Added Weights Lead to Reduced Flight Behavior and Mating Success in Polyandrous Honey Bee Queens (Apis mellifera)

Honey bee queens are exceptionally promiscuous. Early in life, queens perform one to five nuptial flights, mating with up to 44 drones. Many studies have documented potential benefits of multiple mating. In contrast, potential costs of polyandry and the sensitivity of queens to such costs have largely been ignored because they are difficult to address experimentally. To consider one aspect of mating costs to queens, the difficulty of flight, we compared flight behavior and success among a group of control queens and two experimental groups of queens that carried lead weights of two different sizes. For each queen, we assessed the number and duration of all flights and, after egg-laying commenced, the amount of stored sperm and the number of mates in terms of the offspring's patrilineal genetic diversity. Added weights quantitatively decreased the number of flights, the mean duration of flights and consequently the total time spent flying. Mating success in terms of sperm quantity and patrilines detected among the queens' offspring was also negatively impacted by the experimental manipulation. Thus, it can be concluded that the flight effort of honey bee queens during their mating period is adjusted in response to an experimentally increased cost of flying with multiple consequences for their mating success. Our results suggest that queen behavior is flexible and mating costs deserve more attention to explain the extreme polyandry in honey bees.     

Characterization of EVL-I as a protein kinase D substrate

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPβ and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell–cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell–cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.     

Biochemical Characterization of the Prolyl 3-Hydroxylase 1��Cartilage-associated Protein��Cyclophilin B Complex

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.     

Diversity of urea‐degrading microorganisms in open‐ocean and estuarine planktonic communities

Urea is an important and dynamic natural component of marine nitrogen cycling and also a major contributor to anthropogenic eutrophication of coastal ecosystems, yet little is known about the identities or diversity of ureolytic marine microorganisms. Primers targeting the gene encoding urease were used to PCR‐amplify, clone and sequence 709 urease gene fragments from 31 plankton samples collected at both estuarine and open‐ocean locations. Two hundred and eighty‐six amplicons belonged to 22 distinct sequence types that were closely enough related to named organisms to be identified, and included urease sequences both from typical marine planktonic organisms and from bacteria usually associated with terrestrial habitats. The remaining 423 amplicons were not closely enough related to named organisms to be identified, and belonged to 96 distinct sequence types of which 43 types were found in two or more different samples. The distributions of unidentified urease sequence types suggested that some represented truly marine microorganisms while others reflected terrestrial inputs to low‐salinity estuarine areas. The urease primers revealed this great diversity of ureolytic organisms because they were able to amplify many previously unknown, environmentally relevant urease genes, and they will support new approaches for exploring the role of urea in marine ecosystems.     

Inadvertent Propagation of Factor VII Deficiency in a Canine Mucopolysaccharidosis Type I Research Breeding Colony

Issues of cost and genetics can result in inbreeding of canine genetic disease colonies. Beagles often are used to maintain such colonies, providing stock for outcrosses. Factor VII (FVII) deficiency is a hemostatic disorder found at increased frequency in beagles and has been characterized at the DNA level. Deficiency of FVII presents obstacles in colonies founded with beagles. An initial finding of a FVII-deficient pup from a longstanding colony prompted us to evaluate FVII deficiency fully in this colony. Current and archival records and tissues were used to reconstruct the colony pedigree, assess the contribution from beagles, and test samples to document the source and frequency of the mutant FVII allele. As part of this study we developed a PCR-based diagnostic assay that was simpler than what was previously available. Pedigree analysis revealed a founder effect implicating beagles that led to high frequency (55%) of the mutant allele. In addition, affected animals were identified. The complete picture of the clinical effect within the colony remains unclear, but unusual neonatal presentations, including hemoabdomen, have occurred in pups affected with FVII deficiency. Use of a PCR-based diagnostic assay to screen all potential beagle breeding stock will prevent similar occurrences of FVII deficiency in future canine research colonies.Abbreviations: FVII, factor VII; MPS I, mucopolysaccharidosis I; PT, prothrombin timeThe importance of developing clinically relevant large animal models for human genetic diseases is becoming increasingly evident.⁴ For example, preclinical assessments of gene transfer experiments require large long-lived animal models physiologically and genetically comparable to humans. Canine models are ideal because their genome has been sequenced, they are large and long-lived, and because more than 60% of inherited diseases of dogs are known to be homologs of human diseases.⁴The maintenance of genetic diseases in research colonies results, for all practical purposes, in a founder effect by which allelic frequencies in a research colony may be skewed upward from those in the general population, due to founding of the colony by a limited number of animals. This founder effect is due to insufficient genetic outcrosses, resulting from economic constraints and considerations such as the inbreeding needed with a recessive condition. Practically speaking, colony founders may inevitably be incompletely characterized at the genetic level, potentially leading to increased prevalence of an additional genetic disease. When available, practical screening tests (clotting times, cardiac evaluation, and so on) or breed-specific genetic tests should be conducted to reduce additional genetic diseases within a research colony.The occurrence of additional genetic diseases in research colonies can limit or confound the primary research objective and affect the number of research animals used and their health and welfare. Inherited factor VII (FVII) deficiency in beagles is such a condition.⁸ Although largely an asymptomatic defect, this autosomal recessive hemostatic disorder, can lead to excessive bleeding after surgery or trauma, hematoma formation, body cavity bleeding, and persistent uterine and vaginal hemorrhage.²³ Factor VII deficiency also occurs in Alaskan malamutes,¹⁴ mixed breeds,¹³ and Alaskan klee kai dogs.¹¹ Clinical symptoms in canines can be reduced by transfusions with fresh plasma or blood, or administration of recombinant activated human FVII.^9,17 However, treatments are only a temporary solution, because the half-life of FVII protein is only 3 to 4 h and, in canines, treatment with human proteins raises concern about antibody responses to those proteins, thus potentially limiting further therapy. The FVII mutation initially described in beagles (referred to henceforth as the ‘beagle mutation’ in full recognition of its occurrence in additional breeds) is well described.¹ Furthermore the beagle mutation has been documented to cause the FVII deficiency seen in the Alaskan klee kai,¹¹ Airedale terrier, giant schnauzer, and Scottish deerhound.²² The published assay is a restriction digest to test beagles for the causative transitional missense mutation of a guanine-to-adenine located in exon 5 (leading to the G96E mutant protein).¹ However, because the assay relies on an enzyme with multiple sites in the resultant amplicon, interpretation of results can be problematic, potentially requiring direct DNA sequencing for confirmation of the genotype.Herein we present data from a long-standing canine research breeding colony for mucopolysaccharidosis type I (MPS I) which indicate that FVII deficiency should be a primary concern when developing research colonies by using beagle breeding stock. We suspected factor VII deficiency in this colony after an episode of hemoabdomen in a neonate, which was noted shortly after the colony was transferred to a different institution. Using an improved PCR-based diagnostic assay for the beagle FVII mutation, we have documented the history of this mutant allele within the colony in detail and have identified the presence of this allele in other canine colonies.     

Meiotic Recombination in Human Oocytes

Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1) in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of “vulnerable” crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.     

Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln(775) constrains the orientation of the invariant Gln(817) side chain, which forms bidentate bonds with 5'-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98-102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095-13101; Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H., Schlessinger, J., and Bollag, G. (2004) Mol. Cell 15, 279-286). PDE5(Q817A) and PDE5(Q775A) were generated to test the hypotheses that Gln(817) is critical for cyclic nucleotide or inhibitor affinity and that Gln(775) immobilizes the Gln(817) side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5(WT). For PDE5(Q817A), K(m) for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5(Q775A), K(m) for cGMP was weakened approximately 20-fold but was unchanged for cAMP. For PDE5(Q817A), vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln(817) is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5(Q775A). It is concluded that Gln(817) is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln(775), which perhaps restricts rotation of Gln(817) side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil.     

Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes

Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGIalpha-(Delta349-670) and PKGIbeta-(Delta364-685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 A, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and K(D) values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGIalpha-isolated R domain and contraction of the PKGIbeta-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGIbeta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIalpha R domain may partially account for the elongation of wild type PKGIalpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.