Takao Hama

AMINO ACIDS AND PEPTIDES. XIV, LAMININ RELATED PEPTIDES AND THEIR INHIBITORY EFFECT ON EXPERIMENTAL METASTASIS FORMATION

Inhibitory effects of synthetic laminin related peptides on experimental metastasis formation in mice were examined. Of the synthetic peptides, YIGSRG-[amino-poly(ethylene glycol)] hybrid exhibited the most potent inhibitory effect on the metastasis of B16 melanoma BL6.

Flavonoid glucuronides from Helicteres isora

Five flavonoid glucuronides were obtained from the fruit of Helicteres isora, three of which were previously unknown compounds: isoscutellarein 4-methyl ether 8-O-beta-D-glucuronide 6-n-butyl ester. isoscutellarein 4-methyl ether 8-O-beta-D-glucuronide 2, 4-disulfate and isoscutellarein 8-O-beta-D-glucuronide 2,4-disulfate. The structures were determined on the basis of spectroscopy and hydrolysis experiments.

Chromone C-glycosides from Baeckea frutescens

Abstract Five new chromone C-glycosides have been obtained from the leaves of Baeckea frutescens. They are: 6-β-C-glucopyranosyl-5,7- dihydroxy-2-isopropylchromone, 8-β-C-glucopyranosyl-5,7-dihydroxy-2-isopropylchromone, 6-β-C-glucopyranosyl-5,7-dihydroxy- 2-methylchromone, 6-β-C-(2′-galloylglucopyranosyl)-5,7-dihydroxy-2-isopropylchromone and 8-β-C-(2′-galloylglucopyranosyl)-5,7-dihydroxy-2-isopropylchromone.

Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody OBF13

The variable reactivity of OBF13 monoclonal antibody to mouse sperm was studied using a fluorescein activated cell sorter. Sperm from cauda epididymis were incubated with ionophore A23187, subjected to indirect immunostaining and analyzed by a cell sorter. Three peaks showing different fluorescence intensities were observed. These peaks contained (i) not stained (N), (ii) acrosomal cap or anterior region stained (A) and (iii) head stained (H) sperm, respectively. H type sperm showed more intense integral fluorescence than A type sperm. It was also noted that the H type were observed when cauda epididymal sperm were incubated with A23187, but not among non-incubated, or A23187 treated caput epididymal sperm. When sperm were pre-categorized as dead or alive by propidium iodide staining, no A type were observed in the live population. These results suggest that the sperm exhibiting the OBF13 antigen in the acrosome region lost their viability before they accomplished a true acrosome reaction.

[78] Aldehyde dehydrogenase from bakers' yeast

Publisher Summary This chapter describes the assay method, purification, and properties of aldehyde dehydrogenase isolated from bakers yeast. Aldehyde dehydrogenase activity is assayed at 25°C by measuring the rate of formation of nicotinamide adenine dinucleotide dehydrogenase (NADH) at 340 nm. The proteolytic products from the native enzyme are found in the process of the purification of aldehyde dehydrogenase from yeast. The preparation method in the presence of phenylmethylsulfonyl fluoride (PMSF) as a serine esterase inhibitor diminishes the amount of proteolytic degradation products. Fresh bakers yeast is washed twice with three volumes of cold distilled water and stored at -25°C before use. The steps involved in the purification of aldehyde dehydrogenase are (1) the preparation of crude extract, (2) protamine sulfate precipitation, (3) first and second ammonium sulfate fractionation, (4) diethylaminoethyl (DEAE)-sepharose chromatography, (5) gel filtration, and (6) blue sepharose chromatography. All steps are performed at about 4°C. The purified enzyme migrates as a single component when subjected to polyacrylamide electrophoresis in the absence and in the presence of sodium dodecyl sulfate (SDS) and does not show any sign of proteolytically degraded forms.

Presence of β-citryl-L-glutamic acid in the lens : its possible role in the differentiation of lens epithelial cells into fiber cells

The beta-CG concentration in the chicken brain was high during embryonic development and decreased rapidly to a lower level close to hatching, while the concentration in the eyeball which was also high during the embryonic life retained a fairly high level after hatching. The distribution of beta-CG in the bovine eye was determined. About 95% of total beta-CG content in the whole eye was localized in the lens. However, the distribution of beta-CG in the eye varied depending on species. beta-CG was exclusively localized in the lens in the eyes of fish and mammals, but distributed in both lens and retina in frogs. The molecule was localized in the retina rather than the lens in the chicken eye, although the concentrations was extremely low compared to those in the mammalian, amphibian and fish eyes. It was found that beta-CG is present ubiquitously in the lens or retina in various species. The distribution of beta-CG in the bovine lens was determined in the three cortex regions and nucleus. beta-CG was present at the highest concentration in the equatorial cortex, at a moderate concentration in the posterior and anterior cortex, and at the lowest concentration in the nucleus. Similar distribution patterns were also found in the rabbit and rat lens. When embryonic chick lens epithelial cells were cultured in the presence of fetal calf serum, the cells elongated, differentiated into fiber cells and formed lentoid bodies. The cells of lentoid bodies were stained strongly by the anti-beta-CG antibody, while cells around the structures were not. In addition, the beta-CG content in the lenses from the galactose cataractous rat decreased to about 20-30% of that in the normal lens. These findings suggest that beta-CG may play a role in the differentiation of epithelial cells into fiber cells.

Purification and properties of ß-citryl-l-glutamate-hydrolysing enzyme from rat testis particulate

beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiffs-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.

Studies on Ergothioneine. IX. Ergothioneine Stimulates Mouse Spermatozoa and Fertilization in vitro

The effects of ergothioneine on spermatozoa and ova were investigated in vitro and in vivo. Spermatozoa were treated with ergothioneine in vitro, and injected into the uterine cavity of female mice immediately after the induction of superovulation. The ova were recovered 24 hr later and assessed for fertilization. Preincubation of spermatozoa with ergothioneine resulted in a significant increase in the fertilization rate. When ova were inseminated in the same manner in vitro with spermatozoa treated with 0.1 or 1.0 mM of ergothioneine, the penetration rate was significantly increased. These results suggest that ergothioneine is effective in inducing both capacitation and the acrosome reaction of mouse spermatozoa. Ergothioneine at concentrations of 0.1 and 1.0 mM in the preincubation medium was also effective in inducing the acrosome reaction of guinea pig spermatozoa. However, it had no significant effect on the development of 2‐cell ova in vitro.

Involvement of inflammatory leukocytes in hepatic induction of T-kininogen in rat.

Hepatic synthesis as well as plasma levels of T-kininogen, a protein precursor of T-kinin (Ile-Ser-bradykinin), increase in rats following the induction of acute inflammation. Studies have been undertaken to elucidate an involvement of inflammatory leukocytes in the acute-phase response of T-kininogen. Peritoneal exudate cells (PEC) were prepared from rats six days after the intraperitoneal injection of Freunds complete adjuvant. By transfer of these leukocytes into the peritoneal cavity of normal rats, plasma kininogen levels of these recipients increased about threefold after one day. Secretion of kininogen from rat hepatocytes in primary culture was enhanced about twofold by coculturing with PEC. A similar effect was also obtained by adding culture supernatant of these leukocytes into hepatocytes, and the increased levels of kininogen in culture medium of hepatocytes was due to the increased levels of T-kininogen. From these results, it was concluded that leukocytes in the inflammatory site, probably macrophages, release some substance(s) which stimulate(s) the hepatic synthesis of T-kininogen.

Study on 14C-BHA absorption and distribution in rat

BHA was administered to rats at doses of 5 or 500 mg/kg for seven days. 14C-BHA absorption was investigated on the eighth day and compared with animals receiving a single dose of 5 or 500 mg/kg BHA. Absorption of 14C-BHA was proportionately greater at the high dose rats. 14C distribution was 3–6 times higher in the forestomach than in the pyloric region of the stomach. The urinary excretion of BHA conjugates was not dependent on pretreatment with BHA or on the dose.