Kozo Ohtsuki

Evidence for the existence of a soybean resistant protein that captures bile acid and stimulates its fecal excretion.

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.

Comparison of the Glucosinolate−Myrosinase Systems among Daikon (Raphanus sativus, Japanese White Radish) Varieties

Myrosinase is a cytosolic plant enzyme present in daikon ( Raphanus sativus, Japanese white radish) roots that hydrolyzes 4-methylthio-3-butenyl glucosinolate (MTBGLS) into the natural pungent agent 4-methylthio-3-butenyl isothiocyanate (MTBITC), which possesses antimicrobial, antimutagenic, and anticarcinogenic properties. The concentration of MTBGLS, myrosinase activity, and production of MTBITC in seven daikon varieties (one conventional and six heirlooms) were determined to rank the activity of the glucosinolate-myrosinase system and identify critical factors influencing the production of MTBITC. The six heirloom varieties produced 2.0-11.5 times higher levels of MTBITC as compared to the conventional variety, Aokubi, which is consumed by the present Japanese population. The myrosinase was located exclusively in the outer epidermal layer in Aokubi, and MTBGLS was widely distributed throughout the root tissue. Although the skin is a potentially rich source of myrosinase in Aokubi, the skin is usually peeled off in the current practice of preparing daikon for cooking. New practices are therefore proposed for the preparation of daikon tubers that eliminate the peeling of the skin to avoid removing the enzyme needed to convert MTBGLS to the health-beneficial MTBITC. It is also concluded that the consumption of heirloom daikon varieties in addition to changes in food preparation will optimize the health benefits of daikon.

Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing

Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extracted from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3-10 or pH 4-6. The proteases were fractionated into three components by the isoelectric focusing, having pI value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29,000 as measured by SDS-polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg2+, Cu2+, Cd2+, and Zn2+, strongly inhibited these purified enzymes.

β-sitosterol from psyllium seed husk (Plantago ovata Forsk) restores gap junctional intercellular communication in Ha-ras transfected rat liver cells

Abstract: We purified compounds from the husks of psyllium seeds (Plantago ovata Forsk; desert Indian wheat), beginning with an ethanol extraction then followed by HP-20 and silica gel chromatography, which restored gap junctional intercellular communication (GJIC) in v-Ha-ras transfected rat liver epithelial WB-F344 cell line (WB-Ha-ras). GJIC was assessed by a scrape loading dye transfer assay. The active compound was identified as β-sitosterol based on gas chromatography retention times and electron ionization mass spectroscopy (EI-MS) spectrum of authentic β-sitosterol. Authentic β-sitosterol restored GJIC in the tumorigenic WB-Ha-ras GJIC-deficient cells at a dose of 2.4 μM. In addition, a similar phytosterol, stigmasterol, also restored GJIC, albeit at a lower activity. β-sitosterol and stigmasterol increased the level of connexin43 protein (Cx43) and restored phosphorylation of Cx43 to levels similar to the parental nontransfected cell line. We concluded that the restoration of intercellular communication in the GJIC-deficient, tumorigenic WB-Ha-ras cell line by the ethanol soluble fraction of psyllium seed husks is largely due to the presence of the phytosterol, β-sitosterol. We discuss implications for dietary modulation of cancer by β-sitosterol.

Improved chromatographic purification of human and bovine type V collagen sub-molecular species and their subunit chains from conventional crude preparations Application to cell-substratum adhesion assay for human umbilical vien endothelial cell

Two human type V collagen sub-molecular species, designated [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V), were purified chromatographically from a commercially available preparation, in which cystine-rich collagenous contaminants were contained, with a column packed with Fractogel EMD SO3-. From bovine crude preparations, the [alpha 1(V)]2 alpha 2(V) form free from the collagenous contaminants was purified. Type V collagen subunit chains were isolated from each type V collagen molecule by anion-exchange HPLC with a Bakerbond PEI Scout column. The highly purified human type V collagen molecules and their subunit chains were used to examine the inhibitory effect on human umbilical vein endothelial cell proliferation. It was confirmed that the alpha 1(V) chain has inhibitory activity and it was found that the inhibitory effect of the [alpha 1(V)]2 alpha 2(V) form is stronger than that of the alpha 1(V)alpha 2(V)alpha 3(V) form and that the alpha 3(V) chain has no inhibitory activity.

Simple and rapid chromatographic purification of type V collagen from a pepsin digest of porcine intestinal connective tissue, an unmanageable starting material for conventional column chromatography

A chromatographic method is described for purification of type V collagen, a minor constituent in extracellular matrix, from a pepsin digest of porcine intestinal connective tissue. The starting material was a viscous and turbid solution even after centrifugation. Direct application of the sample to a commercially available DEAE-cellulose column resulted in clogging. On the other hand, type V collagen, [alpha1(V)](2)alpha2(V) form, was successfully captured by a filter paper-based DEAE-cellulose column chromatography and purified by a subsequent commercially available cation-exchange medium without clogging. This is a vast improvement over previously described salt fractionation methods.

Direct fractionation of proteins in particle-containing feedstocks by a filter paper pieces-based DEAE-cellulose column chromatography: Rapid, robust and low-cost capturing procedure for protein

Filter paper pieces-based (FPB) DEAE-celluloses was prepared for direct fractionation of proteins in particle-containing feedstocks. FPB DEAE-cellulose has a protein binding capacity equivalent to that of commercially available DEAE-cellulose. Crude extracts from porcine intestine and kiwi fruit pulp, which were unmanageable by commercially available chromatographic media due to rapid clothing, could be directly fractionated with FPB DEAE-cellulose column. In addition, effluents from an FPB DEAE-cellulose column were extensively clarified. The present approach can be used as a rapid, robust and low-cost capturing step for protein from particle-containing feedstocks.

Moderation of chemo–induced cancer by water extract of dried shark fin : anti–cancer effect of shark cartilage

Abstract It is now well recognized that matrix metalloproteinase (MMP) plays significant roles in cancer progression. Shark cartilage contains the MMP inhibitors. On the basis of these findings, oral administration of the shark cartilage has been proposed to suppress cancer progression and improve quality of life of cancer patient. Now, the “shark cartilage therapy” is popular in USA, Japan and other countries. However, there are conflicting data on the effectiveness of the therapy. In addition, knowledge on the active component responsible for the potential anti-cancer effect is limited. To facilitate the further study on the anti-cancer effect of shark cartilage and utilization of shark fin cartilage, a by-product from fisheries industry, moderation of chemo-induced cancer by the water extract of dried shark fin is discussed.