Triple-label beta liquid scintillation counting.

The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three beta-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label beta counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed.     

Poly(adenylic acid)-containing and -deficient messenger RNA of mouse liver

RNA was isolated and fractionated into poly(A)-containing and -deficient classes by oligo(dT) chromatography. Approximately 99% of the poly(A) material bound to the oligo(dT); that which did not bind contained substantially shorter poly(A) chains. All RNA fractions retained an ability to initiate cell-free translation, with the poly(A)-deficient fraction containing half the total translational activity, i.e., mRNA. Two-dimensional polyacrylamide gel analysis of the cell-free translation products revealed three classes of mRNA: 1, mRNA preferentially containing poly(A), including the abundant liver mRNA species; 2, poly(A)-deficient mRNA, including many mid- and low-abundant mRNAs exhibiting less than 10% contamination in the poly(A)-containing fraction fraction; and 3, bimorphic species of mRNA proportioned between both the poly(A)-containing and -deficient fractions. Poly(A)-containing and bimorphic mRNA classes were further characterized by cDNA hybridizations. The capacity of various RNA fractions to prime cDNA synthesis was determined. Compared to total RNA, the poly(A)-containing RNA retained 70% of the priming capacity, while 20% was found in the poly(A)-deficient fraction. Poly(A)-containing, poly(A)-deficient, and total RNA fractions were hybridized to cDNAs synthesized from (+)poly(A)RNA. Poly(A)-containing RNA hybridized with an average R0t 1/2 approximately 20 times faster than total RNA. Poly(A)-deficient RNA hybridized with an average R0t 1/2 approximately 3-4 times slower than total RNA. These R0t 1/2 shifts indicated that in excess of three-quarters of the total hybridizable RNA was recovered in the poly(A)-containing fraction and that less than one-quarter was recovered in the poly(A)-deficient RNA fraction. Abundancy classes were less distinct in heterologous hybridizations. In all cases the extent of hybridization was similar, indicating that while the amount of various mRNA species varied among the RNA fractions, most hybridizing species of RNA were present in each RNA fraction. cDNA to the abundant class of mRNAs was purified and hybridized to both (+)- and (-)poly(A)RNA. Messenger RNA corresponding to the more abundant species was enriched in the poly(A)-containing fraction at least 2-fold over the less abundant species of mRNA, with less than 10% of the abundant mRNAs appearing inthe poly(A)-deficient fraction.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

Teratogenic effects of retinoic acid in pigtail monkeys (Macaca nemestrina). I. General features.

Daily oral administration of 10 mg/kg retinoic acid to pregnant Macaca nemestrina monkeys on days 20 to 44 resulted in a high frequency of craniofacial and musculoskeletal malformations. Craniofacial defects including cleft palate and anomalies of the prinna were common as were ectrodactyly, kyphosis, and muscular-joint contractures. Transposition of the great vessels of the heart occurred in one animal and polycystic kidney and associated urogenital anomalies in another. Shorter treatment periods with similar or higher dosages were not teratogenic and were less fetocidal. Although only relatively long treatment courses were teratogenic, the defects that resulted were morphologically similar to those induced with retinoic acid or other vitamin A compounds in other animal orders.     

Two classes of p38alpha MAP kinase inhibitors having a common diphenylether core but exhibiting divergent binding modes

Two new classes of diphenylether inhibitors of p38alpha MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38alpha ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38alpha inhibitors are discussed.     

2,3-Diarylthiophenes as selective EP1 receptor antagonists

The synthesis and the EP(1) receptor binding affinity of 2,3-diarylthiophene derivatives are described. The evaluation of the structure-activity relationship (SAR) in this series led to the identification of compounds 4, 7, and 12a, which exhibit high affinity for the human EP(1) receptor and a selectivity greater than 100-fold against the EP(2), EP(3), EP(4), DP, FP, and IP receptors and greater than 25-fold versus the TP receptor. These three antagonists present good pharmacokinetics in rats and significant differences in the way they are distributed in the brain.     

Current knowledge of hagfish reproduction: implications for fisheries management

This review briefly summarizes the latest findings on reproductiveendocrinology of Atlantic hagfish (Myxine glutinosa) and implicationsfor fisheries management. In response to a major decline orcollapse of the fisheries (groundfish and anadromous species)industry in the Northeast, species that were once consideredalternative or underutilized have and are being identified thatmay be suitable for commercial harvest, one such example isthe hagfish. Hagfish in recent years have been sought afteras valuable fish not only for their flesh, but also their skin.Currently, there are no regulations governing the harvestingof hagfish along the East Coast. There has been little to noinformation of the life history of hagfish including growthrate, age determination, reproductive biology, life span, andlarval size at hatching. Thus, the level at which a sustainablefisheries for this species can be maintained is unknown. Insome parts of the world, hagfish stocks are being depleted dueto overfishing. In order for fisheries management to manageits hagfish stocks and develop a sustainable commercial hagfishfishery, critical information is needed to assist in determiningthe optimal use of this valuable resource. Key elements of the reproductive system have not been elucidatedin hagfish. However, there is new evidence from recent reproductivestudies that Atlantic hagfish may have a seasonal reproductivecycle. These data include seasonal changes in gonadotropin-releasinghormone (GnRH), gonadal steroids, estradiol and progesterone,corresponding to gonadal reproductive stages along with theputative identity of a functional corpus luteum. This newlyacquired data may provide important information to fisheriesmanagers of the East Coast.     

AGAPE (Automated Genome Analysis PipelinE) for Pan-Genome Analysis of Saccharomyces cerevisiae

The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community.