Evolutionary Divergence in the Catalytic Activity of the CAM-1, ROR1 and ROR2 Kinase Domains

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.     

Using microsatellite diversity in wild Anegada iguanas (<Emphasis Type="Italic">Cyclura pinguis</Emphasis>) to establish relatedness in a captive breeding group of this critically endangered species

Awareness of the genealogical relationships between founder animals in captive breeding programs is essential for the selection of mating pairs that maintain genetic diversity. If captive founder relationships are unknown they can be inferred using genetic data from wild populations. Here, we report the results of such an analysis for six Cyclura pinguis (Sauria: Iguanidae) acquired as adults in 1999 by the San Diego Zoo Institute for Conservation Research to begin a captive breeding program for this critically endangered species. The six founder animals were reportedly hatched in captivity from eggs collected on Anegada in 1985. No records exist, however, as to where on Anegada the eggs were collected or from how many nests they originated. To assist determination of genealogical relationships, we genotyped the six captive founders, their first six offspring, and 33 wild adult iguanas from Anegada at 23 informative microsatellite loci. With these data, we estimated allele frequencies among the wild samples and then estimated the relatedness of the captive population. Using likelihood inference, we determined that three closely related pairs exist among the six captive founders and that each pair is not closely related to the other two. In addition, we were able to assign parentage for all six of the founders’ offspring tested, one of which had been previously misdiagnosed. Using the assigned parentage and inferred relatedness of the six founders, we calculated mean kinship for each of the six founders and their five living offspring. Finally, based on the allelic diversity of the wild iguanas sampled, we conclude that the C. pinguis population on Anegada is not excessively inbred; however, further investigation is warranted.     

Onset and progression of behavioral and molecular phenotypes in a novel congenic R6/2 line exhibiting intergenerational CAG repeat stability

In the present study we report on the use of speed congenics to generate a C57BL/6J congenic line of HD-model R6/2 mice carrying 110 CAG repeats, which uniquely exhibits minimal intergenerational instability. We also report the first identification of the R6/2 transgene insertion site. The relatively stable line of 110 CAG R6/2 mice was characterized for the onset of behavioral impairments in motor, cognitive and psychiatric-related phenotypes as well as the progression of disease-related impairments from 4 to 10 weeks of age. 110Q mice exhibited many of the phenotypes commonly associated with the R6/2 model including reduced activity and impairments in rotarod performance. The onset of many of the phenotypes occurred around 6 weeks and was progressive across age. In addition, some phenotypes were observed in mice as early as 4 weeks of age. The present study also reports the onset and progression of changes in several molecular phenotypes in the novel R6/2 mice and the association of these changes with behavioral symptom onset and progression. Data from TR-FRET suggest an association of mutant protein state changes (soluble versus aggregated) in disease onset and progression.     

Particle morphology characterization and manipulation in biomass slurries and the effect on rheological properties and enzymatic conversion

An improved understanding of how particle size distribution relates to enzymatic hydrolysis performance and rheological properties could enable enhanced biochemical conversion of lignocellulosic feedstocks. Particle size distribution can change as a result of either physical or chemical manipulation of a biomass sample. In this study, we employed image processing techniques to measure slurry particle size distribution and validated the results by showing that they are comparable to those from laser diffraction and sieving. Particle size and chemical changes of biomass slurries were manipulated independently and the resulting yield stress and enzymatic digestibility of slurries with different size distributions were measured. Interestingly, reducing particle size by mechanical means from about 1 mm to 100 μm did not reduce the yield stress of the slurries over a broad range of concentrations or increase the digestibility of the biomass over the range of size reduction studied here. This is in stark contrast to the increase in digestibility and decrease in yield stress when particle size is reduced by dilute-acid pretreatment over similar size ranges.     

The effect of low survey response rates on estimates of alcohol consumption in a general population survey

Background

Response rates for surveys of alcohol use are declining for all modes of administration (postal, telephone, face-to-face). Low response rates may result in estimates that are biased by selective non-response. We examined non-response bias in the NZ GENACIS survey, a postal survey of a random electoral roll sample, with a response rate of 49.5% (n = 1924). Our aim was to estimate the magnitude of non-response bias in estimating the prevalence of current drinking and heavy episodic (binge) drinking.

Methods

We used the “continuum of resistance” model to guide the investigation. In this model the likelihood of response by sample members is related to the amount of effort required from the researchers to elicit a response. First, the demographic characteristics of respondents and non-respondents were compared. Second, respondents who returned their questionnaire before the first reminder (early), before the second reminder (intermediate) or after the second reminder (late) were compared by demographic characteristics, 12-month prevalence of drinking and prevalence of binge drinking.

Results

Demographic characteristics and prevalence of binge drinking were significantly different between late respondents and early/intermediate respondents, with the demographics of early and intermediate respondents being similar to people who refused to participate while late respondents were similar to all other non-respondents. Assuming non-respondents who did not actively refuse to participate had the same drinking patterns as late respondents, the prevalence of binge drinking amongst current drinkers was underestimated. Adjusting the prevalence of binge drinkers amongst current drinkers using population weights showed that this method of adjustment still resulted in an underestimate of the prevalence.

Conclusions

The findings suggest non-respondents who did not actively refuse to participate are likely to have similar or more extreme drinking behaviours than late respondents, and that surveys of health compromising behaviours such as alcohol use are likely to underestimate the prevalence of these behaviours.     

N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor

A series of potent antagonists of the ion channel transient receptor potential A1 (TRPA1) was developed by modifying lead structure 16 that was discovered by high-throughput screening. Based on lead compound 16, a SAR was established, showing a narrow region at the nitro-aromatic R(1) moiety and at the warhead, while the R(2) side had a much wider scope including ureas and carbamates. Compound 16 inhibits Ca(2+)-activated TRPA1 currents reversibly in whole cell patch clamp experiments, indicating that under in vivo conditions, it does not react covalently, despite its potentially electrophilic ketone.     

Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins

The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination – which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1–5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.     

Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production

Methanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzyme M (2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells.     

Fate of Escherichia coli O157:H7 in Manure-Amended Soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21°C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21°C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.