Tadahiro Tadokoro

Nutritive evaluation on chemical components of leaves, stalks and stems of sweet potatoes (Ipomoea batatas Poir).

For the effective use of leaves, stalks and stems of two kinds of sweet potatoes, we determined their chemical components and evaluated their nutritive values. Some parts of this plant, which are not usually used, were found to be rich in nutritive and functional components. In particular, leaves contain a large amount of protein, showing high amino acid score. Any part of sweet potatoes was rich in dietary fiber and in particular, leaves were soluble dietary fiber and stems were insoluble dietary fiber, respectively. Mineral content, particularly iron, and vitamin content such as carotene, vitamin B2, vitamin C and vitamin E were high in leaves in comparison with other vegetables. Furthermore, polyphenol content in leaves was comparatively high. These results suggest that the whole parts of sweet potatoes should be utilized as valuable foodstuffs to cope with future changes in food supply and demand, particularly in developing countries.

Changes in susceptibility of tissues to lipid peroxidation after ingestion of various levels of docosahexaenoic acid and vitamin E.

To examine the effects of dietary docosahexaenoic acid (DHA) on the potential changes in endogenous lipid peroxidation in the liver and kidney, diets containing a fixed amount of vitamin E (VE; RRR-alpha-tocopherol equivalent; 134 mg/kg diet) and a graded amount of DHA at the levels of 0, 1.0, 3.4 and 8.7% of total dietary energy were fed to rats for 14 d (Expt 1). In Expt 2, diets containing a fixed amount of DHA (8.7% of total dietary energy) and a graded amount of VE at the levels of 54, 134 and 402 mg/kg were fed to rats for 15 d. In Expt 1 it was found that endogenous lipid peroxide contents of the liver and kidney, as measured by thiobarbituric acid value and chemiluminescence intensity, were higher, and their alpha-tocopherol contents lower than those of the controls, with a gradual increase and decrease in values respectively as the dietary DHA level increased (Expt 1). However, the contents of water-soluble antioxidants, i.e. ascorbic acid and non-protein-SH (glutathione), increased with increases in the dietary DHA level, while the Se-dependent glutathione peroxidase (EC 1.11.1.9) activities did not change or tended to be lower. When the graded level of VE was given to rats in Expt 2, lipid peroxide contents in the liver and kidney did not change significantly in response to the increasing levels of dietary VE, although their alpha-tocopherol contents were higher than control values, increasing with increases in the dietary VE levels. The lipid peroxide scavengers other than alpha-tocopherol changed similarly to those in Expt 1. The results obtained in Expts 1 and 2 indicate that DHA enhances the susceptibility of the liver and kidney to lipid peroxidation concomitant with higher levels of DHA in these tissues, as shown by the fatty acid composition. In addition, VE is unable to protect membranes of the liver and kidney rich in DHA from lipid peroxidation, even after ingestion of the highest level of VE. However, the liver lipid peroxide content of the group given the highest level of DHA was not as high as expected, based on the peroxidizability index which was calculated from the fatty acid composition of the liver lipid.

Nutrient composition of kopyor coconuts (Cocos nucifera L.)

Kopyor coconuts (matured coconuts with broken meat particles in the watery endosperm due to abnormal formation of the kernel during the development of the fruits) were analysed for their proximate composition, minerals, vitamins, dietary fibres, sugars, organic acids, fatty acid composition and amino acid profile. The chief constituent of kopyor water was sucrose (about 92% of the total sugar); in contrast, the young or normal-mature nut water contained glucose and fructose as the main sugars. α-Tocopherol was detectable in the kopyor water. Total amino acid content of kopyor water was higher than that of the young or normal-mature water. Like the young or normal-mature nut water, kopyor water seemed to be a good source of dietary minerals, with potassium as the predominant one. The relatively high contents of sucrose, glucose, fructose, citric and malic acids might contribute to the deliciousness of kopyor meat. The lipid content of kopyor meat was lower than that of the normal-mature meat, but fatty acid profiles were similar.

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction.

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.

Inhibitory effect of honeybee-collected pollen on mast cell degranulation in vivo and in vitro.

Bee-collected pollen (bee pollen [BP]) has been used as a folk medicine for centuries against various diseases, including allergy. There is no study elucidating how BP exerts such an anti-allergic effect. Since mast cells play a central role in the pathogenesis of various allergic diseases, we investigated the effect of BP on mast cell activation elicited by the Fc immunoglobulin E (IgE) receptor (Fc epsilon RI)-mediated pathways. The in vivo effect of orally administered BP on cutaneous mast cell activation was examined by passive cutaneous anaphylaxis reaction. In vitro mast cell degranulation and IgE binding to mast cells and the status of protein tyrosine phosphorylation were examined using bone marrow-derived mast cells. Daily oral administration of BP to mice significantly reduced the cutaneous mast cell activation elicited by IgE and specific antigens. BP also reduced in vitro mast cell degranulation and tumor necrosis factor-alpha production by inhibiting IgE binding to Fc epsilon RI on mast cells. The inhibitory effect of BP on mast cell degranulation by preventing IgE binding was confirmed by the reduced levels of protein tyrosine phosphorylation, which occurred as downstream events in activated mast cells via Fc epsilon RI. These results first revealed that the anti-allergic action of BP was exerted by inhibiting the Fc epsilon RI-mediated activation of mast cells, which plays important roles, not only in the early phase, but also in the late phase of allergic reactions.

Iron deficiency down-regulates the Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in rats and in COS-1 cells

Iron deficiency (ID) is one of the most commonly known forms of nutritional deficiencies. Low body iron is thought to induce neurologic defects but may also play a protective role against cancer development by cell growth arrest. Thus, ID may affect cellular pathways controlling cell growth and proliferation, the mechanism of which is still not fully understood. The serine/threonine protein kinase Akt and its downstream target, the mammalian Target of Rapamycin (mTOR), is known to play a crucial role in the regulation of cell growth and survival. Therefore, we hypothesized that Akt/mTOR pathway could be influenced by ID. Three-week-old male Wistar-strain rats were divided into 3 groups and the 2 groups had free access to a control diet (C group) or an iron-deficient diet (D group). The third group (PF group) were pair-fed the control diet to the mean intake of the D group. After 4 weeks, rats were killed and their brains were sampled. In separate experiments, COS-1 cells were cultured with or without the iron chelator deferoxamine. Western blots of brain samples and COS-1 lysates were used to analyze the expression and phosphorylation state of Akt, TSC2, mTOR, and S6 kinase proteins implicated in the Akt/mTOR pathway. Using 2 different ID models, we show for the first time that iron deficiency depresses Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity.

Change of Epigenetic Control of Cystathionine Beta-Synthase Gene Expression through Dietary Vitamin B12 Is Not Recovered by Methionine Supplementation

Background/Aims: Vitamin B12 (B-12) is an essential cofactor for methionine synthase, and methionine is critical for the methylation of various biological molecules including DNA. Whether changes in B-12 levels can alter specific gene expression through DNA methylation and whether dietary methionine has any effect on general DNA methylation status still remains controversial. Methods: We raised severely B-12-deficient rats as well severely-B-12 deficient rats but supplemented with 5% methionine. mRNA levels of methionine cycle-related enzymes were analyzed. Results: Gene expression patterns changed under B-12-deficient conditions but were recovered by dietary methionine supplementation to B-12-deficient rats. However, cystathionine β-synthase mRNA levels, which had decreased under B-12-deficient conditions, did not recover with supplementary dietary methionine. The CpG island of the cystathionine β-synthase promoter was hypomethylated in B-12-deficient rats, and showed no recovery after methionine addition. Conclusions: Dietary B-12 can affect epigenetic machinery by regulating DNA methylation status and dietary methionine may have small effects on DNA methylation.

Interaction of activated rab5 with actin-bundling proteins, l- and t-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.

Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis.

Western blot analysis has been a useful method for analysis of expression levels of specific proteins and is conducted after sodium dodecyl sulfate (SDS) or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels usually must be prepared, one of which is stained, leading to the consumption of precious sample. Thus, we developed a convenient and efficient Western blotting method using a stained gel. This simple modification should be beneficial for analyzing samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.

Tuberous sclerosis complex 2 loss-of-function mutation regulates reactive oxygen species production through Rac1 activation.

The products of the TSC1 (hamartin) and TCS2 (tuberin) tumor suppressor genes negatively regulate cell growth by inhibiting mTOR signaling. Recent research has led to the postulation that tuberin and/or hamartin are involved in tumor migration, presumably through Rho activation. Here we show that LEF-8 cells, which contain a Y1571 missense mutation in tuberin, express higher Rac1 activity than tuberin negative and positive cells. We also provide evidence of obvious lamellipodia formation in LEF-8 cells. Since the production of TSC2(Y1571H) cannot form a hetero-complex with hamartin, we further analyzed another mutant, TSC2(R611Q), which also lacks the ability to form a complex with hamartin. Introducing both forms of mutated TSC2 into COS-1 cells increased Rac1 activity as well as cell motility. We also found these two mutants interacted with Rac1. We further demonstrated that the introduction of mutated TSC2 into COS-1 cells can generate higher reactive oxygen species (ROS). These results indicate that loss-of-function mutated tuberin can activate Rac1 and thereby increase ROS production.