Lynn McLean

Pheromonal induction of spatial learning in mice.

Spots to Remember Scent marking is an essential component of communication for most mammals. Individuals remember the location of scent marks and regularly revisit marked sites, presumably to assess the condition and status of the animal doing the marking. It is known that individuals can follow odor or pheromone gradients to locate another individual, but relocating scent marks is a much more difficult task given the small amount of volatile compounds deposited, and their static nature. Roberts et al. (p. 1462) show that a nonvolatile component of male urine, the protein pheromone darcin, stimulates spatial preference and learning in mice. Female mice preferred locations where male urine (or synthesized darcin) had been found, and remembered these spatial locations for 2 weeks post-exposure. A male sex pheromone rapidly stimulates learning of place preference that is remembered for about 2 weeks. Many mammals use scent marking for sexual and competitive advertisement, but little is known about the mechanism by which scents are used to locate mates and competitors. We show that darcin, an involatile protein sex pheromone in male mouse urine, can rapidly condition preference for its remembered location among females and competitor males so that animals prefer to spend time in the site even when scent is absent. Learned spatial preference is conditioned through contact with darcin in a single trial and remembered for approximately 14 days. This pheromone-induced learning allows animals to relocate sites of particular social relevance and provides proof that pheromones such as darcin can be highly potent stimuli for social learning.

Comparison of the biochemical changes in the jejunal mucosa of dogs with aerobic and anaerobic bacterial overgrowth

Subcellular biochemical changes in the jejunal mucosa have been compared in dogs with either aerobic or anaerobic bacterial overgrowth to explore relationships between composition of the flora and mucosal damage. Affected animals comprised 17 German shepherd dogs with chronic diarrhea or weight loss, or both. Analysis of duodenal juice demonstrated aerobic overgrowth in 10 cases, most frequently comprising enterococci and Escherichia coli, and obligate anaerobic overgrowth in 7 cases, most frequently including Clostridia spp. Histologic changes were minimal; however, examination of peroral jejunal biopsy specimens by sucrose density gradient centrifugation revealed specific biochemical abnormalities. In the dogs with aerobic overgrowth, there was a selective loss of brush border alkaline phosphatase activity, and gamma-glutamyl transferase activity was increased, whereas activities of disaccharidases and aminopeptidase N were unaltered. In contrast, anaerobic overgrowth was associated with a reduction in brush border density, indicative of a considerable fall in the glycoprotein-to-lipid ratio of the brush border membrane, whereas brush border enzyme activities were unaltered. There was a loss of peroxisomal catalase activity in dogs with aerobic overgrowth, and an indication of mitochondrial disruption in dogs with anaerobic overgrowth, but little evidence for damage to other subcellular organelles. These findings demonstrate that aerobic and anaerobic overgrowth may be associated with distinct but different mucosal abnormalities particularly affecting the brush border membrane.

Sequential morphologic and biochemical studies of naturally occurring wheat-sensitive enteropathy in Irish setter dogs

This study has investigated the potential role of wheat in the pathogenesis of a naturally occurring enteropathy in Irish setter dogs. At eight months on a cereal-containing diet, jejunal biopsies from affected animals exhibited partial villus atrophy, increased intraepithelial lymphocytes, and distinct biochemical abnormalities in the brush border. Activities of alkaline phosphatase and leucyl-2-naphthylamidase were almost undetectable while disaccharidases were unaltered. Activity of 5′-nucleotidase (basolateral membrane) was low, and reduced malate dehydrogenase reflected a loss of mitochondrial activity, but other organelles were unaffected. Recovery was achieved on a wheat-free diet. Relapse on subsequent wheat challenge was characterized by partial villus atrophy and a selective effect on the brush border: modal density was decreased and there was a severe loss of brush-border alkaline phosphatase activity. These findings document a wheat-sensitive enteropathy in Irish setter dogs and suggest that brush-border alkaline phosphatase is specifically susceptible to damage by wheat.

Regional variation in parvalbumin isoform expression correlates with muscle performance in common carp (Cyprinus carpio)

SUMMARY The mechanical properties of the axial muscles vary along the length of a fishs body. This variation in performance correlates with the expression of certain muscle proteins. Parvalbumin (PARV) is an important calcium binding protein that helps modulate intracellular calcium levels which set the size and shape of the muscle calcium transient. It therefore has a central role in determining the functional properties of the muscle. Transcript data revealed eight specific isoforms of PARV in common carp (Cyprinus carpio) skeletal muscle which we classified as α1 and β1–7. This study is the first to show expression of all eight skeletal muscle PARV isoforms in carp at the protein level and relate regional differences in expression to performance. All of the PARV isoforms were characterised at the protein level using 2D-PAGE and tandem mass spectrometry. Comparison of carp muscle from different regions of the fish revealed a higher level of expression of PARV isoforms β4 and β5 in the anterior region, which was accompanied by an increase in the rate of relaxation. We postulate that changes in specific PARV isoform expression are an important part of the adaptive change in muscle mechanical properties in response to varying functional demands and environmental change.

Characterization of Cauxin in the Urine of Domestic and Big Cats

Cauxin is an abundant protein in feline urine. We have used proteomics strategies to characterize cauxin from the urine of domestic cats and a number of big cat species. Proteins were resolved by gel-based electrophoretic purification and subjected to in-gel digestion with trypsin. The resultant tryptic peptides were mass-measured by matrix-assisted laser desorption ionization time of flight mass spectrometry. Peptides were also resolved by liquid chromatography and analyzed by electrospray ionization and tandem mass spectrometry to generate fragment ion data to infer the amino acid sequence. We identified cauxin polymorphisms and corrected a sequencing artifact in cauxin from the domestic cat. The proteomics data also provided positive evidence for the presence of a cauxin homolog in the urine of big cats (Pantherinae), including the Sumatran tiger, Asiatic lion, clouded leopard, Persian leopard, and jaguar. The levels of cauxin in the urine of all big cats were substantially lower than that in the urine of intact male domestic cats.

The Structure, Stability and Pheromone Binding of the Male Mouse Protein Sex Pheromone Darcin

Mouse urine contains highly polymorphic major urinary proteins that have multiple functions in scent communication through their abilities to bind, transport and release hydrophobic volatile pheromones. The mouse genome encodes for about 20 of these proteins and are classified, based on amino acid sequence similarity and tissue expression patterns, as either central or peripheral major urinary proteins. Darcin is a male specific peripheral major urinary protein and is distinctive in its role in inherent female attraction. A comparison of the structure and biophysical properties of darcin with MUP11, which belongs to the central class, highlights similarity in the overall structure between the two proteins. The thermodynamic stability, however, differs between the two proteins, with darcin being much more stable. Furthermore, the affinity of a small pheromone mimetic is higher for darcin, although darcin is more discriminatory, being unable to bind bulkier ligands. These attributes are due to the hydrophobic ligand binding cavity of darcin being smaller, caused by the presence of larger amino acid side chains. Thus, the physical and chemical characteristics of the binding cavity, together with its extreme stability, are consistent with darcin being able to exert its function after release into the environment.

Avian proteomics: advances, challenges and new technologies.

Proteomics is defined as an analysis of the full complement of proteins of a cell or tissue under given conditions. Avian proteomics, or more specifically chicken proteomics, has focussed on the study of individual tissues and organs of interest to specific researchers. Researchers have looked at skeletal muscle and growth, and embryonic development and have performed initial studies in avian disease. Traditional proteomics involves identifying and cataloguing proteins in a cell and identifying relative changes in populations between two or more states, be that physiological or disease-induced states. Recent advances in proteomic technologies have included absolute quantification, proteome simplification and the ability to determine the turnover of individual proteins in a global context. This review discusses the current developments in this relatively new field, new technologies and how they may be applied to biological questions, and the challenges faced by researchers in this ever-expanding and exciting field.

A Proteome Analysis of the Subcutaneous Gel in Avian Hatchlings

An appropriate level of water loss from eggs is critical to successful hatching. This water may be lost from the egg by evaporation, but where water loss is suboptimal, it is commonly observed that the hatchlings contain substantial amounts of a subcutaneous gel-like fluid. To characterize this fluid, we have analyzed the proteins that are contained within it. The protein complement comprised a small number of proteins in high concentrations. Proteomics analysis of the constituent proteins identified virtually all of these abundant proteins and confirmed that the subcutaneous gel was very similar in protein composition to plasma. However, the subcutaneous gel was substantially depleted of fibrinogen. It is possible that activation of the final stages of the coagulation process might account for the enhanced viscosity, creating a gel-like material that is relatively immobile in the subcutaneous space. This gel may function as a water volume that is partitioned during embryonic development in order to mitigate the effects of high water content of the egg caused by low mass loss during incubation and in some instances might also function as a water reserve to support the hatchling in the first few hours of life free of the shell.

RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies

This study introduces a new reversed-phase liquid chromatography retention time (RT) standard, RePLiCal (Reversed-phase liquid chromatography calibrant), produced using QconCAT technology. The synthetic protein contains 27 lysine-terminating calibrant peptides, meaning that the same complement of standards can be generated using either Lys-C or trypsin-based digestion protocols. RePLiCal was designed such that each constituent peptide is unique with respect to all eukaryotic proteomes, thereby enabling integration into a wide range of proteomic analyses. RePLiCal has been benchmarked against three commercially available peptide RT standard kits and outperforms all in terms of LC gradient coverage. RePLiCal also provides a higher number of calibrant points for chromatographic retention time standardization and normalization. The standard provides stable RTs over long analysis times and can be readily transferred between different LC gradients and nUHPLC instruments. Moreover, RePLiCal can be used to predict RTs for other peptides in a timely manner. Furthermore, it is shown that RePLiCal can be used effectively to evaluate trapping column performance for nUHPLC instruments using trap-elute configurations, to optimize gradients to maximize peptide and protein identification rates, and to recalibrate the m/z scale of mass spectrometry data post-acquisition.

1H, 15N and 13C resonance assignment of darcin, a mouse major urinary protein.

Darcin is an important lipocalin of the urinary MUP family. These beta-barrel structures differ subtly in sequence and function and facilitate communication between members of the mouse population via scent marks. Polymorphism within the family has led to the hypothesis that individual MUPs can also contribute to social and physiological information of the scent owner and thus demonstrates the necessity for structural investigation of these variations. Using conventional triple resonance experiments, 1H 15N and 13C assignment of recombinant N terminal hexa-histidine tagged Darcin has been achieved. The corresponding chemical shifts have been deposited in the BioMagResBank; Accession No. 16840.