Mortality in over 350,000 Insured Swedish Dogs from 1995–2000: II. Breed-Specific Age and Survival Patterns and Relative Risk for Causes of Death

This study continues analysis from a companion paper on over 350,000 insured Swedish dogs up to 10 years of age contributing to more than one million dog-years at risk during 1995–2000. The age patterns for total and diagnostic mortality and for general causes of death (trauma, tumour, locomotor, heart and neurological) are presented for numerous breeds. Survival estimates at five, eight and 10 years of age are calculated. Survival to 10 years of age was 75% or more in Labrador and golden retrievers, miniature and toy poodles and miniature dachshunds and lowest in Irish wolfhounds (91% dead by 10 years). Multivariable analysis was used to estimate the relative risk for general and more specific causes of death between breeds accounting for gender and age effects, including two-way interactions. Older females had tumour as a designated cause of death more often than males in most breeds, but not in the Bernese mountain dog. Information presented in this and the companion paper inform our understanding of the population level burden of disease, and support decision-making at the population and individual level about health promotion efforts and treatment and prognosis of disease events.     

Fetal death persists through recurrent pregnancies in mice following Ljungan virus infection

OBJECTIVES: Laboratory mice infected with Ljungan virus (LV) early in pregnancy suffer from perinatal death. Here we investigate the persistence of that effect through the outcome of consecutive pregnancies in LV‐infected mice. STUDY DESIGN: CD‐1 mice were infected while pregnant and their adult female offspring were followed in parallel with uninfected control mice during repeated pregnancies. Three mating attempts resulted in two or three pregnancies per dam. The outcome of the last pregnancy was carefully monitored. RESULTS: Both the dams infected as adults and their adult female offspring suffered perinatal deaths during the last pregnancy which occurred approximately 6 months after the original LV exposure and acute infection. The non‐infected control animals experienced no perinatal death. CONCLUSIONS: Perinatal death persists across recurrent pregnancies in this mouse model of LV infection, both in animals infected as adults and in females exposed to the virus in utero. This implies that LV persists in mice long after intial infection, and is maintained in a quiescent state but can remain pathogenic in later pregnancies. Birth Defects Res (Part B) 83:507‐510, 2008. © 2008 Wiley‐Liss, Inc.     

Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.     

Lipid interacting regions in phosphate stress glycosyltransferase atDGD2 from Arabidopsis thaliana

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227-245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130-148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227-245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.     

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed 3H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested 3H-SM in the intestinal content increased by 6-fold and the formation of 3H-ceramide decreased markedly, resulting in 98% reduction of 3H-ceramide/3H-SM ratio 1 h after gavage. The absorbed 3H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in 3H-ceramide was identified in distal intestine in KO mice. In feces, 3H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.     

A human protein atlas for normal and cancer tissues based on antibody proteomics

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.     

Six genes strongly regulated by mercury in Pisum sativum roots.

Suppression subtractive hybridisation was used to isolate heavy metal-induced genes from Pisum sativum roots hydroponically exposed to 5 microM HgCl2 and 10 microM EDTA. Six genes were induced out of which one, PsHMIP6B, was novel. The other genes (PsSAMT, PsI2'H, PsNDA, PsAPSR, PsPOD) had not previously been isolated from pea and sequenced. All six genes were also induced after exposure to 5 microM HgCl2 in the absence of EDTA. The induction pattern was in some cases different for the two Hg species, demonstrating a quicker response to-free Hg2+ than Hg-EDTA. The stress-specificity of the gene regulation was investigated by hydroponically adding 5 microM Cd2+. Most Hg-induced cDNAs were also induced by Cd2+ but to a smaller extent than after Hg exposure. In addition, the gene expression was also probed for tissue specificity, which showed that all six genes were expressed in roots and not in leaves.     

Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.