Adoption of shelter dogs in a Brazilian community: assessing the caretaker profile

A survey in Ibiuna, Sao Paulo, Brazil, of caregivers (owners) who adopted shelter dogs assessed length of ownership, proportion of male and female dogs adopted, and owners' characteristics. It addressed breeding, neutering, vaccination, and veterinary care. It used no testing to provide a good “match ”between dog and future owner. Of adopted dogs, 58% were male. Only 36% of owners were located. Mean ownership length was 14.8 months (95% confidence interval = 12.4 to 17.2 months), estimated through a survival analysis method. Of adopted dogs, 40.9% lived with their owners; 34.9% had died (some had lived on the streets); 15.0% were donated; 4.3% ran away; 3.2% were returned to the city shelter. Of interviewees, 57% reported no difficulties with the adoption; 23.1% cited the animal's illness and death as the main difficulty. For contraception, 87 owners (46.7%) chained dogs to prevent contact with other animals; 56.5% were against neutering. Reasons given were compassion (58.1%), unnecessary procedure (11.4%), cost (9.5%), and behavior change (4.8%). This research motivated a design for Ibiuna shelter dog adoption to improve the proportion of successful adoptions.     

Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii

Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline beta-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas-liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of alpha-Rhap-(1 --> 3)-alpha-Rhap-(1 --> 3)-alpha-Manp-(1 --> 2)-Man-ol substituted at O-6 of mannitol with an alpha-Glcp-(1 --> 4)-beta-Galp group. Species containing one and two additional alpha-Glcp-(1 --> 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by approximately 75%, whereas fraction 3 inhibited by approximately 55%.     

Structural and functional characterization of CFE88: evidence that a conserved and essential bacterial protein is a methyltransferase

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.     

Alterations in redox homeostasis and prostaglandins impair endothelial-dependent vasodilation in euglycemic autoimmune nonobese diabetic mice

We report herein the novel observation that alterations in oxidant/antioxidant balance are evident and cause vascular dysfunction in aortae of prediabetic nonobese-diabetic mice (NOD). We found that nitrotyrosine, a biochemical marker of oxidant stress, was higher in the NOD aortae when compared to age-matched non-autoimmune BALB/c controls or the diabetes-resistant NOD congenic strain, NOD.Lc7. The oxidant stress was localized to the intimal and medial layers, and endothelium-dependent relaxation to acetylcholine was decreased in isolated aortic rings from NOD mice. Inhibition of nitric oxide synthesis caused an endothelium-dependent contraction, and treatment with either a selective thromboxane A2/prostaglandin H2 receptor antagonist or a non-isozyme-specific cyclooxygenase inhibitor reversed this effect. Aortic rings from NOD.Lc7 did not display the paradoxical vasoconstriction. Furthermore, the vascular dysfunction was caused by oxidative stress, as treatment with a superoxide dismutase mimetic in vivo or with native antioxidant enzymes ex vivo inhibited the tissue oxidant stress and restored endothelium-dependent relaxation. Endothelial function was also restored by the inhibitors of NAD(P)H oxidase, diphenylene iodonium or apocynin. Our studies indicate that an oxidant stress that occurs prior to the onset of diabetes in this mouse model contributes to endothelial dysfunction independently of overt diabetes.     

Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).     

An inter-platform repeatability study investigating real-time amplification of plasmid DNA

Background  

The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results.     

Rapid spontaneous accessibility of nucleosomal DNA

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only approximately 250 ms before spontaneously unwrapping; unwrapped DNA rewraps within approximately 10-50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.     

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.     

Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.