Structure of Leu5-enkephalin bound to a model membrane as determined by high-resolution NMR

Summary Opioid peptides are thought to interact with the cell membrane in their biological journey to the membrane-bound receptor. Both organic solvents and model membranes have been used previously to determine the stable solution conformations of peptide hormones. Leucine enkephalin has been studied in a number of different environments, but with limited resolution. Here it is shown that leucine enkephalin forms a stable type IV -turn structure in dodecylphosphocholine micelles. We have observed a highly solvent-shielded amide proton with no evidence for a complementary hydrogen bond acceptor. The structural details of the peptide as determined by NMR spectroscopy in solution are described.     

The formation of an azo anion free radical metabolite during the microsomal azo reduction of sulfonazo III.

An ESR spectrum is observed during the anaerobic incubation of the diazonaphthol dye sulfonazo III, with rat hepatic microsomes and NADPH. This spectrum is characterized by a partially resolved 17-line hyperfine pattern and g = 2.0043, as is consistent with the spectrum of an azo anion free radical, [R-N-N-R′]^$\text{?}$. Oxygen, which strongly inhibits microsomal azoreductase, destroys the ESR signal. The oxidation of the azo anion radical metabolite by oxygen to the parent azo dye may account for the oxygen inhibition of microsomal azoreductase.     

Effects of enkephalins on perfusion pressure in isolated hindlimb preparations

A series of studies were conducted to determine the effects of leucine-(leu-) enkephalin and methionine-(met-) enkephalin on perfusion pressure. These experiments utilized isolated perfused femoral arterial preparations in pentobarbital-anesthetized cats. The enkephalins were administered intraarterially into the femoral artery and changes in perfusion pressure recorded. Leu-enkephalin in doses of 1 μg to 320 μg produced significant dose-dependent decreases in perfusion pressure (4.0 ± 1.3% with 1 μg to 19.1 ± 2.1% with 320 μg). Similar declines in perfusion pressure (5.2 ± 2.4% with 1 μg to 21.7 ± 4.1% with 320 μg) were observed following the administration of met-enkephalin. Pretreatment with naloxone (3 mg/kg) antagonized the effects of both enkephalins. Diphenhydramine (2 mg/kg) effectively antagonized the leu-enkephalin elicited decline in perfusion pressure but blocked the effects of met-enkephalin only at lower agonist doses. Propranolol treatment (4 mg/kg) did not alter the pressure responses to either enkephalin. The results of the study show that intraarterially administered enkephalins exert a vasodilatory effect on vasculature in skeletal muscle which may be direct, indirect or both. The differential antagonism of the effects of the two enkephalins suggest that the two opioids act through different receptors or multiple receptors.     

Thirteen new chromosome-7 congenic lines

Thirteen new congenic lines have been produced which have chromosome-7 segments introduced from different strains onto the C57BL/10Sn background. Sublines B10.P(61NX)C,D, and E received chromosome-7 segments from P/J, B10.CE(62NX) from CE/J, B10.SEC(64NX)A,C,E, and F from SEC/1Re, B10.SM(65NX) from SM/J, B10.WB(66NX) from WB/Re, B10.A(67NX) from A/SnGrf, B10.AKR(68NX) from AKR/SnGrf, and B10.K(69NX) from C3H.K. Isograft testing indicated that three sublines, B10.P(61NX)D, B10.CE(62NX)B, and B10.WB(66NX)B are histoisogenic, i.e., histocompatible within each line. With the exception of B10.A(67NX), B10.AK(68NX), and B10.K(69NX), which have not been isografted, the remaining sublines showed residual heterozygosity on isografting. The three histoisogenic lines have undergone F₁ testing and have been found to possess theH-4 ^a allele and new and distinct alleles at theH-1 locus. They have been designated B10.P(61NX)-H-4^a H-1 ^d , B10.WB(66NX)-H-4^a H-1 ^e , and B10.CE(62NX)-H-4^a H-1 ^f . Direct exchange of grafts has indicated the following genotypes: B10.A(67NX)-H-4^a H-1 ^b , B10.AK(68NX)-H-4^a H-1 ^b , and B10.K(69NX)-F-4^a H-1 ^b . The B10.SEC(64NX) and B10.SM(65NX) sublines have not been typed completely forH-4 andH-1. F ₁ testing or direct exchange of skin grafts indicated that B10.P(61NX)-H-4^a H-1 ^d , B10.WB(66NX)-H-4^a H-1 ^e , B10.A(67NX)-H-4^a H-1 ^b B10.AK(68NX)-H-4^a H-1 ^b and B10.K(69NX)-H-4^a H1 ^b possess nonon-H-1 histocompatibility differences from the G57BL/10 background.     

RESPIRATION IN IMMATURE RAT BRAIN MITOCHONDRIA

—Respiration was studied polarographically in mitochondria isolated from immature rat cerebral hemispheres. Respiratory rates are compared as a function of age, substrate, and the requirement for a phosphate acceptor. 1. All respiratory rates are low in the first week of life. These rates increase during the first month and then decline to about the newborn rate by 5 weeks of age. 2. With the NAD-linked substrate pair, glutamate and malate, the changes with age are significant only for the rate of ADP-dependent respiration. With succinate as substrate, significant age-dependent changes in respiration occur only in ADP-independent respiration. 3. In mitochondria from animals less than five weeks of age, the ADP-dependent respiratory rate is significantly greater with the NAD-linked substrate pair than with succinate. In mitochondria from older animals, both ADP-dependent and ADP-independent rates are greater with succinate.     

Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels

Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels.     

HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days—Implications for HIV Remission

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to ‘purge’ the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different ‘reactivation founder’ viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.