Brain microsomal oxidation of delta 8- and delta 9-tetrahydrocannabinol

Brain microsomes of mice, rats, guinea pigs and rabbits catalyzed the oxidation of delta 8- and delta 9-tetrahydrocannabinol to their monohydroxylated metabolites. The most prominent metabolite was the 4'-hydroxylated metabolite on the pentyl side chain of the cannabinoids in all species tested, except that the 5'-hydroxylation of delta 9-tetrahydrocannabinol was most abundant in the guinea pig. These results are quite different from the metabolic profile of the cannabinoids with hepatic microsomes.     

Cardiovascular effects of delta 9- and delta 9(11)-tetrahydrocannabinol and their interaction with epinephrine

The administration of delta-9-tetrahydrocannabinol (delta 9-THC, 0.078-5.0 mg/kg, i.v.) to rats anesthetized with pentobarbital caused as much as a 50% decrease in mean arterial blood pressure, heart rate and respiratory rate in a dose-dependent manner. Delta-9(11)-tetrahydrocannabinol (delta 9(11)-THC) was approximately 8-fold less potent than delta 9-THC in its hypotensive effect and had smaller effects on heart and respiratory rates that were not dose-related at doses below 5 mg/kg. Alternate injections of epinephrine (2 micrograms/kg) with vehicle and increasing cannabinoid doses (1.25-5.0 mg/kg) indicated a potentiation of both the duration of the pressor effect and the magnitude of the reflex bradycardic effect of epinephrine by both delta 9- and delta 9(11)-THC. Epinephrine also produced arrhythmias in rats receiving cannabinoids, but not in rats receiving alternate injections of vehicle. It is concluded that both cannabinoids have adverse effects on the cardiovascular system and adverse interactions with epinephrine in rats anesthetized with pentobarbital.     

Evidence for the different responses of delta9-, delta6- and delta5-fatty acyl-CoA desaturases to cytoplasmic proteins.

Microsomes prepared from the livers of 4-week-old rats were, after extraction with 0.1 M potassium phosphate buffer, pH 7.4, unable to catalyse either the delta6 desaturation of alpha-linolenic acid (9c.12c.15c., 18 : 3) into 6c.9c.12c.15c., 18 : 4 or the delta5 desaturation of eicosatrienoic acid (8c.11c.14c., 20 : 3) into arachidonic acid (5c.8c.11c.14c., 20 : 4). Both these enzymes only showed full activity after incubation of the microsomes with either the 100 000 X g supernatant fraction or with purified bovine catalase. Bovine serum albumin, while capable of restoring 50% of the delta5 desaturase activity has no effect on the delta6 desaturase. In contrast the delta9 desaturase activity of microsomes was never completely lost after extraction with buffer but could be stimulated by optimum concentrations of both bovine serum albumin and catalase. The significance of the different responses of the three desaturases to the cytoplasmic components is discussed.     

Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex

Genotoxic stress activates checkpoint signaling pathways that block cell cycle progression, trigger apoptosis, and regulate DNA repair. Studies in yeast and humans have shown that Rad9, Hus1, Rad1, and Rad17 play key roles in checkpoint activation. Three of these proteins-Rad9, Hus1, and Rad1-interact in a heterotrimeric complex (dubbed the 9-1-1 complex), which resembles a PCNA-like sliding clamp, whereas Rad17 is part of a clamp-loading complex that is related to the PCNA clamp loader, replication factor-C (RFC). In response to genotoxic damage, the 9-1-1 complex is loaded around DNA by the Rad17-containing clamp loader. The DNA-bound 9-1-1 complex then facilitates ATR-mediated phosphorylation and activation of Chk1, a protein kinase that regulates S-phase progression, G2/M arrest, and replication fork stabilization. In addition to its role in checkpoint activation, accumulating evidence suggests that the 9-1-1 complex also participates in DNA repair. Taken together, these findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

Occurrence of 9-deoxy-delta 9,delta 12-13,14-dihydroprostaglandin D2 in human urine

We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.     

9-1-1: PCNA's specialized cousin

All living organisms are vulnerable to DNA damage. Cells respond to this hazard by activating a complex network of checkpoint and repair proteins to preserve genomic integrity. The DNA-encircling, ring-shaped heterotrimeric 9-1-1 complex, a relative of the replication protein PCNA, is a central coordinator of these events. 9-1-1 is loaded to damaged sites where it serves as a platform for the selective recruitment of checkpoint and repair proteins. In this Opinion article, 9-1-1 and proliferating cell nuclear antigen (PCNA) are compared and discussed in light of their respective structures and functions. We propose that the interaction partners of 9-1-1 possess specific 9-1-1-interaction boxes, which discriminate between 9-1-1 and PCNA thereby enabling specific interactions with individual 9-1-1 subunits.     

Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages

Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted.     

Requirements of delta 9 and delta 12 fatty acid desaturation in Neurospora

Microsomes prepared from the wild-type strain and lipid auxotrophs of Neurospora were analyzed for delta 9 - (stearoyl-CoA) and delta 12 - (oleoyl-CoA) desaturase activities. The wild-type delta 9-desaturase was found to have a 20-fold higher specific activity and 2-fold lower activation energy than the delta 12-desaturase. In addition, delta 12-desaturase had higher Km app values for oleoyl-CoA and for NADH than the equivalent values for delta 9-desaturase. These properties were correlated with a rate-limiting role of delta 12-desaturase in the production of 18:2, the major fatty acid of Neurospora. The delta 12-desaturase also exhibited a higher tolerance to pH changes and to cyanide than did the delta 9-desaturase. Both activities could be measured in the same reaction mixture using stearoyl-CoA as the substrate, indicating a coupling of the two enzymes. Enrichment of cellular membranes of the wild-type Neurospora with 18:0 and 18:1, 18:2, 18:3 fatty acids led to the conclusion that the presence of excess substrate in the membrane induces activation of the appropriate desaturase. These experiments also suggested that the membrane fluidity, as determined by the degree of unsaturation of membrane fatty acids, may influence the activities of the desaturating enzymes. Perturbation of the polar head groups of the membrane phospholipids indicated that the correct composition of anionic phospholipids is an absolute requirement for the function of both desaturases. These studies show that the activities of the delta 9-desaturase and the delta 12-desaturase are regulated by a variety of factors and that the delta 12-desaturase is subjected to less stringent controls than the delta 9-desaturase.     

Blocking of the effect of delta1 tetrahydrocannabinol (delta1THC) on spontaneous motor activity in the rat by immunization with a protein conjugate of delta1THC

Rats actively immunized with porcine gamma globulin- hemisuccinate-Δ¹-tetrahydrocannabinol (PγG-HS-Δ¹THC) showed higher spontaneous motor activity after intraperitoneal administration of Δ¹THC at a dose of 10 mg. per kg. than did rats immunized with a control antigen, porcine gamma globulin-hemisuccinate-phenol (PγG-HS-Phenol). The capacity to neutralize the effect of Δ¹THC was found to depend on the degree of immunization; thus, the difference in mean spontaneous motor activity after injection of Δ¹THC was significant in rats which had received five injections of the immunogen over a period of 86 days, and not in those which had received only two injections over a period of 34 days.In view of the observations that Δ¹-tetrahydrocannabinol induces a decrease in spontaneous motor activity in rats, the observed neutralization of the effect of δ¹THC in animals receiving multiple injections of protein conjugates of Δ¹THC may be due to the binding of the drug by anti-THC antibodies (which are expected to be produced on active immunization with these conjugates), thus preventing Δ¹THC from reaching drug-receptor sites.     

Engineering delta 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9-18:0-ACP desaturase

Six amino acid locations in the soluble castor Delta(9)-18:0-acyl carrier protein (ACP) desaturase were identified that can affect substrate specificity. Combinatorial saturation mutagenesis of these six amino acids, in conjunction with selection, using an unsaturated fatty acid auxotroph system, led to the isolation of variants with up to 15-fold increased specific activity toward 16-carbon substrates. The most improved mutant, com2, contained two substitutions (T117R/G188L) common to five of the 19 complementing variants subjected to further analysis. These changes, when engineered into otherwise wild-type 18:0-ACP desaturase to make mutant 5.2, produced a 35-fold increase in specific activity with respect to 16-carbon substrates. Kinetic analysis revealed changes in both k(cat) and K(m) that result in an 82-fold improvement in specificity factor for 16-carbon substrate compared with wild-type enzyme. Improved substrate orientation apparently compensated for loss of binding energy that results from the loss of desolvation energy for 16-carbon substrates. Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of magnitude higher than those of known natural 16-carbon specific desaturases. These data support the hypothesis that it should be possible to reengineer archetypal enzymes to achieve substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of a parental paralog.