Molecular structure and dynamics of the four 10-hydroxynortriptyline isomers.

The three-dimensional structures, molecular conformations, and electrostatic potentials of the R-E-, S-E-, R-Z-, and S-Z-isomers of 10-hydroxynortriptyline were examined by computer graphics, molecular mechanical energy calculations, and molecular dynamics simulations in vacuo and in aqueous solution. Molecular models of the isomers, based on the structure of nortriptyline, were refined by energy minimization and used as starting points in the simulations. R-E- and S-Z-10-hydroxynortriptyline formed intramolecular hydrogen bonds between the side-chain nitrogen atom and the hydroxyl group during the simulations in vacuo, and had the side chain folded over the ring system in the minimum energy conformations. Intramolecular hydrogen bonding was not observed for R-Z- and S-E-10-hydroxynortriptyline, which had extended side chains in the minimum energy conformations and stronger negative molecular electrostatic potentials around the hydroxyl group than the R-E- and S-Z-isomers.     

Covalent binding of o,p'-DDD in rabbit lung and isolated rabbit lung cells

The irreversible binding of o,p'-DDD was examined in isolated lung cells, in lung microsomes and in vivo in male New Zealand White rabbits. Non-ciliated bronchiolar (Clara) cells had the highest capacity to bind o,p'-DDD, followed by alveolar type II cells. A fraction of mixed unidentified lung cells was also able to bind o,p'-DDD while no binding was observed in alveolar macrophages. The activation of o,p'-DDD was shown to be mediated by cytochrome P-450 in both lung microsomes and isolated lung cells. In vivo, the binding was preferentially localized in the lung alveolar and bronchiolar regions. The binding of o,p'-DDD observed in vivo may thus be caused by the capacity of several cell types to activate o,p'-DDD.     

Corticotropin-induced changes in protein labelling: lack of molecular specificity

Previous studies in this and other laboratories have indicated that corticotropin (ACTH) administered to intact or hypophysectomized rats or mice increased the labelling of cerebral proteins by radioactive amino acids, presumably indicative of increased protein synthesis. This study was designed to reveal whether this increased labelling was specific for particular proteins by studying labelling patterns in SDS polyacrylamide gels using a double isotope procedure. Two strains of mice, two different amino acid precursors, leucine and lysine, and both peripheral and central administration of ACTH and amino acids were used. In no case were treatment dependent changes in labelling observed in any region of the gel. This included regions containing S-100 or NSP, two brain specific proteins whose content had been reported to be increased by treatment of rats with an ACTH analog.     

Detection and characterization of four binding sites for opioids in the mouse brain

The binding sites labelled by 3H-dihydromorphine, 3H-ethylketocyclazocine and 3H-D-Ala2-L-Leu5-enkephalin in mouse brain membranes were characterized in cross-competition studies. The data was evaluated by simultaneous non-linear least-squares regression analysis with the "Ligand" program (Munson & Rodbard 1980), that had to be upgraded to handle more than three binding sites. By statistical analysis four different binding sites were identified. Three of the sites probably correspond to the pharmacologically well characterized mu, kappa and delta-opioid receptors, respectively, and their binding capacities relate as 1:1.5:2.5. Classification of the fourth site is more problematic. Using 3H-ethylketocyclazocine it had higher capacity than the others and bound ethylketocyclazocine with a relatively high affinity (Kd = 10 nM), dihydromorphine with a very low affinity (Kd greater than 10(-5)M) and showed no binding of D-Ala2-L-Leu5-enkephalin. In displacement studies, N-allylnorcyclazocine (SKF 10,047), though unselective, bound with highest affinity to the mu and the fourth site. Since naloxone did not bind to this fourth site, it can not be termed an opioid site in a strict sense, but it might have some relevance in view of non-naloxone-reversible effects reported for some opioids.     

Covalent binding of DBCP to proteins in vitro

[¹⁴C] 1,2-Dibromo-3-chloropropane (DBCP) was incubated with rat liver 9000 × g supernatant in order to investigate the covalent binding of DBCP to proteins. Radioactivity incorporated into proteins was dependent on the microsomal oxidase system, but was not prevented by sufficient puromycin to inhibit protein synthesis. From these results DBCP was considered to bind to proteins covalently after activation by microsomal oxidase.     

Toxicity of selected symmetrical hexachlorobiphenyl isomers in the mouse

Five-week-old male mice were given: 0.3, 1, 3, 10, 30, 100, or 300 ppm of 3,4,5,3′,4′,5′-hexachlorobiphenyl (HCB); 10, 30, 100 or 300 ppm of 2,4,5,2′,4′,5′-HCB, 2,3,6,2′,3′,6′-HCB, or 2,4,6,2′,4′,6′-HCB in feed daily for 28 days. HCB residue levels were determined in adipose tissue and liver. There were marked differences in dose response and severity of pathologic effects among the isomers. 3,4,5,3′,4′,5′-HCB was the most toxic isomer causing mortality, and body and organ weight effects at all dose levels, and was the only isomer which produced excess porphyrin accumulation. It was also the most concentrated isomer in fat and liver. 3,4,5,3′,4′,5′-HCB caused subcutaneous edema, enlargement of liver with accentuated hepatic lobular markings, fatty infiltration, hepatocellular swelling and necrosis, and atrophy of the thymus. 2,4,6,2′,4′,6′-HCB and 2,4,5,2′,4′,5′-HCB caused the same lesions but to a lesser degree. Slight cardiomyopathy was observed with 2,4,6,2′,4′,6′-HCB. The decreasing order of overall toxicity was 3,4,5-HCB >> 2,4,6-HCB > 2,4,5-HCB > 2,3,6-HCB.     

Affinities of some common opioid analgesics towards four binding sites in mouse brain

Summary The selectivities of morphine, codeine, l-methadone and d-propoxyphene, towards the binding sites in mouse brain membranes labelled by ³H-dihydromorphine (DHM), ³H-ethylketocyclazocine (EKC) and ³H-D-Ala²-Leu⁵-enkephalin (DALE), were investigated. Of the four binding sites identified, three correspond to mu-, kappaand delta-opioid binding sites or receptors, respectively. The fourth site has a high capacity and binds EKC with a high affinity, DHM with a very low affinity and does not bind DALE. In displacement studies, the relative affinities of morphine and methadone were quite similar towards the tree sites with highest affnity (mu kappa delta). Codeine and d-propoxyphene were mu-selective but did not differentiate between kappa-and delta-sites. At high concentrations I-methadone (K_d=6.7 M), and d-propoxyphene (K_d=40 M) bound to the fourth site, while morphine, codeine and naloxone were practically inactive. The binding selectivities of these drugs were quite different from those of metkephamid and U-50, 488 H, substances that are thought to exert their antinociceptive effects through delta-and kappa-receptors, respectively. It was concluded that while d-propoxyphene and codeine may partly act througg other receptors than morphine, this is probably not the case for l-methadone.     

Covalent binding of benzo[a]pyrene diol epoxide to DNA of mouse skin: in vivo persistence of adducts formation

In the first 9 d after topical application of a single dose of benzo[a]pyrene to the dorsal skin of C3H mice, the half-lives of benzo[a]pyrene diol epoxide-DNA adducts and of DNA were determined to be approximately 5 d. These data indicate that, in proliferating mouse skin, benzo[a]pyrene diol epoxide-DNA lesions are not repaired, but are diluted from the genome at a rate equivalent to DNA turnover (i.e., replication versus degradation). Subsequent to this initial period, benzo[a]pyrene diol epoxide-DNA adduct removal continues, but at a much reduced rate. At 30 d posttreatment with benzo[a]pyrene, approximately 15% of the adducts are still detectable; however, their half-lives had increased to 30 d. Similar experiments with a hairless mouse showed that, although the amount of adduct formation was lower initially, the kinetics of adduct disappearance and persistence were essentially the same as found with the C3H mouse. The data obtained in this work are consistent with the hypothesis that benzo[a]pyrene diol epoxide adducts persist in a subpopulation of skin cells long after their disappearance by DNA turnover would predict.     

In vitro degradation of the four isomers of soman in human serum

Starting from racemic soman (1,2,2-trimethylpropyl methylphosphonofluoridate), the degradation of its four stereoisomers in human serum (25 degrees, pH 8.8), has been studied at the nM level. Phosphylation of serum proteins is eliminated by preincubation of the serum with soman. A capillary gas chromatographic method with nitrogen-phosphorous detection allows the separation of the diastereoisomers. The total hydrolysis (enzymatic and non-enzymatic) rate constants of the isomers can then be resolved indirectly on the basis of the important rate difference between P(+) and P(-) isomers. The enzymatic hydrolysis rate constants are obtained by subtracting, for each isomer, the spontaneous (non-enzymatic) rate constant from the total hydrolysis rate constant. The non-enzymatic part of the hydrolysis is obtained from experiments in serum ultrafiltrate (30,000 NMWL). Enzymatic hydrolysis of C(+) P(+) soman occurs so rapidly that only a lower limit of the rate constant can be given. The other enzymatic rate constants are 0.016 min-1 for C(+)P(-), 0.74 min-1 for C(-)P(+) and 0.028 min-1 for C(-)P(-).     

Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships

1. The present study describes the labelling of the nociceptin (NC) receptor, ORL₁, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [³H]-NC-NH₂, has a specific activity of 24.5 Ci mmol⁻¹
2. Saturation experiments revealed a single class of binding sites with a K_D value of 0.55 nM and B_max of 94 fmol mg⁻¹ of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: K_obs=0.104 min⁻¹; K₁=0.034 min⁻¹; T_1/2=20 min; K₊₁=0.07 min nM⁻¹.
3. Thermodynamic analyses indicated that [³H]-NC-NH₂ binding to the mouse ORL₁ is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(δ), OP2(κ) and OP3(μ).
4. Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]NC(1–13)-NH₂ ([F/G]NC(1–13)-NH₂), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH=NC-NH₂=NC(1–13)-NH₂ >> NC(1–12)-NH₂ > NC(1–13)-OH >> NC(1–11)-NH₂, and for NC and NC(1–13)-NH₂ analogues: [Tyr¹]NC-NH₂ ⩾ [Leu¹]NC(1–13)-NH₂ ⩾ [Tyr¹]NC(1–13)-NH₂ ⩾ [F/G]NC(1–13)-NH₂ >> [Phe³]NC(1–13)-NH₂ > [DF/G]NC(1–13)-NH².
5. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [³H]-NC-NH₂ binding when applied at concentrations up to 10 μM indicating that this new radioligand interacts with a non opioid site, probably the ORL₁ receptor.

     

Covalent grafting of common trihydroxymethylaminomethane in the headgroup region imparts high serum compatibility and mouse lung transfection property to cationic amphiphile

Clinical success of cationic transfection lipids in nonviral gene therapy continues to remain critically dependent on the use of serum compatible cationic amphiphiles efficient in delivering genes into our body cells. To this end, we demonstrate that covalent grafting of simple Tris-base component of the widely used biological Tris buffer in the headgroup region is capable of imparting high serum compatibility and intravenous mouse lung transfection properties to cationic amphiphile.     

The effect of isomers of DDD on the ACTH-induced steroid output, histology and ultrastructure of the dog adrenal cortex

The effect of 3 geometric isomers of DDD on ACTH-induced steroid production, histology, and ultrastructure of the dog adrenal cortex were examined. When administered iv, equal doses of all 3 compounds eventually inhibited ACTH-induced steroid production. The order of onset of inhibition was m,p′-DDD > o,p′-DDD > p,p′-DDD with the inhibition reaching 50% of control values at 27 min, 87 min and between 4–18 hr, respectively. Histologic examination of the various zones of the adrenal cortex showed minimal effects, from any of the isomers, on the zona glomerulosa at times when the internal structure and spacial arrangement of the cells of the zona fasciculata and reticularis were markedly disrupted. Electron micrographs revealed that these DDD isomers exhibited very minimal effects on the mitochondria of the zona glomerulosa. On the other hand, the mitochondria of the zonas fasciculata-reticularis underwent progressive swelling, dissolution, and eventual rupture following DDD treatment. The order of onset of histologic and ultrastructural effects was m,p′-DDD > o,p′-DDD > p,p′-DDD. However, for each isomer the ultrastructural damage was detected prior to the observation of histologic lesions. Thus, there was a marked temporal correlation between the percentage inhibition of ACTH-induced steroid production, the disruption of normal cellular structure and arrangement in the innermost zones of the adrenal cortex, and the severity of the DDD-induced mitochondria damage in the cells of the zonas fasciculata-reticularis by each of the 3 isomers of DDD.     

梭曼4个异构体与乙酰胆碱酯酶相互作用的计算机模拟

采用计算机模拟方法研究了梭曼4个异构体与乙酰胆碱酯酶（AChE）活性中心的作用情况. 结果表明RR构型的梭曼对AChE的作用最强,而在RS, SS和SR构型的梭曼分子中,由于磷原子(P)上的甲基对His440的排斥作用,使其上的咪唑环旋转,削弱了His440促进磷酰化酶的老化作用,同时也得出梭曼上的(CH₃)₃C^＋与Trp84和Phe330残基上的芳香环也有一定的静电相互作用.     

Covalent binding of PCB metabolites to lipids: route of formation and characterization

1. After an oral dose of (14)C-labelled 3,3',4,4'-tetrachlorobiphenyl (CB-77), the conventional germ-free and bile-duct cannulated male Sprague-Dawley rat excreted approximately 80% of the dose in faeces and/or bile within 3 days. 2. For the germ-free and conventional rat, 15% of the dose was excreted via the faeces as metabolites covalently bound to lipids. Bile-duct-cannulated rats excreted similar amounts of lipid-bound metabolites in the bile. The lipid-bound metabolites appear to be formed in the liver and excreted via the bile, and the microflora did not seem essential for the formation of lipid-bound metabolites. 3. The novel CB-77 metabolites had chemical and physical properties similar to those of lipids with regard to solubility and polarity, as determined by partition characteristics on various chromatographic systems. 4. In addition to identification of hydroxylated CB-77 metabolites, several fatty acid esters of hydroxy-chlorobiphenyls were indicated and one hydroxy-tetrachlorobiphenylol palmitoate was identified, but fatty acid esters were minor metabolites. 5. Approximately 70% of the lipid-bound metabolites were present in the fraction that contained phospholipids. The formation of lipid-bound CB-77 metabolites seems a spontaneous reaction rather than an enzymatically catalysed reaction, as indicated by the large number of different lipid-bound metabolites.     

Covalent binding of PCB metabolites to lipids: route of formation and characterization

1. After an oral dose of 14 C-labelled 3,3',4,4'-tetrachlorobiphenyl (CB-77), the conventional germ-free and bile-duct cannulated male Sprague-Dawley rat excreted approximately 80% of the dose in faeces and/or bile within 3 days. 2. For the germ-free and conventional rat, 15% of the dose was excreted via the faeces as metabolites covalently bound to lipids. Bile-duct-cannulated rats excreted similar amounts of lipid-bound metabolites in the bile. The lipid-bound metabolites appear to be formed in the liver and excreted via the bile, and the microflora did not seem essential for the formation of lipid-bound metabolites. 3. The novel CB-77 metabolites had chemical and physical properties similar to those of lipids with regard to solubility and polarity, as determined by partition characteristics on various chromatographic systems. 4. In addition to identification of hydroxylated CB-77 metabolites, several fatty acid esters of hydroxy-chlorobiphenyls were indicated and one hydroxy-tetrachlorobiphenylol palmitoate was identified, but fatty acid esters were minor metabolites. 5. Approximately 70% of the lipid-bound metabolites were present in the fraction that contained phospholipids. The formation of lipid-bound CB-77 metabolites seems a spontaneous reaction rather than an enzymatically catalysed reaction, as indicated by the large number of different lipid-bound metabolites.     

Covalent binding of trichloroethylene to proteins in human and rat hepatocytes

The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte cultures were exposed to vaporized trichloroethylene at 0–10 000 parts per million for up to 2 h. Immunochemical detection of three major dose- and time-dependent trichloroethylene protein adducts at 50, 52 and 100 kDa was demonstrated in the rat hepatocytes, while a single, distinctively different 47 kDa adduct was detected in human hepatocytes. The 50 kDa adduct in rat hepatocytes was found to comigrate on SDS-PAGE with cytochrome P450 2E1 (CYP2E1), while the adduct found in humans did not comigrate with CYP2E1. These data show that reactive metabolites of trichloroethylene can be formed in human and rat hepatocytes and bind covalently to discrete hepatic proteins, and suggests that in rats, but not humans, that one of the targets is CYP2E1.