-reduced steroid metabolites in rat C6 glioma cells, proposing the possibility that 5alpha-reduced neurosteroids may play a potential role in the neuronal regulation of glial cell differentiation.


Serotonergic 5-HT2A receptor stimulation induces steroid 5a-reductase gene expression in rat C6 glioma cells via transcription factor Egr-1

Selective serotonin reuptake inhibitors (SSRIs) are widely used for the treatment of depressive mood disorders and well known to inhibit the reuptake of neurotransmitter serotonin into nerve terminals. Thus, it seems conceivable that these drugs may induce the outflow of serotonin from the synapse as a consequence of inhibiting the reuptake, resulting in the stimulation of glial cells surrounding nerve terminals. On this hypothesis, the effect of serotonin on steroid 5alpha-reductase type 1 (5alpha-R) gene expression in rat C6 glioma cells was examined as one of the in vitro model experiments for investigating the indirect influence of SSRIs on glial cells. Serotonin elevated 5alpha-R mRNA and protein levels through the stimulation of serotonin 5-HT2A receptors, and also elevated Egr-1 mRNA and protein levels prior to 5alpha-R gene expression in the glioma cells. Furthermore, serotonin failed to significantly increase 5alpha-R mRNA levels in the cells preloaded with the antisense oligodeoxynucleotide targeted on Egr-1 gene. These results indicate that serotonin may stimulate 5alpha-R gene expression via transcription factor Egr-1 in glial cells, thus suggesting that serotonin flowing out of the serotonergic synapse may be implicated in SSRI-induced changes in neurosteroid metabolism in brain.

Brain Research. Molecular Brain Research


Down-regulation of immediate early gene egr-1 expression in rat C6 glioma cells by short-term exposure to high salt culture medium.

Influence of high salt culture conditions on the expression of immediate early gene egr-1 in rat C6 glioma cells was investigated by measuring both Egr-1 mRNA and protein levels in the cells exposed to the medium containing high concentrations of NaCl. The exposure to high salt medium reduced Egr-1 mRNA and protein levels, while Egr-1 mRNA levels were not altered by the medium containing either sucrose or glycerol. Veratridine and monensin also reduced Egr-1 mRNA levels, similar in extent to that induced by high salt medium. Imaging analysis indicated that the exposure to high salt medium induced the elevation of Na+ levels within the cells. These results indicate that neither hyperosmotic pressure nor ionic strength of high salt medium contribute to the reduction of Egr-1 expression, and suggest that the elevation of intracellular Na+ concentration is closely associated with the down-regulation of egr-1 gene expression.


Inhibitory effect of fluvastatin on ileal ulcer formation in rats induced by nonsteroidal antiinflammatory drug

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal damage as one of their side effects in humans and experimental animals. Lipid peroxidation plays an important role in NSAID-induced ulceration. The aim of this study was to investigate the inhibitory effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on the ulceration in small intestines of rats. The effects of three HMG-CoA reductase inhibitors, fluvastatin, pravastatin and atorvastatin on ileal ulcer formation in 5-bromo-2-(4-fluorophenyl)-3-(4- methylsulfonylphenyl) thiophene (BFMeT)-treated rats were examined. Antioxidative activity of the inhibitors was measured by a redox-linked colorimetric method. Fluvastatin, which was reported to have antioxidative activity, repressed the ileal ulcer formation in rats treated with BFMeT an NSAIDs. However, the other HMG-CoA reductase inhibitors (pravastatin and atorvastatin) did not repress the ileal ulcer formation. Among these HMG-CoA reductase inhibitors, fluvastatin showed a significantly stronger reducing power than the others (pravastatin, atorvastatin). Fluvastatin having the antioxidaitive activity suppresses ulcer formation in rats induced by NSAIDs.


Simultaneous detection of Bacteroides fragilis group species by leuB-directed PCR

Bacteroides species, saccharolytic Gram-negative obligate anaerobes, are frequently isolated from human infections such as peritonitis, abscesses and bacteremia. Among the species in the genus Bacteroides, the species called "B. fragilis group" are particularly involved in human infections and are medically important because they account for a major part of anaerobic isolates from clinical specimens. The purpose of this study was to develop PCR primers that specifically and simultaneously amplify the beta -isopropylmalate dehydrogenase gene leuB in B. fragilis group species. We determined partial nucleotide sequences of leuB genes and compared them in seventeen strains of nine B. fragilis group species, and the regions that are conserved among Bacteroides strains but different from other species were used as a B. fragilis group-specific PCR primer set, BacLBF-BacLBR. Specificity tests of the primer set using 52 phenotypically characterized strains and 75 isolates from rat feces showed only one case each of false-positive and false-negative. The detection limit of the leuB-directed PCR using BacLBF and BacLBR was 3.9 x 10(3) colony-forming units. These results indicate that leuB amplification using BacLBF andBacLBR is a useful tool for rapid diagnosis of Bacteriodes infection and for rapid differential diagnosis of anaerobic infections.

The Journal of Medical Investigation : JMI


Inhibitory effects of asiatic acid and CPT-11 on growth of HT-29 cells


High salt culture conditions suppress proliferation of rat C6 glioma cells by arresting cell-cycle progression at S-phase

As one of the in vitro model experiments for investigating a possible effect of extracellular environmental stresses on glial cell proliferation, the influence of high salt culture conditions on the growth of rat C6 glioma cells was examined. Exposure to the culture medium containing high concentrations of NaCl reduced the number of viable cells in a concentration-dependent manner without any significant change in their viability. In contrast, proliferation of these cells was not substantially altered by culturing them in hyperosmotic medium containing either sucrose or glycerol, both of which were osmotically almost equivalent to high salt culture medium. On the other hand, expression of the egr-1 gene, an immediate early gene related to the proliferation and differentiation of various cells, was enhanced by culturing glioma cells in high salt medium while the reduction of glial fibrillary acidic protein content, an index of glial cell differentiation, was observed under the same culture conditions. Further studies on the relationship between egr-1 gene expression and the cell cycle showed that cell-cycle progression was arrested at S-phase by culturing glioma cells in high salt medium. Moreover, both resveratrol and CPT-11, which are known to arrest cell-cycle progression, elevated egr-1 mRNA levels in glioma cells. Thus, these observations suggest that high salt culture conditions might suppress the proliferation of rat C6 glioma cells as a consequence of arresting cell-cycle progression at S-phase, resulting secondarily in the compensatory enhancement of egr-1 gene expression.


Inhibitory effects of Centella asiatica on azoxymethane-induced aberrant crypt focus formation and carcinogenesis in the intestines of F344 rats

Effects of the water extract of Centella asiatica Linn. on formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and intestinal tumorigenesis in male F344 rats were investigated. Treatment with the extract significantly decreased the number of larger ACF (with four or more crypts per focus) in the large intestine in the early stage, while the number of methylated DNA adducts was not decreased compared with that in the AOM-treated group. In the post-initiation stage, the extract significantly decreased the total number of ACF and the number of larger ACF, accompanied by a decrease in the 5-bromo-2'-deoxyuridine-labeling index and an increase in the induction of apoptotic cells in the colonic mucosa. The incidences of neoplasms, the numbers of adenocarcinomas in the small intestines and entire intestines, and sizes of neoplasms in the entire intestines in rats fed C. asiatica extract at a dose of 10 mg/kg were smaller than those in rats given AOM alone (p < 0.05). The extract at a dose of 100 mg/kg significantly reduced the multiplicity of neoplasms in the small intestine (p < 0.05). These results suggest that inhibition of the formation of AOM-induced ACF by C. asiatica extract is associated with modification of cell proliferation and induction of apoptosis in colonic crypts and that the extract has a chemopreventive effect on colon tumorigenesis.


Nerve growth factor induces elevation of steroid 5alpha-reductase mRNA levels in rat C6 glioma cells through expression of transcription factor Egr-1

Steroid 5alpha-reductase type 1 (5alpha-R), the enzyme converting progesterone and other steroid hormones to their 5alpha-reduced metabolites, has been shown to be localized in both neuronal and glial cells, and this enzyme in glial cells has previously been reported to be activated either by co-culturing with neuronal cells or by adding the conditioned medium of neuronal cells, thus suggesting that neuronal activity may be implicated in the regulation of neurosteroid metabolism in brain. In the present study, to investigate a potential role of neurotrophic factors in the mechanism regulating the production of neuroactive 5alpha-reduced steroid metabolites, the direct action of NGF on 5alpha-R gene expression was examined by measuring the steady-state levels of 5alpha-R mRNA levels in rat C6 glioma cells. Exposure of the glioma cells to NGF increased both 5alpha-R mRNA and its protein levels, and induced the transient elevation of Egr-1 mRNA levels prior to the expression of 5alpha-R mRNA in the cells. Furthermore, NGF failed to induce any significant elevation of 5alpha-R mRNA levels in the cells pretreated with Egr-1 antisense oligodeoxynucleotides. These findings indicate that NGF induces the elevation of 5alpha-R gene expression in the glioma cells through the expression of transcription factor Egr-1, proposing the possibility that NGF, and probably other neurotrophic factors as well, may play a potential role in the regulation of 5alpha-reduced steroid production as one of the factors mediating the intercellular communication between neuronal and glial cells in the brain.


Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice

We examined the antitumour effect of a combination of betulinic acid (BA) and vincristine (VCR) on murine melanoma B16F10 cells in vitro and in vivo. Betulinic acid, a pentacyclic triterpene, showed a synergistic cytotoxic effect on melanoma cells by combinational use of VCR. Betulinic acid and VCR induced cell cycle arrest at different points (BA at G1 phase and VCR at G2/M phase) and caused apoptosis in B16F10 melanoma cells. In the in vivo study, VCR inhibited metastasis of tumour cells to the lung. The addition of BA to VCR augmented suppression of the experimental lung metastasis of melanoma cells in C57BL/6 mice. The number of lung nodules of more than 1 mm in diameter in mice treated with BA and VCR was less than that in mice treated with VCR alone. These results suggest that BA is an effective supplement for enhancing the chemotherapeutic effect on malignant melanoma.


Role of unbalanced growth of Gram-negative bacteria in ileal ulcer formation in rats treated with a nonsteroidal anti-inflammatory drug

Nonsteroidal anti-inflammatory drugs (NSAIDs) induced formation of intestinal ulcers as side effects, in which an unbalanced increase in the number of gram-negative bacteria in the small intestine plays an important role. To clarify how intestinal microflora are influenced by NSAIDs, we examined the effects of 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl) thiophene (BFMeT), an NSAID, on intestinal motility and on the growth of Escherichia coli and Lactobacillus acidophilus. Transit index, a marker of peristalsis, was not different in BFMeT-treated and solvent-treated rats, indicating that BFMeT increased the number of gram-negative bacteria without suppression of peristalsis. The factors that affect the growth of intestinal bacteria were not found in intestinal contents of BFMeT-treated rats, because the growth of E. coli and that of L. acidophilus in the supernatants of small intestinal contents of BFMeT-treated rats and solvent-treated rats were not different. The mechanism of the increase in the number of gram-negative bacteria is still unclear, but heat-killed E. coli cells and their purified lipopolysaccharide (LPS) caused deterioration of BFMeT-induced ileal ulcers, while they could not cause the ulcers by themselves without the NSAID. Concentration of LPS and myeloperoxidase activity level were elevated correlatively in the intestinal mucosa of rats treated with LPS and BFMeT. These results suggest that an increase in the number of gram-negative bacteria and their LPS in the mucosa induces activation of neutrophils together with the help of NSAID action and causes ulcer formation.


Adrenergic activation of steroid 5a-reductase gene expression in rat C6 glioma cells: Involvement of cyclic AMP/protein kinase A-mediated signaling pathway

Steroid 5alpha-reductase (5alpha-R) is well known as the enzyme converting progesterone and other steroid hormones to their 5alpha-reduced metabolites and has been reported to be localized in both neuronal and glial cells in the brain. Previously, the enzyme activity in glial cells has been shown to be enhanced either by coculturing with neuronal cells or by adding the conditioned medium of neuronal cells, suggesting a possible implication of neuro-glial interactions in the regulation of neurosteroid metabolism in the brain. In the present studies, the effects of adrenergic agonists on 5alpha-R mRNA and protein levels in rat C6 glioma cells were examined as one of the model experiments for investigating the influence of neuronal activity on the expression of 5alpha-R gene in the glial cell. The direct challenge of beta-adrenergic agonists to glioma cells resulted in the rapid and transient elevation of 5alpha-R mRNA levels through the activation of the cyclic AMP (cAMP)/protein kinase A-mediated signaling pathway. Further studies showed that cAMP-induced 5alpha-R mRNA expression was completely abolished by pretreatment of cells with actinomycin D and also indicated that the elevation of 5alpha-R mRNA levels was accompanied by an increase in enzyme protein in the cells. These findings provide strong evidence that the stimulation of beta-adrenergic receptors might induce the transcriptional activation of 5alpha-R gene expression in glial cells, proposing the possibility that neuronal activity might be involved in the production of neuroactive 5alpha-reduced steroids in the brain.


In vitro cytotoxicity of 4-methylcatechol in murine tumor cells: Induction of apoptotic cell death by extracellular pro-oxidant action

Assessment of the in vitro cytotoxicity has recently been become popular as a primary screening method for evaluating the antitumor activities of various chemicals and natural substances. For example, quercetin and related phenolic compounds, present in teas, wines, and other plant products, have been shown to cause their cytotoxic effects on tumor cells in culture, proposing their protective effects against the development of cancer. However, 4-methylcatechol, a metabolite produced in the intestinal tract after ingestion, has been shown to cause the promotion rather than suppression of tumor in rat stomach despite its in vitro cytotoxic activity. To address the inconsistency between its in vivo and in vitro actions, the effect of 4-methylcatechol on the viabilities of murine tumor cells was examined, and 4-methylcatechol was shown to reduce their viabilities through the induction of apoptosis. In addition, since catechol compounds have been shown to have a complex mixture of pro-oxidant and antioxidant actions in the in vitro assay systems, the cytotoxic activity of 4-methylcatechol was reassessed in the presence of either catalase or reduced-form glutathione, and both of them were shown to protect the cells against the damage induced by 4-methylcatechol. Moreover, the generation of hydrogen peroxide was observed by incubating the drug in the growth medium with or without the cells. These findings indicate that, similar to other catechol compounds, 4-methylcatechol may induce the apoptotic death of murine tumor cells through its extracellular pro-oxidant action on the cells.

The Journal of Pharmacology and Experimental Therapeutics


Antimutagenicity of Murdannia loriformis in the Salmonella mutation assay and its inhibitory effects on azoxymethane-induced DNA methylation and aberrant crypt focus formation in male F344 rats

An 80% ethanol extract of Murdannia loriformis, a Thai medicinal plant, was examined for antimutagenic activity and cancer chemopreventive activity. In the Salmonella mutation assay, the extract showed antimutagenicity against 2-amino-3-methylimidazo [4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, 2-aminodipyrido[1,2-a:3',2'-d]imidazole, 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol acetate and reduced their mutagenicities to 31.4-67.9% at the dose of 10 mg/plate. However, it did not inhibit the mutagenicities of 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9 H-pyrido[2,3-b]indole, benzo[a]pyrene,N-ethyl-N'-nitro-N-nitrosoguanidine and 1-nitropyrene. The extract itself showed no mutagenicity. The chemopreventive activity of M. loriformis was examined using azoxymethane (AOM)-induced aberrant crypt focus (ACF) formation in the colon of F344 rats. The extract at doses of 0.1-1.0 g/kg wt significantly inhibited ACF formation in the initiation stage (21-51%), although it was more effective at a lower dose. In the post-initiation stage, the extract also tended to inhibit ACF formation (12-27%) and significantly decreased the number of larger ACFs that have more than 3 aberrant crypts per focus. The extract inhibited the formation of O6-methylguanine and N7-methylguanine in the colonic mucosa and muscular layers but not or increased in the liver. These results indicate that M. loriformis extract has antimutagenic activity toward various known mutagens and that it inhibits AOM-induced ACF formation both in the initiation and post-initiation stages in the rat colon.

The Journal of Medical Investigation : JMI


Inhibitory effects of bitter melon (Momordica charantia Linn.) on bacterial mutagenesis and aberrant crypt focus formation in the rat colon

Antimutagenicity and chemopreventive activity of an 80%-ethanol extract of bitter melon (Momordica charantia Linn.) against the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) was investigated. The bitter melon extract was nonmutagenic and inhibited the mutagenicity of heterocyclic amines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and aflatoxin B1 in the Salmonella mutation assay. To examine the inhibitory effect of bitter melon on AOM-induced ACF formation, male F344 rats were fed various concentrations of the extract (0.1, 0.5, and 1.0 g/kg body weight) for five weeks during the initiation stage. One week after the administration of the plant extract, rats were subcutaneously given AOM at 15 mg/kg body weight once a week for two weeks. Three rats in each group were sacrificed 12 hr after the second AOM injection to analyze DNA adducts, O6-methylguanine (O6-meG) and N7-methylguanine in the liver and colon. The remaining rats were sacrificed 3 weeks after the second AOM injection to observe ACF. To examine the inhibitory effect of the extract on ACF formation in the postinitiation stage, rats were fed the extract at 0.1 and 1.0 g/kg body weight for 12 weeks starting two weeks after the second AOM injection. Treatment with bitter melon extract significantly inhibited ACF formation in the colon during the initiation stage and dose-dependently decreased the average of O6-meG DNA adduct in the colonic mucosa. During the postinitiation stage, bitter melon extract, at 1.0 g/kg body weight, significantly inhibited ACF formation in the colon, especially the formation of ACF with four or more crypts per focus. These findings suggest that bitter melon is a possible chemopreventive agent against colon carcinogenesis.


PCR-dot blot hybridization based on the neuraminidase-encoding gene is useful for detection of Bacteroides fragilis

Bacteroides fragilis is a Gram-negative obligate anaerobe frequently isolated from clinical specimens and sometimes causes severe septicemia in compromised hosts. Increasing interest has been shown in the enterotoxigenicity and drug resistance of B. fragilis in the field of medical microbiology. We previously reported rapid detection of this anaerobe by nested PCR targeting a neuraminidase-encoding gene nanH. In the present study, we synthesized a digoxigenin-labeled oligonucleotide probe, NH1, which is specific for nanH of B. fragilis, and we combined the hybridization assay using NH1 with the nanH-PCR to detect this anaerobe in a bacteremia model mice. In the specificity test, the oligonucleotide probe, NH1, hybridized only to amplification products from B. fragilis. PCR-dot blot hybridization based on nanH enabled detection of cells of B. fragilis in blood samples even when the number was as low as 2 x 10(3) colony-forming units/ml. These findings suggest that PCR-dot blot hybridization targeting nanH is a useful procedure for diagnosis of septicemia caused by B. fragilis when viable cells in blood cannot be detected by the traditional culture techniques.


Expression of Human Lactoferrin in Bacteroides uniformis and its Effect on Azoxymethane-induced Aberrant Crypt Focus Formation in the Rat Colon


Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.


Effects of β-Glucuronidase-Deficient and Lycopene-Producing Escherichia coli Strains on Formation of Azoxymethane-Induced Aberrant Crypt Foci in the Rat Colon

Biochemical and Biophysical Research Communications


Effects of roselle (Hibiscus sabdariffa Linn.), a Thai medicinal plant, on the mutagenicity of various known mutagens in Salmonella typhimurium and on formation of aberrant crypt foci induced by the colon carcinogens azoxymethane and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in F344 rats

The 80% ethanol extract of roselle (Hibiscus sabdariffa Linn.), a medicinal plant in Thailand, was examined for antimutagenic and chemopreventive activity in a colon carcinogenesis model. It reduced about 60-90% of the mutagenicity induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and other heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MelQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline(MelQx),3-amino-1,4-dimet hyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2),2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1),2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), at a concentration of 12.5 mg/plate in the Salmonella mutation assay. The extract showed no mutagenicity and no antibacterial activity below this dose. Mutagenicity of methylazoxymethanol (MAM) acetate, which, like PhIP, is a colon carcinogen,was also efficiently inhibited by the roselle extract. To investigate chemoprevention by roselle in a colon carcinogenesis model, we examined the inhibitory effects of the roselle extract in F344 rats in which aberrant crypt focus (ACF) formation was induced by azoxymethane (AOM) and PhIP. In the initiation stage, the number of AOM-induced ACF in the colon was significantly decreased by roselle (17-25%) compared with that in rats treated with AOM alone. The amount of O6-methylguanine in the colonic mucosa tended to be decreased in the roselle-treated rats. The number of PhIP-induced ACF was also significantly decreased by roselle treatment (22%) at a concentration of 1.0 g/kg body weight in the initiation stage. However, in the post-initiation stage of AOM-induced ACF formation, roselle increased the number of ACF, especially the number of foci which had more than three crypts/focus. These results indicate that roselle has antimutagenic activity against MAM acetate and heterocyclic amines and that it decreases the number of AOM- and PhIP-induced ACF in the initiation stage, although it rather increased the number of ACF in the post-initiation stage.


Activation of 1-nitropyrene by nitroreductase increases the DNA adduct level and mutagenicity

1-Nitropyrene (1-NP) is a mutagenic nitro compound in the environment. We studied correlations between the mutagenicity of 1-NP for three strains of Salmonella typhimurium, the activity of bacterial nitroreductases and the amount of 1-NP-derived DNA adducts. Bacterial strains used in this study were S. typhimurium strains TA98, nitroreductase-less mutant TA98NR and YG1021 carrying a nitroreductase-producing plasmid. The mutagenicity of 1-NP was measured using the Ames assay, and the nitroreductase activities of these strains were assayed by quantification of 1-aminopyrene produced from 1-NP. The DNA adducts were measured by the 32P-postlabeling method. Among the three bacterial strains, strain YG1021 was the highest in mutagenicity of 1-NP, the nitroreductase activity and the DNA adduct level. However, S. typhimurium strain TA98NR had the lowest values of these three parameters. Nitroreductase activity, DNA adduct level and mutagenicity were strongly correlated with each other. These results indicate that bacterial nitroreductase plays an important role in forming the DNA adducts, and that the higher the adduct level the higher the level of mutagenicity.


Effect of Intestinal Bacteria on Formation of Azoxymethane-Induced Aberrant Crypt Foci in the Rat Colon

The effect of intestinal bacteria on formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and DNA adducts in the rat colon was investigated. Male Sprague-Dawley rats were administered cultures of Lactobacillus acidophilus, Bifidobacterium adolescentis, Bacteroides fragilis, Escherichia coli and Clostridium perfringens for five weeks and given injections of AOM at 15 mg/kg body weight at the first and second weeks. The number of ACF five weeks after the start of the experiment was decreased in the rats treated with the cultures or culture supernatants of L. acidophilus and C. perfringens. The half-life of O6-methylguanine (O6-meG) in the L. acidophilus group was shorter than that in the GAM broth group. The half-life of 7-methylguanine did not differ among the groups. These results suggest that the metabolite(s) of L. acidophilus and C. perfringens inhibit(s) the ACF formation in rats treated with AOM and that the inhibitory effect of L. acidophilus is due to the enhanced removal of O6-meG from the colon mucosal DNA.


Inhibitory effects of lemon grass (Cymbopogon citratus Stapf) on formation of azoxymethane-induced DNA adducts and aberrant crypt foci in the rat colon


Branched chain amino acid suppressed insulin-initiated proliferation of human cancer cells through induction of autophagy.

Branched chain amino acid (BCAA) dietary supplementation inhibits activation of the insulin-like growth factor (IGF)/IGF-I receptor (IGF-IR) axis in diabetic animal models. However, the in vitro effect of BCAA on human cancer cell lines under hyper-insulinemic conditions remains unclear. Colon (HCT-116) and hepatic (HepG2) tumor cells were treated with varying concentrations of BCAA with or without fluorouracil (5-FU). The effect of BCAA on insulin-initiated proliferation was determined. Gene and protein expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. BCAA supplementation had no significant effect on cell proliferation and did not show significant synergistic or antagonistic effects with 5-FU. However, BCAA significantly decreased insulin-initiated proliferation of human colon and hepatic cancer cell lines in vitro. BCAA supplementation caused a marked decrease in activated IGF-IR expression and significantly enhanced both mRNA and protein expression of LC3-II and BECN1 (BECLIN-1). BCAA could be a useful chemopreventive modality for cancer in hyperinsulinemic conditions.


日本臨床腸内微生物学会誌

日本臨床腸内微生物学会誌