Cordon bleu promotes the assembly of brush border microvilli

Microvilli are actin-based protrusions found on the surface of diverse cell types, where they amplify membrane area and mediate interactions with the external environment. In the intestinal tract, these protrusions play central roles in nutrient absorption and host defense and are therefore essential for maintaining homeostasis. However, the mechanisms controlling microvillar assembly remain poorly understood. Here we report that the multifunctional actin regulator cordon bleu (COBL) promotes the growth of brush border (BB) microvilli. COBL localizes to the base of BB microvilli via a mechanism that requires its proline-rich N-terminus. Knockdown and overexpression studies show that COBL is needed for BB assembly and sufficient to induce microvillar growth using a mechanism that requires functional WH2 domains. We also find that COBL acts downstream of the F-BAR protein syndapin-2, which drives COBL targeting to the apical domain. These results provide insight into a mechanism that regulates microvillar growth during epithelial differentiation and have significant implications for understanding the maintenance of intestinal homeostasis.     

Behavioral phenotyping of transgenic and knockout mice: practical concerns and potential pitfalls

New technologies in molecular genetics have dramatically increased the number of targeted gene mutations available to the biomedical research community. Many mutant mouse lines have been generated to provide animal models for human genetic disorders, offering insights into anatomical, neurochemical, and behavioral effects of aberrant gene expression. A variety of assays have been developed to identify and characterize phenotypic changes. In the behavioral domain, our phenotyping strategy involves a comprehensive standardized methodological approach that assesses general health, reflexes, sensory abilities, and motor functions. This assessment is followed by a series of complementary tasks in the specific behavioral domain(s) hypothesized to reveal the function(s) of the gene. Our multitiered approach minimizes intersubject variability by standardizing the experimental history for all animals, improves interlaboratory reliability by providing a clearly defined experimental protocol, and minimizes artifactual interpretations of behavioral data by careful preliminary assessments of basic behaviors, followed by multiple tests within the behavioral domain of interest. Despite meticulous attention to experimental protocol, attention to environmental factors is essential. Differences in noise, light, home cage environment, handling, and diet can dramatically alter behavior. Baseline differences in the behaviors of inbred strains used to generate targeted mutant mouse lines can directly influence the behavioral phenotype of the mutant line. Strategies aimed at minimizing environmental variability and contributions of background genes will enhance the robustness of mouse behavioral phenotyping assays.     

Advantages of using same species enzyme for replacement therapy in a feline model of mucopolysaccharidosis type VI

In a feline model of mucopolysaccharidosis type VI (MPS VI), recombinant feline N-acetylgalactosamine-4-sulfatase (rf4S) administered at a dose of 1 mg/kg of body weight, altered the clinical course of the disease in two affected cats treated from birth. After 170 days of therapy, both cats were physically indistinguishable from normal cats with the exception of mild corneal clouding. Feline N-acetylgalactosamine-4-sulfatase was effective in reducing urinary glycosaminoglycan levels and lysosomal storage in all cell types examined except for corneal keratocytes and cartilage chondrocytes. In addition, skeletal pathology was nearly normalized as assessed by radiographic evidence and bone morphometric analysis. Comparison of results with a previous study in which recombinant human 4S (rh4S) was used at an equivalent dose and one 5 times higher indicated that rf4S had a more pronounced effect on reducing pathology than the same dose of rh4S, and in some instances such as bone pathology and lysosomal storage in aorta smooth muscle cells, it was as good as, or better than, the higher dose of rh4S. We conclude that in the feline MPS VI model the use of native or same species enzyme for enzyme replacement therapy has significant benefits.     

Direct activation of the ventral striatum in anticipation of aversive stimuli

The brain "reward" system, centered on the limbic ventral striatum, plays a critical role in the response to pleasure and pain. The ventral striatum is activated in animal and human studies during anticipation of appetitive/pleasurable events, but its role in aversive/painful events is less clear. Here we present data from three human fMRI studies based on aversive conditioning using unpleasant cutaneous electrical stimulation and show that the ventral striatum is reliably activated. This activation is observed during anticipation and is not a consequence of relief after the aversive event. Further, the ventral striatum is activated in anticipation regardless of whether there is an opportunity to avoid the aversive stimulus or not. Our data suggest that the ventral striatum, a crucial element of the brain "reward" system, is directly activated in anticipation of aversive stimuli.     

The effects of grazers on the performance of individuals and populations of scarlet gilia,Ipomopsis aggregata

Summary I. aggregata exhibits considerable powers of regrowth following removal of its primary shoot by herbivores, but we found no evidence of overcompensation (i.e. of significantly higher plant performance where plants were exposed to ungulate herbivory) in a comparison between individuals on grazed and ungrazed sides of exclosure fences, in a comparison between artificially clipped and control plants in one population in the Okanagan National Forest, or in comparisons between grazed and ungrazed plants in 14 natural populations. We tested whether ungulate grazing affects the population size of Ipomopsis aggregata by comparing populations inside and outside deer exclosures at 7 sites in the Western United States. We found consistent, highly significant differences in plant population density on the grazed and ungrazed sides of these exlosure fences. Plant density was a modal 25-fold higher on the protected side of the fence, suggesting that exposure to ungulate grazing increases plant death rates at some stage in the life cycle. Our results show that the presence of ungulate grazers leads to a substantial decrease in plant density despite the fact that grazing on young bolting shoots has very little influence on fruit production. Since this decrease in population density is not correlated with a decrease in the fecundity of individuals, it must instead be due to other direct and indirect effects of ungulate grazers.     

An enzyme-linked immunosorbent assay for N-acetylglucosaminyltransferase-V

The development of an enzyme-linked immunosorbent assay (ELISA) for uridine 5'-diphospho-N-acetyl-glucosamine: alpha mannoside beta 1----6 N-acetylglucosaminyltransferase (GnT-V) is reported. The assay quantitates the enzymatic conversion of the specific synthetic GnT-V acceptor GlcNAc beta 1----2Man alpha 1----6Man beta-R (5) to the product GlcNAc beta 1----2[GlcNAc-beta 1----6]Man alpha 1----6Man beta-R (6) when these oligosaccharide structures were covalently attached to bovine serum albumin which was then coated on microtiter wells. Conversion of 5 to 6 was detected using a polyclonal antiserum raised against the product 6 and from which antibodies cross-reacting with acceptor 5 had been removed by affinity adsorption. GnT-V activity detected by ELISA was linearly proportional to both enzyme concentration and time under appropriate experimental conditions where 50-300 fmol of product was formed per microtiter well. GnT-V activity could be measured by ELISA in Triton X-100 extracts of hamster kidney acetone powder and in human serum. The twofold increase in GnT-V activity which is known to accompany Rous sarcoma virus transformation of baby hamster kidney cells could also be quantitated using the ELISA.     

Spatially structured population dynamics in feral oilseed rape

We studied the population dynamics of feral oilseed rape (Brassica napus) for 10 years (1993-2002) in 3658 adjacent permanent 100 m quadrats in the verges of the M25 motorway around London, UK. The aim was to determine the relative importance of different factors affecting the observed temporal patterns of population dynamics and their spatial correlations. A wide range of population dynamics was observed (downward or upward trends, cycles, local extinctions and recolonizations), but overall the populations were not self-replacing (lambda < 1). Many quadrats remained unoccupied throughout the study period, but a few were occupied at high densities for all 10 years. Most quadrats showed transient oilseed rape populations, lasting 1-4 years. There were strong spatial patterns in mean population density, associated with soil conditions and the successional age of the plant community dominating the verge, and these large-scale spatial patterns were highly consistent from year to year. The importance of seed spilled from trucks in transit to the processing plant at Erith in Kent was confirmed: rape populations were significantly higher on the 'to Erith' verge than the 'from Erith' verge (overall mean 2.83-fold greater stem density). Quadrats in which lambda > 1 were much more frequent in the 'to Erith' verge, indicating that seed immigration can give the spurious impression of self-replacing population dynamics in time-series analysis. There was little evidence of a pervasive Moran effect, and climatic forcing did not produce widespread large-scale synchrony in population dynamics for the motorway as a whole; just 23% of quadrats had significant rank correlations with the mean time-series. There was, however, significant local spatial synchrony of population dynamics, apparently associated with soil disturbance and seed input. This study draws attention to the possibility that different processes may impose population synchrony at different scales. We hypothesize that synchrony in this system is driven by at least three processes: small-scale, local forcing caused by soil disturbance, intermediate-scale forcing as a result of seed input, and large-scale climatic forcing (e.g. winter rainfall) that affects the motorway as a whole.