Presynaptic actions of 4-Aminopyridine and γ-aminobutyric acid on rat sympathetic ganglia in vitro

Responses to bath-applications of 4-aminopyridine (4-AP) and -aminobutyric acid (GABA) were recorded intracellularly from neurones in the rat isolated superior cervical ganglion. 4-aminopyridine (0.1–1.0 mmol/l) usually induced spontaneous action potentials and excitatory postsynaptic potentials (EPSPs), which were blocked by hexamethonium. Membrane potential was unchanged; spike duration was slightly increased. Vagus nerve B-and C-fibre potentials were prolonged. In 4-AP solution (0.1–0.3 mmol/l), GABA (0.1 mmol/l), 3-aminopropanesulphonic acid or muscimol evoked bursts of spikes and EPSPs in addition to a neuronal depolarization. These bursts, which were not elicited by glycine, glutamate, taurine or (±)-baclofen, were completely antagonised by hexamethonium, tetrodotoxin or bicuculline methochloride. It is concluded that: (a) 4-AP has a potent presynaptic action on sympathetic ganglia; (b) presynaptic actions of GABA can be recorded postsynaptically in the presence of 4-AP; and (c) the presynaptic GABA-receptors revealed in this condition are similar to those on the postsynaptic membrane

Genetic Code Mutations: The Breaking of a Three Billion Year Invariance

The genetic code has been unchanging for some three billion years in its canonical ensemble of encoded amino acids, as indicated by the universal adoption of this ensemble by all known organisms. Code mutations beginning with the encoding of 4-fluoro-Trp by Bacillus subtilis, initially replacing and eventually displacing Trp from the ensemble, first revealed the intrinsic mutability of the code. This has since been confirmed by a spectrum of other experimental code alterations in both prokaryotes and eukaryotes. To shed light on the experimental conversion of a rigidly invariant code to a mutating code, the present study examined code mutations determining the propagation of Bacillus subtilis on Trp and 4-, 5- and 6-fluoro-tryptophans. The results obtained with the mutants with respect to cross-inhibitions between the different indole amino acids, and the growth effects of individual nutrient withdrawals rendering essential their biosynthetic pathways, suggested that oligogenic barriers comprising sensitive proteins which malfunction with amino acid analogues provide effective mechanisms for preserving the invariance of the code through immemorial time, and mutations of these barriers open up the code to continuous change

The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world

Biotechnology has empirically established that it is easier to construct and evaluate variant genes and proteins than to account for the emergence and function of wild-type macromolecules. Systematizing this constructive approach, synthetic biology now promises to infer and assemble entirely novel genomes, cells and ecosystems. It is argued here that the theoretical and computational tools needed for this endeavor are missing altogether. However, such tools may not be required for diversifying organisms at the basic level of their chemical constitution by adding, substituting or removing elements and molecular components through directed evolution under selection. Most importantly, chemical diversification of life forms could be designed to block metabolic cross-feed and genetic cross-talk between synthetic and wild species and hence protect natural habitats and human health through novel types of containment

An Expanded Set of Amino Acid Analogs for the Ribosomal Translation of Unnatural Peptides

BACKGROUND: The application of in vitro translation to the synthesis of unnatural peptides may allow the production of extremely large libraries of highly modified peptides, which are a potential source of lead compounds in the search for new pharmaceutical agents. The specificity of the translation apparatus, however, limits the diversity of unnatural amino acids that can be incorporated into peptides by ribosomal translation. We have previously shown that over 90 unnatural amino acids can be enzymatically loaded onto tRNA. METHODOLOGY/PRINCIPAL FINDINGS: We have now used a competition assay to assess the efficiency of tRNA-aminoacylation of these analogs. We have also used a series of peptide translation assays to measure the efficiency with which these analogs are incorporated into peptides. The translation apparatus tolerates most side chain derivatives, a few alpha,alpha disubstituted, N-methyl and alpha-hydroxy derivatives, but no beta-amino acids. We show that over 50 unnatural amino acids can be incorporated into peptides by ribosomal translation. Using a set of analogs that are efficiently charged and translated we were able to prepare individual peptides containing up to 13 different unnatural amino acids. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that a diverse array of unnatural building blocks can be translationally incorporated into peptides. These building blocks provide new opportunities for in vitro selections with highly modified drug-like peptides

A novel Caenorhabditis elegans allele, smn-1(cb131), mimicking a mild form of spinal muscular atrophy, provides a convenient drug screening platform highlighting new and pre-approved compounds

Spinal muscular atrophy (SMA), an autosomal recessive genetic disorder, is characterized by the selective degeneration of lower motor neurons, leading to muscle atrophy and, in the most severe cases, paralysis and death. Deletions and point mutations cause reduced levels of the widely expressed survival motor neuron (SMN) protein, which has been implicated in a range of cellular processes. The mechanisms underlying disease pathogenesis are unclear, and there is no effective treatment. Several animal models have been developed to study SMN function including the nematode, Caenorhabditis elegans, in which a large deletion in the gene homologous to SMN, smn-1, results in neuromuscular dysfunction and larval lethality. Although useful, this null mutant, smn-1(ok355), is not well suited to drug screening. We report the isolation and characterization of smn-1(cb131), a novel allele encoding a substitution in a highly conserved residue of exon 2, resembling a point mutation found in a patient with type IIIb SMA. The smn-1(cb131) animals display milder yet similar defects when compared with the smn-1 null mutant. Using an automated phenotyping system, mutants were shown to swim slower than wild-type animals. This phenotype was used to screen a library of 1040 chemical compounds for drugs that ameliorate the defect, highlighting six for subsequent testing. 4-aminopyridine, gaboxadol hydrochloride and N-acetylneuraminic acid all rescued at least one aspect of smn-1 phenotypic dysfunction. These findings may assist in accelerating the development of drugs for the treatment of SMA.