Rapid glucocorticoid stimulation and GABAergic inhibition of hippocampal serotonergic response: in vivo dialysis in the lizard anolis carolinensis

Central serotonin (5-HT) is activated during stressful situations and aggressive interactions in a number of species. Glucocorticoids secreted peripherally during stressful events feed back on central systems and may affect 5-HT mediation of stress-induced behavioral events. To test the neuromodulatory effect of stress hormone secretion, serotonin overflow was measured from the hippocampus of the lizard Anolis carolinensis. Microdialysis was used to collect repeated samples from anesthetized lizards, with perfusate measured by HPLC with electrochemical analysis. Following initially high levels of 5-HT, concentrations stabilized to basal levels after approximately 2 h. Intracortical infusion of 200 ng/ml corticosterone evoked transient increases in 5-HT release of approximately 400%. The effect of corticosterone on 5-HT overflow appears to be dose dependent as 20 ng/ml stimulated an increase of 200%, whereas 2 ng/ml stimulated a 50% increase. Administration of 0.1 and 1 ng/ml GABA via the dialysis probe significantly inhibited 5-HT overflow by 20 and 40%, respectively. The duration of GABA inhibition is greater than the stimulatory response for glucocorticoids. Short-lived glucocorticoid stimulation of 5-HT release suggests a possible mechanism for endocrine mediation of continuously changing social behavioral events.     

Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues

We report a novel, highly sensitive and selective method for the extraction and quantification of acyl CoA esters from plant tissues. The method detects acyl CoA esters with acyl chain lengths from C4 to C20 down to concentrations as low as 6 fmol in extracts. Acyl CoA esters from standard solutions or plant extracts were derived to their fluorescent acyl etheno CoA esters in the presence of chloroacetaldehyde, separated by ion-paired reversed-phase high-performance liquid chromatography, and detected fluorometrically. This derivitization procedure circumvents the selectivity problems associated with previously published enzymatic methods, and methods that rely on acyl chain or thiol group modification for acyl CoA ester detection. The formation of acyl etheno CoA esters was verified by mass spectrometry, which was also used to identify unknown peaks from chromatograms of plant extracts. Using this method, we report the composition and concentration of the acyl CoA pool during lipid synthesis in maturing Brassica napus seeds and during storage lipid breakdown in 2-day-old Arabidopsis thaliana seedlings. The concentrations measured were in the 3--6 microM range for both tissue types. We also demonstrate the utility of acyl CoA profiling in a transgenic B. napus line that has high levels of lauric acid. To our knowledge, this is the first time that reliable estimates of acyl CoA ester concentrations have been made for higher plants, and the ability to profile these metabolites provides a valuable new tool for the investigation of gene function.     

Mode of reproduction and amplified fragment length polymorphism variation in purple needlegrass (Nassella pulchra): utilization of natural germplasm sources

A dominant plant of the California grasslands, purple needlegrass [Nassella pulchra (Hitchc.) Barkworth] is an important revegetation species in its native range. The amplified fragment length polymorphism (AFLP) method was used to elucidate mode of reproduction and nucleotide variation among 11 natural populations and three selected natural germplasm releases of N. pulchra. A total of 12 co-dominant AFLPs, informative within eight populations, failed to reveal any heterozygous individuals, indicating very high selfing rates (S(H)=1). Estimates of nucleotide diversity within populations ranged from 0 to 0.00069 (0.00035 average), whereas the total nucleotide divergence among populations ranged from 0.00107 to 0.00382 (0.00247 average). Measures of population differentiation (GS) in terms of Shannon-Weaver diversity values and estimated nucleotide substitutions were 0.90 and 0.86, respectively. Although some of the sample populations contained a mixture of true breeding genotypes, most populations could be distinguished unambiguously. Moreover, geographical distance between the natural source populations was significantly correlated with genetic distance (r = 0.60) among the corresponding sample populations. Results indicate that inbreeding, combined with founder effects and/or selection, has contributed to the differentiation of N. pulchra populations. Foundation seed populations of the selected natural germplasm releases were genetically well defined and most similar to natural seed collected near the corresponding source populations. Thus, these commercial germplasm sources will be made practically available and useful for conservation plantings within the intended areas of utilization.     

Molecular phylogenetics of western North American frogs of the Rana boylii species group

Phylogenetic relationships among frogs of the genus Rana from western North America are investigated using 2013 aligned bases of mitochondrial DNA sequence from the genes encoding ND1 (subunit one of NADH dehydrogenase), tRNA(Ile), tRNA(Gln), tRNA(Met), ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), and COI (subunit I of cytochrome c oxidase), plus the origin for light-strand replication (O(L)) between the tRNA(Asn) and tRNA(Cys) genes. The aligned sequences contain 401 phylogenetically informative characters. A well-resolved phylogenetic hypothesis in which the Rana boylii species group (R. aurora, R. boylii, R. cascadae, R. muscosa, and R. pretiosa) is monophyletic is obtained. Molecular sequence divergence suggests that the R. boylii species group is approximately 8 million years old. The traditional hypothesis showing monophyly of the yellow-legged frogs (R. boylii and R. muscosa) is statistically rejected in favor of a hypothesis in which R. aurora, R. cascadae, and R. muscosa form a clade. Reanalyses of published nuclear ribosomal DNA restriction-site data and allozymic data support a monophyletic R. boylii group, but do not effectively resolve relationships among species within this group. Eight populations of R. muscosa form two major clades separated by a biogeographic break in the Sierra Nevada of California. This biogeographic break is broadly concordant with breaks found in four other amphibian and reptilian taxa. The two major clades within R. muscosa are estimated to have diverged approximately 2.2 million years before present. Each of these major clades contains two subgroups showing approximately 1.5 million years divergence, implicating climatic effects of Pleistocene glaciation in vicariance. The four distinct subgroups of R. muscosa separated by at least 1.4 million years of evolutionary divergence are suggested as potential units for conservation.     

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.     

Specific mode of interaction between components of model pulmonary surfactants using computer simulations

Atomistic molecular dynamics simulations and structural bioinformatics tools enable the identification of the exact mode of interaction between model pulmonary surfactant components. Two nanosecond long simulations of the N-terminal region of human surfactant protein-B (SP-B(1-25)) in dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers of different lipid surface densities reveal the preferential affinity of SP-B(1-25) for anionic phospholipids. In particular, arginine 12 and lysine 24 interact strongly and with high specificity with the phosphate group of the DPPG lipids, stabilizing the position, the orientation, and the secondary structure of the peptide in the monolayer. The peptide lies at an oblique angle to the interfacial plane, ranging between 47 degrees and 62 degrees, increasing with decreasing lipid surface density. In DPPC monolayers the interaction is largely determined by hydrophobic interactions. The non-specific nature of DPPC-SP-B(1-25) interactions allows for significant flexibility in the topology of the peptide in the lipid matrix. Bioinformatics tools are employed to generalize the simulation results to the sequences of SP-B(1-25) in other organisms. The importance of specific residues, and the role of the largely helical and amphiphilic nature of the peptide in the functionality of SP-B(1-25) are established. The synergy of classical mechanics tools with bioinformatics methods greatly enhances the molecular-level interpretation of pulmonary surfactant action and facilitates the development of design rules for synthetic surfactant analogues.     

Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination

Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.     

Control of germination and lipid mobilization by COMATOSE,the Arabidopsis homologue of human ALDP

Embryo dormancy in flowering plants is an important dispersal mechanism that promotes survival of the seed through time. The subsequent transition to germination is a critical control point regulating initiation of vegetative growth. Here we show that the Arabidopsis COMATOSE (CTS) locus is required for this transition, and acts, at least in part, by profoundly affecting the metabolism of stored lipids. CTS encodes a peroxisomal protein of the ATP binding cassette (ABC) transporter class with significant identity to the human X-linked adrenoleukodystrophy protein (ALDP). Like X-ALD patients, cts mutant embryos and seedlings exhibit pleiotropic phenotypes associated with perturbation in fatty acid metabolism. CTS expression transiently increases shortly after imbibition during germination, but not in imbibed dormant seeds, and genetic analyses show that CTS is negatively regulated by loci that promote embryo dormancy through multiple independent pathways. Our results demonstrate that CTS regulates transport of acyl CoAs into the peroxisome, and indicate that regulation of CTS function is a major control point for the switch between the opposing developmental programmes of dormancy and germination.     

Visualization of proteins in acrylamide gels using ultraviolet illumination.

Proteins in polyacrylamide gels can be rapidly visualized by soaking in trichloroacetic acid or chloroform followed by illumination with UV light. The UV-light-driven reaction of tryptophan in the presence of trichlorocompounds yields products that emit sufficiently in the visible region to identify the location of the protein bands on the gel. This method can be used to rapidly identify protein bands on a gel in less than 20 min. On thin polyacrylamide gels, 1.0 microg of protein can easily be detected for proteins with typical tryptophan percentages.