Structure-activity relationship studies of pyrogallol as a calcineurin/NFAT signaling suppressor.

Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC50 value of 82.6 µM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.

Resveratrol can induce differentiating phenotypes in canine oral mucosal melanoma cells.

This report described the differentiation induction of canine oral mucosal melanoma (OMM) cells by resveratrol. Exposure of canine OMM cells to resveratrol (maximum dose: 50 µM and treatment period: 72 hr) induced differentiating features like melanocytes, and enhanced chemosensitivity against cisplatin, but alone had no influence on cell viability. Additionally, resveratrol significantly enhanced mRNA expression of key melanoma differentiation markers such as microphthalmia-associated transcription factor (MITF). Of several inhibitors against mitogen-activated protein kinase subtypes, only the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, induced melanocyte-like morphological change and enhanced MITF mRNA expression. Furthermore, resveratrol also suppressed JNK activation in OMM cells by approximately 33%. Overall, these findings suggest that resveratrol induces differentiation in canine OMM cells, due to the inhibition of JNK signaling.

Dietary Oyster (Crassostrea gigas) Extract Ameliorates Dextran Sulfate Sodium-Induced Chronic Experimental Colitis by Improving the Composition of Gut Microbiota in Mice.

Previously, we have reported that the intake of oyster extract (OE), prepared from Pacific oysters (Crassostrea gigas), can attenuate symptoms of dextran sulfate sodium (DSS)-induced acute experimental colitis in mice. Herein, we aimed to evaluate whether OE intake ameliorates chronic experimental colitis induced by repeated DSS administration in mice. Male C57BL/6J (4-week-old) mice were fed either the standard diet AIN93G (control diet) or the control diet containing 5.0% (w/w) OE (OE diet). After 21 days of diet feeding, chronic experimental colitis was induced by three cycles of 2.0% (w/w) DSS solution administration (5 days), followed by distilled water (5 days). Mice fed OE alleviated the shortened colonic length, increased the relative weight of the spleen, colonic histopathological score (regeneration), and blood in the stool score compared with mice fed control diet. A tendency to improve the α-diversity of fecal microbiota, which was exacerbated by colitis, was observed in mice fed OE. Correlation analysis suggested that the anti-colitis effect of OE intake could be related to the valeric acid content and relative abundances of Ruminococcus and Enterococcus in the feces. In conclusion, OE could ameliorate DSS-induced chronic experimental colitis by improving the gut environment, including the microbiota community and SCFA composition.

A mutation in chitin synthase I associated with etoxazole resistance in the citrus red mite Panonychus citri (Acari: Tetranychidae) and its uneven geographical distribution in Japan.

BACKGROUND: High-levels of etoxazole resistance have not yet been frequently reported in Panonychus citri. Although a highly resistant strain was discovered in 2014, etoxazole resistance has not become a significant problem in areas of citrus production in Japan. A target site mutation in chitin synthase 1 (CHS1), I1017F, is a major etoxazole-resistance factor in Tetranychus urticae. To investigate the mechanisms of etoxazole resistance and the dispersal of resistance genes, we analyzed target-site mutations in a highly resistant strain and their geographical distribution in Japan. RESULTS: High-level etoxazole resistance was completely recessive. The I1017F mutation was detected in CHS1 of the highly resistant strain, and its frequency was correlated with the hatchability of eggs treated with etoxazole. Sequencing and variant frequency analyses of local populations by quantitative polymerase chain reaction revealed that I1017F is restricted to the Ariake Sea area of Kyushu Island. Although a new nonsynonymous substitution, S1016L, accompanied by I1017F was found in CHS1 of the highly resistant strain, CRISPR/Cas9 engineering of flies showed that S1016L had no effect on the etoxazole resistance conferred by I1017F. CONCLUSION: I1017F is a major target site mutation that confers high-level etoxazole resistance on P. citri. Dispersion of I1017F possibly was suppressed as a result of the completely recessive inheritance of resistance together with low gene flow between local populations. © 2022 Society of Chemical Industry.

A Redox-Silent Analogue of Tocotrienol May Break the Homeostasis of Proteasomes in Human Malignant Mesothelioma Cells by Inhibiting STAT3 and NRF1.

6-O-Carboxypropyl-alpha-tocotrienol (α-T3E) is a multi-target redox-silent analogue of tocotrienol that exhibits cytotoxicity against many cancer cells, including malignant mesothelioma (MM) cells. α-T3E has several molecular targets to effectively induce cytotoxicity against MM cells; however, the mechanisms underlying this cytotoxicity remain unclear. In the present study, we demonstrated that the α-T3E-dependent disruption of the homeostasis of proteasomes strongly induced endoplasmic reticulum (ER) stress, which resulted in effective cytotoxicity against MM cells. The α-T3E-dependent disruption of the homeostasis of proteasomes depended on decreases in proteasome subunits via the inactivation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2 related factor-1 (NRF1), which inhibited protease activity, such as chymotrypsin-like activity, in proteasomes. The α-T3E-dependent inhibition of this activity also induced severe ER stress and ultimately resulted in effective cytotoxicity against MM cells with chemoresistance. The present results indicate that α-T3E acts as an effective anti-mesothelioma agent by disrupting the homeostasis of proteasomes through the simultaneous inactivation of STAT3 and NRF1.

Oyster (Crassostrea gigas) Extract Attenuates Dextran Sulfate Sodium-Induced Acute Experimental Colitis by Improving Gut Microbiota and Short-Chain Fatty Acids Compositions in Mice.

Drugs for inflammatory bowel diseases can be associated with serious side effects, and the development of alternative candidate resources derived from natural products has attracted considerable attention. Oyster extract (OE) derived from Crassostrea gigas contains glycogen, taurine, and amino acids, and has been assigned diverse health-promoting properties. This study investigated the anti-colitis effect of OE intake on fecal microbiota and its metabolites of acute experimental colitis mouse model induced by dextran sulfate sodium (DSS). C57BL/6J mice (male) were divided into three groups: (1) American Institute of Nutrition (AIN) 93G diet + DSS-untreated, (2) AIN93G diet + DSS-treated, and (3) 5% OE diet + DSS-treated. Mice were fed each diet for 21 days, and then administered 2.5% DSS solution to induce acute colitis for 7 days. In DSS-induced colitis mice, OE decreased body weight loss and increased disease activity index during the DSS-induced period. In addition, OE tended to decrease the colon length shortening and the relative spleen weight and alleviated colonic tissue damage. Moreover, OE improved fecal short-chain fatty acids compositions and altered the structure of fecal microbiota. These results provide insight into the health-promoting property of OE in alleviating DSS-induced acute colitis, providing a basis for the development and use of functional foods.

Effect of lactose hydrolysis on the milk-fermenting properties of Lactobacillus delbrueckii ssp. bulgaricus 2038 and Streptococcus thermophilus 1131.

Yogurt is a well-known nutritious and probiotic food and is traditionally fermented from milk using the symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, yogurt consumption may cause health problems in lactose-intolerant individuals, and the demand for lactose-free yogurt has been increasing. The standard method to prepare lactose-free yogurt is to hydrolyze milk by lactase; however, this process has been reported to influence the fermentation properties of starter strains. This study aimed to investigate the fermentation properties of an industrial starter culture of L. bulgaricus 2038 and S. thermophilus 1131 in lactose-hydrolyzed milk and to examine the metabolic changes induced by glucose utilization. We found that the cell number of L. bulgaricus 2038, exopolysaccharide concentration, and viscosity in the coculture of L. bulgaricus 2038 and S. thermophilus 1131 was significantly increased in lactose-hydrolyzed milk compared with that in unhydrolyzed milk. Although the cell number of S. thermophilus 1131 showed no difference, production of formic acid and reduction of dissolved oxygen were enhanced in lactose-hydrolyzed milk. Further, in lactose-hydrolyzed milk, S. thermophilus 1131 was found to have increased the expression of NADH oxidase, which is responsible for oxygen reduction. These results indicated that glucose utilization promoted S. thermophilus 1131 to rapidly reduce the dissolved oxygen amount and produce a high concentration of formic acid, presumably resulting in the increased cell number of L. bulgaricus 2038 in the coculture. Our study provides basic information on the metabolic changes in starter strains in lactose-hydrolyzed milk, and demonstrates that lactose-free yogurt with increased cell number of L. bulgaricus can be prepared without delay in fermentation and decrease in the cell number of S. thermophilus.

Isolation and characterization of Streptococcus thermophilus possessing prtS gene from raw milk in Japan.

Streptococcus thermophilus is widely used for producing fermented dairy products such as yogurt and cheese. Some S. thermophilus strains possessing the cell-wall protease PrtS show high proteolytic activity and fast acidification properties, which are very useful in industrial starters. However, few S. thermophilus strains possessing the prtS gene have been isolated from the environment. To clarify whether or not S. thermophilus strains possessing the prtS gene are present in Japan, we isolated S. thermophilus from raw milk collected in Japan from 2011 to 2017 and investigated the strains for the presence of prtS by PCR. A total of 172 S. thermophilus strains were isolated, and 59 strains were confirmed to possess prtS. We measured fermentation times of 59 prtS-positive strains in skim milk broth and found that 53 strains showed fast acidification properties, finishing fermentation within 10 hr. However, the remaining 6 prtS-positive strains showed slow acidification properties, and they had several amino acid mutations in PrtS compared with fast acidifying S. thermophilus LMD-9 and 4F44. These results demonstrate that S. thermophilus strains possessing prtS are prevalent in Japan and that some prtS-positive strains could lose their fast acidifying properties through mutations in PrtS.

Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model.

Chronic inflammation is involved in the development of colon cancer by inducing mutations and aberrant DNA methylation in colon epithelial cells. Furthermore, there is growing evidence that colonic microbiota modulates the inflammation response in the host and influences colon tumorigenesis. However, the influence of colonic microbiota on aberrant DNA methylation remains unknown. Here, we show the effect of colonic microbes on DNA methylation and tumorigenicity using a mouse model of human ulcerative colitis. Mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS) showed an increase in degree of colitis, as estimated by body weight, occult blood, and stool consistency/diarrhea at 2 weeks after treatment, but treatment with antibiotics markedly reduced the severity of the colitis. Although mucosal hyperplasia and increased inflammation-related genes were observed in the colonic epithelial cells of the AOM/DSS-treated mice, treatment with antibiotics abrogated these changes. In addition, treatment with antibiotics significantly decreased the number of mucosal nodules from 5.9 ± 5.3 to 0.2 ± 0.6 (P < .01) and area of occupancy from 50.1 ± 57.4 to 0.5 ± 1.4 mm2 (P < .01). Aberrant DNA methylation of three marker CpG islands (Cbln4, Fosb, and Msx1) was induced by AOM/DSS treatment in colonic mucosae, but this increase was suppressed by 50%-92% (P < .05) with antibiotic treatment. Microbiome analysis showed that this change was associated with a decrease of the Clostridium leptum subgroup. These data indicate that antibiotics suppressed tumorigenesis through inhibition of aberrant DNA methylation induced by chronic inflammation.

Kososan, a Kampo medicine, prevents a social avoidance behavior and attenuates neuroinflammation in socially defeated mice.

BACKGROUND: Kososan, a Kampo (traditional Japanese herbal) medicine, has been used for the therapy of depressive mood in humans. However, evidence for the antidepressant efficacy of kososan and potential mechanisms are lacking. Recently, it has been recognized that stress triggers neuroinflammation and suppresses adult neurogenesis, leading to depression and anxiety. Here, we examined whether kososan extract affected social behavior in mice exposed to chronic social defeat stress (CSDS), an animal model of prolonged psychosocial stress, and neuroinflammation induced by CSDS. METHODS: In the CSDS paradigm, C57BL/6J mice were exposed to 10 min of social defeat stress from an aggressive CD-1 mouse for 10 consecutive days (days 1-10). Kososan extract (1.0 g/kg) was administered orally once daily for 12 days (days 1-12). On day 11, the social avoidance test was performed to examine depressive- and anxious-like behaviors. To characterize the impacts of kososan on neuroinflammation and adult neurogenesis, immunochemical analyses and ex vivo microglial stimulation assay with lipopolysaccharide (LPS) were performed on days 13-15. RESULTS: Oral administration of kososan extract alleviated social avoidance, depression- and anxiety-like behaviors, caused by CSDS exposure. CSDS exposure resulted in neuroinflammation, as indicated by the increased accumulation of microglia, the resident immune cells of the brain, and their activation in the hippocampus, which was reversed to normal levels by treatment with kososan extract. Additionally, in ex vivo studies, CSDS exposure potentiated the microglial pro-inflammatory response to a subsequent LPS challenge, an effect that was also blunted by kososan extract treatment. Indeed, the modulatory effect of kososan extract on neuroinflammation appears to be due to a hippocampal increase in an anti-inflammatory phenotype of microglia while sparing an increased pro-inflammatory phenotype of microglia caused by CSDS. Moreover, reduced adult hippocampal neurogenesis in defeated mice was recovered by kososan extract treatment. CONCLUSIONS: Our findings suggest that kososan extract prevents a social avoidant behavior in socially defeated mice that is partially mediated by the downregulation of hippocampal neuroinflammation, presumably by the relative increased anti-inflammatory microglia and regulation of adult hippocampal neurogenesis. Our present study also provides novel evidence for the beneficial effects of kososan on depression/anxiety and the possible underlying mechanisms.

Resistant starch promotes equol production and inhibits tibial bone loss in ovariectomized mice treated with daidzein.

Daidzein is metabolized to equol in the gastrointestinal tract by gut microflora. Equol has greater estrogenic activity than genistein and daidzein, with its production shown to be promoted by dietary fiber. It is known that resistant starch (RS) is not absorbed in the proximal intestine and acts as dietary fiber in the colon. In this study, we investigated the combined effects of daidzein and RS intake on equol production, bone mineral density, and intestinal microflora in ovariectomized (OVX) mice. Female mice of the ddY strain, aged 8 weeks, were either sham operated (n = 6) or OVX. The OVX mice were randomly divided into 5 groups: OVX control (n = 6), OVX fed 0.1% daidzein-supplemented diet (OVX + Dz, n = 8), OVX fed 0.1% daidzein- and 12% RS-supplemented diet (OVX + Dz + RS, n = 8), OVX fed 12% RS-supplemented diet (OVX + RS, n = 8), and OVX who received daily subcutaneous administration of 17 ß-estradiol (n = 6). After 6 weeks, urinary equol concentration was significantly higher in the OVX + Dz + RS group than in the OVX + Dz group. The bone mineral density of the whole tibia was higher in the OVX + Dz +RS group compared with the OVX + Dz group. The occupation ratios of Bifidobacterium spp in the cecal microflora in groups fed RS were significantly higher than those in the other groups. The present study demonstrated that RS may increase the bioavailability of daidzein.

Connexin 32 potentiates vinblastine-induced cytotoxicity in renal cell carcinoma cells.

We have reported that connexin (Cx) 32 gene, a member of gap junction protein family, acts as a tumor suppressor gene in human renal cell carcinoma (RCC). Of solid tumors, RCC is one of the most chemoresistant cancers, and there is no effective cancer chemotherapy against RCC at present. In this study, we examined if the combination of Cx32-dependent tumor-suppressive effect and vinblastine (VBL), a chemotherapeutic agent which has been utilized for clinical RCC treatment, could be effective in enhancing the sensitivity of RCC to VBL treatment. Cx32 expression in a human metastatic RCC cell (Caki-1 cell) significantly enhanced in vitro and in vivo VBL-induced cytotoxicity on the cell. Cx32 expression in the RCC cells potentiated VBL-induced apoptosis compared to the Cx32-negative RCC cells in vitro as well as in vivo. The enhancing apoptosis in the RCC cells by Cx32 mainly depended on the decrease of P-glycoprotein (P-gp), a multidrug resistance gene-1 (MDR-1) product responsible for reduction of VBL accumulation into the cells. We also observed that silencing of Cx32 by short interfering RNA (siRNA) treatment elevated the level of P-gp in Caki-1 cells and that inhibition of P-gp function enhanced VBL-induced apoptosis in the RCC cells. These results suggest that Cx32 is effective to enhance VBL-induced cytotoxicity in Caki-1 cells via the reduction of P-gp. Overall, it seems that the combination of Cx32-dependent tumor-suppressive effect and VBL is promising as a new cancer therapy against RCC.

Soybean isoflavones preserve bone mass in hindlimb-unloaded mice.

It is well known that skeletal unloading induces bone loss. It has been shown that soybean isoflavones are effective in preventing bone loss in osteoporotic animals. We investigated the ability of isoflavones to protect bone loss induced by hindlimb unloading by using a tail-suspension mouse model. Eight-week-old female mice were divided into five groups: (1) normal housed group (Normal), (2) sham unloaded group fed a control diet (Sham-UL), (3) hindlimb unloaded group fed a control diet (UL-C), (4) hindlimb unloaded group fed a 0.25% isoflavone conjugates diet (UL-ISO 0.25), and (5) hindlimb unloaded group fed a 0.5% isoflavone conjugates diet (UL-ISO 0.5). After 3 weeks, bone mineral density (BMD) of the femur was significantly decreased in UL-C, and this bone loss was prevented by isoflavone treatment. Histomorphometric analysis revealed a decrease in the cancellous bone of the distal femur in the UL-C group, and isoflavone prevented this change. Serum corticosterone increased in the UL-C group, and isoflavones inhibited the elevation. These results suggest that isoflavones might be promising food components that provide protection from bone loss and normalize stress-induced serum corticosterone during skeletal unloading.

Expression patterns of connexin 26 and connexin 43 mRNA in canine benign and malignant mammary tumours.

The expression patterns of connexin (Cx) genes, encoding gap junctional proteins, are tissue- and cell-specific and, as their expression is mostly suppressed during carcinogenic processes, they are appropriate for monitoring tumour development. In this study, using reverse transcriptase-coupled polymerase chain reaction (RT-PCR), the expression of Cx mRNAs was examined in seven normal canine mammary glands and in 31 mammary gland tumour samples. Cx26 and Cx43 gene expression was studied in all normal tissues using specific Cx26 and Cx43 primers. When the expression patterns of Cx26 and Cx43 genes were analyzed in several types of canine mammary gland tumours, it was noted that it was the loss of Cx26 expression rather than the occurrence of Cx43 expression that was associated with malignancy. These results suggest that Cx26 plays an important role in tumourigenesis of canine mammary gland.

Connexin 32 down-regulates the fibrinolytic factors in metastatic renal cell carcinoma cells.

Fibrinolytic factors have an important role in tumor progression through the degradation of extracellular matrix. The increased levels of urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR) and type-1 PA inhibitor (PAI-1) are reported in human renal cell carcinoma (RCC). Connexin (Cx) gene, a member of gap junction, is known to act as a tumor suppressor gene. We have reported that Cx32 improves malignant phenotypes of metastatic RCC cells via the inhibition of Src-dependent signaling. In this study, we examined the effect of expression of Cx32 gene on the production of uPA, uPAR and PAI-1, and on the induction of PAI-1 stimulated by hypoxia in a human metastatic RCC cell line, Caki-1 cells. Cx32 expression decreased both mRNA level and production of PAI-1, uPA and uPAR in Caki-1 cells. Cx32 also decreased hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha mRNA level. PP1, a Src inhibitor, significantly decreased PAI-1, uPA, uPAR and HIF-alpha mRNA levels in Caki-1 cells. Furthermore, Cx32 suppressed the induction of HIF-2alpha protein in Caki-1 cells under hypoxia. PAI-1 mRNA level in Cx32-transfected Caki-1 cells was lower than that of mock transfectant under hypoxic conditions. These results suggest that Cx32 might reduce PAI-1, uPA and uPAR production in metastatic RCC cells via the inhibition of Src-dependent induction of HIF-1alpha and HIF-2alpha gene expression and that Cx32 might suppress hypoxia-inducible gene expression under hypoxic conditions.

Identification and characterization of lactococcal and Acetobacter strains isolated from traditional Caucasusian fermented milk.

The fermented milk, so-called "Caspian Sea Yogurt" in Japan, consists of two bacterial strains isolated from traditional Caucasusian fermented milk. In the present study, those strains were identified and characterized. Strain FC was Gram-positive, facultatively anaerobic cocci and strain FA was Gram-negative, aerobic rods. Phylogenetic analysis based on 16S rDNA sequences showed that strain FC formed a cluster with Lactococcus lactis strains and was most closely related to L. lactis subsp. cremoris. Strain FA was included in the genus Acetobacter cluster and was most closely related to A. orientalis. The DNA G+C contents of strain FC and strain FA were 39.2 and 51.6 mol%, respectively. Biochemical tests and DNA-DNA hybridization clarified that strain FC belongs to L. lactis subsp. cremoris and strain FA belongs to A. orientalis. The culture supernatant of lactococcal strain FC inhibited the growth of L. lactis subsp. cremoris DSM 20069T and L. lactis subsp. hordniae JCM 1180T. The inhibitory activity was detected after incubation at 70 degrees C for 60 min or 100 degrees C for 30 min and was stable when the supernatant was adjusted to a pH ranging from 4.9 to 7.5. The antimicrobial activity was lost on treatment with proteolytic enzymes such as proteinase K, trypsin, pronase, and pepsin, although it was not affected by catalase. The gene of lactococcin B (lcnB) homolog was found in the strain FC. From the above results, the strain FC was thought to produce a bacteriocin-like substance.