MIXOTROPHY AND NITROGEN UPTAKE BY PFIESTERIA PISCICIDA (DINOPHYCEAE)

The nutritional versatility of dinoflagellates is a complicating factor in identifying potential links between nutrient enrichment and the proliferation of harmful algal blooms. For example, although dinoflagellates associated with harmful algal blooms (e.g. red tides) are generally considered to be phototrophic and use inorganic nutrients such as nitrate or phosphate, many of these species also have pronounced heterotrophic capabilities either as osmotrophs or phagotrophs. Recently, the widespread occurrence of the heterotrophic toxic dinoflagellate, Pfiesteria piscicida Steidinger et Burkholder, has been documented in turbid estuarine waters. Pfiesteria piscicida has a relatively proficient grazing ability, but also has an ability to function as a phototroph by acquiring chloroplasts from algal prey, a process termed kleptoplastidy. We tested the ability of kleptoplastidic P. piscicida to take up ¹⁵N-labeled NH     , NO     , urea, or glutamate. The photosynthetic activity of these cultures was verified, in part, by use of the fluorochrome, primulin, which indicated a positive relationship between photosynthetic starch production and growth irradiance. All four N substrates were taken up by P. piscicida , and the highest uptake rates were in the range cited for phytoplankton and were similar to N uptake estimates for phagotrophic P. piscicida . The demonstration of direct nutrient acquisition by kleptoplastidic P. piscicida suggests that the response of the dinoflagellate to nutrient enrichment is complex, and that the specific pathway of nutrient stimulation (e.g. indirect stimulation through enhancement of phytoplankton prey abundance vs. direct stimulation by saprotrophic nutrient uptake) may depend on P. piscicida 's nutritional state (phagotrophy vs. phototrophy).     

Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation

This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.     

Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y₁ purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.     

Anti-transforming growth factor ß antibody treatment rescues bone loss and prevents breast cancer metastasis to bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p?=?0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p?=?0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.     

Anti-Transforming Growth Factor ? Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.     

Regulation of oxygen delivery during induced polycythemia in exercising dogs

Previous studies have concluded that polycythemia decreases oxygen delivery primarily because of a large fall in cardiac output associated with a rise in systemic vascular resistance that has been attributed to increased blood viscosity. However, because other studies have shown that polycythemia may not reduce oxygen delivery, an alternative hypothesis is that cardiac output falls in response to a rising oxygen content, thereby maintaining oxygen delivery constant. To determine whether oxygen content participates in the regulation of cardiac output during polycythemia, we studied eight chronically instrumented dogs trained to exercise on a treadmill. The dogs underwent exchange transfusion with packed red blood cells containing methemoglobin, which caused an increase in hematocrit from 35 +/- 1 to 50 +/- 1% and in viscosity, with little change in oxygen content. The expected fall in exercise cardiac output failed to occur after exchange transfusion with red blood cells containing methemoglobin (7.5 +/- 4 vs. 6.8 +/- 0.5 l/min; P = not significant), and there was no rise in systemic vascular resistance. Methylene blue was then administered intravenously to facilitate conversion of methemoglobin to oxyhemoglobin, which increased oxygen content (12.8 +/- 0.9 vs. 18.4 +/- 0.9 vol%; P < 0.01) with no change in hematocrit or viscosity. Resting cardiac output did not change significantly, but there was a significant decrease in exercise output (6.8 +/- 0.5 vs. 5.8 +/- 0.4 l/min; P < 0.05). Thus we conclude that the fall in cardiac output seen in acute polycythemia results in part from the regulation of oxygen delivery and is not due solely to increased blood viscosity.     

Studies on two strains of Plasmodium cynomolgi in New World and Old World monkeys and mosquitoes

Infections that cause the Gombak and Smithsonian strains of Plasmodium cynomolgi were induced in Macaca mulatta, Aotus lemurinus griseimembra, Aotus nancymai, and Saimiri boliviensis monkeys. Transmission of the Gombak strain to Aotus spp. monkeys was obtained by the injection of sporozoites dissected from the salivary glands of experimentally infected Anopheles dirus and by the bites of infected An. dirus and Anopheles farauti mosquitoes. Two S. boliviensis monkeys were infected via the injection of sporozoites dissected from An. dirus. Prepatent periods in New World monkeys ranged from 14 to 44 days, with a median of 18 days. The Smithsonian strain was transmitted via sporozoites to 1 A. lemurinus griseimembra and 9 A. nancymai monkeys. Prepatent periods ranged from 12 to 31 days.     

Observations on the Vietnam Palo Alto strain of Plasmodium vivax in two species of Aotus monkeys

Thirty-three splenectomized Aotus lemurinus griseimembra monkeys with no previous experience with malaria were infected with the Vietnam Palo Alto strain of Plasmodium vivax. The median maximum parasite count was 280,000/microl. Nine splenectomized monkeys with previous infection with Plasmodium falciparum had median maximum parasite counts of 120,000/microl. Splenectomized Aotus nancymai monkeys supported infections at a lower level. Transmission via the bites of Anopheles dirus mosquitoes was obtained in a splenectomized A. lemurinus griseimembra, with a prepatent period of 31 days. It is estimated that between 1.5 x 10(8) and 1.6 x 10(9) parasites can be removed from an infected animal for molecular or diagnostic antigenic studies.