Gene discovery and microarray analysis of cacao (Theobroma cacao L.) varieties

The cacao bean harvest from the relatively under developed tropical tree cacao (Theobroma cacao L.) is subject to high losses in potential production due to pests and diseases. To discover and understand the stability of putative natural resistance mechanisms in this commodity crop, essential for chocolate production, we undertook a gene-discovery program and demonstrated its use in gene-expression arrays. Sequencing and assembling bean and leaf cDNA library inserts produced a unique contig set of 1,380 members. High-quality annotation of this gene set using Blast and MetaFam produced annotation for 75% of the contigs and allowed us to identify the types of gene expressed in cacao beans and leaves. Microarrays were constructed using amplified inserts of the uni-gene set and challenged with bean and leaf RNA from five cacao varieties. The microarray performed well across the five randomly chosen cacao genotypes and did not show a bias towards either leaf or bean tissues. This demonstrates that the gene sequences are useful for microarray analysis across cacao genotypes and tissue types. The array results, when compared with real-time PCR results for selected genes, showed a correlation with differential gene-expression patterns.We intend that the resultant DNA sequences and molecular microarray platform will help the cacao community to understand the basis, likely stability and pathotype resistance range of candidate cacao plants.     

Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

Tome-1, a trigger of mitotic entry,is degraded during G1 via the APC

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.     

The influence of seat configuration on maximal average crank power during pedaling: a simulation study

Manipulating seat configuration (i.e., seat tube angle, seat height and pelvic orientation) alters the bicycle-rider geometry, which influences lower extremity muscle kinematics and ultimately muscle force and power generation during pedaling. Previous studies have sought to identify the optimal configuration, but isolating the effects of specific variables on rider performance from the confounding effect of rider adaptation makes such studies challenging. Of particular interest is the influence of seat tube angle on rider performance, as seat tube angle varies across riding disciplines (e.g., road racers vs. triathletes). The goals of the current study were to use muscle-actuated forward dynamics simulations of pedaling to 1) identify the overall optimal seat configuration that produces maximum crank power and 2) systematically vary seat tube angle to assess how it influences maximum crank power. The simulations showed that a seat height of 0.76 m (or 102% greater than trochanter height), seat tube angle of 85.1 deg, and pelvic orientation of 20.5 deg placed the major power-producing muscles on more favorable regions of the intrinsic force-length-velocity relationships to generate a maximum average crank power of 981 W. However, seat tube angle had little influence on crank power, with maximal values varying at most by 1% across a wide range of seat tube angles (65 to 110 deg). The similar power values across the wide range of seat tube angles were the result of nearly identical joint kinematics, which occurred using a similar optimal seat height and pelvic orientation while systematically shifting the pedal angle with increasing seat tube angles.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Quantification of cockroach allatostatin-like peptide and its myotropic effects in males of the earwig Euborellia annulipes

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to establish a competitive enzyme‐linked immunosorbent assay for quantification of allatostatin‐like peptides in the hindgut of the adult male earwig, Euborellia annulipes. Hindguts of 0‐day males contained significantly more allatostatin‐positive material than those of 8‐day males fed on catfood. However, males starved for the first 8 days of adult life had significantly higher levels of allatostatin‐positive material than those of either 0‐day or of 8‐day fed males. Hindguts from 0‐day old males exhibited lower spontaneous motility in vitro than those from 8‐day males. Hindguts from males at both ages responded to allostatin with reversible, dosage‐dependent decreases in hindgut motility, and responded to proctolin with reversible, dosage‐dependent increases in hindgut motility. When both allatostatin and proctolin were applied to hindgut preparations simultaneously and in equal concentrations, the response varied with the stage of the male. Starvation enhanced hindgut motility and abolished the response to allatostatin, but not to proctolin. These results indicate the presence of material similar to cockroach allatostatins in male earwigs, and that the levels change with age and physiological stage. Furthermore, such peptides may indeed be regulatory neuropeptides and could modulate hindgut contraction. There was an increase in sensitivity to exogenous allatostatin in the hindgut during development from day 0 to day 8 in feeding males, but a loss in sensitivity in response to starvation; sensitivity to exogenous proctolin also increased with age, but such responsiveness was not diminished by starvation.