Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung

Summary Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit antiadult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA.Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 dalton proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.     

Probabilistic Firing of Neurons Considered as a First Passage Problem

A formalized neuron receiving unitary excitatory impulses at random is considered. Each impulse provokes an effect of equal magnitude and of a duration not constant for each impulse, but which varies according to an exponential distribution. The effects sum until a threshold is reached when a response occurs. The distribution of intervals between successive responses is computed and compared with those obtained from a model in which the effects decay exponentially with time. Upon introducing inhibitory impulses also, the theory is applied to data on discharge characteristics of driven and spontaneously active thalamic neurons reported in the literature.     

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y‐chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y‐chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.     

600 ps Molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase

Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen‐bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of α‐helices and β‐strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy‐terminal tail. The most significant flexible region was the highly conserved glycine‐rich loop between β strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of “open” and “closed” conformations of cAPK correspond to the highly flexible residues found during dynamics. © 1999 John Wiley & Sons, Inc. Biopoly 50: 513–524, 1999     

Ezetimibe: A biomarker for efficacy of liver directed UGT1A1 gene therapy for inherited hyperbilirubinemia

As recently demonstrated in patients with factor IX deficiency, adeno-associated virus (AAV)-mediated liver-directed therapy is a viable option for inherited metabolic liver disorders. Our aim is to treat Crigler-Najjar syndrome type I (CN I), an inherited severe unconjugated hyperbilirubinemia, as a rare recessive inherited disorder. Because the number of patients eligible for this approach is small, the efficacy can only be demonstrated by a beneficial effect on the pathophysiology in individual patients. Serum bilirubin levels in potential candidates have been monitored since birth, providing an indication of their pathophysiology. Adjuvant phototherapy to prevent brain damage reduces serum unconjugated bilirubin (UCB) levels in CN I patients to the level seen in the milder form of the disease, CN type II. This therapy increases the excretion of UCB, thereby complicating the use of UCB and conjugated bilirubin levels in serum as biomarkers for the gene therapy we try to develop. Therefore, a suitable biomarker that is not affected by phototherapy is currently needed. To this end, we have investigated whether estradiol, ethinylestradiol or ezetimibe could be used as markers for uridine 5'-di-phospho-glucuronosyltransferase isoform 1A1 (UGT1A1) activity restored by AAV gene therapy in Gunn rats, a relevant animal model for CN I. Of these compounds, ezetimibe appeared most suitable because its glucuronidation rate in untreated control Gunn rats is low. Subsequently, ezetimibe glucuronidation was studied in both untreated and AAV-treated Gunn rats and the results suggest that it may serve as a useful serum marker for restored hepatic UGT1A1 activity.     

Breeding replacement gilts for organic pig herds

In this study, breeding structures and commercial sow lines were evaluated by economic and genetic simulation studies for their suitability to provide the Dutch organic pig sector with replacement gilts. Sow and litter performance from over 2000 crossbred sows from 2006 to 2007 were collected on 11 to 14 Dutch organic pig herds, respectively, and compared with conventional herds. Results showed that organic herds had lower farrowing rates (3.6% to 7.5%), more live born piglets per litter (0.4% to 1.2%) and higher preweaning mortality rates (7% to 13%) compared to conventional herds. These results were used to simulate economic performance of various combinations of breeding structures and sow lines under organic conditions, under the assumption of absence of genotype-environment interactions. Sow and litter performance data under organic conditions (total piglets born/litter, stillborn piglets/litter, mortality until weaning, lactation length, interval weaning-oestrus and sow culling rate) and the costprice calculation for the Dutch organic pig sector were used as input for the economic simulation studies. The expected genetic progress was simulated for three potential breeding structures of the organic sector: organic breeding herds producing F1 gilts (OrgBS), a flower breeding system (FlowerBS) and a two-line rotation breeding system (RotBS). In FlowerBS, an organic purebred sow line is bred, using on-farm gilt replacement. The OrgBS with a Yorkshire × Landrace cross had the highest margin per sow place (€779), followed by RotBS with Yorkshire × Landrace cross (€706) and FlowerBS with Yorkshire sow line (€677). In case that an organic purebred sow population of 5000 sows would be available, FlowerBS gave the highest genetic progress in terms of cost price reduction (€3.72/slaughter pig per generation), followed by RotBS and OrgBS (€3.60/slaughter pig per generation). For FlowerBS, additional costs will be involved for maintaining a dedicated breeding programme. In conclusion, OrgBS using conventional genetics is economically the most viable option for the organic pig sector. However, this structure has clear disadvantages in terms of risks with regard to disease transmission and market demand. FlowerBS using a dedicated purebred organic line will only be cost-effective if sow population size is sufficiently large. RotBS might be a viable alternative, especially in combination with artificial insemination (AI) boars that are ranked according to an organic selection index. Regardless of breeding structure, the Yorkshire sow line gave the highest prolificacy and the highest economic returns on organic herds.     

Mixed effects models with bivariate and univariate association parameters for longitudinal bivariate binary response data

When two binary responses are measured for each study subject across time, it may be of interest to model how the bivariate associations and marginal univariate risks involving the two responses change across time. To achieve such a goal, marginal models with bivariate log odds ratio and univariate logit components are extended to include random effects for all components. Specifically, separate normal random effects are specified on the log odds ratio scale for bivariate responses and on the logit scale for univariate responses. Assuming conditional independence given the random effects facilitates the modeling of bivariate associations across time with missing at random incomplete data. We fit the model to a dataset for which such structures are feasible: a longitudinal randomized trial of a cardiovascular educational program where the responses of interest are change in hypertension and hypercholestemia status. The proposed model is compared to a naive bivariate model that assumes independence between time points and univariate mixed effects logit models.     

Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster

Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.     

Number and sex of offspring of ΔF508 carriers outside cystic fibrosis families

Number and sex of offspring were determined in a group of 7,841 randomly selected blood donors who were screened for the F508 mutation. We did not find any evidence for differences in number or sex ratio of offspring between F508 carriers and non-carriers.