Abnormal binding of factor VIII is linked with the substitution of glutamine for arginine 91 in von Willebrand factor in a variant form of von Willebrand disease

von Willebrand factor (vWf) is a multimeric plasma glycoprotein that functions in hemostasis as the initiator of platelet adhesion to damaged blood vessels and as the carrier of Factor VIII (FVIII). Montgomery et al. (Montgomery, R.R., Hathaway, W.E., Johnson, J., Jacobsen, L., and Muntean, W. (1982) Blood 60, 201-207) reported a variant of von Willebrand disease characterized by the abnormal interaction between FVIII and a defective vWf. To identify the molecular basis of this abnormal interaction, we isolated platelet RNA from members of one of the affected families and determined the nucleotide sequence of the FVIII-binding domain encoded by the vWf mRNA. A single G to A transition at nucleotide 2561 was linked with disease expression and results in the substitution of Gln for Arg91 in mature vWf. A restriction fragment containing this mutation was introduced into a full-length vWf expression vector, and both wild type and mutant vWf were expressed in COS-7 cells. In a solid-phase binding assay, expressed vWf was captured with anti-vWf monoclonal antibody AVW1 and then incubated with 6.25-400 milliunits of recombinant FVIII. After washing, vWf-bound FVIII activity was determined with a chromogenic assay. Mutant vWf showed reduced binding of FVIII compared with wild type, suggesting that the substitution of Gln for Arg91 is the likely basis for the abnormal vWf/FVIII interaction in this von Willebrand disease variant.     

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 microM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) could not replace GTP but prevented the action of GTP. The effects of GTP and GTP gamma S were reversible. Neither GTP nor GTP gamma S induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 microM free Ca2+, a half-maximal Ca2+ no Ca2+ release was observed with 0.1 microM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 microM) were required to evoke Ca2+ release. At 8 microM free Ca2+, even 0.25 microM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 microM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.     

Drought consistently alters the composition of soil fungal and bacterial communities in grasslands from two continents

The effects of short‐term drought on soil microbial communities remain largely unexplored, particularly at large scales and under field conditions. We used seven experimental sites from two continents (North America and Australia) to evaluate the impacts of imposed extreme drought on the abundance, community composition, richness, and function of soil bacterial and fungal communities. The sites encompassed different grassland ecosystems spanning a wide range of climatic and soil properties. Drought significantly altered the community composition of soil bacteria and, to a lesser extent, fungi in grasslands from two continents. The magnitude of the fungal community change was directly proportional to the precipitation gradient. This greater fungal sensitivity to drought at more mesic sites contrasts with the generally observed pattern of greater drought sensitivity of plant communities in more arid grasslands, suggesting that plant and microbial communities may respond differently along precipitation gradients. Actinobateria, and Chloroflexi, bacterial phyla typically dominant in dry environments, increased their relative abundance in response to drought, whereas Glomeromycetes, a fungal class regarded as widely symbiotic, decreased in relative abundance. The response of Chlamydiae and Tenericutes, two phyla of mostly pathogenic species, decreased and increased along the precipitation gradient, respectively. Soil enzyme activity consistently increased under drought, a response that was attributed to drought‐induced changes in microbial community structure rather than to changes in abundance and diversity. Our results provide evidence that drought has a widespread effect on the assembly of microbial communities, one of the major drivers of soil function in terrestrial ecosystems. Such responses may have important implications for the provision of key ecosystem services, including nutrient cycling, and may result in the weakening of plant–microbial interactions and a greater incidence of certain soil‐borne diseases.     

Extrapolating Antifungal Animal Data to Humans—Is It Reliable?

This article aimed to review animal models of antifungals and identifies human literature to assess if the extrapolation of results is reliable. Animal studies have helped identify area under the concentration curve to minimum inhibitory concentration ratio targets for new drugs and formulations such as isavuconazole and delayed-release posaconazole that have translated to successful outcomes in humans. Models have also been influential in the identification of possible combination therapies for the treatment of aspergillosis, such as voriconazole and echinocandins. However, challenges are endured with animal models when it comes to replicating the pharmacokinetics of humans which has been exemplified with the newest itraconazole formulation. Additionally, animal models have displayed a survival benefit with the use of iron chelators and amphotericin for mucormycosis which was not demonstrated in humans. Animal models have been a staple in the development and optimization of antifungal agents. They afford the ability to investigate uncommon diseases, such as invasive fungal infections, that would otherwise take years and many resources to complete. Although there are many benefits of animal models, there are also shortcomings. This is why the reliability of extrapolating data from animal models to humans is often scrutinized.     

Disruption of calcineurin catalytic subunit (cnaA) in Epichloë festucae induces symbiotic defects and intrahyphal hyphae formation

Calcineurin is a conserved calcium/calmodulin‐dependent protein phosphatase, consisting of a catalytic subunit A and a regulatory subunit B, which is involved in calcium‐dependent signalling and regulation of various important cellular processes. In this study, we functionally characterized the catalytic subunit A (CnaA) of the endophytic fungus Epichloë festucae which forms a symbiotic association with the grass host Lolium perenne. We deleted the CnaA‐encoding gene cnaA in E. festucae and examined its role in hyphal growth, cell wall integrity and symbiosis. This ΔcnaA strain had a severe growth defect with loss of radial growth and hyper‐branched hyphae. Transmission electron microscopy and confocal microscopy analysis of the mutant revealed cell wall defects, aberrant septation and the formation of intrahyphal hyphae, both in culture and in planta. The mutant strain also showed a reduced infection rate in planta. The fluorescence of mutant hyphae stained with WGA‐AF488 was reduced, indicating reduced chitin accessibility. Together, these results show that E. festucae CnaA is required for fungal growth, maintaining cell wall integrity and host colonization.     

Lymphoma models for B-cell activation and tolerance. II. Growth inhibition by anti-mu of WEHI-231 and the selection and properties of resistant mutants

Regulation of the growth of murine B-cell lymphomas has been used as a model for tolerance induction. The inhibition by anti-immunoglobulin reagents of the growth of WEHI-231 and several variant clones has now been studied. The parental line is exquisitely sensitive to growth inhibition by heterologous or monoclonal anti-mu or anti-k reagents and ceases to incorporate thymidine within 24-48 hr of exposure to anti-immunoglobulin reagents. Growth inhibition is initially reversible, but prolonged exposure to anti-mu results in cell death. This inhibition is specific for immunoglobulin light and heavy chains since growth is not inhibited by antibodies directed at either class I or class II histocompatibility antigens. In order to study the mechanism of growth inhibition, we have mutagenized WEHI-231 with ethylmethane sulfonate and cloned the surviving colonies in the presence of anti-mu. Such variants, which have been repeatedly recloned, are able to grow normally in the presence of anti-mu up to 100 micrograms/ml. These "resistant" clones, while expressing amounts of surface IgM similar to that observed on WEHI-231, do not differ markedly in their ability to cap their immunoglobulin receptors compared to the parental line but appear to have lost class II antigens. Cell cycle analysis revealed that anti-mu causes a block in the transition of WEHI-231 from G1 to S phase. The relevance of these processes to models of B-cell tolerance induction are discussed.     

Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km^r callus developing on hypocotyl sections. The totipotency of the Km^r callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km^r tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km^r callus and regenerated shoots.Abbreviations npt-II neomycin phosphotransferase II gene - NPT-II neomycin phosphotransferase II - nos nopaline synthase gene promoter - Km^r kanamycin resistant - BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MSD4×2 medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987)     

Artificial selection on sexual aggression: Correlated traits and possible trade-offs

Forced copulation is an extreme form of sexual aggression that can affect the evolution of sex-specific anatomy, morphology, and behavior. To characterize mechanistic and evolutionary aspects of forced copulation, we artificially selected male fruit flies based on their ability to succeed in the naturally prevalent behavior of forced matings with newly eclosed (teneral) females. The low and high forced copulation lineages showed rapid divergence, with the high lineages ultimately showing twice the rates of forced copulation as the low lineages. While males from the high lineages spent more time aggressively pursuing and mounting teneral females, their behavior toward non-teneral and heterospecific females was similar to that of males from the low lineages. Males from the low and high lineages also showed similar levels of male-male aggression. This suggests little or no genetic correlations between sexual aggression and non-aggressive pursuit of females, and between male aggression toward females and males. Surprisingly however, males from the high lineages had twice as high mating success than males from the low lineages when allowed to compete for consensual mating with mature females. In further experiments, we found no evidence for trade-offs associated with high forced mating rates: males from the high lineages did not have lower longevity than males from the low lineages when housed with females, and four generations of relaxed selection did not lead to convergence in forced mating rates. Our data indicate complex interactions among forced copulation success and consensual mating behavior, which we hope to clarify in future genomic work.