Teratologic evaluation of synthetic delta-9-tetrahydrocannabinol in rats.

Delta-9-Tetrahydrocannabinol (THC) was dissolved in propylene glycol and 25, 50, or 100 mg/kg administereed dialy sc to pregnant Charles River Sprague-Dawley rats on days 6-15 of gestation (presence of sperm considered day 1). Maternal weight gain was depressed, but a significant decrease in fetal weight occurred only in the 50 mg/kg group. No malformations were noted, only some abnormalities consisting of several instances of rudimentary 14th rib and soft or spongy spinal cords.     

Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration. Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin. By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces. Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain. Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles. Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation. Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles. Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups. This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+. Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+. Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport. Proton-conducting ionophores and several inhibitors of respiration blocked Rb+ uptake and dissipated a preformed gradient. Preincubation of the vesicles with oxamate or p-chloromrecuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ be, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ compete for uptake when present simultaneously. We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration.     

Preliminary characterization of the delta-9 desaturase of Tetrahymena pyriformis W.

1. In vitro assay conditions have been defined for measurement of delta 9 desaturase activity in Tetrahymena pyriformis W. 2. The reaction depends on the presence of oxygen and a reduced pyridine nucleotide cofactor. FAD supports a low level of enzymatic activity. 3. Both stearyl-CoA and palmityl-CoA are acceptable substrates. Oleate formation is maximal at 30 degrees C. 4. Delta-9 desaturase activity appears to be localized in the microsomal fraction. Delta-6 and/or delta 12 desaturase activities have also been observed. 5. When the specificity of the delta 9 desaturase towards stearyl-CoA and palmityl-CoA was observed at 30 and 16 degrees C it was found that lowering the assay temperature did not affect specificity. Stearyl-CoA was more readily desaturated at both temperatures. 6. Exogenous oleyl-CoA and diisopropylfluorophosphate had little effect on delta 9 desaturase activity. However, cyanide strongly inhibited desaturation and a sensitivity to sulfhydryl-binding reagents has also been demonstrated.     

delta- and gamma-Sarcoglycan localization in the sarcoplasmic reticulum of skeletal muscle.

Sarcoglycans are transmembrane proteins that are members of the dystrophin complex. Sarcoglycans cluster together to form a complex, which is localized in the cell membrane of skeletal, cardiac, and smooth muscle fibers. However, it is still unclear whether or not sarcoglycans are restricted to the sarcolemma. To address this issue, we examined alpha-, beta-, delta-, and gamma-sarcoglycan expression in femoral skeletal muscle from control and dystrophin-deficient mice and rats using confocal microscopy and immunoelectron microscopy. Confocal microscopy of the tissues in cross-section showed that all sarcoglycans were detected under the sarcolemma in rats and control mice. delta- and gamma-sarcoglycan labeling demonstrated striations in the longitudinal section, suggesting that the proteins were expressed in the sarcoplasmic reticulum (SR) or transverse tubules (T-tubules). Moreover, such striations of both sarcoglycans were recognized in the dystrophin-deficient mouse skeletal muscle. Double labeling with phalloidin or alpha-actinin and delta- or gamma-sarcoglycan showed different labeling patterns, indicating that delta-sarcoglycan localization was distinct from that of gamma-sarcoglycan. Immunoelectron microscopy clarified that delta-sarcoglycan was localized in the terminal cisternae of the SR, while gamma-sarcoglycan was found in the terminal cisternae and longitudinal SR over I-bands but not over A-bands. These data demonstrate that delta- and gamma-sarcoglycans are components of the SR in skeletal muscle, suggesting that both sarcoglycans function independent of the dystrophin complex in the SR.     

Biosynthesis of delta-7-cholesten-3-beta-ol, delta-5,7-cholestadien-3-beta-ol, and delta-5-cholesten-3-beta-ol by guinea pig intestinal mucosa in vitro

Methods were developed for the separation and determination of the various 27-carbon sterols of intestinal mucosa by means of thin-layer chromatography. Scrapings of the mucosa of the small intestine of guinea pig and rat were shown to incorporate isotope from (14)C-labeled acetate and mevalonate into sterols in vitro. For each substrate this activity was lowest in mucosa from the proximal third of the small intestine and greatest in mucosa from the more distal regions of the small intestine. The total 27-carbon sterol content of guinea pig mucosa varied only slightly along the length of the small intestine, but the concentration of cholesterol was highest distally. More than 95% of the radioactivity incorporated from acetate-2-(14)C into 27-carbon sterols by guinea pig mucosa in 4 hr was recovered as lathosterol and 7-dehydrocholesterol; less than 5% was in cholesterol. The specific activities of the 27-carbon sterols were consistent with the concept that synthesis proceeds from lathosterol to 7-dehydrocholesterol to cholesterol.     

The inactivation of ornithine transcarbamoylase by N delta-(N''-sulpho-diaminophosphinyl)-L-ornithine.

Phaseolotoxin, a tripeptide inhibitor of ornithine transcarbamoylase, is a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, the causal agent of halo-blight in beans. In vivo the toxin is cleaved to release N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine, the major toxic chemical species present in diseased leaf tissue. This paper reports on the interaction between N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine and ornithine transcarbamoylase. N delta-(N'-Sulpho-diaminophosphinyl)-L-ornithine was found to be a potent inactivator of the enzyme, in contrast with phaseolotoxin, which previously has been reported to inhibit the enzyme reversibly. Inactivation by N delta-(N'-[35S]sulpho-diaminophosphinyl)-L-ornithine resulted in the incorporation of 35S into ethanol-precipitated protein. The stoicheiometry of 35S incorporation was approximately 1 mol/mol of active sites. Inactivation was second-order and a rate constant of 10(6) M-1 X s-1 at 0 degree C in 50 mM-Tris/HCl, pH 9.0, was obtained. Carbamoyl phosphate, a substrate of ornithine transcarbamoylase, protected the enzyme from inactivation. A dissociation constant of 3 microM for the enzyme-carbamoyl phosphate complex was calculated. L-Ornithine, the second substrate for ornithine transcarbamoylase, protected the enzyme only at high concentrations. The results are consistent with N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine being a potent affinity label that binds via the carbamoyl phosphate-binding site of ornithine transcarbamoylase. Cleavage of phaseolotoxin to N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine in vivo appears to be an important function in the physiology of the disease.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Suppressive effect of delta-9-tetrahydrocannabinol on herpes simplex virus infectivity in vitro.

Delta-9-Tetrahydrocannabinol (THC) was found to reduce the infectivity of herpes simplex virus and was without effect against adenovirus type 2 or poliovirus. The effective THC concentration resulting in an 80% decrement in virus viability was dependent upon the presence or absence of serum in the incubation mixture, as a 5% serum concentration decreased the drug activity by approximately 50-fold. THC-mediated inactivation of herpes simplex virus was both time and dose dependent and did not result in virion disassembly or clumping. The THC-related effect was not influenced by the pH of the suspending medium, suggesting that the mechanism of inactivation differed from that associated with the thermal inactivation of the virus. Thus, the data suggest that THC preferentially reduces the infectivity of the enveloped herpes simplex virus, and that this activity is modulated by the presence of serum proteins.     

Use of delta-(alpha-aminoadipoyl) chromogenic amides in screening for aminoadipoyl amidohydrolases.

The synthesis of delta-(alpha-aminoadipoyl) aromatic amides and their use in screening for enzymes able to cleave delta-(alpha-aminoadipoyl) residues off the synthetic amides and cephalosporin C are described. A number of commercially available proteases and peptidases were not active with delta-(alpha-aminoadipoyl) chromogenic amides. Also, most tested microbial strains known to produce acylases did not hydrolyze these compounds. Only one microbial strain, Xanthomonas maltophila, had an appreciable activity toward the racemic form of chromogenic substrates. Activity measured in crude extracts from Xanthomonas cells indicated that this bacterium produces predominantly L-specific aminoadipoyl amidohydrolase and gamma-glutamyl hydrolase. A low level of cephalosporin C and glutaryl-cephalosporin acylase activities was also found.