Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

Adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning

The purpose of this study was to determine whether the adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning. Regional stunning was produced by 15 min of coronary artery occlusion and 3 h of reperfusion (RP) in anesthetized open-chest pigs. In acute protection studies, animals were pretreated with saline, low-dose AMP-579 (15 microg/kg iv bolus 10 min before ischemia), or high-dose AMP-579 (50 microg/kg iv at 14 microg/kg bolus + 1.2 microg.kg(-1).min(-1) for 30 min before coronary occlusion). The delayed preconditioning effects of AMP-579 were evaluated 24 h after administration of saline vehicle or high-dose AMP-579 (50 microg/kg iv). Load-insensitive contractility was assessed by measuring regional preload recruitable stroke work (PRSW) and PRSW area. Acute preconditioning with AMP-579 dose dependently improved regional PRSW: 129 +/- 5 and 100 +/- 2% in high- and low-dose AMP-579 groups, respectively, and 78 +/- 5% in the control group at 3 h of RP. Administration of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.7 mg/kg) blocked the acute protective effect of high-dose AMP-579, indicating that these effects are mediated through A1 receptor activation. Delayed preconditioning with AMP-579 significantly increased recovery of PRSW area: 64 +/- 5 vs. 33 +/- 5% in control at 3 h of RP. In isolated perfused rat heart studies, kinetics of the onset and washout of AMP-579 A1 and A2a receptor-mediated effects were distinct compared with those of other adenosine receptor agonists. The unique nature of the adenosine agonist AMP-579 may play a role in its ability to induce delayed preconditioning against in vivo myocardial stunning.     

Topography of pleural epithelial structure enabled by en face isolation and machine learning

Pleural epithelial adaptations to mechanical stress are relevant to both normal lung function and parenchymal lung diseases. Assessing regional differences in mechanical stress, however, has been complicated by the nonlinear stress–strain properties of the lung and the large displacements with ventilation. Moreover, there is no reliable method of isolating pleural epithelium for structural studies. To define the topographic variation in pleural structure, we developed a method of en face harvest of murine pleural epithelium. Silver-stain was used to highlight cell borders and facilitate imaging with light microscopy. Machine learning and watershed segmentation were used to define the cell area and cell perimeter of the isolated pleural epithelial cells. In the deflated lung at residual volume, the pleural epithelial cells were significantly larger in the apex (624 ± 247 μm²) than in basilar regions of the lung (471 ± 119 μm²) (p < 0.001). The distortion of apical epithelial cells was consistent with a vertical gradient of pleural pressures. To assess epithelial changes with inflation, the pleura was studied at total lung capacity. The average epithelial cell area increased 57% and the average perimeter increased 27% between residual volume and total lung capacity. The increase in lung volume was less than half the percent change predicted by uniform or isotropic expansion of the lung. We conclude that the structured analysis of pleural epithelial cells complements studies of pulmonary microstructure and provides useful insights into the regional distribution of mechanical stresses in the lung.     

Contribution of subsurface peat to CO2 and CH4 fluxes in a neotropical peatland

Tropical peatlands play an important role in the global carbon cycling but little is known about factors regulating carbon dioxide (CO₂) and methane (CH₄) fluxes from these ecosystems. Here, we test the hypotheses that (i) CO₂ and CH₄ are produced mainly from surface peat and (ii) that the contribution of subsurface peat to net C emissions is governed by substrate availability. To achieve this, in situ and ex situ CO₂ and CH₄ fluxes were determined throughout the peat profiles under three vegetation types along a nutrient gradient in a tropical ombrotrophic peatland in Panama. The peat was also characterized with respect to its organic composition using ¹³C solid state cross‐polarization magic‐angle spinning nuclear magnetic resonance spectroscopy. Deep peat contributed substantially to CO₂ effluxes both with respect to actual in situ and potential ex situ fluxes. CH₄ was produced throughout the peat profile with distinct subsurface peaks, but net emission was limited by oxidation in the surface layers. CO₂ and CH₄ production were strongly substrate‐limited and a large proportion of the variance in their production (30% and 63%, respectively) was related to the quantity of carbohydrates in the peat. Furthermore, CO₂ and CH₄ production differed between vegetation types, suggesting that the quality of plant‐derived carbon inputs is an important driver of trace gas production throughout the peat profile. We conclude that the production of both CO₂ and CH₄ from subsurface peat is a substantial component of the net efflux of these gases, but that gas production through the peat profile is regulated in part by the degree of decomposition of the peat.     

PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

Cardioprotection through autophagy: Ready for clinical trial?

Interventions that reduce infarct size in animal models have largely failed to improve outcome in patients suffering acute myocardial infarction (MI), or 'heart attack'. Our group recently reported a reduction of infarct size by chloramphenicol treatment in a porcine in vivo model of acute MI, through a mechanism involving the induction of autophagy. Since 2005 several studies have implicated autophagy as a target for cardioprotection.     

Melittin-induced membrane permeability: A nonosmotic mechanism of cell death

Summary Derived from honeybees, melittin is a 26-amino acid, α-helical, membrane-attack protein that efficiently kills mammalian cells. To investigate the contribution of colloid-osmotic effects to the mechanism of cell death, we studied the effect of melittin on lymphocyte membrane permeability and cell volumes. Melittin concentrations of 0.5 to 2.0 μM induced release of membrane permeability markers without total disruption of the cell membrane. At these melittin concentrations, electrical-impedance cytometry demonstrated melittin-induced changes in red blood cell volumes (P<0.01), but no change in lymphocyte cell volumes (P>0.05). Streaming video microscopy, obtaining images of melittintreated lymphocytes at 80-ms intervals, demonstrated a loss of optical density (P<0.001) suggesting a flattening of the cell but no significant increase in cell perimeter (P>0.05). Real-time multiparameter flow cytometry of melittin-treated lymphocytes confirmed simultaneous loss of the cytoplasmic marker, calcein, and uptake of the DNA dye, ethidium homodimer, but demonstrated no increase in forward light scatter. Transmission-electron microscopy of melittin-treated lymphocytes showed normal cell volumes but discontinuities in the cell membrane suggesting direct membrane toxicity. We conclude that melittin causes lymphocyte death by a “leaky patch” mechanism that is independent of colloid-osmotic effects.     

Modification of an existing chromosomal inversion to engineer a balancer for mouse chromosome 15

Chromosomal inversions are valuable genetic tools for mutagenesis screens, where appropriately marked inversions can be used as balancer chromosomes to recover and maintain mutations in the corresponding chromosomal region. For any inversion to be effective as a balancer, it should exhibit both dominant and recessive visible traits; ideally the recessive trait should be a fully penetrant lethality in which inversion homozygotes die before birth. Unfortunately, most inversions recovered by classical radiation or chemical mutagenesis techniques do not have an overt phenotype in either the heterozygous or the homozygous state. However, they can be modified by relatively simple procedures to make them suitable as an appropriately marked balancer. We have used homologous recombination to modify, in embryonic stem cells, the recessive-lethal In(15)21Rk inversion to endow it with a dominant-visible phenotype. Several ES cell lines were derived from inversion heterozygotes, and a keratin-14 (K14) promoter-driven agouti minigene was introduced onto the inverted chromosome 15 in the ES cells by gene targeting. Mice derived from the targeted ES cells carry the inverted chromosome 15 and, at the same time, exhibit lighter coat color on their ears and tails, making this modified In(15)21Rk useful as a balancer for proximal mouse chromosome 15.     

Intercellular adhesion molecule-1 (ICAM-1) is involved in the cytolytic T lymphocyte interaction with a human synovial cell line

The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)-synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL-synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM-1 and LFA-3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I-directed cytolytic activity. The cell surface molecules utilized by the anti-SYN.SPP and anti-JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA-1 (CD11a) significantly inhibited CTL-mediated lysis of both target cells. An interesting observation was that the anti-SYN.SPP CTL line appeared to utilize the ICAM-1 and not the LFA-3 target cell molecule. In contrast, the anti-JY CTL line utilized the LFA-3 and not the ICAM-1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell-cell interaction molecules.