Coinfection of SCID-hu Thy/Liv mice with human herpesvirus 6 and human immunodeficiency virus type 1

Human herpesvirus 6 (HHV-6) has been proposed as a potential cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) disease. We used the SCID-hu Thy/Liv mouse model to evaluate the in vivo interactions between HHV-6 and HIV-1. Our results demonstrate that HHV-6 and HIV-1 can simultaneously replicate in the human thymus in vivo. In this model, however, the presence of one virus appears not to modify the replication or cytopathicity of the other.     

Long-term response of the biotic community to fluctuating water levels and changes in water quality in Cootes Paradise Marsh,a degraded coastal wetland of Lake Ontario

During the early 1900s, more than 90% of the surface area of Cootes Paradise Marsh was covered with emergent vegetation; currently, less than 15% of the surface is covered with aquatic vegetation and the remainder is wind-swept, turbid, open water. The loss of emergent cover is significantly correlated with mean annual water levels that increased more than 1.5 m over the past 60 years. Species diversity and the percent cover of the submerged macrophtye community also declined dramatically after the 1940s, coincident with decreased water clarity and increased nutrients from pollution by sewage and stormwater effluent. Phosphorus levels in the marsh dropped ten-fold after the sewage plant was upgraded to a tertiary-treatment facility in 1978; however, there was no measurable improvement in water clarity, in spite of a decrease in chlorophyll concentrations. Long-term changes in the composition of the planktonic, benthic and fish communities accompanied changes in water clarity, nutrient status and macrophyte cover. Phytoplankton changed from a community dominated by diverse taxa of green algae and diatoms during the 1940s, to a less diverse community dominated by a few taxa of green and blue-green algae in the 1970s, then to a much more diverse community recently, including many taxa of green algae, diatoms and chrysophytes; however, because water turbidity continues to be high, and algae tolerant of low light levels are now very abundant. Daphnia, which were prominent during the 1940s (especially in the vegetated sites) were replaced in the 1970s by smaller zooplankton such as the cladoceran, Bosmina, and several rotifer species including Brachionus, Asplanchna and Keratella. In the recent survey conducted in 1993 and 1994, small-bodied forms still dominate the turbid open-water areas, while medium-sized cladocerans such as Moina were common near macrophyte beds. Generally, total herbivorous zooplankton biomass tended to be highest next to Typha beds and declined with increasing distance from the plants. Conversely, biomass of edible algae at these sites increased with distance from the macrophytes. Species diversity of aquatic insects declined dramatically over the past 40 years, from 57 genera (23 families and 6 orders) in 1948, to 9 genera (6 families and 3 orders) in 1978, to only 5 genera (3 families and 2 orders) in 1995. The diverse benthic community present 5 decades ago has now been replaced by a community consisting primarily of chironomid larvae, oligochaetes and other worms associated with low-oxygen environments. These successional changes illustrate the impact of natural (fluctuating water levels) and anthropogenic (deterioration in water quality) stressors on the character of the biotic communities, and reveal the complex interactions among the various trophic levels and the abiotic environment as degradation and remediation proceeded.     

Spatial Organization of EphA2 at the Cell-Cell Interface Modulates Trans-Endocytosis of EphrinA1

EphA2 is a receptor tyrosine kinase (RTK) that is sensitive to spatial and mechanical aspects of the cell’s microenvironment. Misregulation of EphA2 occurs in many aggressive cancers. Although its juxtacrine signaling geometry (EphA2’s cognate ligand ephrinA1 is expressed on the surface of an apposing cell) provides a mechanism by which the receptor may experience extracellular forces, this also renders the system challenging to decode. By depositing living cells on synthetic supported lipid membranes displaying ephrinA1, we have reconstituted key features of the juxtacrine EphA2-ephrinA1 signaling system while maintaining the ability to perturb the spatial and mechanical properties of the membrane-cell interface with precision. In addition, we developed a trans-endocytosis assay to monitor internalization of ephrinA1 from a supported membrane into the apposing cell using a quantitative three-dimensional fluorescence microscopy assay. Using this experimental platform to mimic a cell-cell junction, we found that the signaling complex is not efficiently internalized when lateral reorganization at the membrane-cell contact sites is physically hindered. This suggests that EphA2-ephrinA1 trans-endocytosis is sensitive to the mechanical properties of a cell’s microenvironment and may have implications in physical aspects of tumor biology.     

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.     

Novel substituted pyrrolidines are high affinity histamine H3 receptor antagonists

Pre-clinical characterization of novel substituted pyrrolidines that are high affinity histamine H₃ receptor antagonists is described. These compounds efficiently penetrate the CNS and occupy the histamine H₃ receptor in rat brain following oral administration. One compound, (2S,4R)-1-[2-(4-cyclobutyl-[1,4]diazepane-1-carbonyl)-4-(3-fluoro-phenoxy)-pyrrolidin-1-yl]-ethanone, was extensively profiled and shows promise as a potential clinical candidate.     

C-terminal tail phosphorylation of N-formyl peptide receptor: differential recognition of two neutrophil chemoattractant receptors by monoclonal antibodies NFPR1 and NFPR2

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni(2+)-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45-75 kDa species (peak M(r) approximately 60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of M(r) approximately 40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting approximately 60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337-346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.     

Childhood and adolescent depression: why do children and adults respond differently to antidepressant drugs?

Childhood and adolescent depression is an increasingly problematic diagnosis for young people due to a lack of effective treatments for this age group. The symptoms of adult depression can be treated effectively with multiple classes of antidepressant drugs which have been developed over the years using animal and human studies. But many of the antidepressants used to treat adult depression cannot be used for pediatric depression because of a lack of efficacy and/or side effects. The reason that children and adolescents respond differently to antidepressant treatment than adults is poorly understood. In order to better understand the etiology of pediatric depression and treatments that are effective for this age group, the differences between adults, children and adolescents needed to be elucidated. Much of the understanding of adult depression has come from studies using adult animals, therefore studies using juvenile animals would likely help us to better understand childhood and adolescent depression. Recent studies have shown both neurochemical and behavioral differences between adult and juvenile animals after antidepressant treatment. Juvenile animals have differences compared to adult animals in the maturation of the serotonergic and noradrenergic systems, and in dose of antidepressant drug needed to achieve similar brain levels. Differences after administration of antidepressant drug have also been reported for adrenergic receptor regulation, a physiologic hypothermic response, as well as behavioral differences in two animal models of depression. The differences between adults and juveniles not only in the human response to antidepressants but also with animals studies warrant a specific distinction between the study of pediatric and adult depression and the manner in which new treatments are pursued.     

Probing the gamma2 calcium-binding site: studies with gammaD298,301A fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site

To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the fibrinogen variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal fibrinogen. Notably, the influence of calcium on polymerization was the same for the variant and for normal fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.