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.     

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.     

R15 alpha 1 and R 15 alpha 2 peptides from Aplysia: comparison of bioactivity, distribution, and function of two peptides generated by alternative splicing

The mRNA precursor encoded by the R15 gene is alternatively spliced in different neurons to form two related variants, R15-1 and R15-2 mRNA. One of the peptides encoded by the R15-2 mRNA, the R15 alpha 1 peptide, is expressed in the endogenously bursting neuron R15 and mediates some of its central and peripheral synaptic actions. In this study we found that the R15 alpha 2 peptide, which is encoded by the R15-1 mRNA, is synthesized in other neurons in the abdominal ganglion and is also bioactive. The R15 alpha 1 and R15 alpha 2 peptides were found to exert many similar actions on the cardiovascular, digestive, respiratory, and reproductive systems. However, the differences between many of the pharmacological effects of the R15 alpha 1 and R15 alpha 2 peptides indicate that alternative splicing in this system results in two functionally different peptides. Widespread immunoreactivity was found for an antibody directed against the R15 alpha 2 peptide, both in the central nervous system and the periphery. But because of the shared sequence with the R15 alpha 1 peptide, the antibody cross-reacts with the R15 alpha 1 peptide. To distinguish immunocytochemically between the two peptides, we also raised a second antibody that recognizes only the R15 alpha 1 peptide. This antibody labeled the cell body of only one neuron in the central nervous system, R15, although widespread immunoreactivity was found in axons and varicosities in the periphery.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2.

The c-kit ligand, KL, and its receptor, the proto-oncogene c-kit are encoded, respectively, at the steel (Sl) and white spotting (W) loci of the mouse. Both Sl and W mutations affect cellular targets in melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. Although identified as a soluble protein, the predicted amino acid sequence of KL indicates that it is an integral transmembrane protein. We have investigated the relationship between the soluble and the cell associated forms of KL and the regulation of their expression. We show that the soluble form of KL is generated by efficient proteolytic cleavage from a transmembrane precursor, KL-1. An alternatively spliced version of KL-1, KL-2, in which the major proteolytic cleavage site is removed by splicing, is shown to produce a soluble biologically active form of KL as well, although with somewhat diminished efficiency. The protein kinase C inducer phorbol 12-myristate 13-acetate and the calcium ionophore A23187 were shown to induce the cleavage of both KL-1 and KL-2 at similar rates, suggesting that this process can be regulated differentially. Furthermore, proteolytic processing of both the KL-1 and KL-2 transmembrane protein products was shown to occur on the cell surface. The relative abundance of KL-1 and KL-2 is controlled in a tissue-specific manner. Sld, a viable steel allele, is shown to encode a biologically active secreted mutant KL protein. These results indicate an important function for both the soluble and the cell associate form of KL. The respective roles of the soluble and cell associated forms of KL in the proliferative and migratory functions of c-kit are discussed.     

A review of Cheilothela (Ditrichaceae)

Cheilothela Lindb. is recognized as a monotypic genus forC. chloropus (Brid.) Lindb. The South American and New Zealand taxa are considered to be synonymous and placed in the resurrectedChrysoblastella Williams asC. chilensis (Mont.) Reim.Chrysoblastella is segregated fromCheilothela on the basis of numerous sporophytic characters as well as their phytogeography.Cheilothela longirostre Fleisch., from Java, is misplaced in either genus and a new genus,Strombulidens, is erected to accommodate it.Strombulidens is considered most closely related toWilsoniella on the basis of their unique peristomes.