Lactic acid induces HSPA1A expression through ERK1/2 activation.

Heat shock protein (HSP) A1A protects cells from various stressors. The concentrated liquid of the traditional Japanese rice black vinegar Kurozu increased HSPA1A expression in normal rat liver RLN-10 cells. Lactic acid, the primary component of concentrated Kurozu, induced HSPA1A expression in a concentration-dependent manner. Induction with 4 m m lactic acid increased HSPA1A expression by three times compared with that in the absence of lactic acid. The induction was inhibited by staurosporine or a selective MEK1/2 inhibitor (SL327). The phosphorylation of ERK1/2 was increased by lactic acid. These results suggest that lactic acid induces HSPA1A expression by activating ERK1/2. As well as lactate, 3,5-dihydroxybenzoic acid (DHBA), a ligand for G protein-coupled receptor 81 (GPR81), also induced HSPA1A at lower concentrations than lactate. The increased effect of DHBA on HSPA1A expression as compared with lactate may be related to the higher affinity of DHBA for GPR81 than of lactate.

Effect of dietary fermented mushroom bed on egg production in laying hens.

Egg productivity is declined by stress. It has been reported that some food supplements can improve the egg productivity due to a reduction of environmental stress. We evaluated the effect of fermented waste mushroom bed (FWMB) as a dietary additive on egg productivity. Hens were fed control food (control group, n = 100) or 3% FWMB-added food (FWMB group, n = 100) for 16 months. The number of eggs, soft-shelled eggs, and broken eggs were recorded for 15 months. We also evaluated stress-related markers (ovotransferrin, lipid peroxide, and the heterophil-to-lymphocyte ratio). The FWMB group had slightly increased egg production compared with control hens. The FWMB group produced significantly less broken and soft-shelled eggs than the control group. All stress-related markers were significantly lower in the FWMB group than in the control group. Gut flora was also affected by FWMB feeding. The increased egg production and decreased proportion of broken and soft-shelled eggs might be related to the prevention of stressful conditions by FWMB.

Growth suppression and cell death by pyridoxal is dependent on p53 in the human breast cancer cell line MCF-7.

Vitamin B6 compound, pyridoxine (PN), has shown antitumor action. Our previous experiments showed that PN induces expression of insulin-like growth factor binding protein-3 to arrest proliferation and induce cell death. This induction is inhibited by the p53-specific inhibitor pifithrin-α. Here, we report that another B6 compound, pyridoxal (PL), strongly inhibited MCF-7 cell growth compared to PN. PL induced the G0/G1 arrest and the accumulation of subG1 population. Although p53 mRNA was not changed by PL, 0.5 mM PL increased the protein level in MCF-7 cells. The cell growth suppression by 0.5 mM PL did not occur when p53 expression was knocked down using siRNA. Together, these data suggest that PL accumulate p53 and PL-induced cell growth suppression is dependent on p53 in MCF-7 breast cancer cells.

Ethanol extract of Brazilian propolis ameliorates cognitive dysfunction and suppressed protein aggregations caused by hyperhomocysteinemia.

Homocysteine (Hcy) has been proposed to be a risk factor for cognitive dysfunction. We investigated the effects and the underlying mechanisms of action of propolis, which has antioxidant activity on Hcy-induced oxidative stress in vitro and in vivo. For the in vitro assays, neuroblastoma SH-SY5Y and glioblastoma U-251MG cells were cultured with Hcy and various concentrations of propolis. Cell death and reactive oxygen species production were significantly suppressed by propolis in dose-dependent manner, compared with Hcy alone. For the in vivo assays, mice were fed a propolis-containing diet and Hcy thiolactone in water. Cognitive function was evaluated using the Morris water maze test. Propolis suppressed cognitive dysfunction caused by hyperhomocysteinemia. Accumulation of aggregated protein in brain was accelerated in hyperhomocysteinemia, and the accumulation was suppressed by propolis. Hyperhomocysteinemia, however, did not enhance the oxidative stress in brain. In vitro amyloid formation assay showed that Hcy accelerated lysozyme aggregation and propolis inhibited the aggregation.

Association of blood pressure and dietary intake of Sunomono, Japanese vinegared side dishes, in community-dwelling Japanese: A cross-sectional study.

Objective: Vinegar has been reported to have a hypotensive effect. We aimed to investigate the relationship between the consumption of vinegar-based side dishes and blood pressure. Research methods & procedures: This cross-sectional study included 746 individuals (257 men and 489 women) aged ≥40 years from Tarumizu, Kagoshima, Japan. Nutrient intake was estimated based on the brief-type self-administered diet history questionnaire. The intake frequency of vinegar-based side dishes (Sunomono and pickles) was determined using a self-administered diet history questionnaire. Participants who did not consume vinegar-based side dishes for a month were defined as having no Sunomono or pickle eating habit. Blood pressure was categorized into four groups according to the Japanese Society of Hypertension Guidelines for the Management of Hypertension. The association between the intake of vinegar-based side dishes and blood pressure categories was analyzed using ordinal logistic regression analysis adjusted for age, body mass index, smoking history, excessive alcohol intake, living situation, energy intake, protein intake, sodium intake, potassium intake, and seaweed intake. Results: Approximately 13.6% men and 6.1% women had no Sunomono eating habits. In men, eating Sunomono, but not pickles, was significantly related to blood pressure categories (estimate, -0.702; 95% CI, -1.122 to -0.310), whereas more frequent consumption of Sunomono did not show an improvement in the blood pressure category. The relationship between eating Sunomono and blood pressure categories was not recognized in women. Conclusion: This was the first study assessing the association between consumption of vinegar-based side dishes and blood pressure categories. We highlighted the effect of Sunomono consumption on blood pressure categories in men. Consumption of Sunomono may improve blood pressure in men.

The Association between Dietary Variety and Physical Frailty in Community-Dwelling Older Adults.

The aim of this cross-sectional study was to examine the association between diet variety and physical frailty in community-dwelling older adults. Data of 577 older adults (mean age: 74.0 ± 6.3 years, women: 62.5%) were analyzed. Diet variety was assessed using the Food Frequency Score (FFS) (maximum, 30 points). The FFS assessed the one-week consumption frequency of ten foods (meat, fish/shellfish, eggs, milk & dairy products, soybean products, green & yellow vegetables, potatoes, fruits, seafood, and fats & oil). Physical frailty was assessed using Fried's component (slowness, weakness, exhaustion, low physical activity, and weight loss). The participants were classified into frail, pre-frail, and non-frail groups. The prevalence of physical frailty was 6.6%. This study found significant associations between physical frailty and low FFS after adjusting for covariates (odds ratio (OR) 0.90, 95% confidence interval (CI) 0.84-0.97, p < 0.01). The optimal cutoff point of the FFS for physical frailty was ≤16 points. FFS lower than the cutoff point were significantly associated with physical frailty after adjusting for covariates (OR 3.46, 95% CI 1.60-7.50, p < 0.01). Diet variety assessed using the FFS cutoff value of ≤16 points was related to the physical frailty status in community-dwelling older adults.

Association of Protein and Magnesium Intake with Prevalence of Prefrailty and Frailty in Community-Dwelling Older Japanese Women.

We examined the association between nutrient intake and prefrailty. Data from 815 older people (63% women) who participated in a community-based health check survey (Tarumizu Study) were analyzed. Prefrailty were defined using five parameters (exhaustion, slowness, weakness, low physical activity, and weight loss). Participants with one or more components were considered to belong to the prefrailty group. Nutrition intake was estimated from a validated brief-type self-administered diet history questionnaire. Among the participants, 154 men (52%) and 278 women (54%) were found to be in a status of prefrailty. In men, there were no significant associations between nutrient intake and prefrailty. In women, carbohydrate intake was slightly higher in prefrailty group. Vitamins K, B1, B2, folic acid, pantothenic acid, phosphorus, potassium, calcium, magnesium, iron, zinc, and copper intake was significantly lower in the prefrailty group. Among the nutrients, magnesium was identified as a significant covariate of prefrailty using a stepwise regression method. In women adjusted ORs (95%CI, p value) for prefrailty in the first, second, third, and fourth quartiles of magnesium intake were 1.00 (reference), 0.52 (0.29-0.92, 0.024), 0.51 (0.28-0.95, 0.033), and 0.38 (0.19-0.74, 0.005), respectively, by multivariate logistic regression analysis (variates: age, body mass index, energy intake, supplement use, osteoporosis, magnesium, and protein intake). Protein intake did not related to prefrailty. Protein intake might be sufficient to prevent prefrailty in the present study. We propose magnesium to be an important micronutrient that prevents prefrailty in community-dwelling older Japanese women.

Relationship between Oral Hypofunction, and Protein Intake: A Cross-Sectional Study in Local Community-Dwelling Adults.

Few studies have investigated the relationship between nutritional status and comprehensive assessment of oral hypofunction, especially protein intake-related sarcopenia. Thus, we explored these relationships in a large-scale cross-sectional cohort study using the seven-item evaluation for oral hypofunction and Diet History Questionnaire for nutritional assessment. We used the data from 1004 individuals who participated in the 2019 health survey of the residents of Tarumizu City, Kagoshima Prefecture, Japan for analysis. We found that individuals with oral hypofunction were significantly older with a lower skeletal muscle index. Although there were few foods that had a significant difference between the groups with and without oral hypofunction, the consumption of beans and meats was significantly lower in women and men in the oral hypofunction group, respectively. According to the lower limit of the tentative dietary goal defined in Japan, comprehensive evaluation of oral hypofunction was significantly and independently associated with protein intake in both men and women (odds ratio, 1.70; 95% confidence interval, 1.21-2.35). In conclusion, we found that oral hypofunction was associated with targeted protein intake for sarcopenia and frailty prevention in middle-aged and older community-dwelling adults. Comprehensive evaluation of oral function with intervention in cases of hypofunction could inform clinicians to better prevent sarcopenia.

Change in VEGF expression in mouse mammary gland during reproductive cycle.

During pregnancy, the mammary epithelium and its supporting vasculature undergo extensive growth and proliferation in preparation for lactation, which is thought to be dependent on vascular endothelial growth factor (VEGF). We investigated the expression of VEGF, using immunohistochemistry and immunoblotting, in the mouse mammary gland during the reproductive cycle. Immunohistochemical studies localized VEGF protein predominantly in the cytoplasm of the mammary epithelium and revealed it to be strongly expressed in late pregnancy and early lactation. In addition, immunoblot analysis revealed a 45-kD VEGF band, corresponding to the homodimer of the VEGF-A164 isoform, with increased expression towards the end of pregnancy but no additional increase with the onset of lactation. As the post-lactation period advanced, a dramatic decrease in VEGF was observed in the regressed mammary epithelium. The expression of VEGF suggests that mammary epithelium-derived VEGF may be involved in pregnancy-associated mammary growth and differentiation and angiogenesis, and regulate vascular permeability during lactation in an autocrine or paracrine manner.

The Induction of Heat Shock Protein 70 after Oral Administration of Concentrated Brewed Rice Vinegar Kurozu in Mice.

Heat shock protein 70 (HSP70) is induced by various stresses. Since HSP70 has a protein refolding activity and an anti-inflammatory activity, the HSP70 induction will help cells from harmful acute stresses. Feeding a diet containing concentrated brewed rice vinegar Kurozu (CK) diet for 5 wk resulted in an increase of HSP70 in the brains of mice. In the present study, we evaluated whether oral feeding of 25 µL CK induces HSP70 mRNA in brain and other tissues. HSP70 mRNA was significantly increased in the esophagus, small intestine, liver, and brown adipose tissue within 1 h after the oral administration of CK. A weaker induction of HSP70 mRNA was demonstrated in the stomach, large intestine, and brain. HSP70 mRNA induction returned to basal levels within 3 h after feeding. We doubted that the induction of HSP70 mRNA was caused by manual restraint of the mice during CK administration. Manual restraint of the mice did not influence HSP70 mRNA expression in intestine 1 h after these treatments. Our results suggest that transient HSP70 mRNA induction by oral feeding of CK was not caused by retention stress. There are some compounds in CK that increase HSP70 mRNA in various tissues.

Immunohistochemical localization of nitric oxide synthase (NOS) in mouse mammary gland during reproductive cycle.

Nitric oxide (NO) has been proposed as a mediator of postnatal mammary gland development. We investigated the immunohistochemical localization of three isoforms of nitric oxide synthase (NOS) enzymes, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), in the mouse mammary gland during reproductive cycle. The NOS isoforms detected in normal mouse mammary glands were the constitutive forms of NOS (nNOS and eNOS). nNOS was localized to the alveoli, myoepithelia, lactiferous ducts and blood vessel endothelia, while eNOS was localized in the alveoli, lactiferous ducts and blood vessel endothelia. The strongest immunoreactivity for both constitutive NOS isoforms was observed in pregnant mice. The differential staining intensity of NOS enzymes in the mammary gland led us to conclude that nitric oxide in the mouse mammary gland is mainly synthesized by constitutive NOS isoforms, and suggest that NO has functional roles in post-pubertal growth and differentiation of the mammary gland.

Promoting effects of Chinese pangolin and wild pink medicines on the mammary gland development in immature mice.

The effects of the mixture of crude aqueous extracts from Chinese pangolin and wild pink (C+P), traditional Chinese medicine, on the proliferation and differentiation of mammary gland epithelium in intact and ovariectomized immature mice were investigated by light and electron microscopy and BrdU immunohistochemistry. Although there were no significant differences in mammary gland fat pad and parenchyma areas between the intact experimental groups, the numbers of duct branchings and buds were significantly larger in the C+W treated mice than in the control mice. The ratio of BrdU immunopositive cells to total epithelial cells was higher in C+W treated intact mice. Ultrastructurally, epithelial cells of the mammary buds and ducts possessed an oval and lucent nucleus, and ribosomes increased in number or developed to a greater degree in C+W treated intact mice than in the control mice. Conversely, there were no significant differences in any measurements of mammary gland between the experimental groups of ovariectomized mice. BrdU immunoreactive cells were never seen and the ultrastructure of mammary epihelial cells indicated the inactive cell phase in both ovariectomied mice. In comparison between the intact and overiectomized mice, the mammary fat pad area was larger in the ovariectomized mice than in the intact mice, although another four measurements were larger in the intact groups. These observations suggest that administration with C+W could promote the development of mammary glands via ovary in immature mice.

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper.

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.

Perchloric acid-soluble protein is expressed in enterocytes and goblet cells in the intestine and upregulated by dietary lipid.

We previously identified perchloric acid-soluble protein (PSP) in the rat liver, kidney, brain and lung, and reported that it appeared to be related to repression of cell proliferation. In the present study, we clarified that PSP was expressed in the intestine, and found that the amino acid sequence of the intestinal PSP was consistent with those of other PSPs present in other tissues. An immunohistochemical study revealed that PSP was expressed in enterocytes and goblet cells, but not in other cell types among the lamina propria epithelial cells. A comparison of the expressions of PSP and proliferating cell nuclear antigen demonstrated that the proliferating cells did not express PSP. Intestinal PSP expression was induced by approximately 3-fold by oral administration of dietary fat. These findings indicate that the proliferation repression activity may be related to renewal of the intestinal epithelium, and that PSP is one of the fatty acid-inducible proteins.

Increases in pig major acute-phase protein in wasting pigs brought to the abattoir.

Plasma proteins of wasting pigs were quantitatively and qualitatively compared with those of normal fattening pigs. Higher expression of a 120 kDa protein was observed in the plasma of wasting pigs by SDS-PAGE. This protein was identified as pig major acute-phase protein (Pig-MAP) by proteomic analysis using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The plasma concentration of Pig-MAP in wasting pigs was 7-fold higher than that of normal ones.

The Brewed Rice Vinegar Kurozu Increases HSPA1A Expression and Ameliorates Cognitive Dysfunction in Aged P8 Mice.

Kurozu is a traditional Japanese rice vinegar. During fermentation and aging of the Kurozu liquid in an earthenware jar over 1 year, a solid residue called Kurozu Moromi is produced. In the present study, we evaluated whether concentrated Kurozu or Kurozu Moromi could ameliorate cognitive dysfunction in the senescence-accelerated P8 mouse. Senescence-accelerated P8 mice were fed 0.25% (w/w) concentrated Kurozu or 0.5% (w/w) Kurozu Moromi for 4 or 25 weeks. Kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while Kurozu Moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. We hypothesize that concentrated Kurozu has an antioxidant effect; however, the level of lipid peroxidation in the brain did not differ in senescence-accelerated P8 mice. DNA microarray analysis indicated that concentrated Kurozu increased HSPA1A mRNA expression, a protein that prevents protein misfolding and aggregation. The increase in HSPA1A expression by Kurozu was confirmed using quantitative real-time PCR and immunoblotting methods. The suppression of amyloid accumulation by concentrated Kurozu may be associated with HSPA1A induction. However, concentrated Kurozu could not increase HSPA1A expression in mouse primary neurons, suggesting it may not directly affect neurons.

Positive Association of Plasma Homocysteine Levels with Cardio-Ankle Vascular Index in a Prospective Study of Japanese Men from the General Population.

AIM: Observational studies have reported that elevated homocysteine (Hcy) levels are associated with the risk of cardiovascular disease (CVD). However, interventions that lower Hcy do not provide a corresponding risk reduction. Therefore, the causal role of Hcy in CVD remains unclear. This 5-year prospective study investigated the associations of Hcy levels, folate intake, and host factors with arterial stiffness among the general Japanese population. METHODS: We prospectively recruited 658 participants (40-69 years old) from the general population during regular health checkup examinations. Arterial stiffness was evaluated using the cardio-ankle vascular index (CAVI) at baseline and the 5-year follow-up. Folate intake was estimated using a structured questionnaire. Genotyping was used to evaluate the MTHFR C677T and MS A2756G gene polymorphisms. Ultrafast liquid chromatography was used to measure total plasma Hcy levels. Association between these variables and CAVI values was evaluated using general linear regression and logistic regression models that were adjusted for atherosclerosis-related factors. RESULTS: Men had higher Hcy levels and CAVI values and lower folate intake than women (all, p＜0.001). At baseline, Hcy, folate intake, and the two genotypes were not associated with CAVI values for both sexes. Among men, Hcy levels were positively associated with CAVI values at the 5-year follow-up (p=0.033). Folate intake and the two genotypes were not associated with the 5-year CAVI values. CONCLUSION: Plasma Hcy may be involved in arterial stiffness progression, as monitored using CAVI, among men.

Nuclear transfer of perchloric acid-soluble protein by endoplasmic reticulum stressors.

Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.

Perchloric acid-soluble protein regulates cell proliferation and differentiation in the spinal cord of chick embryos.

The role of perchloric acid-soluble protein (PSP) was investigated in chick embryos. Fluorescently labeled anti-chick liver (CL)-PSP IgG was injected into the yolk sac in ovo at embryonic day 3, and became localized in neuroepithelial cells. Within 12 h, morphological changes were observed in 37.5% of anti-CL-PSP IgG-injected embryos, and the neuroepithelial cells formed a wavy line. No significant changes were observed in embryos injected with non-immune IgG or PBS. Increased expression of PCNA and decreased expression of neuronal class III beta-tubulin were observed in the spinal cord after anti-CL-PSP IgG injection. These results suggest that PSP controls the proliferation and differentiation of neuroepithelial cells in chick embryos.

Recombinant perchloric acid-soluble protein suppresses the immunoglobulin production of human-human hybridoma HB4C5 cells.

Because perchloric acid-soluble protein (PSP) has been conserved evolutionally in various species from Escherichia coli to humans, it may reflect an involvement in basic cellular regulation. However, the precise function of PSP is currently unknown. In this study, we examined the direct effect of PSP on the production of immunoglobulin (Ig) using human B, HB4C5, NAT-30, and U266 cells because it has been reported that subcutaneous administration of PSP affects rodent immune systems. Suppression of Ig productivity and decrement of the cell viability was recognized only in HB4C5 cells by the addition of PSP into the medium. On the other hand, PSP had no effect on Ig productivity and cell viability in NAT-30 and U266 cells. In addition, PSP was clearly incorporated by HB4C5 but not by the other cells. These results suggest that the Ig production suppressed by PSP, which has been previously reported to inhibit protein synthesis, contributed to the incorporation of PSP into the HB4C5 cells.