The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.     

Identifying the predator complex of <Emphasis Type="Italic">Homalodisca vitripennis</Emphasis> (Hemiptera: Cicadellidae): a comparative study of the efficacy of an ELISA and PCR gut content assay

A growing number of ecologists are using molecular gut content assays to qualitatively measure predation. The two most popular gut content assays are immunoassays employing pest-specific monoclonal antibodies (mAb) and polymerase chain reaction (PCR) assays employing pest-specific DNA. Here, we present results from the first study to simultaneously use both methods to identify predators of the glassy winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). A total of 1,229 arthropod predators, representing 30 taxa, were collected from urban landscapes in central California and assayed first by means of enzyme-linked immunosorbent assay (ELISA) using a GWSS egg-specific mAb and then by PCR using a GWSS-specific DNA marker that amplifies a 197-base pair fragment of its cytochrome oxidase gene (subunit I). The gut content analyses revealed that GWSS remains were present in 15.5% of the predators examined, with 18% of the spiders and 11% of the insect predators testing positive. Common spider predators included members of the Salticidae, Clubionidae, Anyphaenidae, Miturgidae, and Corinnidae families. Common insect predators included lacewings (Neuroptera: Chrysopidae), praying mantis (Mantodea: Mantidae), ants (Hymenoptera: Formicidae), assassin bugs (Hemiptera: Reduviidae), and damsel bugs (Hemiptera: Nabidae). Comparison of the two assays indicated that they were not equally effective at detecting GWSS remains in predator guts. The advantages of combining the attributes of both types of assays to more precisely assess field predation and the pros and cons of each assay for mass-screening predators are discussed.     

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.     

Exploring human interaction and diet effects on the behavior of dogs in a public animal shelter

This study examined the effects of 2 manipulations-a brief, regular period of human contact and diet-on the behavior of dogs confined in a public animal shelter. A behavioral battery designed to assess reactions to novel situations, and a test of responsiveness to an unfamiliar human were administered both prior to (pretest) and immediately following (posttest) the 8-week intervention period. Overall, the regular periods of increased human contact together with a diet that contained augmented levels of digestible protein, fat, calories, and animal-derived ingredients reduced signs of behavioral reactivity from pretest to posttest. In some cases, the comparison diet appeared more effective, but only for dogs receiving minimal human interaction. The results indicate that a combination of human interaction and high quality diet may positively affect the behavior of dogs in animal shelters.     

Species traits and channel architecture mediate flow disturbance impacts on invertebrate drift

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Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants displey GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination.     

The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70

The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.     

Two-temperature LATE-PCR endpoint genotyping

Background  

In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.     

Quality prediction of cell substrate using gene expression profiling

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.     

The effects of natural substrate discontinuities on the quadrupedal gait kinematics of free‐ranging Saimiri sciureus

Wild primates encounter complex matrices of substrates that differ in size, orientation, height, and compliance, and often move on multiple, discontinuous substrates within a single bout of locomotion. Our current understanding of primate gait is limited by artificial laboratory settings in which primate quadrupedal gait has primarily been studied. This study analyzes wild Saimiri sciureus (common squirrel monkey) gait on discontinuous substrates to capture the realistic effects of the complex arboreal habitat on walking kinematics. We collected high‐speed video footage at Tiputini Biodiversity Station, Ecuador between August and October 2017. Overall, the squirrel monkeys used more asymmetrical walking gaits than symmetrical gaits, and specifically asymmetrical lateral sequence walking gaits when moving across discontinuous substrates. When individuals used symmetrical gaits, they used diagonal sequence gaits more than lateral sequence gaits. In addition, individuals were more likely to change their footfall sequence during strides on discontinuous substrates. Squirrel monkeys increased the time lag between touchdowns both of ipsilaterally paired limbs (pair lag) and of the paired forelimbs (forelimb lag) when walking across discontinuous substrates compared to continuous substrates. Results indicate that gait flexibility and the ability to alter footfall patterns during quadrupedal walking may be critical for primates to safely move in their complex arboreal habitats. Notably, wild squirrel monkey quadrupedalism is diverse and flexible with high proportions of asymmetrical walking. Studying kinematics in the wild is critical for understanding the complexity of primate quadrupedalism.