Dermatan sulfate-proteoglycans (DS-PGs) were extracted from rabbit, rat and bovine defatted livers by magnesium chloride extraction and DEAE-cellulose chromatography, and then submitted successively to Asahipak GS-520 gel filtration chromatography, Asahipak ES-502N anion exchange chromatography, and cellulose acetate membrane electrophoresis. The disaccharide composition of the glycosaminoglycan chains was determined by differential digestion by chondroitinase ABC, AC, ACII and/or B followed by HPLC for analysis of the resulting unsaturated disaccharides. The hepatic dermatan sulfate chains contained disulfated disaccharide units; Di-diSB and Di-diSE. The hepatic DS-PGs were divided into two groups; Di-diSE-poor DS-PGs and Di-diSE-rich DS-PGs. The iduronic acid content of Di-diSE-poor dermatan sulfate chains was higher than that of Di-diSE-rich ones.
Dermatan sulfate excreted in normal human urine was isolated and characterized by TLC and cellulose acetate strip electrophoresis after cetylpyridinium chloride precipitation and pronase digestion. In these separation methods, dermatan sulfate and chondroitin sulfate were extracted and then monitored by sensitive HPLC methods with post column fluorometric derivatization coupled with chondroitinase ABC, ACII and B digestion. From the results, we demonstrated that human urinary dermatan sulfate contains iduronic acid as its major uronic acid (80-90% of total uronic acid), and is composed mainly of repeated mono-sulfated disaccharide units [Di-4S (structure shown in Fig. 1), 89%] and small numbers of di-sulfated disaccharide units (Di-diSB, 7% and Di-diSE, 1%).
The complete molecule or the ligand-binding domain-containing region of each of the three subtypes of human retinoic acid receptors (hRARα, hRARβ and hRARγ) was expressed in Escherichia coli. The expressed recombinant RARs (rRARs : rRARα/E, rRARβ/E and rRARγ) showed nearly the same magnitude of binding affinity toward [3H]retinoic acid (RA) as hRARs extracted from human cells (Ka values : 6.0×109 M-1 for rRARα/E and 2.7×1010 M-1 for both rRARβ/E and rRARγ). Therefore, the ligand-binding selectivity of the rRARs toward RA and synthetic retinoids (4-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetrametyl-2-naphthalenylcarbamoyl)benzoic acid (Am80), (E)-4-[3-(3, 5-di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid (Ch55)) was examined. Am80 bound rRARα/E preferentially and showed no binding activity toward rRARγ, which is consistent with the case of hRARγ. Ch55 bound all three subtypes of rRARs, preferentially rRARβ/E. These results suggest that the intrinsic nature of the binding of each retinoid can be investigated by usage of the rRARs. However, rRARs show quantitatively different ligand-selectivity from that of hRARs : RA showed higher binding affinity toward rRARs than both Am80 and Ch55, but Ch55 binds all three subtypes of hRARs stronger than RA and Am80, which binds hRARβ stronger than RA.
Squalene epoxidase is a microsomal membrane-associated enzyme that acts as an important regulator in the sterol biosynthetic pathway. In this study, the enzymatic properties of squalene epoxidase from Saccharomyces cerevisiae were examined. Unlike Candida squalene epoxidase, S. cerevisiae squalene epoxidase required NADPH for enzyme reaction. However, S. cerevisiae enzyme reaction did not require FAD or autologous S105 fraction. Unlike rat squalene epoxidase, the activity of S. cerevisiae was reduced by Triton X-100, a nonionic detergent. Terbinafine, an inhibitor of fungal squalene epoxidase, inhibited the enzyme in a non-competitive manner, while NB-598, an inhibitor of mammalian squalene epoxidase, barely inhibited it in a partially non-competitive manner. Thus, the properties of squalene epoxidase from S. cerevisiae were different from those of squalene epoxidase from rats and Candida, which were previously known. We propose that a species difference of squalene epoxidase exists not only between animals and fungi but between Candida and Saccharomyces.
A 122 kDa RNase from eggs of Xenopus laevis was purified by sequential chromatography on Sephadex G-75, DEAE-cellulose, heparin-Sepharose and TSK gel G3000SW columns, and gave a single 60 kDa band on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The RNase composed of two 60 kDa subunits is able to recognize pyrimidine bases specifically. The pH optimum of the RNase was 7.5 in Tris-HCl buffer. The enzyme activity was abolished by treatment at 80°C for 5 min and pH 2 or 12 for 1 h. Since egg lectins with RNase activity obtained from Rana catesbeiana and R. japonica and bovine pancreatic RNase A show about 30% protein homology and these three proteins are 12-14 kDa heat-stable RNases, [K. Titani, K. Takio, M. Kuwada, K. Nitta, F. Sakakibara, H. Kawauchi, G. Takayanagi and S. Hakomori, Biochemistry, 26, 2189 (1987); Y. Kamiya, F. Oyama, R. Oyama, F. Sakakibara, K. Nitta, H. Kawauchi, Y. Takayanagi and K. Titani, J. Biochem. (Tokyo), 108, 139 (1990)], the data suggest that the X. laevis egg RNase is a unique protein compared with RNases from not only amphibians, but also mammals.
HeLa cells were synchronized by a double-thymidine block. After removal of thymidine, the cells immediately caused the uptake of [3H]thymidine into DNA and reached a half-maximum. The duration of the cell cycle was 23 h, and definite changes in cell density were observed between 12 and 13 h and between 35 and 36 h after removal of thymidine. Thus, the initiation time of S phase could be fixed. A trypsin-like proteinase appearing at around 17 h 17 min after removal of thymidine and correlated with the onset of the second S phase, tryptase 17 : 17 [cf., M. Muramatu et al., Biochim. Biophys. Acta, 1087, 87 (1990)], was obtained. 4-tert-Butylphenyl and 4-biphenyl esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA) and amidinopiperidine-4-alkanoic acids, trypsin inhibitors, strongly inhibited the tryptase activity, and these esters exert different effects on the cell cycle of HeLa cells at concentrations showing complete inhibition or maximal inhibitory effect on the tryptase. Both esters of GMCHA elongated the onset of the second S phase for 3 h. Esters of amidinopiperidine-4-acetic and 4-propionic acids showed a similar effect at lower concentrations than GMCHA esters. 4-tert-Butylphenyl esters of amidinopiperidine-4-propionic acid and butyric acids strongly suppressed the second S and M phases by probably affecting the G1 late phase, since they have no effect on the first S and M phases. The addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester 0 min after removal of thymidine into the cells completely suppressed the first S and M phases. Addition 10 min after removal of the arrest had no effect on the first S and M phases, but completely suppressed the second S and M phases, suggesting that this ester inhibits the onest of DNA synthesis.
The effects of ethyl all-cis-5, 8, 11, 14, 17-icosapentaenoate (EPA-E), highly purified ethyl ester of icosapentaenoic acid (EPA), on rabbit platelets were studied. In in vitro, highly purified EPA (62.5-3000 μM) suppressed the platelet aggregation induced by collagen, arachidonic acid (AA) and adenosine diphosphate (ADP). In ex vivo, a single administration of EPA-E (300 and 1000 mg/kg, p.o.) and repeated administrations (30 and 300 mg/kg/d, p.o.) for 2 weeks showed no effects on collagen-, AA- and ADP-induced platelet aggregation. Repeated administrations (30 and 300 mg/kg/d, p.o.) for 4 weeks suppressed the collagen-induced platelet aggregation, but not the AA- and ADP-induced platelet aggregation. Repeated administrations for 4 weeks also suppressed thromboxane B2 (TXB2) formation induced by collagen, but a single administration and repeated administrations for 2 weeks failed to inhibit TXB2 formation. The EPA level in the platelet phospholipids increased slightly with a single administration, and increased markedly with repeated administrations for 2 and 4 weeks. The AA level in the phospholipids showed practically no changes with a single and repeated administrations. These results suggested that highly purified EPA-E could reduce platelet aggregability by the change of the EPA level in the platelet phospholipids and should allow for a reasonable period of administration.
The protective effects various dithiocarbamates such as N-benzyl-D-glucamine dithiocarbamate (BGD), N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD), N-p-carboxybenzyl-D-glucamine dithiocarbamate (CBGD), and diethyldithiocarbamate (DDTC) on cis-diamminedichloroplatinum (DDP)-induced toxicity in mice were studied. The mice were injected i.v. with a chelating agent (1 mmol/kg) immediately or 5 min after i.v. injection of DDP (15 or 20 mg/kg). The lethal toxicity of DDP (20 mg/kg) was completely prevented by treatment with HBGD or CBGD immediately after DDP. The survival time of mice treated with HBGD or CBGD 5 min after DDP tended to be longer than that treated with BGD or DDTC. Significant increases in blood urea nitrogen (BUN) level and plasma aspartate aminotransferase (AST) activity were observed 3 d after DDP injection. The increase in BUN level was completely prevented only by HBGD and CBGD among these chelating agents, while increase in AST activity was significantly prevented by treatment with these two agents. Treatment with HBGD or CBGD immediately after DDP (20 mg/kg) completely protected against DDP-induced diarrhea. These chelating agents significantly decreased the platinum (Pt) contents in the kidney and liver after DDP administration. Treatment with HBGD or CBGD was the most effective in decreasing the renal Pt content, resulting in maximum protection against DDP-induced renal damage. The antitumor efficacy of DDP (15 mg/kg) in the colon 26 carcinoma-bearing mice was not affected by HBGD administration.
Silver ion causes a number of toxic effects, it decreases the activities of lactate dehydrogenase and glutathione peroxidase, and the peroxidation of membrane lipids. Silver ions complex strongly to sulfhydryl groups and the effects of preinduction of metallothionein, a sulfur-rich protein, on silver administration in rats were examined. The preinduction of metallothionein decreased the mortality of mice following silver administration. A major portion of the incorporated silver in the liver was bound to the basal membrane and cellular components, and the distribution of silver into cytosol was small. The preinduction of metallothionein decreased the amount of bound silver in the membrane and increased the distribution of silver in the cytosol; in addition, it depressed silver-induced lipid peroxidation in liver.
To clarify the absorption mechanism of 1, 3-bis(2-ethoxycarbonylchromon-5-yloxy)-2-((S)-lysyloxy)propane dihydrochloride (N-556), a prodrug for the oral delivery of disodium cromoglycate (DSCG), a study was made using rats. N-556 gave the highest plasma level of DSCG following its injection into the loop at the upper part of the small intestine. N-556 was stable in acidic washings of gastric contents, but rapidly hydrolyzed to M1 with twin ethyl residues on DSCG in the washings of the small intestinal contents. N-556 and M1 were hydrolyzed to DSCG via M2 having a mono ethyl residue in the homogenate of the small intestinal mucosa. The oral absorption of M1 following its administration in 50% (v/v) propylene glycol solution was essentially the same as that of N-556. That of M1 administered in aqueous suspension was low. After the oral adiministration of N-556, a small amount of M2 and a trace of M3 having L-lysyl residue were detected in the portal plasma, but no hydrolytic intermediate except DSCG could be found in the general plasma. The major absorption mechanism of N-556 may thus be concluded as follows : N-556 given orally is transferred to the small intestine in essentially intact form. N-556 is then rapidly diffused to an aqueous layer on the surface of the mucosal membrane and hydrolyzed to M1. The resultant M1 is transported to the mucosal membrane and hydrolyzed to DSCG via M2. DSCG generated in the mucosal membrane is used for general circulation through the portal blood and liver. A small amount of M2 in the mucosal membrane is transported to the portal blood and hydrolyzed to DSCG in the portal blood and/or liver to make DSCG available for the general circulation.
Forty-five ellagitannins and related compounds were intraperitoneally injected into mice once, 4 d before intraperitoneal inoculation of S-180 cells, and their antitumor activities were evaluated. When an antitumor-active tannin was defined as one producing a 70% increase or more in the mean life span of mice or one regressor out of six mice, twenty-one ellagitannins were active. Among monomeric ellagitannins, tellimagrandin II was most active. Most of the oligomeric ellagitannins, consisting of tellimagrandins I and II as the monomer unit, had a significant antitumor activity. Macrocyclic ellagitannins were all active. Oenothein B, among them, had the most potent antitumor activity. In contrast, ellagitannins containing a casuarictin or potentillin moiety in their molecules, except for extensively oligomerized ones, showed very low or negligible activity. These results suggest that tannins need the ellagitannin monomer units, having galloyl groups at the O-2 and O-3 positions on the glucose core(s), such as tellimagrandins, in order to exhibit a strong antitumor activity.
A simple and efficient method for identification of Duboisia leichhardtii F. MUELL, D. myoporoides R. BR. and their interspecific hybrid was established by analyzing restriction fragment length polymorphism (RFLP) profiles using non-radioactive rice ribosomal DNA as a probe. This procedure was shown to be applicable to both fresh and dried samples, and also suitable for detection of somatic hybrid at the callus stage after protoplast fusion.
Sustained-release hydrogel suppositories prepared with water-soluble dietary fibers, xanthan gum and locust bean gum, were evaluated as a vehicle for rectal administration of indomethacin (IMC) in rabbits. The drug plasma levels were compared with those after rectal administration of commercial suppositories. When the commercial suppositories were given to rabbits, the plasma concentration reached the maximum level at 30 min after administration followed by a quick reduction, while no sharp peak of plasma levels was seen with the hydrogel suppositories. In particular, the plasma levels observed with the hydrogel suppositories of 1% (w/v) gum concentration were sustained much longer than those after dosing with the commercial suppositories; the mean residence times had higher values without a decrease in the area under the plasma concentration vs. time curves. Histopathological study showed good biological safety of the hydrogel suppositories to the rectal mucosa.These results suggested that the IMC hydrogel suppositories prepared with xanthan gum and locust bean gum were a practical rectal preparation with prolonged action and reduced side effects.
Transport mechanisms of p-aminohippurate (PAH) were investigated in rat renal brush-border membrane vesicles. The uptake of PAH was stimulated by an inside-positive membrane potential created by K+ and valinomycin. This potential-stimulated uptake of PAH was inhibited by various anion transport inhibitors and was saturable. In addition, PAH uptake in the presence of valinomycin was linearly increased in proportion to log[K+]out/[K+]in. On one hand, PAH uptake was stimulated by [14C]PAH/PAH or [14C]PAH/Cl- exchange, and the [14C]PAH/PAH exchange was insensitive to the membrane potential. The uptake by the exchanger was also inhibited by anion transport inhibitors, but the potential-stimulated uptake of PAH was more sensitive to furosemide and 4, 4'-diiso-thiocyano-2, 2'-disulfonic stilbene. On the contrary, [14C]PAH/PAH exchange was more sensitive to urate than the potential-stimulated uptake of PAH. These findings indicate that PAH is transported by two distinct transport systems in rat renal brush-border membranes, a potential-sensitive transport system and an anion exchanger.
Optimal conditions for the crystallization and subsequent freeze-drying of an aqueous cephalothin sodium (CET-Na) solution of supersaturated concentrations to obtain granular crystalline CET-Na have been discussed, as have the qualities of the product thus obtained.In general, CET-Na in supersaturated aqueous solution is barely recrystallized, even when in its frozen stage. Our previous report revealed that when the solution is kept at low temperatures for a long duration, the molecules begin to change structurally to as condensed a state as that of liquid crystals, and such change facilitates a spontaneous nucleation and seed-independent crystal growth in the frozen solution.These findings prompted the authors to investigate optimal CET-Na concentrations and thermal histories during the crystallization and subsequent freeze-drying process without seeding.It has subsequently been found out that optimization of the latter conditions gives granular agglomerated crystalline CET-Na contaminated with neither the amorphous nor the quasi-crystalline form. The optimized conditions are : 25-28% CET-Na concentrations; storage before the freezing process at 0°C for 2 h, and subsequent storage at 20-25°C for 1 h; cooling in the freezing process at a rate not faster than 0.5°C/min; warming of the solution for facilitating crystallization prior to vacuum application for drying at -4°C.Under these conditions, the freeze-dried product of CET-Na in granular form has been successfully obtained in a shorter freeze-drying cycle, exhibiting a faster reconstitution time than those of CET-Na prepared according to seeded crystallization followed by a conventional freeze-drying.
The mechanism of granularly agglomerated crystallization during freezing of cephalothin sodium (CET-Na) in aqueous solution has been discussed. Our previous report noted that strict control of the following is important in order to obtain crystalline granular agglomerates of CET-Na : (1) thermal history of the solution before receiving freezing, (2) cooling rate in freezing, and (3) aging temperature level in the crystal growth step. In order to clarify the physico/chemical meanings of the individual controls, further investigations have been made : 1) with varying thermal history, aqueous 30-% CET-Na solutions were prepared for storage, first at 0°C with its supersaturation and secondly at 25°C, in an unsaturated state for observing any structural changes by viscosimetry, refractometry, and surface tensiometry; 2) morphological changes in ice crystals during freezing at varying cooling rates, as well as those during the crystal growth step, were observed by polarized-light cryomicroscopy and scanning electron microscopy; 3) melting, as well as crystal growth, at several aging temperature levels were observed by electrical conductometry and also by the above-mentioned techniques.These investigations have disclosed that the structural properties of both the liquid and frozen solutions should be thermally well-controlled in order to realize each of the following : 1) the condensed structure of CET-Na solute in its liquid solution is just like a cluster level before freezing; 2) ice crystals formed during freezing are less changable morphologically with varying temperatures; 3) CET-Na crystal growth is facilitated by an increased mobility of the solute due to the partial thawing of ice crystals : it therefore is important that the aging temperature is adjusted to a level at which ice crystals show negligible changes in surface area, with the degree of supersaturation in its liquid portion still kept high.Using a freezer-dryer of about 1000 vials of 14-ml size, supersaturated CET-Na solutions have been successfully controlled to achieve the above-described optimal structures in these three steps, the control yielding the freeze-dried product of granular CET-Na crystals almost quantitatively.
The immunopharmacological activities of a fungal (1→3)-β-D-glucan, OL-2, isolated from "Leiwan" Omphalia lapidescens were examined. Intraperitoneal (i.p.) administration of OL-2 to ICR mice induced a significant number of peritoneal exudate cells (PEC) and whilte blood cells over the period of a few days. Spleen cell numbers were also increased by i.p. administration of OL-2 at about a week. These changes reverted to the normal level within a month. Responses of spleen cells and bone marrow cells (BM) to colony stimulating factors (CSF) were augmented by OL-2 administration assessed by cell proliferation assay. Sera from OL-2 administered mice contained an increased concentration of colony stimulating activity. Gene expressions of interleukin-1β, interleukin-6, and tumor necrosis factor α in the spleen were also increased. These results suggested the activation of hematopoietic responses, and would well relate to the incremental increase in PEC, white blood cell and spleen cell numbers. OL-2 also increased the serum concentration of fibronectin and complement component C-3. However, OL-2 did not show adjuvant activity to SRBC and antitumor activity against the solid form of Sarcoma 180 by i.p. administration. Yet, OL-2 did not interfere with the antitumor activity of SSG against the same tumor system. These facts suggested that OL-2 could enhance nonspecific host defense mechanisms by enhancing hematopoietic responses, but would not enhance or inhibit the specific immunity mediated by lymphocytes. On the contrary, negligible changes in cell numbers were observed by OL-2 on AKR mice, which are complement C5-deficient mice, suggesting the significant contribution of complement systems to the immunopharmacological activity of OL-2.
Alginate gel beads containing nifedipine (NP) were prepared using a gelation of alginate with calcium cations. The dissolution and absorption of NP from alginate gel beads were evaluated as a controlled-release formulation of NP. The release of NP from alginate gel beads was affected by the composition of uronic acid in alginate, and by the NP content in alginate gel beads. NP absorption after oral administration to beagle dogs of alginate gel beads prepared by air-drying was significantly lower than that after the administration of NP powder alone, due to the limited release of NP from the alginate gel beads in the gastrointestinal tract. On the other hand, the alginate gel beads prepared by freeze-drying improved the absorption of NP because of the increasing disintegration of alginate gel beads with decreasing structural strength. However, this method had poor reproducibility, compared with air-dried alginate gel beads. The gel beads with added alginate propylene glycol ester (PGA) swelled and released calcium ions rapidly, even in water. This is because PGA gels weakly to the calcium cation. Consequently, it was observed that NP release from the PGA gel beads was highly accelerated compared to the release from alginate gel beads. The higher serum level of NP with large variance was obtained after the oral administration of the PGA gel beads. Gel beads consisting of a 1 : 1 ratio of PGA to alginate had intermediate characteristics between the alginate and PGA gel beads in respect to NP release and absorption. Furthermore, the oral administration of the PGA-alginate gel beads was found to reproduce a good sustained-release profile of serum level of NP comparable to that of a commercial sustained-release NP tablet. These results suggest that the PGA-alginate gel beads are useful for a controlled-release formulation of NP.
The effects of thinner and its main components, toluene, xylene, methanol, and ethyl acetate, on reproductive and accessory reproductive organs in male rats were studied. The vapour from these organic solvents was inhaled twice a day for 7 d. Following inhalation of thinner vapour for 7 d, the weights of the testes and prostate fell and acid phosphatase activity in the prostate and plasma testosterone levels were significantly decreased compared with the control group. Both ethyl acetate and xylene caused a decrease in the weight of the testes and accessory reproductive organs, as well as reducing acid phosphatase activity in the prostate and plasma testosterone levels. In contrast, toluene and methanol had no effect on organ weights, circulating testosterone levels, or on enzyme activity. Body weight was decreased by inhalation of thinner or ethyl acetate vapour. Spermatozoa levels in the epididymis were decreased by inhalation of ethyl acetate and xylene vapour. These results suggest that thinner, particularly the components ethyl acetate and xylene, interfere with the functions of the testes and accessory reproductive organ; toluene has no effect on these functions.
The suppression by cannabidiol (CBD) of the liver microsomal drug-metabolizing enzyme activities in female rats was demonstrated and its mechanism was examined. Pretreatment of rats with CBD (10 mg/kg, i.p.) caused temporary decreases in contents of cytochrome P450 (P450) and b5 and NADPH-cytochrome c (P450) reductase activity compared with values from the vehicle control group. p-Nitroanisole O-demethylase, aniline hydroxylase, d-benzphetamine N-demethylase and Δ9-tetrahydrocannabinol 11-hydroxylase were also decreased by the CBD pretreatment. The latter two activities took a longer time to return to control levels than the former two. However, the CBD pretreatment, which reduced the protein level of P450 UT-2 (CYP2C11) in adult male rats, did not decrease the protein level of P450 F-1 (CYP2C6) or F-2 (CYP2C12) in liver microsomes from female rats. These results suggest that the mechanisms by which CBD suppresses liver microsomal drug-metabolizing enzyme activities are different in male and female rats.
Elevation of intracellular sodium ion concentration in human erythrocyte induced by the cardiac glycoside, proscillaridin (1), and its four derivatives (2-5) was measured using 23Na NMR spectrometry. In this examination, there was a significant correlation between the time to half maximum inotropic effect and the time to maximum of Na+ concentrations in human erythrocyte, determined by 23Na NMR.
After the dehydrogenation polymer of p-coumaric acid, a synthetic lignin, was intravenously injected into mice, the serum was collected immediately, 15 min, 1 h, 5 h, and 24 h after the injection. The serum thus obtained was added to the assay medium containing MT-4 cells infected with HTLV-IIIB (an HIV-1 strain). Inhibition of the cytopathogenicity of HIV by these serum preparations was assayed by the MTT method. The result revealed that lignins could be a promising class of anti-HIV agents with an unidentified unique mode of action.