Bronchoalveolar lavage fluid processing. Effect of membrane filtration preparation on neutrophil recovery

Two common methods for the preparation of bronchoalveolar lavage (BAL) fluid for cytologic examination, cytocentrifugation and membrane filtration, have been found to yield different results in the quantitation of lymphocytes. To compare these two methods for the quantitation of neutrophils, the differential counts from 640 consecutive clinical specimens were analyzed retrospectively. The percentage of neutrophils resulting from the preparation of the BAL fluids by the two methods were highly correlated (r2 = .72). However, cytocentrifugation yielded consistently higher neutrophil percentages than did membrane filtration (means for all samples: 18.5 +/- 1.0% vs. 14.7 +/- 0.9%; P less than .001). To investigate the source of the variation in neutrophil quantitation by the two methods, two series of mixing experiments were performed in which neutrophil-rich cell suspensions were added to BAL fluids. Determination of the cellular differentials before and after mixing the cell suspensions demonstrated that membrane filtration preparation tends to lose neutrophils while cytocentrifugation accurately recovers neutrophils. Thus, accurate quantitation of the two cells recovered by BAL may require use of both cytocentrifugation and membrane filtration.     

Intraspecific variability and timing in ancestral ecology reconstruction: a test case from the cape flora

Thamnochortus (ca. 32 species) is an ecologically diverse genus of Restionaceae. Restionaceae comprise a major component of the southern African Cape flora, wherein eco-diversification might have been important in the generation of high levels of species richness. In an attempt to reconstruct the macroecological history of Thamnochortus, it was found that standard procedures for character state optimization make two inappropriate assumptions. The first is that ancestors are monomorphic (i.e., ecologically uniform) and the second is that eco-diversification follows, or is slower than, lineage diversification. We demonstrate a variety of coding schemes with which the assumption of monomorphy can be avoided. For unordered discrete ecological characters, presence coding and generalized frequency coding (GFC) are suboptimal because they occasionally yield illogical assignments of no state to ancestors. Polymorphism coding or use of the program DIVA are preferable in this respect but are applicable only with parsimony. For continuous eco-characters (e.g., a rainfall gradient, where individual species occur in ranges), GFC and MaxMin coding provide equally valid solutions to optimizing ranges with parsimony. However, MaxMin can be extended to likelihood approaches and is therefore preferable. With respect to rates and timing, all algorithms currently employed for ancestral ecology reconstruction bias toward slow rates of eco-diversification relative to lineage diversification. An alternative to this bias is provided by DIVA, which biases toward accelerated rates of eco-diversification and thus inferences of ecology-driven speciation. We see no way of choosing between these biases; however, phylogeneticists should be aware of them. Applying these methods to Thamnochortus, we find there to be important differences in details, yet general congruence, regarding the historical ecology of this clade. We infer the most recent common ancestor of Thamnochortus to have been a post-fire resprouting species distributed on rocky, well-drained, sandstone-derived soils at lower-middle elevations, in regions of moderate levels of yearly (primarily winter) rainfall. This species would have been distributed in habitats much like those of the southwestern Cape mountains today. Major ecological trends include shifts to lower rainfall regimes and shifts from sandstone to limestone-derived alkaline soils at lower altitudes.     

Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2

The C-terminal catalytic domains of the 11 mammalian phosphodiesterase families (PDEs) are important drug targets. Five of the 11 PDE families contain less well-characterized N-terminal GAF domains. cGMP is the ligand for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand for PDE10. Structurally related tandem GAF domains signalling via cAMP are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2. Because current high-resolution crystal structures of the tandem GAF domains of PDE2 and cyaB2 do not reveal how cNMP specificity is encoded, we generated chimeras between the tandem GAF domains of cyaB1 and PDE2. Both bind the ligand in the GAF B subdomains. Segmental replacements in the highly divergent beta1-beta3 region of the GAF B subdomain of cyaB1 by the corresponding PDE2 regions switched signalling from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated that, for this switch in purine specificity, only 11% of the sequence of the cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were unable, however, to switch the purine specificity of the PDE2 tandem GAF domain from cGMP to cAMP in reverse constructs, i.e. by replacement of PDE2 segments with those from the cyaB1 GAF tandem domain. The data provide a novel view on the structure-function relationships underlying the purine specificity of cNMP-binding GAF domains and indicate that, as potential drug targets, they must be characterized structurally and biochemically one by one.     

Computational modeling of factors that modulate the unique FeNO bonding in {FeNO}6 heme–thiolate model complexes

A density functional theory account of the changes in FeNO bonding that occur in response to both bonded and nonbonded structural perturbations is reported for a series of {FeNO}(6) heme-thiolate model complexes. Using [Fe(porphine)(SCH(3))NO] as the reference complex, we constructed models to mimic equatorial (cis), distal, and proximal influences of protein environments. Overall, the results from these calculations reveal that the Fe-NO and N-O bond strengths change in the same direction upon variations in structure and environment. These bonding changes are manifested in unique direct correlations between the Fe-NO and N-O vibrational frequencies and bond lengths, as evidenced by their positive slopes (slopes of the familiar inverse or backbonding correlations are negative). The electronic origin of the direct correlations appears to derive from the electron density distribution in high-energy molecular orbitals. This variability modulates the FeNO antibonding character throughout the triatomic FeNO moiety. The results of this study suggest that the stabilities and reactivities of {FeNO}(6) centers in heme-thiolate enzymes can be modulated over a significant range through a variety of bonded and nonbonded means.     

The Drosophila homolog of the human AF10 is an HP1-interacting suppressor of position effect variegation

In chromosomal rearrangements of acute myeloid leukaemia patients the mixed lineage leukaemia (MLL) gene, a human homolog of the Drosophila gene trithorax, is frequently fused to AF10. Here we describe the identification and a functional characterization of the Drosophila homolog dAF10. We show that dAF10 functions in heterochromatin-dependent genomic silencing of position effect variegation, a phenomenon associated with chromosomal rearrangements that cause mosaic expression of euchromatic genes when relocated next to heterochromatin. We also demonstrate that dAF10 can associate with the heterochromatin protein 1 (HP1) in vitro and in vivo. The results indicate that dAF10 is an HP1-interacting component of the heterochromatin-dependent gene silencing pathway, which either contributes to the stability of the heterochromatin complex or to its function.