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The biggest problem in sports is the use of performance-enhancing drugs, even at the high school level. The hazards of anabolic steroid use include legal risk, dependence, mood swings, liver tumors and blood-filled cysts.

We previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for the formation of its parallel two-stranded coiled coil. Here, we demonstrate that the intrinsic secondary structure of the trigger site is largely stabilized by an intrahelical salt bridge. Removal of this surface salt bridge by a single amino acid mutation induced only minor changes in the backbone structure of the GCN4 leucine zipper dimer as verified by nuclear magnetic resonance. The mutation, however, substantially destabilized the dimeric structure. These findings support the proposed hierarchic folding mechanism of the GCN4 coiled coil in which local helix formation within the trigger segment precedes dimerization.

We have recently identified a distinct 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils but not in trimers, tetramers, or pentamers. This coiled-coil trigger pattern was demonstrated to be indispensable for the assembly of the oligomerization domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum and the leucine zipper domain of the yeast transcriptional activator GCN4. With the aim to extend our knowledge on trigger sequences we have investigated the human macrophage scavenger receptor type A oligomerization domain as a representative of three-stranded coiled coils. We prepared a variety of recombinant N- and C-terminal deletion mutants from the full-length oligomerization domain by heterologous gene expression in Escherichia coli and assessed their ability to form trimeric coiled-coil structures by circular dichroism spectroscopy and analytical ultracentrifugation. Deletion mapping identified a distinct seven-residue sequence that was absolutely required for proper coiled-coil formation, supporting our previous results that heptad repeats alone are not sufficient for oligomerization. The finding that all fragments containing this particular sequence exhibited similar thermal stabilities indicates primarily a stabilizing function of the coiled-coil trigger. Based on sequence similarity, we suggest that functionally related sites are present in other three-stranded coiled-coil proteins.

The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5' promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5' and 3' untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.

Xylamine (N-2'-chloroethyl-N-ethyl-2-methylbenzylamine), a nitrogen mustard that irreversibly inhibits norepinephrine uptake, cyclizes in solution to form an aziridinium ion. The first-order rate constants for cyclization at 23 degrees and 37 degrees are 0.12 min-1 and 0.40 min-1, respectively. The aziridinium ion is relatively stable at 23 degrees but hydrolyzes at 37 degrees with a half-time of 70 min. A dimeric compound was indirectly shown to form at 1 mM xylamine through a reaction between the parent mustard and its aziridinium ion. A similar reaction between the 2-hydroxyethylamine and the aziridinium ion does not take place at pH 7.4. The aziridinium ion, its hydrolysis product, and the dimer were synthesized to evaluate directly their effects on norepinephrine uptake in rabbit thoracic aorta. The aziridinium ion was as potent as xylamine as an irreversible uptake inhibitor, and the effects of both compounds were sodium-dependent. The dimer was a weak competitive inhibitor of norepinephrine uptake, with an IC50 of about 10 microM. The 2-hydroxyethylamine, at 100 microM, competitively inhibited only 20% of control norepinephrine accumulation. These results demonstrate that the aziridinium ion is responsible for xylamine's uptake blocking activity and that the other xylamine derivatives do not influence this action.

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.

Background: Immunologic mechanisms of desensitization are still incompletely understood. Safer methods of immunotherapy with reduced risks of anaphylaxis need to be developed. Objective: To study the effects of conventional venom immunotherapy (VIT) on phospholipase A"2(PLA"2)-specific T cells and on T-cell reactivity to short and long synthetic peptides that map the PLA"2 molecule. Method: Proliferation of a CD4^+ cell-enriched peripheral blood mononuclear cell fraction and cytokine secretion by T cell lines from patients hypersensitive to bee venom and undergoing VIT in response to PLA"2 and PLA"2 synthetic peptides were measured. Results: T-cell proliferation in response to three synthetic peptides, 40 to 60 amino acids long and mapping the entire PLA"2 molecule with an overlap of 10 residues (1 to 59, 51 to 99, and 90 to 134) steadily increased during the first 14 weeks of VIT corresponding to the treatment period with incremental doses of antigen. These results are in contrast to the low proliferation indices obtained with short (15 amino acid-long) peptides, and the inability to characterize the immunodominant region of the molecule with short peptides. At the end of VIT (after 3 to 5 years), there was correspondingly, a marked decrease in T cell responsiveness to PLA"2 and to its long synthetic peptides. This response was paralleled by a shift in the pattern of cytokine secretion by T cell lines from a T"H"0-type to a T"H"1-type pattern. Conclusion: After a transient increase in T-cell proliferation, late VIT was characterized by T-cell hyporesponsiveness to allergen and by modulation of cytokine secretion from a T"H"0-type to a T"H"1-type pattern. Because of their capacity to recruit multiple T-cell epitopes, long peptides mapping the entire PLA"2 molecule appear to be efficient T cell stimulators and may represent potential candidates for peptide immunotherapy. (J Allergy Clin Immunol 1997;100:96-103.)

The human hepatic asialoglycoprotein receptor is a noncovalent hetero-oligomer composed of two homologous subunits, H1 and H2, with an as yet unknown stoichiometry. Ligand specificity and binding affinity depend on the arrangement of the subunits in the complex. An 80-amino acid segment connecting the transmembrane and the carbohydrate binding domains contains heptad repeats characteristic of alpha-helical coiled coil structure. We expressed and purified corresponding peptides, H1S and H2S, and confirmed by circular dichroism spectroscopy that they can assume alpha-helical conformation. Oxidative cross-linking of amino-terminal cysteines generated specific covalent oligomers, indicating that separately H1S forms trimers and H2S tetramers. Upon mixing, covalent heterotetramers were formed with a preferred stoichiometry of 2 H1S and 2 H2S peptides. These results suggest that the stalk segments of the receptor subunits oligomerize to constitute an alpha-helical coiled coil stalk on top of which the carbohydrate binding domains are exposed for ligand binding. We propose that the functional asialoglycoprotein receptor is a 2:2 heterotetramer.

Xylamine (N-2-chloroethyl-N-ethyl-2-methylbenzylamine) irreversibly inhibits [3H]norepinephrine accumulation by rabbit aortic rings in vitro. Inhibition is observed when aortic rings are exposed to xylamine, washed and [3H]norepinephrine accumulation is measured in media free of xylamine. This dose-dependent effect, with an I50 of 10(-7) M, is prevented by coincubation of xylamine with the reversible uptake inhibitors cocaine and amphetamine in the incubation bath during exposure of the tissue to xylamine. The action of xylamine is also sodium dependent, since when tissue was exposed to xylamine in sodium-free Krebs' solution, xylamine had no action. Reduction of potassium ion concentration and the presence of ouabain also protected against xylamine inhibition. These results indicate that xylamine action involves interaction with the neuronal uptake system in a manner very similar to norepinephrine.

The effects of xylamine (N-2-chloroethyl-N-ethyl-2-methylbenzylamine) on catecholamine content and uptake in rat brain were examined after systemic administration. Four hours after doses of 10 to 50 mg/kg i.p. of xylamine, norepinephrine uptake in synaptosomal preparations from cortex was reduced, but dopamine uptake in striatal preparations was unaffected. The levels of norepinephrine in the cortex and hypothalamus were depressed over this dose range, whereas dopamine levels in the striatum again were unaltered. The depletion of norepinephrine from the cortex and hypothalamus was also seen histologically by fluorescence histochemistry. When brain tissue was examined 10 days after a single 25 mg/kg dose, the depletion persisted in cortex and hypothalamus, and although there was a decrease in fluorescence intensity there was no clear evidence of a decreased number of catecholamine-containing processes that would have indicated neurotoxic effects. There was also no gliosis (an indicator of neuronal degeneration) in any of the brain areas or damage to the blood brain barrier. Thus, in contrast to 6-hydroxydopamine, xylamine appears to have selective action on norepinephrine systems and does not appear to be a nonselective neurotoxin.