Microwave frequency comb Doppler reflectometer applying fast digital data acquisition system in LHD.

We succeeded in increasing the radial observation points of the microwave frequency comb Doppler reflectometer system from 8 to 20 (or especially up to 45) using the high sampling rate of 40 GS/s digital signal processing. For a new acquisition system, the estimation scheme of the Doppler shifted frequency is constructed and compared with the conventional technique. Also, the fine radial profile of perpendicular velocity is obtained, and it is found that the perpendicular velocity profile is consistent with the E × B drift velocity one.

Increased synthesis of hyaluronic acid by mouse mammary carcinoma cell variants with high metastatic potential.

Variant subpopulations of FM3A mouse mammary carcinoma cells that have increased lung-colonizing potential were obtained previously by sequentially harvesting pulmonary metastases, culturing their cells in vitro, and reestablishing the metastases in vivo. In the present study, glycosaminoglycan production by the parental and variant cells was studied after metabolic labeling of cultures by [14C]glucosamine for 24 hr. Analysis of the products indicated that the rate of incorporation of the labeled precursor into hyaluronic acid in the high-metastatic variant cells was 27 to 54 times the rate in the low-metastatic variant cells and that the increase in hyaluronic acid synthesis was not associated with an increase in the rate of synthesis of other glycosaminoglycans. Both the cell layers and media of high-metastatic variants contained a much higher proportion of radioactivity in hyaluronic acid than did the corresponding fractions of low-metastatic cell lines. The results provide a basis for further investigation of the potential role of hyaluronic acid in control of the behavior of epithelial tumor cells during metastasis.

Appearance of a proteoglycan in developing sea urchin embryos.

It was shown that a proteoglycan is synthesised by embryos of a Japanese sea urchin, Hemicentrotus pulcherrimus. This proteoglycan appears as a single peak on sucrose density gradient ultracentrifugation throughout the development. About half of the mucopolysaccharide moiety in this proteoglycan was found to be dermatan sulphate and the rest to be chondroitinase-resistant mucopolysaccharides. Evidence is presented to show that both types of mucopolysaccharide do not exist in a free form but reside as an integral part of the proteoglycan. The linkage between mucopolysaccharide and protein moieties of the proteoglycan appeared not to be an O-glycosidic bond, which is common among other proteoglycans such as proteochondroitin sulphate and proteodermatan sulphate.

Highly sulfated proteochondroitin sulfates synthesized in vitro by rat glomeruli.

A previous report from this laboratory (Kobayashi, S., Oguri, K., Kobayashi, K. and Okayama, M. (1983) J. Biol. Chem. 258, 12051-12057) indicated that isolated rat glomeruli synthesized three species of sulfated glycoconjugates in vitro, namely, sulfated glycoproteins, proteoheparan sulfates and proteochondroitin sulfates. In the present study, the proteochondroitin sulfates, which showed the greatest incorporation of [35S]sulfate among the three sulfated glycoconjugates, were isolated and characterized. Radiolabeled tissue proteochondroitin sulfates were clearly separated on Sepharose CL-6B into three components with partition coefficients (Kd) of 0.16, 0.22 and 0.58, and medium proteochondroitin sulfates were separated into two components with Kd values of 0.33 and 0.62. When the chondroitin sulfate chains released by alkaline borohydride treatment were analyzed by digestion with chondroitinase AC-II, chondroitinase ABC, chondro-6-sulfatase and chondro-4-sulfatase, the results showed that all the samples contained glucuronosyl-N-acetylgalactosamine (chondroitinase AC-II-susceptible sequences, 72-86%) and iduronosyl-N-acetylgalactosamine (chondroitinase ABC-susceptible sequences, 14-28%), containing 4-sulfated N-acetylgalactosamine (50-70%) and 4,6-disulfated N-acetylgalactosamine (30-50%). On two-dimensional electrophoresis on cellulose acetate, all samples gave a single spot which closely coincided with chondroitin sulfate E of squid cartilage in electrophoretic mobility. These results indicated that the chains were highly sulfated chondroitin sulfates containing glucuronic acid and iduronic acid residues.

Synthesis and pharmacological activity of a phosphate ester of delta8-tetrahydrocannabinol.

A water-soluble phosphate ester of delta8-tetrahydrocannabinol (delta8-THC) was synthesized and its pharmacological activities were examined. The cataleptogenic and thiopental sleep-potentiating effects of delta8-THC phosphate in the mouse was approximately 10 and 7% of those of delta8-THC, respectively. However this phosphate showed almost the same potency and a longer duration of hypothermic effect, as compared with delta8-THC in the mouse. The acute toxicity of this phosphate was far lower than that of delta8-THC. delta 8-THC phosphate was difficultly hydrolyzed by alkaline phosphatase or mouse liver homogenate in vitro. The mode of action of the phosphate derivative is discussed in connection with this enzymatically difficult hydrolysis.

Picrotoxin as a potent inducer of rat hepatic cytochrome P450, CYP2B1 and CYP2B2.

The induction by the central stimulant picrotoxin of hepatic drug-metabolizing enzymes was studied in rats. The hepatic content of P450 and the activity of benzphetamine N-demethylation increased gradually after administration of picrotoxin dissolved in drinking water (2 mg/mL), to three-times higher levels than the initial values at the third day of treatment. The increase in benzphetamine N-demethylase activity by picrotoxin was somewhat higher than the increase produced by phenobarbital. Supporting these results, immunoblot analysis showed that CYP2B1 and 2B2 proteins in the liver microsomes were increased by picrotoxin Picrotoxinin and picrotin, which are components of the picrotoxin molecule, had the same ability to induce the hepatic activity of benzphetamine N-demethylation. The liver microsomal activities of testosterone 16 alpha- and 16 beta-hydroxylation were enhanced significantly after treatment with picrotoxinin and picrotin. However, benzo[a]pyrene 3-hydroxylation, aniline 4-hydroxylation, and testosterone hydroxylations at the 2 alpha- and 7 alpha-positions were not increased by picrotoxinin and picrotin treatment. In addition to monooxygenase, significant induction of glutathione S-transferase activity for 1-chloro-2,4-dinitrobenzene and UDP-glucuronyltransferase activity for 4-hydroxybiphenyl and 4-nitrophenol was also observed by pretreatment of picrotoxin. These results clearly indicate that picrotoxin is an inducer of phenobarbital-inducible liver enzymes.

Purification and characterization of dog liver microsomal epoxide hydrolase.

An epoxide hydrolase (mEH) in liver microsomes was purified to apparent homogeneity from a dog treated with phenobarbital. The purified enzyme had a minimum molecular weight of 47,000 as determined by SDS-PAGE. The dog mEH activity was characterized by use of a substrate, 7-glycidoxycoumarin (GOC), and some effectors of this enzyme. In vitro activators, metyrapone, and isoquinoline, stimulated the microsomal activity, but the former had no such effect on the purified enzyme in case of this substrate. All mEH inhibitors, 1,1,1-trichloropropene 2,3-oxide (TCPO), cyclohexene oxide, and 2-bromo-4'-nitroacetophenone (BrNAP), suppressed hydrolase activity. The NH2-terminal amino acid sequence of the purified enzyme was highly homologous (90%) to the sequences deduced from a cDNA clone of rat enzyme. Antiserum to the purified enzyme raised in rabbits cross-reacted with rat and guinea pig epoxide hydrolases. No gender-difference in this enzyme in liver microsomes was observed in dogs.

Membrane-intercalated proteoglycan of a stroma-inducing clone from Lewis lung carcinoma binds to fibronectin via its heparan sulfate chains.

A Lewis lung carcinoma-derived low metastatic clone, P29, with a capacity to induce a fibrotic stromal response of host tissue, exhibits tumorigenesis depending on an interstitial matrix formed by the induced stromal cells. Using this clone, in the present study we isolated and characterized a membrane-intercalated proteoglycan that mediates interaction between the tumor cells and interstitial matrix. The tumor cells were cultured in the presence of [3H]glucosamine and [35S]sulfate or [35S]methionine, and hydrophobic proteoglycans were isolated by chromatography on DEAE-Sephacel and then Octyl-Sepharose CL-4B. Proteoglycans with high affinity to the octylresidue were obtained from the cell layer but not to any significant extent from the medium. By CsCl density gradient centrifugation, they were separated into bottom, middle, and top subfractions, which were shown to consist of homogeneous species with estimated M(r) values of 270,000 (named CPGIIIB), 200,000 (CPGIIIM), and 195,000 (CPGIIIT), respectively, by gel filtration on Sepharose CL-4B. These proteoglycans were intercalated into phosphatidylcholine liposomes, suggesting that they are all membrane-intercalated proteoglycans. Analyses of their glycosaminoglycans with chondroitinase ABC and heparitinase I plus II demonstrated that they all contain heparan sulfate as a major glycosaminoglycan (58-85%) and chondroitin 4-sulfate as a minor one (15-42%). Of these three proteoglycans, only CPGIIIB proteoglycan bound specifically to fibronectin-Sepharose 4B under physiological conditions. Molecular analyses of this proteoglycan by Sepharose CL-4B or SDS-PAGE before and after treatments with glycosaminoglycan degradation enzymes or trifluoromethanesulfonic acid demonstrated that CPGIIIB proteoglycan is a hybrid proteoglycan having heparan sulfate and chondroitin 4-sulfate chains on the same core protein with an M(r) of 40,000. Affinity chromatographies of the CPGIIIB proteoglycan on fibronectin-Sepharose 4B after treatments with these enzymes demonstrated that it bound to fibronectin via its heparan sulfate chains. On the basis of the above results, we propose that the CPGIIIB proteoglycan mediates the interaction between the tumor cells and interstitial matrix.

The localization of sulfated glycoconjugates synthesized by the corneal epithelium of chick embryos.

The localization of sulfated glycoconjugates in the corneal epithelium of 19-d-old chick embryo was investigated biochemically using epithelia labeled in vitro with [35S]sulfate, which exhibited autoradiographically a similar distribution of silver grains to that labeled in ovo. The radiolabeled tissues were dissociated into single cells by incubation in 0.25% trypsin containing 0.02% EDTA at 37 degrees C for 40 min. The proteoglycans and sulfated glycoproteins which were associated with the cells and those released into the dissociation medium were separated by DEAE-Sepharose CL-6B and analyzed on Sepharose CL-6B and SDS-PAGE. About 86% of the proteoglycans was released into the dissociation medium and more than 50% of the cell-associated ones was affected by trypsin. This indicates that the proteoglycans are mostly localized in an extracellular compartment. On the other hand, the extent of release of sulfated glycoproteins into the medium on dissociation of tissues was distinctly different depending upon their molecular weight (Mr): almost all of the sulfated glycoproteins of the family with Mr 48,000-70,000 (32% of the total sulfated glycoproteins) were recovered as intact molecules with the cells, whereas approximately 50% of those with Mr 70,000-150,000 (36%) and about 70% of those with Mr over 150,000 (28%) were released into the dissociation medium. These results indicate that the family with Mr 48,000-70,000 is localized intracellularly and that with Mr 70,000-150,000 in a compartment poorly affected by trypsin; in contrast to those, that with Mr more than 150,000 is localized in an extracellular compartment like the proteoglycans.

Sulfated glycoproteins synthesized by the corneal epithelium of chick embryo having the same molecular weights as cytokeratin polypeptides.

The previous study from this laboratory demonstrated that the corneal epithelium of 19-d-old chick embryo synthesizes two classes of sulfated glycoconjugates consisting of sulfated glycoproteins and proteoglycans (Yonekura, H., Oguri, K., Nakazawa, K., Shimizu, S., Nakanishi, Y., & Okayama, M. (1982) J. Biol. Chem. 257, 11166-11175). The present study demonstrated that when the sulfated glycoproteins labeled metabolically with [35S]sulfate and [3H]glucosamine were analyzed by SDS-PAGE, the 70,000 component (accounting for approximately 30% of the 35S and 35% of the 3H of the total sulfated glycoprotein) co-migrated with five major proteins with apparent molecular weights (Mrs) of 70,000, 66,000, 58,000, 51,000, and 48,000, which together accounted for about 57% of the total tissue protein. All five proteins cross-reacted with an antibody against human sole keratin, indicating that they are cytokeratin polypeptides of the corneal epithelium. Amino acid analysis demonstrated that they had high contents of glycine, serine, glutamic acid, leucine, and aspartic acid. Two-dimensional tryptic peptide maps indicated that they were all different. Analysis of radiolabeled materials released by alkaline borohydride treatment of the sulfated glycoproteins which were synthesized in the presence and absence of tunicamycin and co-purified with the five cytokeratin polypeptides, revealed that they contained both N- and O-glycosidically linked sulfated oligosaccharides. All the results obtained in the present study indicate that the five sulfated glycoproteins are similar, if not identical, to the cytokeratin polypeptides. This is consistent with the result in the accompanying paper that these sulfated glycoproteins are localized intracellularly.

Potentiation of physical dependence by conjugation at the 6-position of nalorphine.

The experiments concerned the effects of glucuronate or sulfate conjugation at the 6-position of nalorphine on the analgesic and antagonistic activities and also on the development of tolerance and physical dependence. Nalorphine-3-and 6-sulfate ester were synthesized for the first time. The analgesic effect of nalorphine-6-sulfate and -glucuronide was higher than that of nalorphine when assessed in the acetic acid writhing test. However, these 6-conjugates exhibited less potent agonistic activity in the test with guinea-pig ileum muscle strip and revealed no analgesic effect in the tail pinch test. The antagonistic activity of these 6-conjugates to morphine analgesia was lower on their s.c. injection, but higher on i.c.v. injection than that of nalorphine. The development of tolerance to the analgesia caused by nalorphine was not affected by the 6-modifications. Frequent withdrawal signs were seen in mice treated chronically with anlorphine-6-conjugates by challenging with naloxone while mice treated with nalorphine showed no such signs. This potent enhancing effect of 6-conjugation on the development of physical dependence was suggested to be also the case with morphine. These changes of potency due to conjugation were interpreted as due to the altered interaction with multiple opioid receptors.

Simple and practical sandwich-type enzyme immunoassay for human oxidatively modified low density lipoprotein using antioxidized phosphatidylcholine monoclonal antibody and antihuman apolipoprotein-B antibody.

OBJECTIVES: To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis. DESIGN AND METHODS: A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL). RESULTS: This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3-7.7%), accuracy (recovery: 90.6-103.8%), simplicity and rapidity (<4 h). Clinical performance of the assay was assessed by measurement of the Ox-LDL in the plasma of normal subjects (10.8 +/- 2.8 U/mL, mean +/- SD) and patients with coronary heart disease (CHD) (19.7 +/- 10.2). The present EIA had a sensitivity of 79% and a specificity of 75% for CHD. CONCLUSIONS: We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay.

Significant suppression of rat liver aldolase B by a toxic coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl.

A toxic coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl (PenCB), significantly suppresses the expression of liver aldolase B in rats. Hepatic aldolase activity in PenCB-treated rats was significantly reduced to about 50% of that in free- and pair-fed control groups. The reduced aldolase activity following PenCB-treatment was due to the marked suppression of the expression of aldolase B shown by immunoblot analysis after SDS-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. The suppression of rat liver aldolase B could be a key biochemical lesion caused by PenCB.

Characteristic glucuronidation pattern of physiologic concentration of morphine in rat brain.

Formation of conjugated metabolites from morphine at a very low level in brain was studied in vitro in rats. Incubation of a low concentration of 3H-morphine with brain homogenate followed by two successive high-performance liquid chromatographic analyses showed that endogenous morphine is converted by brain enzymes to its 3- and 6-glucuronides (M-3-G and M-6-G), and codeine glucuronide (Cod-G). However, the formation of morphine-6-sulfate was likely to be low if it was produced at all. All of the cerebral hemisphere, brain stem and cerebellum were capable of producing M-3-G, M-6-G and Cod-G, although there were differences in selectivity. The capacity of the brain for glucuronide formation was far less than that of the liver, but UDP-glucuronosyltransferase in brain was much more selective in forming M-6-G and Cod-G than liver enzymes.

Enhanced binding of morphine and nalorphine to opioid delta receptor by glucuronate and sulfate conjugations at the 6-position.

Effect of the modification of morphine and nalorphine by glucuronate and sulfate conjugations at the 3- and 6-positions on the binding to opioid receptors was examined in a particulate fraction of rat brain. Competing potencies of both drugs against [3H]morphine and [3H]leucine enkephalin bindings were extremely decreased by either glucuronate or sulfate conjugation at the 3-position. On the other hand, the potencies of morphine and nalorphine against [3H]leucine enkephalin binding were considerably enhanced by the conjugations at the 6-position, whereas the potencies against [3H]morphine binding were decreased. These altered interactions of the conjugates at the 6-position with the two ligands were attributed to their enhanced binding to delta-receptor and reduced binding to mu-receptor by Hill plot and modified Scatchard analysis. Resulted comparable and simultaneous interactions with mu- and delta- receptors were assumed to be a cause of the enhanced mu-receptor-directed analgesia of morphine and elevated same receptor-directed antagonistic effect of nalorphine, which have been found previously in our laboratory.

A highly toxic PCB produces unusual changes in the fatty acid composition of rat liver.

The changes in lipid metabolism produced by a coplanar PCB were studied in rats. Male Wistar rats were given a single intraperitoneal injection of 3,3',4,4',5-pentachlorobiphenyl at a dose of 25 mg/kg. After 5 days of administration, total hepatic lipids were treated with 1 M KOH in methanol at 75 degrees C and the liberated fatty acids were analyzed by HPLC after conversion to fluorescent derivatives. In comparison with free-fed and pair-fed control groups, the proportion of arachidonic acid in the PenCB-treated rats was reduced by about 50%, while oleic and linoleic acids increased significantly. We also examined the individual glycerophospholipids, separated by TLC, to see if they were affected by alteration in the fatty acid composition of the whole liver. In all glycerophospholipids, the proportion of arachidonic acid was reduced significantly to the same degree while linoleic acid increased. Changes in the activity of desaturase isozymes have been postulated to explain this unusual lipid metabolism following administration of a toxic PCB and this may contribute to its toxicity.

Significant induction of a 54-kDa protein in rat liver with homologous alignment to mouse selenium binding protein by a coplanar polychlorinated biphenyl, 3,4,5,3',4'-pentachlorobiphenyl and 3-methylcholanthrene.

A 54-kDa protein in rat liver cytosol was significantly induced by treatment with 3,4,5,3',4'-pentachlorobiphenyl (25 mg/kg, single i.p.) and 3-methylcholanthrene (20 mg/kg, once a day for 3 days, i.p.). The protein exhibited pI of 6.8 on two-dimensional gel electrophoresis. The amino acid sequences of peptide fragments from the protein digested in situ were highly similar to a selenium binding protein in mice and to the isoform acetaminophen binding protein in mice. The present result clearly demonstrates that a coplanar polychlorinated biphenyl and 3-methylcholanthrene are responsible for induction of selenium binding protein homologues. The physiological role of the mouse proteins, however, is not yet elucidated.

A coplanar PCB induces a selenium binding protein as a major cytosolic protein in rat liver.

We obtained evidence that a toxic coplanar polychlorinated biphenyl (PCB) induces a counterpart of murine 56kDa selenium binding protein in rat liver cytosol. A 54kDa protein in the liver cytosol was significantly induced by 3,3',4,4',5-pentachlorobiphenyl and proved to be a major cytosolic protein in the rat liver. The protein exhibited pI of 6.8 on two-dimensional gel electrophoresis. The amino acid sequence of peptide fragments from the protein digested in situ, was highly similar to a 56kDa selenium binding protein and similar to an acetaminophen binding protein in mice.

Involvement of cytochrome b5 in the metabolism of tetrachlorobiphenyls catalyzed by CYP2B1 and CYP1A1.

The role of cytochrome b5 in the cytochrome P450 (CYP)-dependent hydroxylation of tetrachlorobiphenyl (TCB) isomers was examined using a reconstituted mixed function oxygenase (MFO) system containing purified CYP2B1 or 1A1, and rat liver microsomes. Hydroxylations of 2,2',5,5'- and 3,3',4,4'-TCBs were catalyzed mainly by CYP2B1 and 1A1, respectively, in the reconstituted MFO system and those of 2,3',4',5- and 2,3',4,4'-TCBs were mediated by both cytochrome P450 systems. The activity toward 2,2',5,5'- and 2,3',4',5-TCB was significantly increased 6.5- and 5.5-fold, respectively, by addition of cytochrome b5 in the reconstituted MFO system containing of CYP2B1. Either hydroxylation activity toward 2,3',4,4'-TCB with the CYP2B1 system was very low or decreased by addition of cytochrome b5. These results suggest that the involvement of cytochrome b5 to the hydroxylation of TCBs is dependent on the TCB congener being metabolized, and the cytochrome P450 isoform involved in its metabolism.

Species-specific alteration of hepatic glucose 6-phosphate dehydrogenase activity with coplanar polychlorinated biphenyl: evidence for an Ah-receptor-linked mechanism.

We examined the in vivo effect of a highly toxic coplanar polychlorinated biphenyl (PCB) on the hepatic activity of glucose 6-phosphate dehydrogenase (G6PDH) in aryl hydrocarbon (Ah)-responsive (C57/BL) and -less-responsive (DBA) strains of mice. The activity in the C57BL strain was moderately increased by 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in a dose dependent manner. However, this was not observed in DBA mice although greater doses were injected. 2,2',5,5'-Tetrachlorobiphenyl (PCB 52) with a non-planar structure did not increase G6PDH activity. The increase in G6PDH activity with PCB 126 was also seen in rats, but not in guinea pigs. The activity in the latter species was decreased rather than increased. These results suggest that the induction of hepatic G6PDH by coplanar PCB is mediated by a mechanism involving the Ah receptor, and the response was highly species-specific.