ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : I. Method and Morphology

Golgi apparatus were released without fixatives from rat hepatocytes by gentle homogenization, concentrated by differential centrifugation, and purified by sucrose gradient centrifugation. Examination of sections of purified fractions by electron microscopy showed fields of morphologically intact units of Golgi apparatus consisting of stacks of parallel flattened cisternae, secretory vesicles, and small vesicular profiles. Negative staining of unfixed pellets revealed a complex network of anastomotic tubules continuous with platelike structures and secretory vesicles. These structures corresponded, respectively, to the small vesicular profiles and parallel flattened cisternae with attached secretory vesicles of sectioned material. Small fragments of granular endoplasmic reticulum were often closely associated with the peripheral tubules, suggesting sites of continuity in intact hepatocytes.     

Developmental biology of the early Cambrian cnidarian Olivooides

Fossilized embryos afford direct insight into the pattern of development in extinct organisms, providing unique tests of hypotheses of developmental evolution based in comparative embryology. However, these fossils can only be effective in this role if their embryology and phylogenetic affinities are well constrained. We elucidate and interpret the development of Olivooides from embryonic and adult stages and use these data to discriminate among competing interpretations of their anatomy and affinity. The embryology of Olivooides is principally characterized by the development of an ornamented periderm that initially forms externally and is subsequently formed internally, released at the aperture, facilitating the direct development of the embryo into an adult theca. Internal anatomy is known only from embryonic stages, revealing two internal tissue layers, the innermost of which is developed into three transversally arranged walls that partly divide the lumen into an abapertural region, interpreted as the gut of a polyp, and an adapertural region that includes structures that resemble the peridermal teeth of coronate scyphozoans. The anatomy and pattern of development exhibited by Olivooides appears common to the other known genus of olivooid, Quadrapyrgites, which differs in its tetraradial, as opposed to pentaradial symmetry. We reject previous interpretations of the olivooids as cycloneuralians, principally on the grounds that they lack a through gut and introvert, in embryo and adult. Instead we consider the affinities of the olivooids among medusozoan cnidarians; our phylogenetic analysis supports their classification as total‐group Coronata, within crown‐Scyphozoa. Olivooides and Quadrapyrgites evidence a broader range of life history strategies and bodyplan symmetry than is otherwise commonly represented in extant Scyphozoa specifically, and Cnidaria more generally.     

The role of sequence context,nucleotide pool balance and stress in 2′-deoxynucleotide misincorporation in viral,bacterial and mammalian RNA

The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 10³ to 10⁵ ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections.     

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.     

Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: I. Pigment Composition

The use of n-octyl-beta-d-glucopyranoside along with sodium dodecyl sulfate improves the retention of chlorophyll (Chl) by chlorophyll-protein complexes (CPs) prepared from thylakoids of Euglena gracilis Klebs var bacillaris Cori and yields several additional complexes. Thylakoids from wild-type (WT) cells, solubilized in these detergents and subjected to polyacrylamide gel electrophoresis at 0 degrees C, yield the following CPs, in order of relative molecular weight, containing the pigments shown in parentheses with their respective molar ratios where determined: CP Ia (Chl a, diadinoxanthin and beta-carotene; 100:12:5); CP I (Chl a and beta-carotene; 100:6-12); CPx (Chl and carotenoids); LHCP(2) (light-harvesting CP oligomer) (Chl a, Chl b, diadinoxanthin and neoxanthin; 12:4:3:1); CPy (Chl a, diadinoxanthin and beta-carotene; 100:14:8); CPa (Chl a and beta-carotene; 100:18-25) and LHCP (monomer) (Chl a, Chl b, diadinoxanthin and neoxanthin; 12:6:4:1). The LHCP complexes retain up to 40% of the total Chl and 80% of the Chl b in the thylakoids. CP Ia contains only a trace of Chl b (Chl a/b [mol/mol] = 62). The lower amount of Chl b in Euglena (about 10% of Chl a + b) compared to higher plants (about 30% of Chl a + b) is probably a consequence of the lower Chl b (relative to Chl a) in the LHCPs of Euglena rather than of fewer LHCPs being present. G(1)BU, Gr(1)BSL, and O(4)BSL, mutants of bacillaris low in Chl b (1-2% of Chl a + b), lack the CP Ia, LHCP, and LHCP(2) found in wildtype (WT); G(1) and O(4) also lack CPy. The mutants contain reduced amounts of Chl a (two-thirds of WT in Gr(1) and one-third in G(1) and O(4)) and neoxanthin (20-40% of WT) but retain levels of beta-carotene and diadinoxanthin close to those in cells of WT. The CPs remaining in the mutants have pigment compositions very similar to their counterparts from WT.