Intraflagellar Transport Gene Expression Associated with Short Cilia in Smoking and COPD

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.     

N-Cycloalkanoyl-L-phenylalanine derivatives as VCAM/VLA-4 antagonists

A systematic structure-activity relationship investigation of the lead compound 1 resulted the identification of several N-[(substituted alkyl)cycloalkanoyl]-4-[((2,6-dichlorophenyl)carbonyl)amino]-L-phenylalanine derivatives as potent VCAM/VLA-4 antagonists. The data are consistent with a model of these compounds in which these alkanoylphenylalanines reside in a compact gauche (-) bioactive conformation.     

Focused library approach for identification of new N-acylphenylalanines as VCAM/VLA-4 antagonists

A structure-based focused library approach was employed in an effort to identify more lipophilic replacements for the N-benzylpyroglutamyl group of the VCAM/VLA-4 antagonist 2. This effort led to the discovery of two new classes of potent antagonists characterized by the N-(alpha-phenylcyclopentanoyl- and the N-(2,6-dimethylbenzoyl)-derivatives 60 and 64.     

Imide and lactam derivatives of N-benzylpyroglutamyl-L-phenylalanine as VCAM/VLA-4 antagonists

A series of imides and lactams derived from 4-amino-N-benzylpyroglutamyl-L-phenylalanine was prepared and evaluated for activity as VCAM/VLA-4 antagonists. Imides were more potent than the corresponding lactams; several had subnanomolar IC50s in an ELISA based assay and were also highly effective at blocking VLA-4 expressing Ramos cell binding to VCAM coated plates.     

Angiogenic alterations associated with circulating neoplastic DNA in ovarian carcinoma

OBJECTIVES: Forty percent of women with ovarian carcinoma have circulating free neoplastic DNA identified in plasma. Angiogenesis is critical in neoplastic growth and metastasis. We sought to determine whether circulating neoplastic DNA results from alterations in the balance of angiogenesis activators and inhibitors. METHODS: Sixty patients with invasive ovarian carcinomas with somatic TP53 mutations that had been characterized for circulating neoplastic DNA had carcinoma analyzed for microvessel density using immunohistochemistry with CD31 and for the expression of VEGF, ANGPT1, ANGPT2, PTGS2, PLAU, THBS1, CSF1, PIK3CA, HIF1A, IL8, MMP2, and MMP9 message by real-time quantitative polymerase chain reaction. The expression of each gene was calculated relative to GAPDH expression for each neoplasm. Patient plasma had been tested for circulating neoplastic DNA using a ligase detection reaction. RESULTS: MMP2 expression was significantly correlated with free plasma neoplastic DNA (P = .007). Microvessel density was not correlated with plasma neoplastic DNA or BRCA1/2 mutation status. The expression pattern of other angiogenic factors did not correlate with plasma neoplastic DNA but correlated with each other. BRCA1/2 mutated carcinomas had significantly different expression profiles of angiogenesis activators and inhibitors in comparison to sporadic carcinomas. CONCLUSIONS: MMP2 expression is associated with the presence of circulating neoplastic DNA in women with ovarian carcinoma. These data are consistent with the proinvasive properties of MMP2 and suggest that the presence of circulating neoplastic DNA indicates a more aggressive malignant phenotype. Carcinomas with germ line BRCA1/2 mutations had a lower angiogenic profile than those without mutations.     

Structure of the O-antigen polysaccharide present in the lipopolysaccharide of Cronobacter dublinensis (subspecies lactaridi or lausannensis) HPB 3169

Cronobacter dublinensis (formerly Enterobacter sakazakii) HPB 3169 is a pathogenic Gram-negative bacterium that produces a smooth-type lipopolysaccharide in which the antigenic O-polysaccharide component was determined to be a repeating pentasaccharide unit composed of L-rhamnose; 2-acetamido-2-deoxy-D-glucose; 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-glucose; and 3-deoxy-manno-oct-2-ulosonic acid in the respective molar ratio 2:1:1:1. Chemical and 2D NMR analyses of the O-polysaccharide and a pentasaccharide derived by the mild acid hydrolysis of the ketosyl linkage of the Kdo (3-deoxy-D-manno-2-octulosonic acid) residue in the O-polysaccharide established that the O-antigen is a high molecular mass unbranched polymer of a repeating pentasaccharide unit and has the structure [see formula in text] where Bu is a (R)-3-hydroxybutanoyl substituent. The O-antigen is structurally similar to that of the recently reported Cronobacter sakazakii strain G706 (designated as serotype O5), except that in strain G706 the d-Qui3N is in its N-acetyl form, in contrast to its presence as a 3-deoxy-3-(R)-3-hydroxybutyramido derivative in the C. sakazakii HPB 3169 strain O-antigen.     

From static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility

Crystallographic structure and deuterium accessibility comparisons of CcO in different redox states have suggested conformational changes of mechanistic significance. To predict the intrinsic flexibility and low energy motions in CcO, this work has analyzed available high-resolution crystallographic structures with ProFlex and elNémo computational methods. The results identify flexible regions and potential conformational changes in CcO that correlate well with published structural and biochemical data and provide mechanistic insights. CcO is predicted to undergo rotational motions on the interior and exterior of the membrane, driven by transmembrane helical tilting and bending, coupled with rocking of the β-sheet domain. Consequently, the proton K-pathway becomes sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the pathway, associated with conserved Thr-359 and Lys-362 residues. The D-pathway helices are found to be relatively rigid, with a highly flexible entrance region involving the subunit I C-terminus, potentially regulating the uptake of protons. Constriction and dilation of hydrophobic channels in RsCcO suggest regulation of the oxygen supply to the binuclear center. This analysis points to coupled conformational changes in CcO and their potential to influence both proton and oxygen access.     

Whole genome SNP discovery and analysis of genetic diversity in Turkey (Meleagris gallopavo)

ABSTRACT: BACKGROUND: The turkey (Meleagris gallopavo) is an important agricultural species and the second largest contributor to the world's poultry meat production. Genetic improvement is attributed largely to selective breeding programs that rely on highly heritable phenotypic traits, such as body size and breast muscle development. Commercial breeding with small effective population sizes and epistasis can result in loss of genetic diversity, which in turn can lead to reduced individual fitness and reduced response to selection. The presence of genomic diversity in domestic livestock species therefore, is of great importance and a prerequisite for rapid and accurate genetic improvement of selected breeds in various environments, as well as to facilitate rapid adaptation to potential changes in breeding goals. Genomic selection requires a large number of genetic markers such as e.g. single nucleotide polymorphisms (SNPs) the most abundant source of genetic variation within the genome. RESULTS: Alignment of next generation sequencing data of 32 individual turkeys from different populations was used for the discovery of 5.49 million SNPs, which subsequently were used for the analysis of genetic diversity among the different populations. All of the commercial lines branched from a single node relative to the heritage varieties and the South Mexican turkey population. Heterozygosity of all individuals from the different turkey populations ranged from 0.17-2.73 SNPs/Kb, while heterozygosity of populations ranged from 0.73-1.64 SNPs/Kb. The average frequency of heterozygous SNPs in individual turkeys was 1.07 SNPs/Kb. Five genomic regions with very low nucleotide variation were identified in domestic turkeys that showed state of fixation towards alleles different than wild alleles. CONCLUSION: The turkey genome is much less diverse with a relatively low frequency of heterozygous SNPs as compared to other livestock species like chicken and pig. The whole genome SNP discovery study in turkey resulted in the detection of 5.49 million putative SNPs compared to the reference genome. All commercial lines appear to share a common origin. Presence of different alleles/haplotypes in the SM population highlights that specific haplotypes have been selected in the modern domesticated turkey.     

Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum

Plasmodium falciparum, the most virulent agent of human malaria, undergoes both asexual cycling and sexual differentiation inside erythrocytes. As the intraerythrocytic parasite develops it increases in size and alters the permeability of the host cell plasma membrane. An intriguing question is: how is the integrity of the host erythrocyte maintained during the intraerythrocytic cycle? We have used water window cryo X-ray tomography to determine cell morphology and hemoglobin content at different stages of asexual and sexual differentiation. The cryo stabilization preserves native structure permitting accurate analyses of parasite and host cell volumes. Absorption of soft X-rays by protein adheres to Beer–Lambert’s law permitting quantitation of the concentration of hemoglobin in the host cell compartment. During asexual development the volume of the parasite reaches about 50% of the uninfected erythrocyte volume but the infected erythrocyte volume remains relatively constant. The total hemoglobin content gradually decreases during the 48 h cycle but its concentration remains constant until early trophozoite stage, decreases by 25%, then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but then undergoes a dramatic shape change. Our cryo X-ray tomography analysis reveals that about 70% of the host cell hemoglobin is taken up and digested during gametocyte development and the parasite eventually occupies about 50% of the uninfected erythrocyte volume. The total volume of the infected erythrocyte remains constant, apart from some reversible shrinkage at stage IV, while the concentration of hemoglobin decreases to about 70% of that in an uninfected erythrocyte.     

Reexamining Obesigenic Families: Parents’ Obesity‐related Behaviors Predict Girls’ Change in BMI

Objective: It has been shown that girls from families in which mothers and fathers had high dietary intake and low physical activity (i.e., obesigenic families) were at increased risk of obesity from ages 5 to 7 years. This follow‐up study uses additional data collected when girls were 9 and 11 years old to examine whether girls from obesigenic families continued to show greater increases in BMI over time and reported unhealthy dietary and activity patterns. Research Methods and Procedures: Families from the original cohort were reexamined when girls were 9 and 11 years of age. Parents’ and girls’ BMI, dietary intake, and physical activity and girls’ percentage body fat and television viewing were assessed. Results: In comparison with girls from non‐obesigenic families, girls from obesigenic families showed greater increases in BMI and BMI z score from ages 5 to 7 years that were maintained across ages 7 to 11 years. Furthermore, girls from obesigenic families had higher percentage body fat at ages 9 and 11 years. These results were independent of parents’ BMI. Additional findings showed that girls from obesigenic families had diets higher in percentage fat and had higher levels of television viewing than girls from non‐obesigenic families. Discussion: The environment that parents create, by way of their own dietary and physical activity behaviors, may have a lasting negative effect on children's weight trajectories and their emerging obesity risk behaviors, such as their dietary patterns. These findings further highlight the importance of the family in establishing children's obesity risk and the necessity of targeting parents of young children in obesity prevention efforts.