Leukotriene C4 binding to rat lung membranes

A high affinity binding site for leukotriene C4 (LTC4), one component of slow reacting substance of anaphylaxis, has been identified in a membrane preparation from rat lung. As measured by a filtration technique, [3H]LTC4 binding was saturable, specific, reversible, and heat-sensitive. In the presence of 20 mM CaCl2, the dissociation constant (KD) was 41 +/- 9 nM and the maximum number of binding sites (Bmax) was 31 +/- 10 pmol/mg of protein. Specificity was demonstrated by competition studies in which LTC4 had a Ki of 40 nM against specifically bound [3H]LTC4, whereas leukotriene D4 (LTD4) had a Ki of 4 microM. The stereoisomers (5R, 6R) LTC4, (5S, 6S) LTC4, and (5R, 6S) LTC4 had Ki values 3-, 15-, and 25-fold higher than that of natural (5S, 6R) LTC4. Leukotrienes E4 and B4, several prostaglandins and fatty acids, glutathione, and platelet activating factor were even less effective with Ki values above 10 microM. A slow reacting substance of anaphylaxis antagonist, FPL 55712, which, in some systems, distinguishes LTC4- from LTD4-induced contractions, was a weak competitor with a Ki of 16 microM. Serine-borate complex which inhibits gamma-glutamyl transpeptidase, an enzyme responsible for LTC4 metabolism, did not alter binding. In addition, 100 microM FPL 55712 did not reduce metabolism. These observations suggest that the binding observed for LTC4 may represent association with a physiological receptor for this molecule which has a relatively low affinity for LTD4.     

Errata

Chromosomal proteins extracted from calf thymus by 0.35 M NaCl were insoluble at the low salt concentrations needed initially for ion-exchange chromatography. However, the addition of high-affinity carboxymethyl-dextran rendered the precipitated proteins soluble, permitting them to be fractionated by elution from DEAE-Sephacel. In this way, the separation of HMG-1 from HMG-2 was achieved, as well as a partial fractionation of the low mobility group proteins, under conditions that did not disturb the native conformation of the proteins.     

Sequence and expression of murine cDNAs encoding Xlr3a and Xlr3b, defining a new X-linked lymphocyte-regulated Xlr gene subfamily

Using a subtractive cDNA approach we have identified two nearly identical genes, Xlr3a and Xlr3b (X-linked lymphocyte regulated), expressed at a consistently high level in 14 out of 14 murine plasmacytoma cell lines, at a high level in 1 out of 8 B-lymphoma cell lines, and at a very low level in 2 out of the 8 B-lymphoma cell lines. The messages are not detected in 10 pre-B-lymphoma cell lines. These genes express 2.0-kb mRNAs that encode 226-amino-acid proteins that are extremely basic, with an estimated pI of 8.1 and 9.0, respectively. By sequence comparison they are homologous to Xlr1, an acidic nuclear protein that is produced in lymphoid cell lines corresponding to the late stages of lymphocyte differentiation. Xlr2 is a highly homologous gene that is expressed in differentiating male germ cells. Xlr3a and Xlr3b are members of a new subfamily in the Xlr multigene family. Like Xlrl, they are up-regulated during B-cell terminal differentiation in normal and neoplastic B-cells, and cross-hybridize with a message in testis RNA. Also, like Xlrl, they do not cross-hybridize with human genomic DNA.     

Prostaglandins, cyclic nucleotides and cell function

Based upon the ability of the E-prostaglandins to stimulate cyclic AMP formation in a dose-related manner and the correlation between this property and their affinity for a membraneous receptor, the action of these prostaglandins was proposed to be expressed largely via cyclic AMP. The failure of the F-prostaglandins to demonstrate significant activity in these two parameters led to the suggestion that they must act at another receptor via a different mediator. The recent isolation of a receptor unique to PGF_2α and the demonstrated ability of this prostaglandin to increase tissue cyclic GMP levels are consistent with this concept that the E-prostaglandins and PGF_2α play distinctly different roles in cell regulation.     

Endothelial cell prostacyclin production induced by activated neutrophils

A bovine aortic endothelial cell (EC) line released prostacyclin (greater than 1 pmol/10(+5) EC cells) when incubated with fMet-Leu-Phe (FMLP)-stimulated rat and human neutrophils (PMNs). This prostaglandin (PG) I2 was shown to come from the ECs and not from the PMNs by radioactive, high-performance liquid chromatography, and immunochemical criteria. Both FMLP-stimulated rat peritoneal and human peripheral PMNs as well as their stimulated cell-free supernatants and unstimulated sonicates could elicit the release of PGI2 from ECs. Since phorbol myristate acetate stimulated PMN adherence but elicited little PGI2 release from ECs, the PGI2 stimulation in ECs is unrelated to PMN adhesion. The addition of catalase and superoxide dismutase to FMLP-stimulated PMNs enhanced rather than reduced PGI2 formation, indicating that activated oxygen products of the PMN are not responsible for the induction of PGI2. Incubation of ECs with leukotriene (LT) B4, LTC4, or LTD4 did not trigger PGI2 release nor did aspirin pretreatment of the PMNs reduce the PGI2 induction. These data suggest that arachidonic acid metabolites of the PMNs were not responsible for the PGI2 induction. Available data indicates that the PMN factor that stimulates PGI2 from ECs is either released concomitantly with the azurophilic granules or is closely related to this event.     

Complete plastid genome sequences of Drimys, Liriodendron, and Piper : implications for the phylogenetic relationships of magnoliids

Background  

The magnoliids with four orders, 19 families, and 8,500 species represent one of the largest clades of early diverging angiosperms. Although several recent angiosperm phylogenetic analyses supported the monophyly of magnoliids and suggested relationships among the orders, the limited number of genes examined resulted in only weak support, and these issues remain controversial. Furthermore, considerable incongruence resulted in phylogenetic reconstructions supporting three different sets of relationships among magnoliids and the two large angiosperm clades, monocots and eudicots. We sequenced the plastid genomes of three magnoliids, Drimys (Canellales), Liriodendron (Magnoliales), and Piper (Piperales), and used these data in combination with 32 other angiosperm plastid genomes to assess phylogenetic relationships among magnoliids and to examine patterns of variation of GC content.     

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.     

Discovery of Novel Herpes Simplexviruses in Wild Gorillas,Bonobos, and Chimpanzees Supports Zoonotic Origin of HSV-2

Viruses closely related to human pathogens can reveal the origins of human infectious diseases. Human herpes simplexvirus type 1 (HSV-1) and type 2 (HSV-2) are hypothesized to have arisen via host-virus codivergence and cross-species transmission. We report the discovery of novel herpes simplexviruses during a large-scale screening of fecal samples from wild gorillas, bonobos, and chimpanzees. Phylogenetic analysis indicates that, contrary to expectation, simplexviruses from these African apes are all more closely related to HSV-2 than to HSV-1. Molecular clock-based hypothesis testing suggests the divergence between HSV-1 and the African great ape simplexviruses likely represents a codivergence event between humans and gorillas. The simplexviruses infecting African great apes subsequently experienced multiple cross-species transmission events over the past 3 My, the most recent of which occurred between humans and bonobos around 1 Ma. These findings revise our understanding of the origins of human herpes simplexviruses and suggest that HSV-2 is one of the earliest zoonotic pathogens.