Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.     

Starch and the Control of Kernel Number in Maize at Low Water Potentials

After reproduction is initiated in plants, subsequent reproductive development is sometimes interrupted, which decreases the final number of seeds and fruits. We subjected maize (Zea mays L.) to low water potentials (psi(w)) that frequently cause this kind of failure. We observed metabolite pools and enzyme activities in the developing ovaries while we manipulated the sugar stream by feeding sucrose (Suc) to the stems. Low psi(w) imposed for 5 d around pollination allowed embryos to form, but abortion occurred and kernel number decreased markedly. The ovary contained starch that nearly disappeared during this abortion. Analyses showed that all of the intermediates in starch synthesis were depleted. However, when labeled Suc was fed to the stems, label arrived at the ovaries. Solute accumulated and caused osmotic adjustment. Suc accumulated, but other intermediates did not, showing that a partial block in starch synthesis occurred at the first step in Suc utilization. This step was mediated by invertase, which had low activity. Because of the block, Suc feeding only partially prevented starch disappearance and abortion. These results indicate that young embryos abort when the sugar stream is interrupted sufficiently to deplete starch during early ovary development, and this abortion results in a loss of mature seeds and fruits. At low psi(w), maintaining the sugar stream partially prevented the abortion, but invertase regulated the synthesis of ovary starch and partially prevented full recovery.     

Regulation of PKR by HCV IRES RNA: Importance of Domain II and NS5A

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of ∼ 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.     

Colorimetric plasma assay for the bentiromide test (BT-PABA) for exocrine pancreatic insufficiency

Bentiromide is a synthetic peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid, which has been used as a test for exocrine pancreatic function. Following oral administration, bentiromide is hydrolyzed by chymotrypsin to yield free p-aminobenzoic acid (PABA) which is absorbed, conjugated and excreted in the urine. The PABA conjugates reach their peak levels in blood in 90-120 min. Healthy individuals have higher levels of PABA than patients with pancreatic insufficiency. A simple, accurate, and precise method for the determination of PABA in blood has been developed and validated. The plasma (1 ml) is deproteinized by perchloric acid. The conjugates are hydrolyzed and the total PABA is determined colorimetrically by the Bratton-Marshall test. The standard curve in plasma is linear up to 8 micrograms/ml of PABA. A similar semimicro method using 200 microliter of plasma suitable for pediatric samples shows comparable results. Average analytical recovery is 97% and precision studies of pooled within-run and total between-run showed CV% of 5.0 and 5.7%, respectively.     

Evidence of Habitat Structuring Aedes albopictus Populations in Réunion Island

Arbovirus vector dynamics and spread are influenced by climatic, environmental and geographic factors. Major Chikungunya and Dengue fever outbreaks occurring the last 10 years have coincided with the expansion of the mosquito vector Aedes albopictus to nearly all the continents. We characterized the ecological (larval development sites, population dynamics, insemination and daily survival rates) and genetic (diversity, gene flow, population structure) features of two Aedes albopictus populations from distinct environments (rural and urban) on Réunion Island, in the South-West Indian Ocean. Microsatellite analysis suggests population sub-structuring Ae. albopictus populations. Two genetic clusters were identified that were significantly linked to natural versus urban habitats with a mixed population in both areas. Ae. albopictus individuals prefer urban areas for mating and immature development, where hosts and containers that serve as larval development sites are readily available and support high population densities, whereas natural environments appear to serve as reservoirs for the mosquito.     

Long-term trends and causal factors associated with Microcystis abundance and toxicity in San Francisco Estuary and implications for climate change impacts

The impacts of climate change on Microcystis blooms in San Francisco Estuary are uncertain because factors associated with the abundance and distribution of Microcystis blooms since their inception in 1999 are poorly understood. Discrete and continuous data collected between 2004 and 2008 were used to assess what factors controlled bloom initiation and persistence, if there was an impact of the bloom on mesozooplankton abundance and toxicity or dissolved organic carbon concentration, and how these might vary with climate change. Microcystis abundance was greater in dry years than wet years and both total microcystins concentration and the microcystins content of mesozooplankton tissue increased with abundance. The bloom began in the upstream portions of the estuary and spread farther west during dry years. Bloom initiation required water temperature above 19°C and surface irradiance in the visible range above 100 W m^?2. The bloom persisted during a wide range of water quality conditions but was closely correlated with low turbidity. The intensity of Microcystis blooms will likely increase with climate change due to increased water temperature and low streamflow during droughts. Elevated water temperature earlier in the spring could also extend the duration of Microcystis blooms by up to 3 months.     

Forest nitrogen sinks in large eastern U.S. watersheds: estimates from forest inventory and an ecosystem model

The eastern U.S. receives elevated rates of Ndeposition compared to preindustrial times, yetrelatively little of this N is exported indrainage waters. Net uptake of N into forestbiomass and soils could account for asubstantial portion of the difference between Ndeposition and solution exports. We quantifiedforest N sinks in biomass accumulation andharvest export for 16 large river basins in theeastern U.S. with two separate approaches: (1)using growth data from the USDA ForestService's Forest Inventory and Analysis (FIA)program, and (2) using a model of forestnitrogen cycling (PnET-CN) linked to FIAinformation on forest age-class structure. Themodel was also used to quantify N sinks in soiland dead wood, and nitrate losses below therooting zone. Both methods agreed that netgrowth rates were highest in the relativelyyoung forests on the Schuylkill watershed, andlowest in the cool forests of northern Maine. Across the 16 watersheds, wood export removedan average of 2.7 kg N ha^–1 yr^–1(range: 1–5 kg N ha^–1 yr^–1), andstanding stocks increased by 4.0 kg N ha^–1yr^–1 (–3 to 8 kg N ha^–1 yr^–1). Together, these sinks for N in woody biomassamounted to a mean of 6.7 kg N ha^–1yr^–1 (2–9 kg N ha^–1 yr^–1), or73% (15–115%) of atmospheric N deposition. Modeled rates of net N sinks in dead wood andsoil were small; soils were only a significantnet sink for N during simulations ofreforestation of degraded agricultural sites. Predicted losses of nitrate depended on thecombined effects of N deposition, and bothshort- and long-term effects of disturbance. Linking the model with forest inventoryinformation on age-class structure provided auseful step toward incorporating realisticpatterns of forest disturbance status acrossthe landscape.     

Molecular Characterization of Potential Microcystin-Producing Cyanobacteria in Lake Ontario Embayments and Nearshore Waters

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 μg MC-LR_eq liter⁻¹. MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns.     

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34(+) cells and megakaryocyte progenitors in culture

Background aimsExpansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia.MethodsWe measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34⁺ cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design.ResultsThese results revealed an elaborate array of cytokine individual effects complemented by a large number of synergistic and antagonistic interaction effects. Notably, strong interactions with SCF were observed with most cytokines and its concentration level was the most influential factor for the expansion and differentiation kinetics of CB CD34⁺ cells. A response surface methodology was then applied to optimize the concentrations of the selected cytokines. The newly developed cocktail composed of SCF, thrombopoietin (TPO) and FL increased the expansion of Mk progenitors and maintained efficient expansion of clonogenic progenitors and CD34⁺ cells. CB cells expanded with the new cocktail were shown to provide good short- and long-term human platelet recovery and lymphomyeloid reconstitution in NOD/SCID mice.ConclusionsCollectively, these results define a complex cytokine network that regulates the growth and differentiation of immature and committed hematopoietic cells in culture, and confirm that cytokine interactions have major influences on the fate of hematopoietic cells.