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Masahiko NANAMI, Kiyoshi ZAITSU, Yosuke OHKURA
1993 Volume 16 Issue 2 Pages
99-102
Published: February 15, 1993
Released on J-STAGE: April 10, 2008
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The preparation of insulins labeled with a fluorescent moiety at a definite position (Gly(A1) or Lys(B29)) on the molecule is described. Gly(A1)- and Lys(B29)-S-acetylthioglycoloyl-insulins and Gly(A1)-2(or 3)-acetylmercapto-3-carboxypropanoyl-insulin were deacetylated and then reacted with 7-chloro-4-sulfobenzoxadiazole, a fluorogenic reagent, to afford the corresponding fluorescence labeled insulins. The preparative separation of the fluorescence labeled insulins from the insulin derivatives that remained unreacted was carried out by anion-exchange high-performance liquid chromatography on a TSKgel DEAE-2SW column.
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Hideaki IIZUKA, Takehiko YAJIMA
1993 Volume 16 Issue 2 Pages
103-106
Published: February 15, 1993
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It was observed in our laboratory that methoxyacetaldehyde induced strong fluorescence with L-tryptophan in the presence of NaNO
2 at pH 2.75. The reaction mixture was heated at 80°C for 15 min and the fluorescence was measured at the excitation wavelength, 253 nm and the emission, 450 nm. Amino acids other than L-tryptophan did not produce fluorescence under the conditions employed. A good linear working curve was observed between 25 pmol/50 μl and 500 pmol/50 μl of L-tryptophan. The limit of determination for L-tryptophan was 10 pmol/50 μl. The coefficient of variation of the measurements (n=5) was 1.5% for 250 pmol/50 μl of L-tryptophan. The proposed method was successfully applied to the analyses of tryptophan in a pooled human serum. Total tryptophan was determined after deproteinization with trichloroacetic acid, and free tryptophan was determined using the ultrafiltrate of a pooled human serum. Analytical recoveries of total and free tryptophan were 98.8% and 99.1%, respectively. The fluorescent products of L-tryptophan and methoxyacetaldehyde were characterized as β-carboline and 1-methoxymethyl-β-carboline by the use of liquid chromatography-mass spectrometry (LC-MS) and HPLC.
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Haruyoshi MATSUYAMA, Masahiro TOMITA, Ryo TAGUCHI, Hiroh IKEZAWA
1993 Volume 16 Issue 2 Pages
107-111
Published: February 15, 1993
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Purified sphingomyelinase of Streptomyces A9107 (NRRL 15100) was modified with ss-PEG, methoxypolyethyleneglycol succinimidyl succinate, without loss of activity toward sphingomyelin in the mixed micelles with detergents such as sodium deoxycholate and Triton X-100 or toward a water-soluble, synthetic substrate, HNP, 2-(N-hexadecanoylamino)-4-nitrophenyl phosphocholine. The hemolytic and sphingomyelin-hydrolyzing activities of the enzyme toward intact bovine erythrocyte membrane were completely abolished by modification with ss-PEG, whereas the activity toward membranous sphingomyelin in the erythrocyte ghosts was fully retained. The enzyme activity toward liposomal sphingomyelin (large and small unilamellar vesicles) was significantly decreased by PEG-modification. The thermostability of sphingomyelinase was greatly increased by modification with ss-PEG, whereas the sensitivity of the enzyme to proteolysis was not influenced by that modification.
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Yasusuke MASUDA, Masanobu OZAKI
1993 Volume 16 Issue 2 Pages
112-115
Published: February 15, 1993
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Octa heme peptide, an enzymic digestion product of Candida Krusei cytochrome c, was found to catalyze nitro and azo reduction in the presence of NADPH and NADPH-cytochrome P-450 reductase under anerobic conditions. The reduction was dependent on the concentrations of heme peptide and reductase, and inhibited by carbon monoxide and oxygen. Comparison of the activities of the reductase-heme peptide system with liver microsomes revealed that heme peptide was as effective as cytochrome P-450 in nitro reduction, although less effective in azo reduction. Thus, cytochrome c heme peptide may serve as an artificial substitute for cytochrome P-450 at least in the reductive metabolism of xenobiotics.
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Yuichi KAWAI, Shigeru OHNISHI, Mihoko UEHARA, Takao HAMA, Tadanori MAY ...
1993 Volume 16 Issue 2 Pages
116-119
Published: February 15, 1993
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We prepared four monoclonal antibodies raised against mouse spermatozoa. The ability of the hybridoma culture supernatants to inhibit in vitro mouse fertilization was determined. One of these monoclonal antibodies, termed A-1, strongly inhibits sperm penetration into zona pellucida of egg in a concentration-dependent fashion. On the other hand, even in the presence of the same concentration of A-1 antibody, sperm binding to comulus-free eggs was slightly inhibited and sperm-egg fusion was not inhibited at all. Immuno-fluorescence studies with A-1 showed that it recognized an antigen(s) localized in the acrosome of mouse spermatozoa. In the case of unfixed sperm, the acrosome-intact sperm (i.e., sperm bound to egg zona pellucida) and unbound sperm were slightly immunostained with A-1 antibody (1.6% and 9.0%, respectively). All the full acrosome reacted sperm (i.e., sperm penetrating into the perivitelline space) were not immunoreacted. On the other hand, when sperm were fixed with methanol, approximately 80% of unbound sperm were immunoreacted with this antibody. Furthermore, almost all of zona-binding sperm were immunostained, whereas acrosome reacted, penetrating sperm were not. These data suggested that the antigen recognized by A-1 was located inside the plasma membrane of spermatozoa, and appeared on the surface of the spermatozoa during the period of penetration into egg zona pellucida. After sperm penetration into the perivitelline space this antigen must be released from the sperm.
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Munetoshi KATO, Takaji IRISAWA, Mutumi MURAMATU
1993 Volume 16 Issue 2 Pages
120-124
Published: February 15, 1993
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The inhibitory effects of various aromatic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), potent trypsin inhibitors, on the growth of Escherichia coli were examined and the effects were compared with those of well known synthetic trypsin inhibitors. Various GMCHA esters strongly inhibited the growth of E. coli and their effects were markedly affected by the species and position of substitution on the phenyl nucleus of the GMCHA phenyl esters. No correlation was observed between the effects on the growth of E. coli and K
i's for trypsin. Inhibitory effects of benzamidine, phenylmethane sulfonylfluoride and diisopropyl fluorophosphate were less than 10% at 200 μM. 4-tert-Butylphenyl ester of GMCHA (GMCHA-OPh'Bu), a representative of various GMCHA esters, dose-dependently inhibited the growth of E. coli and the growth inhibition was preceded by a dose- and time-dependent inhibition of DNA synthesis. Tosyl-L-lysine chloromethyl ketone (TLCK) and pentamidine isethionate, potent trypsin inhibitors, also dose-dependently inhibited the growth of E. coli and the DNA synthesis. However, their effects were transient and disappeared after a short while. These results indicate that the effects of TLCK and pentamidine isethionate differ from those of GMCHA esters. GMCHA-OPh'Bu and pentamidine isethionate also inhibited RNA and protein synthesis.
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Mitsuo TANAKA
1993 Volume 16 Issue 2 Pages
125-127
Published: February 15, 1993
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The inhibitory effects of Cu
2+ on the hydrolysis of cholesteryl [4-
14C]oleate droplets in rat peritoneal macrophages were studied. Macrophages rapidly incorporated cholesteryl [4-
14C]oleate droplets and hydrolyzed at a linear rate over 9h.When macrophages were preincubated in the medium containing Cu
2+, the hydrolysis of cholesteryl oleate within macrophages was markedly inhibited by Cu
2+ and eventually led to the accumulation of cholesteryl oleate in macrophages. The inhibitory effect on the hydrolysis of cholesteryl oleate was dependent on the concentration of Cu
2+. Also, the activity of acid cholesteryl ester hydrolase (acid CEH) in lysosomal fractions isolated from rat peritoneal macrophages with Cu
2+ showed a marked decrease. This decrease also occurred in a dose dependent manner.These results suggest that Cu
2+ inhibits the hydrolysis of cholesteryl ester in macrophages, and that the accumulation of cholesteryl ester in macrophages of the early atheroscleotic lesion is responsible for the decrease of lysosomal acid CEH activity.
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Chiaki KAMEI, Kenji TASAKA
1993 Volume 16 Issue 2 Pages
128-132
Published: February 15, 1993
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Effects of intracerebroventricular (i.c.v) injection of histamine (Hi) and its related compounds on prolongation of the response latency ensuing after a long interruption of learning were studied by testing the active avoidance response in old rats. After Hi application at doses of 50 and 100 ng, the response latency became significantly shorter than that determined in the pre-injection periods, suggesting that Hi facilitates memory retrieval in old rats. H
1-agonists, 2-methylHi and 2-thiazolylethylamine, effected a dose-related shortening of response latency as seen after Hi application, whereas H
2-agonists, 4-methylHi and impromidine, failed to prompt the response latency. Simultaneous i.c.v. injection of pyrilamine, a H
1-antagonist and Hi abolished the Hi-induced shortening of response latency. Furthermore, intraperitoneal administration of histidine at doses of 200 and 500 mg/kg significantly shortened the response latency. Neither (R)-α-methylHi nor thioperamide caused a significant effect indicating that H
3-receptor may not be involved in Hi-induced facilitation of memory retrieval. Based on these findings, it may be concluded that Hi takes an active part in facilitating memory recall via H
1-receptor in old rats.
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Yuichi KAWAI, Akihiko YOSHIDA, Shinsaku NAKAGAWA, Takao HAMA, Tadanori ...
1993 Volume 16 Issue 2 Pages
133-136
Published: February 15, 1993
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Bredinin, an immunosuppressive agent, had a teratogenic effect and a decreasing effect in litter size in mice and in rats. We investigated the direct effect of bredimin on the development of mouse preimplantation embryos in vitro. Bredinin strongly inhibited the first differentiation step from the morula to the blastocyst stage, whereas the steps from the two-cell embryo to the morula stage were not inhibited. At 10
-6 or 10
-5M of bredinin, only one or none of the morula embryos developed to a blastocyst after a 48 h culture; while approximately 90% of the embryos developed to blastocysts in the absence of bredinin. To determine whether the bredinin inhibition was reversible, the morula embryos were cultured with 10
-5 M of bredinin for 48 h. The embryos were transferred to bredinin-free culture conditions, and their development was observed. The inhibitory effect of bredinin was reversible. The expansion of zona-free blastocyst was also inhibited by bredinin. When blastocyst embryos were cultured in the presence of bredinin (10
-6 M), the mean size of the embryos was reduced to half the initial size. In addition, the steps of hatching from the zona pellucida and attaching to the substratum were significantly inhibited by bredinin. Trophoblast outgrowth was completely inhibited by 10
-6 M bredinin; however, the area of trophoblast outgrowth was expanded to 18-fold the initial size after a 96 h culture in the absence of bredinin. Thus, the decrease in the number of offspring is due to the direct effect of bredinin on mice embryos.
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Takeshi OHYA
1993 Volume 16 Issue 2 Pages
137-141
Published: February 15, 1993
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The reactions of malondialdehyde (MDA) with n-hexylamine (HA) in the presence of alkanals at a neutral pH were investigated. Two new compounds, 1, 1, 1 adduct (4a) and fluorescent compound (3a), were isolated from the reaction of MDA, HA and acetaldehyde. Compounds 4a and 3a were identified as 2-formyl-3-hexylamino-3-methylpropanal and 1-hexyl-5-hexyliminomethylene-4-methyl-1, 4-dihydropyridine-3-carbaldehyde, respectively. Similar compounds (4b and 3b) were obtained from the reaction of MDA, HA and propanal. Compound 4 was obtained in a high yield. In addition, the reactivity of MDA towards phenylethylamine (PEA) in the presence or absence of alkanals was investigated. The results indicated that MDA was of low reactivity in the absence of alkanals at neutral pH. However, when alkanals coexisted, MDA showed high reactivity towards PEA.
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Tetsuro SAKURAI, Hiroyuki YAMADA, Ken-ichi SAITO, Yoshihiro KANO
1993 Volume 16 Issue 2 Pages
142-145
Published: February 15, 1993
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In an attempt to elucidate the physiological activities of (6E, 12E)-tetradecadiene-8, 10-diyne-1, 3-diol diacetate (TDEYA), which was detected as a hydrolysis form (TDEY) in the plasma after oral administration of a decoction of Atractylodes rhizome in rats, we examined the inhibitory effects of various enzymes which are considered to participate in the regulation of body fluid levels and inflammatory reactions.TDEY and TDEYA did not show inhibitory effects on carbonic anhydrase (CA) or angiotensin converting enzyme (ACE) at concentrations less than 1.0×10
-3 M. However, both acetylene compounds inhibited Na
+, K
+ adenosine triphosphatase (Na
+, K
+ -ATPase) weakly and xanthine oxidase (XO) strongly.From the results of several acetylene compounds examined on XO inhibition, it is clear that the active structure of the compounds is due to the presence of conjugated triple and double bonds.In the in vivo experiment of TDEYA, urine volume, urinary electrolytes and uric acid excretion showed no significant differences from the control. However, the administration of TDEYA to rats tended to increase xanthine excretion.
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Yasuhisa FUJIBAYASHI, Kouichi WADA, Hideyuki TANIUCHI, Yoshiharu YONEK ...
1993 Volume 16 Issue 2 Pages
146-149
Published: February 15, 1993
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The retention mechanism of
62Cu-pyruvaldehyde bis(N
4-methylthiosemicarbazone) (
62Cu-PTSM) in the murine brain was evaluated. For this purpose, stable Cu-PTSM was subjected to electron spin resonance spectrometry (ESR) and high performance liquid chromatography (HPLC) analysis to determine the valence state, coordination structure and tissue metabolism. In murine brain homogenate, ESR and HPLC analysis indicated the reduction and cleavage of Cu(II)-PTSM to Cu(I). This virtually irreversible reduction was specifically initiated by the mitochondrial enzymatic system in the murine brain.
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Yoshiaki MATSUMOTO, Yoshiteru WATANABE, Ikuo YAMAMOTO, Mitsuo MATSUMOT ...
1993 Volume 16 Issue 2 Pages
150-153
Published: February 15, 1993
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The bioavailability of morphine after rectal administration using three types of suppositories containing morphine hydrochloride (10 mg) in different added forms was evaluated in rabbits. Three types of suppositories were constructed with a base material (Witepsol H-15) : a conventional suppository containing morphine hydrochloride mixed with a base material, a hollow-type suppository containing morphine in an aqueous solution in its cavity, and a hollow-type suppository containing morphine as a powder (hydrochloride salt) in its cavity. The plasma concentrations of morphine and its metabolites, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G), were determined.The mean AUC
0-6 of morphine after rectal administration of the hollow-type suppository containing powdered morphine was significantly higher than that after the administration of a conventional suppository, whereas the mean AUC
0-6 of M-3-G was lower than that after administration of a conventional suppository. The mean AUC
0-6 of M-6-G by a hollow-type suppository containing powdered morphine was higher than that by a conventional suppository. Although the mean AUC
0-6 ratio of M-3-G to morphine after administration of a conventional suppository was three times larger than that of a hollow-type suppository containing powdered morphine, the mean AUC
0-6 ratios of M-6-G to morphine after use of the three types of suppositories were all approximately 1.0.This study demonstrates that the hollow-type suppository containing powdered morphine is a more effective rectal dosage vehicle than the conventional suppository.
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Masahiko OHSAKO, Yasuhiro MATSUMOTO, Shigeru GOTO
1993 Volume 16 Issue 2 Pages
154-157
Published: February 15, 1993
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The transport of aspirin (ASP) and its metabolites (salicylic acid (SA), salicyluric acid (SAU), gentisic acid (GA) and gentisuric acid (GAU)) through human erythrocyte membrane was investigated. ASP permeated rapidly into the erythrocytes and the concentration dwindled gradually after the maximum concentration was attained almost within one minute. It was suggested that SA is released from the erythrocytes after ASP transported into the erythrocytes is hydrolyzed in them. In both an inward and outward direction, the transport rates of SA and GA were rapid, while those of SAU and GAU were lower by conjugating glycine. It was suggested that GAU remains for a long time in a living body. The rate of transport of GA and GAU were markedly obstructed by the band 3 protein inhibitor 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate, although the transport rates of SA and SAU were obstructed only slightly. It was suggested that the transport of GA and GAU are mediated through band 3 protein.
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Kiyoshi MIHARA, Mihoko MORI, Takami HOJO, Yoshinobu TAKAKURA, Hitoshi ...
1993 Volume 16 Issue 2 Pages
158-162
Published: February 15, 1993
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The disposition characteristics of model macromolecules such as dextran (70kDa), bovine serum albumin (BSA), and their charged derivatives were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery patterns and tissue accumulation of radiolabeled compounds were evaluated under filtering or nonfiltering conditions. In the filtering kidney, cationic macromolecules such as diethylaminoethyl-dextran (DEAE-dex) and cationized BSA (cBSA) accumulated in the kidney to a great extent whereas anionic and neutral macromolecules such as BSA, carboxymethyl-dextran (CM-dex), and dextran showed only small uptake. DEAE-dex and cBSA were distributed to both the medulla and cortex regions of the kidney and their recoveries in the kidney decreased as the injected dose increased. Similar tissue uptake was observed in the nonfiltering kidney perfusion system suggesting that they were mainly taken up by the kidney from the renal capillary side based on electrostatic interaction. In addition, the steady-state distribution volumes of cationic macromolecules calculated from venous outflow patterns were larger than those of the intravascular volume estimated from the distribution volumes of neutral and anionic macromolecules, suggesting their reversible interaction with the vascular wall. On the other hand, dextran derivatives with molecular weight distribution were excreted into urine based on glomerular permselectivity; i.e., cationic DEAE-dex and anionic CM-dex showed enhanced and restricted urinary excretion, respectively, compared with neutral dextran. In contrast, no significant excretion was observed for BSA and cBSA. The utility of the isolated rat kidney perfusion experiment for studying the renal disposition of macromolecular drugs was thus demonstrated.
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Taro OGISO, Masahiro IWAKI, Tadatoshi TANINO, Kayo OKUYAMA, Satoshi UN ...
1993 Volume 16 Issue 2 Pages
163-167
Published: February 15, 1993
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In an attempt to clarify the possible mechanism of the interaction between theophylline (TP) and mexiletine (ME), the elimination kinetics and in vitro metabolism of TP and its metabolites were investigated in rats. The plasma elimination of TP, 1, 3-dimethyluric acid (1, 3-DMU) and 1-methyluric acid (1-MU) was significantly delayed by the intravenous (i.v.) administration of ME. The oral administration of ME also decreased the elimination rate of TP to the same extent as the i.v. dosing. The in vitro metabolic experiment showed that ME significantly inhibited the metabolic conversion of TP to 1, 3-DMU and, 1, 3-DMU to 1-MU, and slightly inhibited the conversion of TP to 3-methylxanthine, these processes being mediated by microsomal enzymes, with no inhibition of xanthine oxidase. Our results indicated that ME could inhibit the metabolic conversion of TP and its metabolite in rat, as reported in man.
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Takahiro UCHIDA, Cheon Koo LEE, Noboru SEKIYA, Shigeru GOTO
1993 Volume 16 Issue 2 Pages
168-171
Published: February 15, 1993
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The novel binary vehicle system consisting of ethanol (EtOH) and Panasate 800 as tricaprylin was applied to five types of nonsteroidal anti-inflammatory drugs, and its enhancing effect on drug permeation across hairless mouse skin in vitro was assessed. The permeability of all drugs was remarkably increased by the treatment of skin with EtOH/Panasate 800 binary systems compared with either EtOH or Panasate 800 alone, and the effect reached a maximum in EtOH/Panasate 800 (40/60) system. With regard to this binary system, the skin permeation ratio or flux of the drug increased in the following order : salicyluric acid (SU)>salicylic acid (SA)>alclofenac (ALC)>ketoprofen (KP)>ibuprofen (IBU).The one-layer skin model was applied concerning the above skin permeation profiles of the five drugs. Diffusion parameter (D'), partition parameter (K'), and permeation constant (K
p=D'×K') were calculated using the Laplace-transformed equations with the aid of MULTI (FILT).It was well demonstrated that the reductin of lag time and the increase of flux caused by EtOH/Panasate 800 binary vehicle systems was due to an increase of D' value by Panasate 800 and K' value by EtOH, respectively, in the skin. Especially, the EtOH/Panasate 800 (40/60) binary vehicle system produced the largest K
p value in each drug.In relation to all the EtOH/Panasate 800 binary vehicle systems, the logarithms of calculated K
p were found to be in inverse proportion to the logarithms of a n-octanol/water partition coefficient (P) of the drug which appeared in previous literature.
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Yoshimi SHIRAI, Kiyomi SOGO, Kazumitsu YAMAMOTO, Kenji KOJIMA, Hiroshi ...
1993 Volume 16 Issue 2 Pages
172-177
Published: February 15, 1993
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In order to prepare fine granules of sparfloxacin (SPFX), a new quinolone anti-bacterial drug that shows masking of the bitter taste of SPFX and dissolutes at a rapid rate, various film-coated fine granules containing 20% SPFX and 0-52% low-substituted hydroxypropylcellulose(L-HPC) in the cores, were prepared by a spray method. Mixtures of ethylcellulose (EC), hydroxypropylmethylcellulose (HPMC), titanium dioxide and sucrose stearate in weight ratios of X : Y : 2 : 1 (X+Y=6) were used as film materials.The degree of masking of the bitter taste by water-insoluble film, mainly consisting of EC and HPMC, increased by increasing the content ratio of EC to HPMC and the amount of films, but was also slightly affected by the amount of L-HPC in the cores, which were coated with either EC or EC/HPMC (4/2). On the other hand, the dissolution rate increased with an increased amount of L-HPC in the cores and with a decreasing ratio of EC to HPMC in the films. Increasing the amount of L-HPC in the cores, which induced a considerable expansion of the fine granules owing to their taking up of water from the dissolution medium, resulted in burstin of the film after a short lag time. The bioavailability of the film-coated fine granules containing 20% SPFX and 52% L-HPC in the cores and 10% EC/HPMC (4/2) in the coating film, which masked the bitter taste of SPFX and showed the optimal release characteristics, was equivalent to that of conventional tablets containing 100 mg SPFX in beagle dogs.
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Takashi KATSU, Satoshi NAKAO, Sadaaki IWANAGA
1993 Volume 16 Issue 2 Pages
178-181
Published: February 15, 1993
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An antimicrobial peptide, tachyplesin I, isolated from hemocytes of the horseshoe crab, Tachypleus tridentatus, increased the K
+ permeability of Staphylococcus aureus and Escherichia coli cells, concomitantly reducing cell viability. At a higher concentration range, this peptide also enhanced the permeability of human erythrocytes. Tachyplesin decreased the phase transition temperature of an artificial membrane composed of dipalmitoylphosphatidylglycerol and, further, broadened it extensively, while it did not affect that of dipalmitoylphosphatidylcholine membrane. The latter result related closely to the fact that this peptide acted weakly on erythrocytes in which acidic lipids constitute a minor portion. Tachyplesin altered the normal discoid shape of human erythrocytes to a crenated form, suggesting that the peptide accumulated predominantly in the outer half of the membrane bilayer and destabilized the membrane structure, thus causing the change in permeability. The mode of action of tachyplesin was compared with that of gramicidin S, a peptide forming an amphiphilic structure analogous to tachyplesin.
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Ichiro YAMAKAWA, Sumio WATANABE, Yoko MATSUNO, Masayuki KUZUYA
1993 Volume 16 Issue 2 Pages
182-187
Published: February 15, 1993
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The release behavior of insulin from a plasma-irradiated sandwitch (PIS) device using poly-DL-lactic acid (PLA) was studied. The controlled release device can be obtained by oxygen plasma irradiation (radiofrequency discharge operating at 13.56 MHz) on the outer layer of the sandwitch device which was fabricated from an insulin-PLA matrix tablet as a core material and a mixture of plasma-degradable polyoxymethylene (POM) and biodegradable PLA as a wall material. The release test indicated that insulin was released through the micropores formed by the vaporization of POM, and that the release behavior of insulin was affected largely by the molecular weight of PLA used as the outer layer rather than the plasma operational condition. The release of insulin can be controlled by the use of PLA with an average molecular weight of 11000 as the outer layer of the PIS device. Insulin from the PIS device maintained normal blood glucose levels for 10 d in diabetic rats as an implantable dosage form. The duration of insulin effectiveness was relatively short considering the degradation rate of PLA, indicating that the degradation characteristics of biodegradable PLA were not well reflected in the PIS device.
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Yukio SATO, Hiroshi YAMAKOSE, Yasuo SUZUKI
1993 Volume 16 Issue 2 Pages
188-194
Published: February 15, 1993
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The study was designed to show wehether band 3, anion exchange protein in human erythrocytes, is responsible for hemolysis in hypotonic solution. From ESR spectra of the spin labeled erythrocytes, it was found that the change of the osmotic condition had no effect on membrane fluidity at pH 7.4. At pH 6.1, however, the fluidity at the central part of the lipid bilayer was dependent on osmotic stress. In this case, the fluidity change as a function of KCl concentration was similar to the hemolysis curve. From circular dichroism (CD) spectra of erythrocyte ghosts, it was found that the conformational and/or associative changes of the membrane proteins correlated with the hypotonic hemolysis. In contrast to the osmotic stress, sodium dodecyl sulfate (SDS) changed the fluidity of the membranes but not the conformation of the membrane proteins regardless of the medium pH. In this case, the changes in the fluidity corresponded to the hemolysis curves in SDS solutions. Further, by using anion transport inhibitors, 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate, eosin 5-isothiocyanate and eosin-5-maleimide, a correlation between the effect on the hypotonic hemolysis and the confomational and/or associative change of band 3 was suggested. These results demonstrate that band 3 is responsible for the hypotonic hemolysis.
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Kunio KOBAYASHI, Koichi YOSHIMOTO, Kazumasa HIRAUCHI, Kiyohisa UCHIDA
1993 Volume 16 Issue 2 Pages
195-198
Published: February 15, 1993
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We tried to measure glycated proteins by a novel method based on colorimetry of 2-keto-glucose which is released from the glycated protein (ketoamine) on heating with hydrazine. Reaction conditions were optimized with glycated human serum albumin (glc HSA) as a model compound. Ketoamine reacted quantitatively with hydrazine on heating at 100°C for 0.5 h, followed by heating with phenylhydrazine at 60°C for 1 h. Glucose interference with the assay was eliminated by preincubation of the sample with glucose oxidase at 37°C for 0.5 h. Time courses for the coloration of glc HSA and human serum showed a profile similar to that of N-p-tolyl-D-isoglucosamine under optimized reaction conditions. The lower limit for the assay of glc HSA was 0.7 μM. The serum level of glycated proteins measured by the present method correlated well with that (fructoamine value, μM) measured by the conventional method (nitroblue tetrazolium-reducing method) (r=0.92, n=35). In conclusion, the present method is a novel, highly sensitive and reliable one for measuring glycated proteins in biological samples.
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Tetsuo MORITA, Fuminori MIKAMI, Asako KANAGAWA, Hiroshi UEKI
1993 Volume 16 Issue 2 Pages
199-200
Published: February 15, 1993
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We have found that release of hepatic lipase activity is stimulated from liver slices by epidermal growth factor (EGF) into the medium in a time-dependent manner. This novel effect of EGF was markedly decreased by various tyrosine kinase inhibitors, such as α-cyano-3-ethoxy-4-hydroxy-5-phenyl-thiomethyl cinnamamide, amiloride and biochanin A. The stimulation by EGF was also suppressed, however, by dibutyryl cyclic adenosine monophosphate, and 3-isobutyl-1-methylxanthine, a cyclic nucleotide dependent phosphodiesterase inhibitor.These findings show that the stimulatory release of the enzyme activity by EGF is associated with the activation of tyrosine kinase activity and with the intracellular cyclic adenosine monophosphate level.
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Tadayori SHIMIZU, Toshiyuki MASUZAWA, Yoshihisa IWAMOTO, Yasutake YANA ...
1993 Volume 16 Issue 2 Pages
201-202
Published: February 15, 1993
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The mitogenicity of chemically synthesized lipid A analogs, 2, 3-acyloxyacylglucosamine-4-phosphate (A-103) and 2, 3-acyloxyacylgalactosamine-4-phosphate (A-113), was compared. Synthetic lipid A analogs of the disaccharide-type (506), the monosaccharide-type (A-103) suspended in RPMI-1640 medium supplemented with triethylamine, and Salmonella typhimurium LT-2 lipopolysaccharide (LPS) were copable of increasing the incorporation of [
3H]thymidine into splenocytes of C57BL/6 mice at various doses ranging from 1.0 to 100 μg/ml. However, the mitogenic activity of A-113 at these doses was markedly weaker than the activity of the above materials. When the A-103 and A-113 were suspended in RPMI-1640 medium supplemented with ethanol, the mitogenic activity of A-113 also showed lower activity than that of A-103 in the splenocytes of C57BL/6 mice. These findings indicate that the mitogenic activity of synthetic lipid A of the monosaccharide-type is affected by a kind of composed sugar.
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Kenji YAMADA, Akinori SHIMIZU, Akihiro OHTA
1993 Volume 16 Issue 2 Pages
203-206
Published: February 15, 1993
Released on J-STAGE: April 10, 2008
JOURNAL
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The effects of dimethylpyrazine isomers on reproductive and accessory reproductive organs in male rats were studied. Following the administration of 2, 5-dimethylpyrazine (100 mg/kg, s.c.), the weight of prostate and seminal vesicles, as well as testosterone levels in plasma were significantly decreased. One isomer of dimethylpyrazine, 2, 6-dimethylpyrazine (100 mg/kg, s.c.), affected only the seminal vesicles, while 2, 3-dimethylpyrazine had no influence on accessory reproductive organs. Following administration of 100 mg/kg of 2, 5-dimethylpyrazine, acid phosphatase activity in the prostate and fructose content in the seminal vesicles were also significantly decreased compared with tissues from vehicle-treated animals. Weight of the testes and acid phosphatase activity therein were not affected following the administration of 2, 5-dimethylpyrazine, nor were numbers of spermatozoa in the epididymis. These results showed that 2, 5-dimethylpyrazine induced decrease in prostate and seminal vesicle weight by inhibiting testosterone uptake and reducing plasma testosterone levels.
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Yasushi NAKAMURA, Tomoaki MATSUO, Kayoko SHIMOI, Yoshiyuki NAKAMURA, I ...
1993 Volume 16 Issue 2 Pages
207-209
Published: February 15, 1993
Released on J-STAGE: April 10, 2008
JOURNAL
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Though various antimutagens with desmutagenic activities have been found in our daily foods of plant origin, the numbers of antimutagens with bio-antimutagenic activities found so far are limited.In the present study, a compound with potential bio-antimutagenic activity to Escherichia coli B/r WP2 was newly isolated from cauliflower, Brassica oleracea L. var. botrytis, and its chemical structure was identified to be S-methyl methane thiosulfonate by NMR and MS analysis.
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Keiji WADA, Keiko SASAKI, Ken-ichi MIURA, Masaaki YAGI, Yoko KUBOTA, T ...
1993 Volume 16 Issue 2 Pages
210-212
Published: February 15, 1993
Released on J-STAGE: April 10, 2008
JOURNAL
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The leaves of (Ginkgo)___- (biloba)___- L. and aqueous extract from them shortened the sleeping time induced in mice by anesthetics (hexobarbital, α-chloralose and urethane, i.p.). Two characteristic terpenoids in (G.)___- (biloba)___-, bilobalide and ginkgolide A, significantly shortened the sleeping time induced by anesthetics.A toxic substance, 4-O-methylpyridoxine (MPN), responsible for "gin-nan food poisoning" isolated from the seed of (G.)___- (biloba)___-, was not detected from the extract of the leaves of (G.)___- (biloba)___-. Therefore, the Ginkgo biloba extract has no toxicities for MPN.
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Yoko MATSUMOTO, Eiichi YAMADA, Jiro HIRANO, Midori OSHIGE, Masayoshi I ...
1993 Volume 16 Issue 2 Pages
213-215
Published: February 15, 1993
Released on J-STAGE: April 10, 2008
JOURNAL
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A highly inhibitory action of hybrid liposomes composed of 43 mol% DLPC / 57 mol% Triton X-100 against the growth of human lymphoma-human lymphocyte B hybridoma was enhanced by including adriamycin or aclarubicin.
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