Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

Background  

Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation.     

pO2-dependent NO production determines OPPC activity in macrophages

Stimulated macrophages produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) using molecular O₂, L-arginine, and NADPH. Exposure of macrophages to hypoxia decreases NO production within seconds, suggesting substrate limitation as the mechanism. Conflicting data exist regarding the effect of pO₂ on NADPH production via the oxidative pentose phosphate cycle (OPPC). Therefore, the present studies were developed to determine whether NADPH could be limiting for NO production under hypoxia. Production of NO metabolites (NOx) and OPPC activity by RAW 264.7 cells was significantly increased by stimulation with lipopolysaccharide (LPS) and interferon γ (IFNγ) at pO₂ ranging from 0.07 to 50%. OPPC activity correlated linearly with NOx production at pO₂ > 0.13%. Increased OPPC activity by stimulated RAW 264.7 cells was significantly reduced by 1400 W, an iNOS inhibitor. OPPC activity was significantly increased by concomitant treatment of stimulated RAW 264.7 cells with chemical oxidants such as hydroxyethyldisulfide or pimonidazole, at 0.07 and 50% O₂, without decreasing NOx production. These results are the first to investigate the effect of pO₂ on the relationship between NO production and OPPC activity, and to rule out limitations in OPPC activity as a mechanism by which NO production is decreased under hypoxia.     

Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences

Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were determined from nine species in the Drosophila saltans species group. The partition homogeneity test and partitioned Bremer support were used to measure incongruence between phylogenetic hypotheses generated from individual partitions. Individual loci were generally congruent with each other and consistent with the previously proposed morphological hypothesis, although they differed in level of resolution. Since extreme conflict between partitions did not exist, the data were combined and analyzed simultaneously. The total evidence method gave a more resolved and highly supported phylogeny, as indicated by bootstrap proportions and decay indices, than did any of the individual analyses. The cordata and elliptica subgroups, considered to have diverged early in the history of the D. saltans group, were sister taxa to the remainder of the saltans group. The sturtevanti subgroup, represented by D. milleri and D. sturtevanti, occupies an intermediate position in this phylogeny. The saltans and parasaltans subgroups are sister clades and occupy the most recently derived portion of the phylogeny. As with previous morphological studies, phylogenetic relationships within the saltans subgroup were not satisfactorily resolved by the molecular data.      

Lysogeny in the oceans: Lessons from cultivated model systems and a reanalysis of its prevalence

In the oceans, viruses that infect bacteria (phages) influence a variety of microbially mediated processes that drive global biogeochemical cycles. The nature of their influence is dependent upon infection mode, be it lytic or lysogenic. Temperate phages are predicted to be prevalent in marine systems where they are expected to execute both types of infection modes. Understanding the range and outcomes of temperate phage–host interactions is fundamental for evaluating their ecological impact. Here, we (i) review phage-mediated rewiring of host metabolism, with a focus on marine systems, (ii) consider the range and nature of temperate phage–host interactions, and (iii) draw on studies of cultivated model systems to examine the consequences of lysogeny among several dominant marine bacterial lineages. We also readdress the prevalence of lysogeny among marine bacteria by probing a collection of 1239 publicly available bacterial genomes, representing cultured and uncultivated strains, for evidence of complete prophages. Our conservative analysis, anticipated to underestimate true prevalence, predicts 18% of the genomes examined contain at least one prophage, the majority (97%) were found within genomes of cultured isolates. These results highlight the need for cultivation of additional model systems to better capture the diversity of temperate phage–host interactions in the oceans.     

Evasive behaviour of a frog in response to bat predation

Chorusing frogs (Physalaemus pustulosus) visually detect hunting bats (Trachops cirrhosus) and models of T. cirrhosus on all but the darkest nights. Detection is apparently communicated rapidly, since all frogs in the area can quit calling withing less than a second of a T. cirrhosus arrival at the pond. Physalaemus pustulosus choruses remain silent longer following trials when a T. cirrhosus model is passed overhead than following normal shutdowns or those caused by a model of a small insectivorous bat. They often do not reduce calling in response to the normal activities of small bats.     

Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera

Nucleotide sequences from a 434-bp region of the 16S rRNA gene were analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps, sawflies) to examine the patterns of variation within the gene fragment and the taxonomic levels for which it shows maximum utility in phylogeny estimation. A hierarchical approach was adopted in the study through comparison of levels of sequence variation among taxa at different taxonomic levels. As previously reported for many holometabolous insects, the 16S data reported here for Hymenoptera are highly AT-rich and exhibit strong site-to-site variation in substitution rate. More precise estimates of the shape parameter (alpha) of the gamma distribution and the proportion of invariant sites were obtained in this study by employing a reference phylogeny and utilizing maximum-likelihood estimation. The effectiveness of this approach to recovering expected phylogenies of selected hymenopteran taxa has been tested against the use of maximum parsimony. This study finds that the 16S gene is most informative for phylogenetic analysis at two different levels: among closely related species or populations, and among tribes, subfamilies, and families. Maximization of the phylogenetic signal extracted from the 16S gene at higher taxonomic levels may require consideration of the base composition bias and the site-to-site rate variation in a maximum-likelihood framework.      

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Stent implantation through a self-expanding stent

Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster

A plasmid library of Acinetobacter calcoaceticus HindIII fragments was constructed, and clones that complemented an Escherichia coli pabA mutant were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were obtained. We infer that complementation of E. coli pabA mutants was the result of the expression of the amphibolic anthranilate- synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that the plasmid insert carried the entire trpGDC gene cluster. In E. coli minicells, the plasmid insert directed the synthesis of polypeptides of 44,000, 33,000, and 20,000 daltons, molecular masses that are consistent with the reported molecular masses of phosphoribosylanthranilate transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase component II, respectively. A 3,105- bp nucleotide sequence was determined. Comparison of the A. calcoaceticus trpGDC sequences with other known trp gene sequences has allowed insight into (1) the evolution of the amphibolic trpG gene, (2) varied strategies for coordinate expression of trp genes, and (3) mechanisms of gene fusions in the trp operon.