Intratracheal instillation of a novel NO/nucleophile adduct selectively reduces pulmonary hypertension

Brilli, Richard J., Brian Krafte-Jacobs, Daniel J. Smith,Dominick Roselle, Daniel Passerini, Amos Vromen, Lori Moore, CsabaSzabó, and Andrew L. Salzman. Intratracheal instillation ofa novel NO/nucleophile adduct selectively reduces pulmonary hypertension. J. Appl. Physiol. 83(6):1968-1975, 1997.We examined the pulmonary and systemichemodynamic effects of administering soluble nitric oxide (NO) donorcompounds (NO/nucleophile adducts, i.e., NONOates) directly into thetrachea of animals with experimentally induced pulmonary hypertension.Steady-state pulmonary hypertension was created by using thethromboxane agonist U-46619. Yorkshire pigs were randomly assigned toone of four groups: group 1,intratracheal saline (control; n = 8);group 2, intratracheal sodiumnitroprusside (n = 6);group 3, intratracheal ethylputreanineNONOate (n = 6); andgroup 4, intratracheal2-(dimethylamino)-ethylputreanine NONOate (DMAEP/NO;n = 6). Pulmonary and systemichemodynamics were monitored after drug instillation.Group 4 had significant reductions in pulmonary vascular resistance index (PVRI) at all time points comparedwith steady state and compared with group1 (P < 0.05), whereas systemic vascular resistance index did not change. The meanchange in mean pulmonary arterial pressure in group4 was 33.1 ± 1.2% compared with +6.4 ± 1.3% in group 1 (P < 0.001), and the mean change inmean arterial pressure was 9.3 ± 0.7% compared with acontrol value of 0.9 ± 0.5%(P < 0.05). Groups 2 and 3 hadsignificant decreases in both PVRI and systemic vascular resistanceindex compared with steady state and with group1. In conclusion, intratracheal instillation of apolar-charged tertiary amine NONOate DMAEP/NO results in the selectivereduction of PVRI. Intermittent intratracheal instillation of selectiveNONOates may be an alternative to continuously inhaled NO in thetreatment of pulmonary hypertension.

     

Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using an anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was tested under two models, autosomal dominant with reduced penetrance and affecteds only. Homogeneity testing on the two-point data gave evidence of heterogeneity at APOC2 under the affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities > = 50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families.     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

Insight into the structure of amyloid fibrils from the analysis of globular proteins

The conversion from soluble states into cross-β fibrillar aggregates is a property shared by many different proteins and peptides and was hence conjectured to be a generic feature of polypeptide chains. Increasing evidence is now accumulating that such fibrillar assemblies are generally characterized by a parallel in-register alignment of β-strands contributed by distinct protein molecules. Here we assume a universal mechanism is responsible for β-structure formation and deduce sequence-specific interaction energies between pairs of protein fragments from a statistical analysis of the native folds of globular proteins. The derived fragment–fragment interaction was implemented within a novel algorithm, prediction of amyloid structure aggregation (PASTA), to investigate the role of sequence heterogeneity in driving specific aggregation into ordered self-propagating cross-β structures. The algorithm predicts that the parallel in-register arrangement of sequence portions that participate in the fibril cross-β core is favoured in most cases. However, the antiparallel arrangement is correctly discriminated when present in fibrils formed by short peptides. The predictions of the most aggregation-prone portions of initially unfolded polypeptide chains are also in excellent agreement with available experimental observations. These results corroborate the recent hypothesis that the amyloid structure is stabilised by the same physicochemical determinants as those operating in folded proteins. They also suggest that side chain–side chain interaction across neighbouring β-strands is a key determinant of amyloid fibril formation and of their self-propagating ability.     

Dispersal, philopatry and intergroup relatedness: fine-scale genetic structure in the white-breasted thrasher, Ramphocinclus brachyurus

Dispersal is a fundamental process influencing evolution, social behaviour, and the long-term persistence of populations. We use both observational and genetic data to investigate dispersal, kin-clustering and intergroup relatedness in the white-breasted thrasher, Ramphocinclus brachyurus, a cooperatively breeding bird that is globally endangered. Mark-resighting data suggested sex-biased dispersal, with females dispersing over greater distances while males remained philopatric. Accordingly, spatial autocorrelation analysis showed highly significant fine-scale genetic structure among males, but not among females. This fine-scale genetic structuring of the male population resulted in very high levels of relatedness between dominant males at neighbouring nests, similar to that seen within cooperative groups in many species where kin selection is cited as a cause of cooperation. By implication, between-group as well as within-group cooperation may be important, potentially creating a feedback loop in which short-distance dispersal by males leads to the formation of male kin clusters that in turn facilitate nepotistic interactions and favour further local recruitment. The strength of spatial autocorrelation, as measured by the autocorrelation coefficient, r, was approximately two to three times greater than that reported in previous studies of animals. Relatively short dispersal distances by both males and females may have a negative impact on the white-breasted thrasher's ability to colonize new areas, and may influence the long-term persistence of isolated populations. This should be taken into account when designating protected areas or selecting sites for habitat restoration.     

Ignoring linkage disequilibrium among tightly linked markers induces false-positive evidence of linkage for affected sib pair analysis

Most multipoint linkage programs assume linkage equilibrium among the markers being studied. The assumption is appropriate for the study of sparsely spaced markers with intermarker distances exceeding a few centimorgans, because linkage equilibrium is expected over these intervals for almost all populations. However, with recent advancements in high-throughput genotyping technology, much denser markers are available, and linkage disequilibrium (LD) may exist among the markers. Applying linkage analyses that assume linkage equilibrium to dense markers may lead to bias. Here, we demonstrated that, when some or all of the parental genotypes are missing, assuming linkage equilibrium among tightly linked markers where strong LD exists can cause apparent oversharing of multipoint identity by descent (IBD) between sib pairs and false-positive evidence for multipoint model-free linkage analysis of affected sib pair data. LD can also mimic linkage between a disease locus and multiple tightly linked markers, thus causing false-positive evidence of linkage using parametric models, particularly when heterogeneity LOD score approaches are applied. Bias can be eliminated by inclusion of parental genotype data and can be reduced when additional unaffected siblings are included in the analysis.     

Electron microscopy of helical filaments: rediscovering buried treasures in negative stain

Although negative stain electron microscopy is a wonderfully simple way of directly visualizing protein complexes and other biological macromolecules, the images are not really comparable to those of objects seen in everyday life. The failure to appreciate this has recently led to an incorrect interpretation of RecA‐family filament structures.