A novel screening for inhibitors of a pleiotropic drug resistant pump, Pdr5, in Saccharomyces cerevisiae.

Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters. Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5. An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (delta syr1/erg3 null mutation). The sensitivity of the indicator strain (delta syr1/erg3 delta pdr5 delta snq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively. The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin. The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (approximately 0.5 microg/ml). In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506. These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.

The crystal structure of the complex of non-peptidic inhibitor of human neutrophil elastase ONO-6818 and porcine pancreatic elastase.

The crystal structure of a new inhibitor of human neutrophil elastase (HNE), N-[2-[5-(tert-butyl)-1,3,4-oxadiazol-2-yl]-(IRS)-1-(methylethyl)-2-oxoethyl]-2-(5-amino-6-oxo-2-phenyl-6H-pyrimidin-1-ly)acetamide (ONO-6818, 1) complexed to porcine pancreatic elastase (PPE) has been determined at 1.86 A resolution. Analytical results provided evidence of a 1:1 complex in which the electrophilic ketone of 1 covalently bound to O gamma of Ser195 at the active site of PPE. The role of the unique electron-withdrawing ketone of 1 has been elucidated.

Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity.

We report the solution structure of T140, a truncated polyphemusin peptide analogue that efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its specific binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel beta-sheet and a type II' beta-turn. A protuberance is formed on one side of the beta-sheet by the side-chain functional groups of the three amino acid residues (L-3-(2-naphthyl)alanine, Tyr5 and Arg14), each of which is indispensable for strong anti-HIV activity. These findings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1.

Purification and some properties of an inhibitor for a yeast pleiotropic drug resistant pump from Kitasatospora sp. E-420.

A strain producing an inhibitor for pleiotropic drug resistant 5 (Pdr5) was isolated using our original screening system in yeast. The strain was classified and named as Kitasatospora sp. E-420. The purified inhibitor (molecular weight = 1,193 by FAB-MS) inhibited a Pdr5-mediated efflux of cycloheximide and cerulenin. The intracellular accumulation of a fluorescent dye, rhodamine 123, by the inhibitor was also confirmed. Some physicochemical data suggested that the Pdr5-specific inhibitor was different from an immunosuppressant, FK506, reported as the only inhibitor of Pdr5 in vivo.

Congestive heart failure caused by aortocaval fistula after nephrectomy.

We treated a 66-year-old man and a 69-year-old woman with high output cardiac failure due to aortocaval fistula. They underwent nephrectomy 40 and 36 years ago, respectively. They suffered from heart failure for a very long time. However, the etiology was not elucidated until recently. The aortogram revealed massive shunt from the aorta to the inferior vena cava via the right renal artery. Three-dimensional computerized tomography clearly delineated the aortocaval fistula. Surgical closure of the fistula promptly improved heart failure. We stress the importance of history taking concerning nephrectomy and the importance of abdominal auscultation in high output cardiac failure.

A case of an intraocular foreign body due to graphite pencil lead complicated by endophthalmitis.

We report a case of an 8-year-old boy who presented with an intraocular foreign body composed of graphite pencil lead. The patient had been accidentally poked in the right eye with a graphite pencil. Primary care consisted of corneal suturing and lens extraction. Two pieces of the pencil lead remained in the vitreous cavity following surgery, and 2 days later the patient developed endophthalmitis. Pars plana vitrectomy was performed immediately and the intraocular foreign bodies were removed through the scleral wound. Cultures of the vitreous fluid revealed no bacterial organisms. X-ray fluoroscopic analysis of the vitreous detected 1 ppm of aluminum (a constituent of the pencil lead). Although the clinical presentation indicated probable bacterial endophthalmitis, the detection of elemental aluminum within the vitreous cavity also suggested the possibility of further retinal toxicity due to some dissolving of the pencil lead.

Interaction of mutagenic tryptophan pyrolysate with d(CGATCG)2: intercalation model based on NMR experiments.

The solution structure of the complex of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), a potent mutacarcinogen isolated from tryptophan pyrolysate, with the hexamer duplex d(CGATCG)2, was analysed by 1H-NMR spectroscopy and molecular dynamic calculation. Trp-P-1 was intercalated between the CpG base pairings in a suitable manner to form the guanosine-Trp-P-1 adduct which corresponds to the major reactant of Trp-P-1 with DNA.

Synthesis of structural analogues of leukotriene B4 and their receptor binding activity.

Structural analogues of leukotriene B4 (LTB4) were designed based on the plausible conformation of LTB4 (1). Joining C-7-C-9 of the conformer A or B into an aromatic ring system led to the discovery of benzene analogues 2, 4 and 6a. Joining C-4-C-9 of the conformer C or D into an aromatic ring system led to the discovery of analogues 3, 5 and 7. The compounds examined in this study were evaluated as to their inhibition of [3H] LTB4 binding to human neutrophils, and by a secondary intact human neutrophil functional assay for agonist/antagonist activity. The first analogues prepared, compounds 2-7, demonstrated moderate potency in the LTB4 receptor binding assay. The modification of these compounds by the introduction of another substituent into the aromatic ring produced a marked increase in receptor binding (28c, IC50 = 0.020 microM; 38c, IC50 = 0.020 microM; 52a, IC50 = 0.020 microM; 52b, IC50 = 0.018 microM). Most of these structural analogues of LTB4 demonstrated agonist activity. Of the analogues prepared in this study, only compound 57 demonstrated weak LTB4 receptor antagonist activity, at 10 microM.

Trisubstituted benzene leukotriene B4 receptor antagonists: synthesis and structure-activity relationships.

A series of trisubstituted benzenes which demonstrate leukotriene B4 (LTB4, 1) receptor affinity was prepared. Previous trisubstituted benzenes from our laboratory showed high affinity to the LTB4 receptor but demonstrated agonist activity in functional assays. Compound 3a, the initial lead compound of this new series, showed only modest affinity (IC50 = 0.20 microM). However, 3a was a receptor antagonist with no demonstrable agonist activity up to 30 microM. Further modification of the lipid tail and aryl head groups region led to the discovery of 3b (ONO-4057). This compound, free of agonist activity, possesses high affinity to the LTB4 receptor (Ki = 3.7 +/- 0.9 nM).

Coronary vasomotor response to acetylcholine late after angioplasty.

To elucidate the changes in coronary vasomotion in a previously balloon-dilated segment, we examined the vasoconstricting response of previously balloon-dilated and non-dilated segments to acetylcholine. Acetylcholine was administered into coronary arteries cumulatively (left and right coronary artery: 10-100 micrograms) in 15 patients (age: 60 +/- 3 years, 12 males and 3 females) at 7.4 +/- 1.5 months after successful percutaneous transluminal coronary angioplasty (PTCA). In PTCA segments with no restenosis, does-dependent constriction in response to acetylcholine was observed in only 1 patient. In non-PTCA segments of PTCA and non-PTCA arteries, 12 patients showed dose-dependent vasoconstriction in response to acetylcholine. Coronary spasm, which was defined as a more than 75% reduction in coronary diameter compared with that after isosorbide dinitrate, was provoked in one PTCA segment (7%). In non-PTCA segments of PTCA and non-PTCA arteries, 15 of 44 arteries (34%) demonstrated coronary spasm in 9 of 15 patients (60%, p < 0.005 vs PTCA segment). In conclusion, PTCA segments free of restenosis showed no hyper-reactivity to acetylcholine, while non-PTCA segments showed hypercontractility in response to acetylcholine. Coronary balloon angioplasty may alter the coronary vasomotor reaction to acetylcholine several months after angioplasty.

[Sevoflurane inhalation reduced bronchospasm after extubation].

A 73-year-old female was scheduled for left upper lobectomy. She had no history of asthma or chronic obstructive pulmonary disease. During the operation, respiratory sound was clear. As spontaneous breathing was regular and stable after the reversal of neuromuscular blockade, an endotracheal tube was extubated. But soon after the extubation, wheezing and gasping respiration occurred and she complained of dyspnea. Therefore, we administered aminophylline and steroid intravenously, and the patient's lungs were ventilated manually with 100% oxygen. But after 10 minutes, there was no improvement in symptoms, and sevoflurane inhalation was started immediately. Inspiratory sevoflurane concentration was 4% at first, and was decreased to 2%. About 20 minutes after starting sevoflurane inhalation, wheezing was reduced. Sevoflurane may be useful in the treatment of bronchospasm after extubation.

[Bacterial intracranial aneurysm associated with infective endocarditis: a case showing enlargement of aneurysm size].

The authors report a case of bacterial intracranial aneurysm associated with infective endocarditis. A 48-year-old male was admitted on March 26, 1994, with complaints of difficulty in speaking and mild swelling of the right leg following mild fever. On examination he showed motor aphasia and mild weakness of the right upper and lower limbs. Cardiac auscultation revealed a grade 3/6 holosystolic murmur. Laboratory data revealed signs of infection through white blood cell count and CRP. Enterococcus faecalis was isolated from the blood culture at the time of admission. A computerized tomographic (CT) scan and magnetic resonance (MR) imaging showed a round mass with perifocal edema. Angiography revealed an aneurysm from the precentral artery of the left middle cerebral artery. A mycotic aneurysm due to bacterial endocarditis was diagnosed. The patient was treated with high doses of antibiotics. However, angiography 2 weeks after the initial study demonstrated the enlargement of the aneurysm and severe narrowing of the angular artery. On April 19, excision of the aneurysm was performed. Operative findings showed degeneration and thickening of the walls of the aneurysm. After the operation, antibiotic therapy was continued. The patient was asymptomatic upon discharge and has continued to do well. Repeated angiography on September 12 showed no further aneurysm. There is a danger of rupture in mycotic aneurysm due to bacterial endocarditis. It is important to repeat angiography and to manage the primary disease. If an aneurysm enlarges with serial angiography, it should be treated surgically without further delay.

Unique binding of a novel synthetic inhibitor, N-[3-[4-[4-(amidinophenoxy)carbonyl]phenyl]-2-methyl-2-propenoyl]- N-allylglycine methanesulfonate, to bovine trypsin, revealed by the crystal structure of the complex.

Trypsin and N-[3-[4-[4-(amidinophenoxy)carbonyl]phenyl]-2-methyl-2-propenoyl]- N-allylglycine methanesulfonate (1), a newly designed and orally active synthetic trypsin inhibitor, were cocrystallized. The space group of the crystal is P2(1)2(1)2(1) with cell constants a = 63.74 A, b = 63.08 A, and c = 69.38 A, which is nearly identical to that of the orthorhombic crystal of guanidinobenzoyltrypsin. The structure was refined to a crystallographic residual R = 0.176. The refined model of the 1-trypsin complex provides the structural basis for the reaction mechanism of 1. On the basis of the present X-ray results, it is proposed that the potent inhibitory activity of 1 is mainly due to the formation of an acylated trypsin through an "inverse substrate mechanism" and its low rate of deacylation.

[Prostacyclin mimetics with non-prostanoid structures].

In searching for new drugs, we have developed receptor binding assays for prostanoids. Among the various compounds that we have tested, we have found that hydronaphthalene derivatives can interact with some prostanoid receptors. Modification of such compounds produced several pure prostacyclin agonists (ONO-AP-227 and ONO-AP-437) and a unique prostacyclin agonist with inhibitory activity against thromboxane synthase (ONO-AP-500-02). These compounds showed specific binding to the IP receptor with Ki values less than 0.2 microM, without binding to EP and TP receptors. These compounds also inhibited human platelet aggregation with IC50 values of 0.03-0.24 microM. These compounds inhibited ex vivo platelet aggregation in dogs or rats in the dose range of 1 to 30 mg/kg. Furthermore, ONO-AP-500-02 inhibited ex vivo thromboxane formation at doses similar to those inhibiting platelet aggregation in rats. These results, taken together, suggest that chemical syntheses of compounds targeting prostanoid receptors can produce unique prostanoid agonists or antagonists, and rational syntheses might be possible for compounds with different pharmacological actions such as inhibition of thromboxane synthase.

[Inhibitory effects of prostaglandin E1.alpha-cyclodextrin (PGE1.CD) on dimethylnitrosamine-induced acute liver damage in rats].

The effects of PGE1.CD on dimethylnitrosamine (DMN)-induced acute liver damage with intravascular coagulation in rats were biochemically and histopathologically investigated. PGE1.CD was administered i.v. from 30 min before to 24 hr after DMN-intoxication (pretreatment) and from 30 min after or from 4 hr after to 24 hr after DMN-intoxication (post-treatment). Pretreatment with PGE1.CD (0.2-2 micrograms/kg/min) dose-dependently suppressed the decrease of platelet counts and the elevation of blood biochemical parameters (PT, HPT, GOT, GPT, LDH, LAP, T-Bil) caused by DMN-intoxication. PGE1.CD (0.5 microgram/kg/min and over) significantly suppressed the DMN-induced histopathological changes (occurrence of hemorrhage and necrosis). Post-treatment with PGE1.CD (2 micrograms/kg/min) also suppressed the liver damage. Furthermore, pretreatment with PGE1.CD (2 micrograms/kg/min) not only suppressed the disruption of hepatocytes, but also prevented the damages of sinusoidal endothelial cells and lysosomal membrane, and it reduced the increase of lipid peroxidation. PGE1.CD (1 microgram/kg/min and over) significantly suppressed the decrease of hepatic tissue blood flow caused by DMN-intoxication. These results demonstrate that PGE1.CD has therapeutically efficacy against DMN-induced acute liver damage in rats; Therefore, it will be clinically useful for the treatment of severe hepatitis such as fulminant hepatitis with intravascular coagulation in the sinusoid.

Application of an alpha-sidechain length-specific monoclonal antibody to immunoaffinity purification and enzyme immunoassay of 2,3-dinor-6-keto-prostaglandin F1 alpha from human urine.

2,3-Dinor-6-keto-prostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha) is a major urinary metabolite of PGI2 (prostacyclin) and one of the most reliable parameters of PGI2 production in vivo. A mouse was immunized with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha conjugated to bovine serum albumin for preparation of a monoclonal antibody which recognized the difference in the alpha-sidechain length of 6-keto-PGF1 alpha and its 2,3-dinor-metabolite. A sensitive and specific enzyme immunoassay was developed by the solid-phase competition method with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha labeled with peroxidase protein. The detection range of the assay was 14-1200 fmol (IC50 = 120 fmol). The cross-reactivities of the antibody with 6-keto-PGF1 alpha, 6,15-diketo-13,14-dihydro-PGF1 alpha, and other arachidonate metabolites were less than 0.01%. An immunoaffinity column was prepared by coupling the anti-2,3-dinor-6-keto-PGF1 alpha antibody to BrCN-activated Sepharose 4B. Human urine was applied to an octadecylsilyl silica cartridge, and the extract was applied to the immunoaffinity column. This procedure allowed an efficient separation of 2,3-dinor-6-keto-PGF1 alpha from unidentified urinary substances which interfered with immunoassay. Validity of the results obtained by the enzyme immunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.

Complete obstruction of right proximal pulmonary artery: a case report.

A fifty-five-year-old woman had worried about dyspnea for three years. A chest x-ray appeared normal except for hypovascularity of the upper lung field. On angiograms, a wedge-shaped obstruction was observed at the left proximal pulmonary artery, and well-developed collateral circulation from the right thoracic artery to the internal costal arteries was observed. On the exercise test, exertional dyspnea developed with tachycardia and a decreased saturation of arterial oxygen.