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1-(5-Dimethylamino-1-naphthalenesulphonyl)-(S)-3-aminopyrrolidi ne (DNS-Apy) has been synthesized for the separation of carboxylic acid enantiomers by high-performance liquid chromatography (HPLC) and sensitive detection. The reagent reacts with carboxylic acids at room temperature in the presence of activation agents 2,2'-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP). The maximum emission of the diastereomeric amide derived from (S)-phenylpropionic acid and ketoprofen derivatives of DNS-Apy was at 530 nm with excitation at 340 nm. The emission wavelength shifted towards the blue and the fluorescence intensities increased with increasing acetonitrile concentration in the medium. The diastereomers derived from anti-inflammatory drugs were efficiently resolved with a reverse-phase column using water:acetonitrile mixture as mobile phase. All of the racemate of arylpropionic acid derivatives gave equal fluorescence intensity of the two enantiomers with the exception of ketoprofen derivatives where the intensity of the first eluting enantiomer was half that of the second.

Regional distribution of D-amino acids in rat brain was studied by the modified highly sensitive analytical method which was previously developed. The method includes fluorogenic derivatization of each amino acid, isolation of each amino acid by reverse-phase HPLC, followed by enantiomeric separation with Pirkle-type chiral stationary phases. D-Amino acid contents were determined in the cerebrum, cerebellum, hippocampus, medulla oblongata, pituitary gland and pineal gland. D-Aspartic acid was observed in the pineal gland (3524 +/- 263 nmol/g, data are for male rats of 6 weeks of age) and the pituitary gland (80.5 +/- 9.0 nmol/g). D-Serine was found in various regions of the brain except for the cerebellum and medulla oblongata. D-Alanine was observed exclusively in the pituitary gland (25.9 +/- 4.4 nmol/g), whereas D-leucine was found in the pineal gland (3.4 +/- 0.4 nmol/g) and the hippocampus (1.6 +/- 0.07 nmol/g). No other D-amino acids were detected in the brain. The contents of D-aspartic acid in the pituitary gland and D-serine in the pineal gland were higher in female rats. In contrast the contents of D-alanine in the pituitary gland and D-leucine in the pineal gland and the hippocampus were higher in males. Postnatal changes of D-aspartic acid and D-leucine in the pineal gland and D-alanine in the pituitary gland were also investigated. The results described in this paper suggested that distinct regulatory mechanisms exist for individual D-amino acids in the corresponding region of rat brain.

Dobutamine stress echocardiography has become a diagnostic tool for the evaluation of coronary artery disease and the detection of myocardial viability. In the diagnosis of significant coronary artery disease, it provides similar accuracy to exercise stress thallium-201 myocardial perfusion scintigraphy. Dobutamine stress echocardiography is also a promising modality for predicting the recovery of hibernating myocardium from contractile dysfunction after coronary angioplasty or bypass surgery. This article, reviews our recent clinical experience with the detection of coronary artery disease and viable myocardium by dobutamine stress echocardiography.

Specific polyclonal antibody was raised against D-aspartate (D-Asp) which had been conjugated to glutaraldehyde and was purified by affinity chromatography. Immunohistochemical staining of rat pineal gland with the antibody demonstrated the presence of D-Asp in the cytoplasm of pinealocytes, the predominant cell type in this gland. D-Asp immunoreactivity was more evident in the distal region than in the proximal region of the gland. Pinealocytes in the distal region are presumably involved in the synthesis and secretion of the pineal hormone, melatonin, and the results of staining may indicate some yet unknown role of D-Asp in the regulation of melatonin secretion.

Two cDNA clones of rat hepatic hydroxysteroid sulfotransferase (ST) (ST-40 and ST-20) were isolated and expressed in Escherichia coli cells. Several histidine residues in their coding regions are highly conserved in the ST superfamily, and histidine mutants were constructed by site-directed mutagenesis. The substitution of alanine or lysine for the histidine at position 98 in the ST-40 enzyme resulted in a loss of ST activities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). The mutation of histidine 98 into alanine abolished the specific binding to 3'-phosphoadenosine 5'-phosphate agarose, suggesting that the residue is located at a critical position in the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site. In the ST-20 enzyme, the replacement of histidine 98 with alanine also resulted in the loss of ST activity toward its preferential substrate, CS. In the ST-40 enzyme, the mutation at histidine 256 into alanine markedly reduced CS-ST activity, but DHEA-ST activity was not changed. Furthermore, selective decrease in CS-ST activity was also observed in the alanine mutant at lysine 254 or at asparagine 255 of the ST-40 enzyme. Kinetic analysis on the ST-40 and its mutant at asparagine 255 indicated that the Km value for CS was significantly increased in the mutant without any change in the Km values for 3'-phosphoadenosine 5'-phosphosulfate and DHEA. Inhibition studies demonstrated that DHEA-ST activity was competitively inhibited by AD, but not by CS in the ST-40 enzyme, whereas triethylamine, a noncompetitive inhibitor of hydroxysteroid ST, inhibited DHEA-ST activity in the ST-40 enzyme but did not inhibit CS-ST activity in either ST-40 or ST-20 enzymes. These data provide evidence that DHEA and CS bind to different sites, which probably function in a different manner in the ST-40 enzyme.

The highly sensitive detection of four near-infrared (near-IR) fluorescent dyes, methylene blue, pyridine 1, oxazine 1 and 3,3'-diethylthiadicarbocyanine iodide (DTDCI) by high-performance liquid chromatography (HPLC) with post-column peroxyoxalate chemiluminescence (PO-CL) detection utilizing bis(4-nitro-2-(3, 6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and hydrogen peroxide was examined. The detection limits for methylene blue, pyridine 1, oxazine 1 and DTDCI were 120, 27, 31, 0.19 fmol on-column at a signal-to-noise ratio of 2, respectively. The sensitivity for DTDCI was 250 times that obtained by HPLC with the conventional fluorescent detection.

We have immunolocalized phenol sulfotransferase (PST)G, an isoform of PST in sustentacular cells which reside in the dorso-medial portion of the nasal cavity of the mouse. The same topographical pattern of gene expression has been reported for some olfactory neuron-specific genes. When several established (phenol-containing) odorants were used as substrates, mouse nasal tissue cytosol showed a significant level of PST activity, as does mouse liver cytosol. This study is the first to demonstrate that gene expression in the olfactory sustentacular cells is also organized zonally, and indicates the involvement of sulfo-conjugation in olfactory perireceptor processes, such as odorant clearance and xenobiotic detoxification.

Boron trifluoride-etherate (BF3), one of the Lewis acids, was found to be an efficient acid in the Edman sequencing method, affording the retention of the N-terminal amino acid configuration at the cyclization/cleavage reaction from peptides. Examples for the sequencing of D-Leu-Gly and L-Pro-L-Leu are described, utilizing 7-N,N-dimethylaminosulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) as a fluorogenic Edman reagent. The peptide labelled with DBD-NCS was cyclized/cleaved with 1% BF3 in dichloroethane containing 0.02% ethanethiol at 50 degrees C for 5 min. The resultant DBD-thiazolinone (TZ)-amino acid was separated on a phenylcarbamoylated cyclodextrin column with fluorometric detection at 524 nm with excitation at 387 nm. DBD-TZ-amino acids thus obtained showed remarkable retention of their configuration of alpha-carbon atoms of amino acids (DBD-TZ-L-Leu; 3%, -D-Pro; 6%). Even after 30 min of the cyclization/cleavage reaction at 50 degrees C, DBD-TZ-amino acids retained their configuration to the same degree. On the other hand, DBD-TZ-amino acid obtained by cyclization/cleavage reaction with TFA at 50 degrees C for 5 min was racemized to a great extent (DBD-TZ-L-Leu; 31%, -D-Pro; 48%). BF3 should be the most recommendable acid at the cyclization/cleavage reaction for amino acid sequence and configuration determination of peptides containing D-amino acid residues.

We investigated the role of myocardial ischemia in acute myocardial infarction and cardiac death in 253 patients with asymptomatic coronary disease (206 men, 47 women, mean age : 55 ± 8 years). Patients were divided into two groups : those with angina pectoris with no history of myocardial infarction (AP group, 93 patients) and those with a history of myocardial infarction (MI group, 160 patients). We also examined the usefulness of exercise electrocardiographic and Holter electrocardiographic findings as prognostic indicators of cardiac events. After 24-hour Holter electrocardiograms were obtained in both groups, patients were assigned to subgroups with or without silent myocardial ischemia (SMI) based on the presence or absence of transient ST-segment depression. Prognostic indicators were evaluated by multiple regression analysis. Cardiac events occurred in 26 (10. 3%) of 253 patients ; in 6 patients these events were fatal. The incidence of cardiac events was significantly higher in the SMI group than in the non-SMI group (16. 4% versus 5. 6%, p < 0.05). SMI was identified as a significant prognostic indicator in the overall population (p = 0.0088), as were the number of diseased coronary arteries in the AP group (p = 0.0152), and SMI (p = 0.0022) in the MI group. There were 3 deaths related to cardiac events in each group. The mean time from onset of angina pectoris to death was 73 ± 41 months compared with 33 ± 43 months in the MI group. Our findings suggest that the severity of the coronary lesion and SMI were important predictors of major cardiac events, and that the mechanism of the onset of cardiac events was different in the AP and MI groups.

In this paper, a new method is described for N-terminal amino acid sequencing of peptides using the fluorescent reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS). Sequence determination is carried out by identifying thiazolinone (TZ) amino acids, which are generally unstable and difficult to detect. The employed system can easily and quickly derive TZ amino acids using the Edman reaction with DBD-NCS; these amino acids are also stable enough to be efficiently detected by high-performance liquid chromatography. Resultant detection limits for DBD-TZ amino acids range from 50 fmol to a sub-picomole level (S/N = 3). This system successfully analyzed sequences of Leu5-enkephalin (25 pmol) and angiotensin I (100 pmol) using fluorometric detection at 524 nm with excitation at 387 nm.

A potential use for phenylisothiocyanate (PITC), the most popular Edman reagent, is presented for analysis of the amino acid sequence and configuration in peptides containing D/L-amino acids. After derivatization with PITC of the N-terminal amino acid (L-Tyr) of an enkephalin analogue, [D-Ala2, D-Leu5]-enkephalin, followed by cleavage/cyclization with trifluoroacetic acid at 50 degrees C for 5 min, the liberated 2-anilino-5-thiazolinone-L-Tyr (ATZ-L-Tyr) was further treated with 20% aqueous trifluoroacetic acid at 50 degrees C for 10 min. The resultant phenylthiohydantoin (PTH)-L-Tyr was then separated on a chiral stationary phase (a penylcarbamoylated cyclodextrin column), retaining its configuration. The residual sequence and configurations of the peptide (D-Ala-Gly-L-Phe-D-Leu) were also determined by separating the corresponding PTH-D- or L-amino acids on chiral columns. This method may be applicable to an automatic Edman sequence analyzer for the configuration determination of peptides containing D/L-amino acids.

The concentrations of D-amino acids and their enantiomeric ratios in wind samples were determined by high-performance liquid chromatography with fluorometric detection. We used Pirkle type chiral stationary phases and fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) for simplicity and high sensitivity. The amino acids determined were D-enantiomers of alanine (Ala), aspartic acid, glutamic acid, isoleucine (Ile) and leucine (Leu), D-Asparagine, -glutamine and -lysine were not detected. D-Leu was detected in red and rose wine samples, and D-Ile was determined only in rose wine. In contrast, neither D-Leu nor Ile were detected in white wine samples. The concentration of D-Ala was the most prominent among these amino acids with the highest content of approximately 180 microns (D/D + L ratio; 25%) in rose wine.

Tetraalkylammonium salts having n-propyl to n-amyl side chains inhibited rat liver sulfotransferase (ST) activities toward dehydroepiandrosterone and cortisol, but not ST activity toward 2-naphthol, whereas trialkylamines having ethyl to n-amyl side chains inhibited ST activity toward dehydroepiandrosterone, but not ST activities toward cortisol and 2-naphthol. A comparison of I50 values, which represent inhibitor concentration resulting in 50% inhibition of dehydroepiandrosterone ST activity, revealed that the values for the tetraalkylammonium salts were 0.015 to 0.017 mM, whereas the values for the trialkylamines were 0.20 to 0.33 mM. Introduction of hydrophilic groups such as hydroxyl, thiol, nitrile and acetamide groups or substitution by methyl and allyl groups in the alkyl side chains markedly diminished the inhibitory effect of triethylamine. These data indicate that ethyl to n-amyl side chains are a prerequisite for the alkylamine-type inhibitor. Tertiary amine drugs such as imipramine, dimenhydrinate, cyclizine, chlorpromazine and promethazine inhibited ST activities toward dehydroepiandrosterone and cortisol similar to the tetraalkylammonium salts, although the drugs were weaker inhibitors of hydroxysteroid ST activities. These results imply that in addition to trialkylamine side chains, the other portion of the drugs may participate in the inhibition of hydroxysteroid ST activities.

Triethylamine, which was used as an elution solvent for column chromatography to purify chemically synthesized 3'-phosphoadenosine 5'-phosphosulfate (PAPS), was a potent inhibitor of rat liver sulfotransferase (ST) activities toward androsterone and dehydroepiandrosterone, but not ST activities toward cortisol and 2-naphthol. Examination of fourteen primary, secondary and tertiary amines revealed that a secondary amine, di-n-butylamine, and three tertiary amines, triethylamine, tri-n-propylamine and tri-n-butylamine, specifically inhibited ST activities toward androgen.

Chemical modifications of the potent A2 adenosine receptor agonist 2-(1-hexyn-1-yl)adenosine (7, 2-HA) at the 5'-position have been carried out to find more potent and selective A2 agonists. These analogues were evaluated for adenosine A1 and A2 receptor binding affinity in rat brain tissues and antihypertensive effects in spontaneously hypertensive rats (SHR). Among the series of compounds, 2-(1-hexyn-1-yl)adenosine-5'-N-cyclopropyluronamide (16d) had the most potent affinity to the A2 receptor with a Ki of 2.6 nM, which is essentially the same as that of the parent agonist, 2-HA. However, the most selective agonist for the A2 receptor was 2-(1-hexyn-1-yl)adenosine-5'-N-methyluronamide (16b) with a Ki of 11 nM and a 162-fold selectivity. The N-alkyl substituents of 5'-uronamide derivatives did not seem to potentiate the A2 binding affinity but drastically reduced the A1 affinity compared with the parent 2-HA. Therefore, the A1/A2 selectivity was consequently increased. Other 5'-deoxy-5'-substituted derivatives of 2-HA such as the chloro (20), carboxamide (27, 28), sulfonamide (29), urea (30), and thiourea (22) analogues were also prepared. Among these nucleosides, no active compounds with potent or selective affinities to both receptors were found except 20. Although glycosyl conformations and sugar-puckering of these nucleosides were studied by 1H NMR spectroscopy, there were no positive correlations between active and inactive agonists. 2-(1-Hexyn-1-yl)adenosine-5'-uronamide (16a) and 16d had a potent hypotensive effect at ED30 values of 0.18 and 0.17 micrograms/kg, respectively, upon iv administration to anesthetized SHR.

A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified 122-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pI = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypeptide in rat liver cytosol. Immunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.

Liver sections prepared from male and female rats were immunohistochemically stained with the antisera against phenol sulfotransferase G (P-STG), an isoenzyme of phenol ST as well as androsterone-sulfating ST (AD-ST), an isoform of hydroxysteroid ST. Localization of these isoenzymes in liver is sex-dependent and is markedly different between the two. P-STG is preferentially localized in the hepatocytes proximal to the central vein in females, whereas it is present in all the hepatocytes throughout the liver in males. On the other hand, AD-ST is evident in the hepatocytes proximal to the portal triad in males, while in females it is synthesized and localized in all the hepatocytes. The polarized sex-related localization of these ST isoenzymes appears to correlate with differential hormonal regulation of the enzymes.

Some properties of androsterone (AD)-sulfating sulfotransferase (ST) present in female rat livers were characterized. Based on the substrate specificities of the enzyme preparation obtained by anion exchange chromatography and 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity chromatography, AD-ST was supposed to be among isoenzymes of hydroxysteroid STs. The identity of the AD-ST with the isoenzymes of hydroxysteroid ST, however, remains unclear at present. The enzyme preparation revealed a wide range of native molecular weight with a major Mr of some 600000. The AD-ST did not appear to have a homogeneous isoelectric point, because the enzymatic activity was spread over a wide range of the pH gradient, centering around pH 6.6 on chromatofocusing. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the AD-ST showed a subunit with Mr of 30000, which was similar to the hydroxysteroid STs purified previously. Under denaturing conditions the subunit was demonstrated to be composed of three protein species containing distinct pI values (pI 6.1, 6.7 and 7.2). The AD-ST was thus supposed to be an oligomer with high molecular weight, in which the subunits of different pI values are assembled in various association numbers.

Bilirubin and phenol UDP-glucuronosyltransferases (UGTs) are located on the same chromosome and comprise the UGT1 gene complex. A 1,763-bp cDNA probe (UGT1*0) specific for rat liver bilirubin UGT was used to localize the UGT1 complex locus (Ugt1a1) to chromosome region 9q35→q36 by fluorescence in situ hybridization. This assignment is the first report on the location of a gene of the rat UGT1 complex using high-resolution banded metaphase chromosomes.

We have previously demonstrated the activity(ies) of phospholipid transacylase in Escherichia coli extract (Homma & Nojima (1982) J. Biochem. 91, 1093-1101), which catalyzed a new type of reaction of acyl transfer from diacylphospholipids to lysophospholipids. In this communication we report the specificities and characteristics of this enzyme activity. The activity catalyzed a reversible transfer of an acyl group between diacylphospholipids and lysophospholipids. The acyl group in the 1-position of the glycerol backbone was selectively transferred, and palmitic acid was the only fatty acid species transferred. Presumably, neutral lipids do not serve as substrates. The transacylase was firmly associated with the envelope fraction of E. coli. Neither potassium chloride nor urea was effective in solubilization of the activity and only about half of the activity was solubilized with Triton X-100. This observation was consistent with the equal distribution of the activity between the outer membrane and the inner membrane of E. coli. Functional aspects of this phospholipid transacylase are also discussed.

Fundamental studies on power transmission by using laser beam have been performed. The experimental results on a plasma direct converter (PDC) from laser energy to electricity are reported. We found that the conversion efficiency of laser energy to plasma varies with target materials and reaches the highest value of 70% for a NaCl target, and the conversion efficiency from energy of expanding plasma to electricity shows no variation with target materials. It is found that the conversion efficiency from laser energy to electricity is about 16% when a NaCl target is used.

Metabolism of platelet-activating factor (PAF) in rabbit plasma or in rabbit platelets was studied. [C3H3]-Labeled PAF was degraded into lysoPAF and choline in plasma. An agonist of PAF, NT071 was not degraded in the plasma. Albumin protects the degradation of PAF in plasma deprived of albumin but not the degradation of lysoPAF. These findings indicate that PAF may be metabolized in plasma by acetylhydrolase and then by lysophospholipase D. PAF was converted to phosphatidylcholine (PC) in washed rabbit platelets. The radioactivities in PC was recovered in the fraction of lysoPC after mild alkaline treatment, suggesting that the product is 1-alkyl-2-acyl-glycerophosphocholine. The binding of PAF and lysoPAF to rabbit platelets, rabbit erythrocytes and liposomal membranes were next examined. The binding of PAF to various membranes was inhibited by albumin. Albumin also suppressed the activation of platelets by PAF. A monomeric form of PAF, which is free from albumin, may react with target cell membrane and also be degraded by catabolic enzymes. The binding of lysoPAF to platelets, erythrocytes and liposomes was more effectively inhibited by albumin than that of PAF. The affinity of PAF to lipid bilayers may be higher than that of lysoPAF.

The nucleotide sequence of the pldA gene of Escherichia coli K-12, which codes for detergent-resistant phospholipase A (DR-phospholipase A), located in the outer membrane, was determined and the amino acid sequence of DR-phospholipase A was deduced. DR-phospholipase A contains 269 amino acids, resulting in a protein with a molecular weight of 30,809. It does not contain any cysteine residues and seems to be synthesized first as a precursor with a typical signal peptide composed of 20 amino acids. The NH2-terminus of the mature protein is glutamine, a polar amino acid, while other outer membrane proteins so far determined have a nonpolar amino acid there. The hydropathy profile of the deduced amino acid sequence of DR-phospholipase A was studied. Most of the region was rather hydrophilic and there were no stretches of hydrophobic amino acids. Computer analysis showed that there are no homologies between DR-phospholipase A and other extracellular phospholipases whose amino acid sequences are known. The candidates for the promoter region of the pldA gene, the 5'-flanking region, have a significantly high AT content, while the AT content of the coding region is about the same as the average AT content of the E. coli chromosome. A typical rho-independent transcription termination site is also present at the 3'-flanking region. This is the first example of the primary structure of a membrane-bound phospholipase.

The GTPase activities of rabbit platelet membrane were stimulated by platelet activating factor (PAF) in a receptor-mediated manner. The activities of the GTPase were investigated in the platelets which had been pretreated with tetradecanoyl phorbol acetate (TPA), dibutyryl cAMP, and PAF. The specific binding of PAF to intact platelet cells was also determined in these treated cells. In platelets which had been pretreated with PAF and then specifically desensitized to PAF, higher concentrations were required for stimulation of the receptor-coupled GTPase. In addition the extent of stimulation of the GTPase by PAF was also decreased. By contrast thrombin stimulation of GTPase activity was unaffected by this process. In platelets pretreated with high levels of dibutyryl cAMP (greater than 4 mM), the specific binding of PAF was reduced to nearly 50% of the control. Although this specific binding apparently was not inhibited by lower concentrations of dibutyryl cAMP (less than 2 mM), PAF could stimulate the receptor-coupled GTPase only to a much lower extent in these treated cells. TPA had virtually no effect on PAF specific binding. However, higher concentrations were needed for stimulation of the GTPase. On the other hand, the extent of PAF stimulation of the GTPase was not altered. Interestingly in the TPA-treated platelet membrane, thrombin stimulated GTPase activity to a higher level than that in untreated platelet membrane. Thus, TPA, dibutyryl cAMP, and desensitization affected the PAF receptor binding and the receptor-coupled GTPase activities in a characteristic fashion. The molecular mechanisms of these effects are discussed.

A rapid method for the separation and purification of uridine diphosphate-glucuronosyltransferases (GT) was developed with the use of chromatofocusing on a high-performance liquid chromatograph. GT isoenzymes solubilized from hepatic microsomes of Wistar rats were separated on a Mono P column, a pre-packed column for chromatofocusing. Using 4-nitrophenol, testosterone and androsterone as substrates, four fractions with different GT activities were separated in a pH gradient from 9.5 to 7.0. Two isoenzymes, testosterone GT and androsterone GT were purified to apparent homogeneity. They were eluted at pH 8.9 and 8.0 and had subunit molecular weight values of 50000 and 52000, respectively. Approximately 10 mg of solubilized microsomal proteins was applied and the elution was completed within 2 h. Addition of N-nitrodiethylamine, an in vitro activator of GT activity, enhanced the GT activity toward 4-nitrophenol in the three fractions. This chromatographic analysis confirmed the absence of androsterone GT isoenzyme in LA Wistar rats, a mutant strain in terms of androsterone glucuronidation.

When 2-[14C]acyl lysophosphatidylethanolamine was incubated with the envelope fraction of E. coli in the presence of Mg2+ ion, phosphatidylethanolamine, acylphosphatidylglycerol and free fatty acid were produced. When 2-[14C]acyl lysophosphatidylglycerol was examined similarly, six phospholipids as well as free fatty acid were produced. These were dilysocardiolipin, lysocardiolipin, phosphatidylglycerol, cardiolipin, bis(monoacylglycero)phosphate and acylphosphatidylglycerol; they were identified by thin layer chromatography, acetolysis and mild alkaline hydrolysis. Studies with an E. coli mutant which is deficient in cardiolipin synthase showed that dilysocardioilipin, lysocardiolipin and cardiolipin were synthesized by cardiolipin synthase. Bis(monoacylglycero)phosphate as well as acylphosphatidylglycerol was produced by acylphosphatidylglycerol synthase. While phosphatidylglycerol and cardiolipin were produced predominantly from 2-acyl lysophosphatidylglycerol, almost the same amounts of dilysocardiolipin, lysocardiolipin and bis(monoacylglycero)phosphate were produced from the 1-acyl and 2-acyl isomers. Metabolites of 2-[14C]acyl lysophosphatidic acid were also examined. Phosphatidic acid, acylphosphatidylglycerol, free fatty acid and monoglyceride were produced, together with a small amount of diglyceride.

Detergent-resistant phospholipase A (DR-phospholipase A) of E. coli is a 28K-dalton protein and is exclusively located in the outer membrane. We cloned the pldA gene of E. coli, which is responsible for the activity of DR-phospholipase A. Strains bearing the plasmid which contained the pldA gene yielded a large amount of the outer membrane protein with a molecular weight of about 28K daltons and overproduced 20 to 65 times as much DR-phospholipase A activity as the wild type strain. Experiments with minicells and maxicells revealed that the pldA-containing plasmid was coding for a 28K protein. These results strongly indicated that pldA is the structural gene for DR-phospholipase A. There was apparently no difference with respect to the association of the enzyme with the envelope fraction between the overproducer and the wild type strain. The overproduced enzyme was properly transported to the outer membrane. Neither the growth rate nor the phospholipid composition of the overproducer was remarkably different from in the wild type strain. Thus, the overproduction of DR-phospholipase A apparently caused no phenotypic variations. E. coli has very excessive ability to transport and integrate the outer membrane protein.

N-Nitrosodiethylamine (NEN) and N-nitrodiethylamine (NEA) are carcinogens and in vitro activators of hepatic UDP-glucuronosyltransferase (GT) toward 2-aminophenol (AP) and 4-nitrophenol (NP). In this communication, they were intraperitoneally administered to male Wistar rats for 7 days and GT activities were determined towards AP, NP, phenolphthalein (PH) and testosterone (TS). Administration of 30 or 20 mg/kg dose of NEN caused marked decrease of liver and body weights, and did not affect hepatic GT activities. Injection of 10 mg/kg dose of NEN did not diminish liver and body weights, and increased the maximally activated GT activities toward AP and NP. In contrast, 30 mg/kg dose of NEA, did not affect either liver and body weights or GT activities. N-Nitrosodimethylamine (NMN), which is a carcinogen and a weak in vitro AP GT activator, was more toxic than NEN, and 3.6 mg/kg dose of NMN appears to induce GT toward NP and AP. Administration of 46.5 mg/kg N-nitrosodibutylamine (NBN), which is a carcinogen but not a GT activator, did not affect GT activities or liver body weights.

Hooded Gunn rats which have low-activity of 4-nitrophenol uridine diphosphate-glucuronosyltransferase (GT) and defect in bilirubin GT were crossed with albino LA Wistar rats with a defect in androsterone GT. From F2 and F3 progeny were selected Gunn-LA Wistar rats which have defective bilirubin and androsterone GTs and low-active 4-nitrophenol GT. These rats had either hooded or albino coat color. In order to establish a uniform genetic background, albino male Gunn-LA Wistar rats were crossed with heterozygous albino female Gunn-LA Wistar rats in terms of bilirubin GT. The offsprings were all albino and were classified to Gunn-LA Wistar rats and heterozygous Gunn-LA Wistar rats by assaying their hepatic GT activities or by the appearance of jaundice. The linkage relationships between GT and coat color genes are discussed.

Two kinds of E. coli K-12 mutants for lysophospholipase L2 (located in the inner membrane) were isolated, using an improved version of the colony autoradiographic method developed by Raetz; these were, 1) strains carrying an elevated level of the enzyme and 2) strains defective or temperature-sensitive in the enzyme. Characterization of the crude lysates of these mutants revealed that the differences of lysophospholipase L2 activity are not due to the presence or absence of regulatory factors. Evidence was obtained, by using these mutants, that this lysophospholipase L2 transfers the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol, forming acyl phosphatidylglycerol.

The authors have devised an apparatus for monitoring the spontaneous locomotor activity of rats, with which the patterns of locomotor activity under the condition of light-dark cycle (light period: twelve hours from 6 a.m., dark period: the other half of the day) were analysed. In addition, changes in locomotor activities due to forced exercise (swimming) were recorded. The results obtained were as follows: 1. The patterns of locomotor activities in rats recorded by the monitor showed the same patterns as reported already by other researchers. The authors believe that this new apparatus can be used practically as a locomotor activity monitor. 2. Due to exhaustive exercise by swimming, changes of the ultradian rhythm in rats were observed. 3. By loading of swimming, locomotor activities in rats decreased significantly, especially in the dark period. These results suggest that this newly devised apparatus is useful as a locomotor activity monitor, and that the fatigue in rats caused by physical load can be shown as the change in locomotor activities.

The incorporation of 2-acyl lysophospholipids into Escherichia coli , and their metabolism were studied. 2-[ 14 C]Acyl lysophosphatidylethanolamine could penetrate into E. coli cells and was mainly incorporated into phosphatidylethanolamine. 2-Acyl lysophosphatidylethanolamine was partially degraded, but some of it was incorporated into membrane phospholipids by acylation. 2-Acyl lysophosphatidylcholine also entered cells and was acylated to phosphatidylcholine. The acylation of 2-acyl lysophospholipid by the envelope fraction was also studied. Fatty acids were incorporated into 2-acyl lysophospholipids by the envelope fraction in the presence of ATP and Mg 2+ , and the incorporation was stimulated by acyl carrier protein, but not by coenzyme A. No acylation was observed with acyl coenzyme A as acyl donor. The acylation activities of the inner and outer membranes were examined. Pathways for degradation and modification of membrane phospholipids in E. coli are proposed.