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Structure-activity relationship studies of several morphinan derivatives were conducted to obtain dual antagonists for μ- and δ-opioid receptors. We discovered peripherally restricted dual antagonists for μ/δ-opioid receptors as a new chemotype with a morphinan scaffold, which are orally available and do not easily pass the blood–brain barrier. As we expected, some of these compounds inhibit opioid-induced constipation and emesis/vomiting with limited potential to interfere the analgesic effects of morphine. Among them, naldemedine was selected as a potential drug candidate.

NR2B subunit containing N-methyl-d-aspartate (NMDA) receptor is an attractive target for chronic pain due to its involvement in disease states and its limited distribution in the central nervous system. Cyclohexanol-based leads 6a and 6c were identified as potent NR2B-selective NMDA antagonists utilizing a scaffold hopping approach. Further optimization of this series through replacement of the amide in the leads with an isoxazole and efforts to optimize the pharmacokinetic profiles led to the discovery of orally available brain penetrants 7k and 7l, which demonstrated analgesic activity in the mouse formalin test at early and late phases.

Cultured broth of Saccharopolyspora erythraea shows a strong red fluorescence. The main fluorescent component was identified as uroporphyrin I by FAB-MS and 1H-NMR. This was confirmed by a feeding experiment using [5-13C]aminolevulinic acid.

The activity of phospholipase D from the Savoy cabbage on alkyl phosphorylcholines containing alkyl groups of C14 to C22 was examined. Apparent Km and maximal velocity values in the presence of diethyl ether were 3.6–8.3 mM and 0.14–0.29 μmol/(min∙mg protein), respectively. The maximal velocities for alkyl phosphorylcholine homologues were lower than the value for pig phosphatidylcholines, but higher than those for lysophosphatidylcholines and synthetic phosphatidylcholine with saturated acyl chains. Results suggest that solubilities of the substrates in the organic solvent play an important role in activity observed with phospholipase D.Key words: phospholipase D, alkylphosphorylcholines, apparent Km values, Vmax values, diethyl ether.

In five experiments of the same design using silages made from wilted Italian ryegrass (dry matter 22.7–40.9%), the effects of caproic acid (C6) (10 mmol kg−1) with and without 6N-HCl (20 ml kg−1), applied at ensiling or at opening, in preventing aerobic deterioration of silages after opening silos were investigated. Application of C6 either at ensiling or at opening was partially effective in preventing temperature increase, pH rise and breakdown of organic acids during the 7-day aerobic exposure period after opening the silos. Addition of HCl reduced the pH of silages irrespective of the time of application. However, no improvement of the effect of C6 due to HCl supplementation was observed as far as the prevention of the aerobic deterioration was concerned. Treatment with C6+ HCl at ensiling inhibited silage fermentation more extensively than C6 alone. Viable counts of yeasts and moulds in the silages treated with C6 or C6+ HCl at ensiling tended to be lower than those in the control silages. Considerable increases in the counts of moulds were found in the control silages during the aerobic exposure period.

A combination method consisting of separation by HPLC using gel permeation columns and selective detection of cholesterol or choline-containing phospholipids was applied to the identification and quantitation of subclasses of high density lipoproteins in human serum. The frequency distribution of peaks in the elution patterns on detection of choline-containing phospholipids in a group (n = 71) of normal males and females showed five maxima. Their particle sizes were determined from the relation between Stokes' diameter and elution volume to be as follows: 122.0 +/- 2.8 A, 110.1 +/- 2.1 A, 97.5 +/- 1.8 A, 86.7 +/- 1.3 A, and 76.3 +/- 16 A. The two larger fractions were found to correspond to HDL2 subclasses, i.e., HDL2b and HDL2a. The other three peaks were found to reflect the subclasses of HDL3 and choline-containing phospholipids in the very high density lipoprotein fraction which sedimented at the bottom on ultracentrifugation at the density of 1.21. Moreover, the existence of these five subclasses in the HDL fraction was confirmed by rechromatography using this combination HPLC method. Our results for the particle sizes of HDL subclasses were confirmed by electron microscopy.

Hemolytic activities of the mixed micelle of phosphatidyl choline with either sodium cholate or sodium deoxycholate were investigated.Phosphatidyl choline showed a strong inhibitory effect on the hemolytic activity of both bile salts. It formed a mixed micelle with both bile salts, but its inhibitory activity was greater with sodium cholate than with sodium deoxycholate.

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.

On-line detection of cholesterol in each human serum lipoprotein class could be performed by combining a bioreactor system prepared by immobilizing cholesterol ester hydrorase and cholesterol oxidase on the same matrix with a metal free size-exclusion chromatographic system separating different classes of lipoproteins.

Radiotracer studies of calcium adsorption to dipalmitoylphosphatidyl-alkanolamine monolayers measured at various temperatures showed that the binding constant of calcium increased with temperature up to around 30°C but then decreased on exceeding this critical temperature. The temperature dependent ionic structure of ampholytic phospholipid monolayers are discussed.

A convenient method for the quantitation of choline-containing phospholipids in each lipoprotein fraction has been developed by combining separation by high-performance liquid chromatography with gel permeation columns and selective detection by enzymatic reaction in the post-column effluent. The elution patterns monitored by choline-containing phospholipids were compared with those monitored by cholesterol. The elution patterns of choline-containing phospholipids were found to give much more information about the distribution of lipoproteins according to their particle-size differentiation than analyses done by cholesterol. This choline-containing phospholipid monitoring method not only resolves lipoprotein peaks of the major classes (chylomicron + VLDL, LDL, HDL2 and HDL3) quantitatively, but also detects the presence of abnormal lipoproteins containing a large amount of choline-containing phospholipids. We could detect these abnormal lipoproteins using a small amount of whole serum (10–20 μl) from patients with various liver diseases. Our examination of HDL subclasses using this technique showed that the HDL fraction was composed of several subfractions due to their particle-size differentiation.

A determination method for choline-containing phospholipids in serum lipoproteins was established by application of the HPLC method [Hara et al. (1980) J. Biochem. 87, 1863-1865; (1981) 89, 879-887] developed for cholesterol quantitation. The concentration of choline-containing phospholipids in the fraction separated by HPLC using gel permeation columns was determined through colorimetric detection using a commercial enzymatic kit. The optimum conditions for enzymatic reaction in the flow diagram were examined. The concentration calculated from the A500 peak area was found to reflect very precisely the concentration of choline-containing phospholipids in all lipoprotein fractions, and the quantitation of each lipoprotein fraction can be performed with only 10-20 microliters of whole serum. Monitoring the elution patterns by detecting choline-containing phospholipids can give much more information about lipoprotein distributions according to particle size than analyses done by detecting cholesterol.

Surface pressures and surface viscosities of dipalmitoylphosphatidylethanolamine and its polar group analogues were measured at the hexane/water interface as a function of bulk pH. The monolayers expanded as the bulk pH was shifted from neutral to alkaline and gave rise to an increase in surface pressure at a constant area, and the surface viscosity was simultaneously reduced at high pH. For the increase in surface pressure, theoretical values were calculated using the Gouy-Chapman equation for the electrical double layers produced by shifting pH, and good agreements were obtained with the measured ones from which a simple mechanism was deduced for the increase in pressure. The ionic dissociation characteristics of amino groups of the lipids were discussed taking p K a values given in the above calculations into account. The reduction of the surface viscosity was thought to be attributable to disintegration of zwitter ionic structure in the condensed monolayers.

The surface pressures and the surface viscosities of lecithin, cephalin and its analogs were measured at the air—water and the oil—water interfaces. It was found that the surface viscosity of the phospholipids used in this study was very high, and was comparable to those of some polymer films at the oil—water interface as well as at the air—water interface under the conditions where the monolayers were condensed. The plateaus indicating the phase transitions in monolayers were clearly observed on the pressure-area curves at the oil—water interface in all of the specimens studied. It was found that the phase transitions exactly corresponded to the abrupt increases in surface viscosity. From the results thus obtained, an intermolecular ionic linkage between neighboring molecules in the monolayers is discussed.

Nephrotoxicity of some aromatic nitro-amino compounds were evaluated by urinary enzyme activities and renal histopathological changes. Male Fischer 344 rats were intraperitoneally injected with aniline, p-aminophenol, acetyl-p-aminophenol, p-chloroaniline, p-chloronitrobenzene, p-anisidine, or p-nitroaniline at 1.0 mmol/kg. In the rats injected with p-aminophenol, necrosis of renal tubular epithelial cells and remarkable elevation of urinary N-acetyl-beta-D-glucosaminidase (NAG) and gamma-glutamyltranspeptidase (gamma-GTP) activities were observed. Injection with p-chloroaniline caused significant elevation of the urinary NAG and gamma-GTP activities. p-Anisidine and p-nitroaniline induced swelling of the tubular epithelial cells and a significant elevation in urinary NAG activities in rats, which was also caused by p-chloronitrobenzene. However, administration of aniline or acetyl-p-aminophenol did not change either the urinary enzymes or renal histopathology. These results indicate that p-aminophenol is a highly nephrotoxic substance, and that nephrotoxicity of p-chloroaniline, p-chloronitrobenzene, p-anisidine and p-nitroaniline exceed that of acetyl-p-aminophenol which has been known to cause a renal damage.

A simple and rapid method has been developed for the separation of apolipoproteins in high density lipoprotein (HDL) fractions by high performance liquid chromatography (HPLC) with gel permeation columns (G3000SW TSK GEL). The HPLC pattern monitored by A280 for a mixed solution of the HDL fraction (10 microliter) and an eluent buffer (200 microliter, 0.1 M sodium phosphate buffer containing 0.1% SDS) incubated at 60 degrees C for 5 min showed two completely separated peaks which corresponded to the major components of human HDL, apolipoprotein A-I and A-II. Moreover, quantitation of apolipoprotein A-I by our method was found to correlate well with that by a single radial immunodiffusion (SRID) assay.

A simple and rapid method for the determination of cholesterol in human serum lipoproteins (VLDL, LDL, HDL2, and HDL3) was described. The contents of cholesterol of lipoprotein fractions from a very small amount of serum (20 microliter) can be determined by the enzymatic assay after separation by high performance liquid chromatography in less than 50 min. The method is very useful for studying lipoprotein metabolism and related diseases.

Cardiolipin was prepared from the frozen heart muscle of fin whale according to Pangborn's method. Every step in the course purification was pursued by T.L.C., and the sample after the last step showed only single spot corresponding to cardiolipin on T.L.C. From chemical analysis it's characteristics were as follows : P (%) -4.2%, N (%)

A simple and rapid method for the quantitation of cholesterol in human serum lipoproteins (VLDL, LDL, HDL2, and HDL3) was developed (1). The content of cholesterol in each lipoprotein fraction was determined by means of a commercial enzymatic reaction kit after separation by high performance liquid chromatography with gel permeation columns. The quantitation of cholesterol could be performed with only 10-20 mu l of serum in less than 50 min by measuring A550 after passing the mixed eluate and enzyme solution through an on-line reactor system of a high-speed chemical derivatization liquid chromatograph. The precision, reproducibility and sensitivity of the quantitation of cholesterol with this method were acceptable, and the values of HDL-cholesterol determined by this method correlated well with those found by the heparin-manganese chloride precipitation method (r=0.958, n=93).

We succeeded in the separation of human serum high density lipoprotein into their subfractions (HDL2 and HDL3) by high performance liquid chromatography with gel permeation columns (1, 2). The cholesterol contents in HDL2 and HDL3 for normal men and women (n = 42) and for the patients with atherosclerotic heart disease (n = 20) were measured by this separation method. The results in our experiment agree closely with those reported by other investigators using the ultracentrifugal method.

The solubility of phosphatidylcholine (PC) was studied by the spectroscopic analysis and the measurement of the solubility. The qualitative analysis of infrared absorption spectra confirmed the existence of two types of hydrogen bondings between chloroform and PC, one between chloroform and the C=O group of PC and the other between chloroform and the phosphorylcholine group of PC. The quantitative analysis of the C-D stretching vibration bands of the chloroform-d solution of PC showed that the latter hydrogen bonding mainly contributes to the solubility and that PC dissolves in chloroform to form a complex consisting of a few or more molecules of chloroform and one molecule of PC. We discussed in this report about the molecular organization of PC in chloroform solution.

Aromatic nitro-amino compounds are used as raw materials of dyes, rubber, pesticides and drugs. A common toxicological reaction to these compounds is the formation of methemoglobin followed by cyanosis and anemia. Little is known about renal damage in workers occupationally exposed to aromatic nitro-amino compounds. Recently, certain urinary enzyme activities have been measured as an index of renal damage. In animal experiments, the activity of urinary N-acetyl-{beta}-D-glucosaminidase (NAG) was increased in rats given p-aminophenol. The authors assay urinary enzyme activities of workers handling aromatic nitro-amino compounds, and discuss the nephrotoxic effects of these compounds used industrially.

The chemical synthesis of two new glycerophosphatide analogues containing steroid groups, i.e., 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3′β-cholesterol and 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-20′-(3β-hydroxy norpregn-5-ene) is described.

The effect of dietary protein quality on chlorobenzene metabolism and its toxicity was studied. Male weanling Wistar rats were fed diets composed of purified whole egg protein (PEP), wheat gluten, or soybean protein isolate (SPI) at an 18% protein level. After being fed for 3 weeks, the rats were injected with chlorobenzene (1.0mmol/kg body weight) intraperitoneally. In the rats fed the SPI diet, chlorobenzene administration caused hepatic microscopic changes and the elevation of serum GOT and GPT activities. On the other hand, the rats fed the PEP or the gluten diet did not show these toxic symptoms. The hepatic glutathione levels and urinary 4-chlorophenylmercapturic acid excretion of the rats fed SPI diet were markedly lower than those of the other rats. It is considered that the hepatic glutathione level was not sufficient for the conversion of chlorobenzene administered to the mercapturic acid in the rats fed the SPI diet. These results indicate that chlorobenzene metabolism and its toxicity are modified by dietary protein quality, especially the level of dietary sulfur-containing amino acids.

Immunologically pure lipoprotein fractions of human serum were subjected to high performance liquid chromatography (HPLC) on a combination system of aqueous gel permeation columns. Each lipoprotein fraction appeared at a distinct elution volume as a single homogeneous peak. Elution was carried out with 0.1 M Tris-HCl buffer solution, pH 7.4, or 0.15 M NaCl solution and no significant differences in elution volume and elution pattern were found. The lipoprotein mixtures (total lipoprotein fraction d less than 1.210) of healthy and pathological subjects were subjected to HPLC and showed characteristic elution patterns which could be of diagnostic significance.

Effects of chlorobenzene-induced alteration in glutathione levels on cysteine metabolism in rat liver were investigated. Male Wistar rats were intraperitoneally injected with chlorobenzene (0.2, 0.5, 1.0 or 2.0 mmol/kg body weight). Both hepatic glutathione and cysteine levels were dose-dependently decreased by the chlorobenzene 6 h after the injection. However, at 24 h, the glutathione in the rats with chlorobenzene increased significantly as compared to that in the rats without chlorobenzene. Concomitant to the elevation in glutathione levels, hepatic glutathione synthesis activities were increased by the chlorobenzene by 68-111%. On the other hand, no significant difference between the rats with and without chlorobenzene was observed as regards cysteine levels at 24 h. Hepatic glutamate, glycine, methionine and serine levels were unaltered but hepatic taurine levels were significantly decreased by the chlorobenzene at both 6 and 24 h. Chlorobenzene administration had no effect on hepatic cystathionine synthase and cystathionase activities. These results indicate that a transient loss of hepatic glutathione, caused by the administration of chlorobenzene, resulted in an acceleration of glutathione synthesis and an increase of cysteine demand in the liver.

1. thermal phase transitions were detected in monolayers of synthetic phospholipids by surface viscosity measurements. 2. The following transition temperatures were found for pure films: 35.5 °C for dipalmitoylphosphatidylethanolamine, 31 °C for dipalmitoylphosphatidyl-n-propanolamine , 29 °C for dipalmitoylphosphatidyl-i-propanolamine , and 8 °C for dipalmitoylphosphatidylcholine. 3. In mixed films of each of three phospholipids consisting of primary amine and dipalmitoylphosphatidylcholine, the transition temperatures are lowered as the proportions of the latter are increased, according to the law of melting point depression. The results were considered to show that they were separated into two phases in the solid films. 4. In mixed films of primary amine-containing phospholipids only, it is inferred that they exist in a homogeneous single phase even in the solid films. 5. It was deduced that the choline group cannot group cannot be introduced into the two-dimensional ionic lattice formed by primary amine and phosphate, because of a steric hindrance caused by their different end group sizes.

1. 1. Three kinds of dipalmitoyl dl -1-phosphatidyl-alkanolamines were synthesized in a pure state and the monolayer properties of these compounds and of the synthetic phosphatidylcholine were measured. Their conformations in monolayer in relation to the effects of the chemical structures of polar groups upon their surface parameters were discussed. 2. 2. Distinct differences in limiting areas were observed in the phosphatidylethanolamine group. This could be interpreted by assuming the parallel orientation of the polar moieties to the interface as predicted by M. M. Standish and B. A. Pethica (Trans. Faraday Soc., 64 (1968) 1113). It was also suggested that in phosphatidyl choline the polar moiety was more inclined to the interface at least at ordinary room temperature and near the isoelectric point. 3. 3. These conformations, however, are largely altered by and dependent on temperature. For example, phosphatidylethanolamine at 40° behaves in monolayer like phosphatidyl choline at 25°. 4. 4. Effects of UO22+ and Th4+ on π-A curves and ΔV-A curves were interpreted by assuming the above conformation for polar moieties in monolayer.