Embryonic development of the oligochaete Enchytraeus coronatus: an SEM and histological study of embryogenesis from one-cell stage to hatching

We describe the embryonic development of the soil-living oligochaete Enchytraeus coronatus (Enchytraeidae, Oligochaeta, Annelida). Enchytraeus coronatus is a direct developer. It follows the typical spiral cleavage mode of development that is highly conserved among annelids and a large number of other lophotrochozoan taxa that are collectively named "Spiralia." Scanning electron microscopy (SEM) was combined with light microscopic analysis of wholemounted and sectioned embryos, differentially processed through histological stainings, to reconstruct and document cellular movements and organogenesis from early cleavage stages until hatching. With the help of these data we have established a scheme of morphologically defined stages in order to facilitate future studies on the molecular and histological level that will allow a detailed cross-species comparison among annelids and other phyla.     

A high-resolution linkage-disequilibrium map of the human major histocompatibility complex and first generation of tag single-nucleotide polymorphisms

Autoimmune, inflammatory, and infectious diseases present a major burden to human health and are frequently associated with loci in the human major histocompatibility complex (MHC). Here, we report a high-resolution (1.9 kb) linkage-disequilibrium (LD) map of a 4.46-Mb fragment containing the MHC in U.S. pedigrees with northern and western European ancestry collected by the Centre d'Etude du Polymorphisme Humain (CEPH) and the first generation of haplotype tag single-nucleotide polymorphisms (tagSNPs) that provide up to a fivefold increase in genotyping efficiency for all future MHC-linked disease-association studies. The data confirm previously identified recombination hotspots in the class II region and allow the prediction of numerous novel hotspots in the class I and class III regions. The region of longest LD maps outside the classic MHC to the extended class I region spanning the MHC-linked olfactory-receptor gene cluster. The extended haplotype homozygosity analysis for recent positive selection shows that all 14 outlying haplotype variants map to a single extended haplotype, which most commonly bears HLA-DRB1*1501. The SNP data, haplotype blocks, and tagSNPs analysis reported here have been entered into a multidimensional Web-based database (GLOVAR), where they can be accessed and viewed in the context of relevant genome annotation. This LD map allowed us to give coordinates for the extremely variable LD structure underlying the MHC.     

Cutoff size need not strongly influence molecular dynamics results for solvated polypeptides

The correct treatment of van der Waals and electrostatic nonbonded interactions in molecular force fields is essential for performing realistic molecular dynamics (MD) simulations of solvated polypeptides. The most computationally tractable treatment of nonbonded interactions in MD utilizes a spherical distance cutoff (typically, 8-12 A) to reduce the number of pairwise interactions. In this work, we assess three spherical atom-based cutoff approaches for use with all-atom explicit solvent MD: abrupt truncation, a CHARMM-style electrostatic shift truncation, and our own force-shifted truncation. The chosen system for this study is an end-capped 17-residue alanine-based alpha-helical peptide, selected because of its use in previous computational and experimental studies. We compare the time-averaged helical content calculated from these MD trajectories with experiment. We also examine the effect of varying the cutoff treatment and distance on energy conservation. We find that the abrupt truncation approach is pathological in its inability to conserve energy. The CHARMM-style shift truncation performs quite well but suffers from energetic instability. On the other hand, the force-shifted spherical cutoff method conserves energy, correctly predicts the experimental helical content, and shows convergence in simulation statistics as the cutoff is increased. This work demonstrates that by using proper and rigorous techniques, it is possible to correctly model polypeptide dynamics in solution with a spherical cutoff. The inherent computational advantage of spherical cutoffs over Ewald summation (and related) techniques is essential in accessing longer MD time scales.     

Towards elucidating the energy of the first excited singlet state of xanthophyll cycle pigments by X-ray absorption spectroscopy

The first excited singlet state (S(1)) of carotenoids (also termed 2A(g)(-)) plays a key role in photosynthetic excitation energy transfer due to its close proximity to the S(1) (Q(y)) level of chlorophylls. The determination of carotenoid 2A(g)(-) energies by optical techniques is difficult; transitions from the ground state (S(0), 1A(g)(-)) to the 2A(g)(-) state are forbidden ("optically dark") due to parity (g <-- //--> g) as well as pseudo-parity selection rules (- <-- //--> -). Of particular interest are S(1) energies of the so-called xanthophyll-cycle pigments (violaxanthin, antheraxanthin and zeaxanthin) due to their involvement in photoprotection in plants. Previous determinations of S(1) energies of violaxanthin and zeaxanthin by different spectroscopic techniques vary considerably. Here we present an alternative approach towards elucidation of the optically dark states of xanthophylls by near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The indication of at least one pi* energy level (about 0.5 eV below the lowest 1B(u)(+) vibronic sublevel) has been found for zeaxanthin. Present limitations and future improvements of NEXAFS to study optically dark states of carotenoids are discussed. NEXAFS combined with simultaneous optical pumping will further aid the investigation of these otherwise hardly accessible states.     

The impact of pedigree structure on heritability estimates for pulse pressure in three studies

OBJECTIVES: Pulse pressure (PP) is a measure of large artery stiffness and has been shown to be an important predictor of cardiovascular morbidity and mortality. The aims of the present study were to investigate the heritability of PP in three studies, the Diabetes Heart Study (DHS), the Insulin Resistance Atherosclerosis Family Study (IRAS FS), and the NHLBI Family Heart Study (FHS), to estimate the residual heritability after inclusion of a common set of covariates, and to investigate the impact of pedigree structure on estimating heritability. METHODS AND RESULTS: DHS is primarily a sibling pair nuclear family study design, while both IRAS FS and FHS have large pedigrees. Heritability estimates of log-transformed PP were obtained using variance component models. After adjusting for age, gender, ethnicity/center, height, diabetes status, and mean arterial pressure (MAP), heritability estimates of PP were 0.40 +/- 0.08 , 0.22 +/- 0.05, and 0.19 +/- 0.03 in DHS, IRAS FS, and FHS, respectively. The heritability estimate from DHS was significantly different from both IRAS FS and FHS (both p values <0.05). A random re-sampling technique (modified bootstrap) was used to explore the heritability in the IRAS FS and FHS data when these pedigrees were trimmed to mimic the DHS pedigree structure. The re-sampling method (mimicking a sibling pair nuclear family design in all studies) yielded PP heritability estimates of 0.37, 0.34, and 0.27 in DHS, IRAS FS, and FHS, respectively. There was no significant difference among the heritability estimates from the three studies based on the re-sampling method. CONCLUSION: We have shown that PP has a moderately heritable component in three different studies. These data illustrate the influence of pedigree structure can have on estimating heritability. Thoughtful comparisons of heritability estimates must consider study design factors such as pedigree structure.     

Regional load bearing of the feline acetabulum

OBJECTIVE: To test if the caudal acetabulum of the cat is unloaded at mid-stance as has been asserted. MATERIALS AND METHODS: A kinematic study of five healthy cats was performed to ascertain the mid-stance angle of the hip joint, and the orientation of the pelvis and femur. Femora and pelves from 10 feline cadavers were loaded at physiological load at the mid-stance angle. Impression material placed within the acetabulum was extruded from areas of load bearing. Digital images before and after loading were used to assess whether three areas of the acetabulum, cranial, central and caudal, were fully, partially or non-load bearing. RESULTS: There was a significant difference in load bearing in the three regions (p < 0.001, Kruskal-Wallis test), with a significant difference in load bearing between the cranial and central thirds (p < 0.001) and the cranial and caudal thirds (p < 0.001) but no difference between the central and caudal thirds of the acetabulum (Mann-Whitney-U test). CONCLUSIONS: The load bearing areas of the feline acetabula are the caudal and central thirds. CLINICAL RELEVANCE: The caudal acetabulum of the cat is loaded, therefore the recommendations for simple fractures being treated conservatively needs to be reconsidered.     

Ablation of a critical surfactant protein B intramolecular disulfide bond in transgenic mice

The 79-amino acid, mature SP-B peptide contains three intramolecular disulfide bonds shared by all saposin-like proteins. This study tested the hypothesis that the disulfide bond formed between cysteine residues 35 and 46 (residues 235 and 246 of the SP-B proprotein) is essential for proper function of SP-B. To test the role of this bridge in SP-B function in vivo, a construct was generated in which cysteine residues 235 and 246 of the human SP-B proprotein were mutated to serine and cloned under the control of the 3.7-kilobase hSP-C promoter (hSP-B(C235S/C246S)). In two transgenic mouse lines, expression of the mutant peptide in the wild-type murine SP-B background was invariably lethal in the neonatal period. In four additional lines, survival was inversely related to the level of transgene expression. To test the ability of the mutant peptide to functionally replace the wild-type protein, transgenic mice were crossed into the SP-B null background. No animals that expressed hSP-B(C235S/C246S) in the murine SP-B-/- background survived the neonatal period. hSP-B(C235S/C246S) proprotein accumulated in the endoplasmic reticulum and was not processed to the mature, biologically active peptide. The results of these studies demonstrate that the intramolecular bridge between residues 235 and 246 is critical for intracellular trafficking of SP-B and suggest that overexpression of mutant SP-B in the wild-type background may be lethal.     

Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice

Innate immunity plays an important role in pulmonary host defense against Pneumocystis carinii, an important pathogen in individuals with impaired cell-mediated immunity. We investigated the role of GM-CSF in host defense in a model of P. carinii pneumonia induced by intratracheal inoculation of CD4-depleted mice. Lung GM-CSF levels increased progressively during the infection and were significantly greater than those in uninfected controls 3, 4, and 5 wk after inoculation. When GM-CSF gene-targeted mice (GM-/-) depleted of CD4+ cells were inoculated with P. carinii, the intensities of infection and inflammation were increased significantly compared with those in CD4-depleted wild-type mice. In contrast, transgenic expression of GM-CSF directed solely in the lungs of GM-/- mice (using the surfactant protein C promoter) dramatically decreased the intensity of infection and inflammation 4 wk after inoculation. The concentrations of surfactant proteins A and D were greater in both uninfected and infected GM-/- mice compared with those in wild-type controls, suggesting that this component of the innate response was preserved in the GM-/- mice. However, alveolar macrophages (AM) from GM-/- mice demonstrated impaired phagocytosis of purified murine P. carinii organisms in vitro compared with AM from wild-type mice. Similarly, AM production of TNF-alpha in response to P. carinii in vitro was totally absent in AM from GM-/- mice, while GM-CSF-replete mice produced abundant TNF in this setting. Thus, GM-CSF plays a critical role in the inflammatory response to P. carinii in the setting of impaired cell-mediated immunity through effects on AM activation.