RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX.

We used in vitro selection (SELEX) to isolate RNA 'aptamers' to S-adenosyl methionine (SAM). Individual aptamer sequences conform to the structural element noted previously for adenosine binding in selections for aptamers to ATP and NAD+. When we compare the patterns of sequence conservation among 65 adenosine-binding sequences to the published structure of the adenosine aptamer, we find that the most highly conserved nucleotides contact the bound adenosine directly, and that one conserved nucleotide outside the binding pocket is in position to stabilize nucleotides within the binding pocket. The aptamer's ability to bind diverse adenosine-containing cofactors is easily understood in terms of its mode of binding, which leaves the 5'position exposed to solvent. We propose that aptamers that bind their targets away from the reactive moiety may be particularly well suited for catalysis. Finally, we estimate that one sequence in 10(11) may be able to form this structural motif, and that there may be many other adenosine-binding motifs that have escaped detection because of their lower representation in the starting random pools.     

Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin.

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature.The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems.A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.     

Mutations that detoxify an aberrant T4 membrane protein

We have investigated suppressors of the bacteriophage T4 rIIB toxic polypeptide encoded by the rIIB frameshift mutation FC238. We have found suppressors that eliminate the toxic polypeptide by creating new translational termination codons, that diminish the toxicity of the polypeptide by altering the amino acid sequence of the toxic protein, that alter the rIIA protein so as to influence toxicity, and that diminish the amount of toxic polypeptide by reducing the quantity of gene expression from the rIIB (FC238) gene. We propose that the toxicity of the FC238 polypeptide derives from its peculiar, bipartite structure and high membrane avidity. Suppressors that detoxify the FC238 polypeptide by missense probably disturb the bipartite structure and/or the affinity for the membrane. The distribution of transition mutations obtained with a variety of mutagens contributes to an appreciation of intrinsic mutability differences. Lastly, although suppressors of FC238 toxicity might emerge in phage genes other than rIIB and rIIA, none have been found.     

Glucocorticoid receptor gene polymorphisms in premenopausal women with major depression.

Glucocorticoid receptor gene polymorphisms are associated with glucocorticoid hypersensitivity and visceral obesity. Perturbations in HPA axis sensitivity to glucocorticoids implicated in the pathogenesis of major depression may result from functional alterations in the glucocorticoid receptor gene. We 1) examined the prevalence of genotype distribution of specific polymorphisms of the glucocorticoid receptor gene (Bcl1, N363S, rs33388, rs33389) in a subset of women from the P.O.W.E.R. Study (which enrolled 21- to 45-year-old premenopausal women with major depression and healthy controls) and 2) explored whether such polymorphisms were associated with visceral obesity and insulin resistance. Women with major depression had a higher body mass index, a higher waist:hip ratio, and more body fat than did controls. No differences were observed in plasma and urinary cortisol or in insulin sensitivity. The G/G genotype of the Bcl1 polymorphism was significantly more common (p<0.03) in women with major depression (n=52) than in controls (n=29). In addition, GG homozygotes (depressed n=10; controls n=2) had higher waist:hip ratios than did non-GG carriers (p<0.02). N363S, rs33388, and rs33389 polymorphisms were not different between groups. In conclusion, premenopausal women with both major depression and the GG genotype of the Bcl1 polymorphism had greater abdominal obesity compared with non-GG carriers.     

Vascular involvement in relapsing polychondritis.

Review of four cases of relapsing polychondritis (RP) seen at one hospital in the 12-year period 1963 to 1974 revealed that one patient had aortic insufficiency with large artery involvement, two others had involvement of medium and large arteries and the fourth may have had mucocutaneous vasculitis. Valvular disease has occurred in 9% of all cases of RP reported in the literature and, if vasculitis beyong the aortic root is included, 25% of cases of RP manifested inflammatory vascular disease. The frequency of pseudotumour of the orbit and cochlear-labyrinthine dysfunction is also high and may be a manifestation of vasculitis.     

Dimethyl Fumarate Ameliorates Lewis Rat Experimental Autoimmune Neuritis and Mediates Axonal Protection

BackgroundDimethyl fumarate is an immunomodulatory and neuroprotective drug, approved recently for the treatment of relapsing-remitting multiple sclerosis. In view of the limited therapeutic options for human acute and chronic polyneuritis, we used the animal model of experimental autoimmune neuritis in the Lewis rat to study the effects of dimethyl fumarate on autoimmune inflammation and neuroprotection in the peripheral nervous system.ConclusionsWe conclude that immunmodulatory and neuroprotective dimethyl fumarate may represent an innovative therapeutic option in human autoimmune neuropathies.     

Tryptic proteol ysis of coupling factor-latent ATPase from Mycobacterium phlei. Theoretical modeling of structure-function relationships

Trypsin treatment of solubilized coupling factor-latent ATPase from Mycobacterium phlei alters its subunit structure and functional properties. This coupling factor exhibits ATPase activity following trypsin treatment. Concurrently, both the ability of the enzyme to rebind to membranes depleted of coupling factor and its capacity for coupled phosphorylation are lost. The native alpha (64 000 dalton) subunit undergoes limited proteolytic digestion, and the delta (14 000 dalton) subunit is partially lost. During the course of tryptic proteolysis, the coupling factor molecule may exist in one of ten unique structural states (e.g. the native, ATPase-inactive molecule exists in the ααα state). Rigorous analysis of the experimental data by theoretical modeling provided information concerning the intermediate structural states leading to the fully ATPase-activated α″α″α″ state under different conditions of trypsin treatment. The theoretical models of structure-function relationships that best-represented the experimental data predicted that the native coupling factor molecule contains three copies of the α (64 000 dalton) form of the alpha subunit, that the α″ (58 000 dalton) alpha subunit species contributes maximally and the α′ (61 000 dalton) form about half-maximally to ATPase activity, that membrane rebinding ability is proportional to the number of native alpha subunits in the enzyme, and that at least one native α subunit/molecule is required for full expression of coupled phosphorylation. These results indicate an essential role for the alpha subunit in the regulation of ATPase activity and in the ability of the solubilized coupling factor to rebind to depleted membranes.     

Using California Gnatcatcher to Test Underlying Models in Habitat Conservation Plans

Abstract: Habitat Conservation Plans are a widely used strategy to balance development and preservation of species of concern and have been used in southern California, USA, to protect the coastal California gnatcatcher (Polioptila californica). Few data exist on gnatcatcher abundance and distribution, and existing data have problems with issues of closure (i.e., sampling occurs in a short enough time period such that abundance or distribution are not changing), detectability, and proper attention to probability-based sampling schemes. Thus, a habitat model has been relied upon in reserve design. California gnatcatchers are the flagship and umbrella species of many plans and we provide the first estimates that incorporate probabilistic sampling and test predictions from the habitat model. Probability of occurrence was 26% (SĚ = 0.06); however, occupancy varied by modeled habitat quality with slopes <40%, warm, and wet sagebrush habitat having higher occupancy probabilities. Interpreting abundance and occupancy probabilities by vegetation type was complicated by error detected in Geographic Information System vegetation metadata files. The slope (1.08, SĚ = 0.66), temperature (0.79, SĚ = 0.70), and precipitation (—2.62, SĚ = 1.21) variables associated with habitat models were stronger influences on occupancy than was patch size (0.48, SĚ = 0.66). Previous models weight patch size equal to slope and climate. Our work demonstrates that probabilistic sampling can be carried out on a large scale and the results provide better information for managers to make decisions about their reserves.