Passive hemolytic plaque assay for detecting antibody-producing cells in fish
No abstract available at this time
Book chapter↗
SEARCH · USGS Science
Results for “Cells”
Search indexed USGS publications on groundwater, aquifers, geologic maps, mineral resources and earthquakes. Explore source records by subject and place.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Reduction in the numbers of antibody-producing cells in rainbow trout, Oncorhynchus mykiss, exposed to sublethal doses of phenol before bath immunization
No abstract available at this time
Book chapter↗
Hormonal control of gill Na+, K+ and -ATPase and chloride cell function
No abstract available at this time
Book chapter↗
Costimulatory receptors in a teleost fish: Typical CD28, elusive CTLA4
T cell activation requires both specific recognition of the peptide-MHC complex by the TCR and additional signals delivered by costimulatory receptors. We have identified rainbow trout sequences similar to CD28 (rbtCD28) and CTLA4 (rbtCTLA4). rbtCD28 and rbtCTLA4 are composed of an extracellular Ig-superfamily V domain, a transmembrane region, and a cytoplasmic tail. The presence of a conserved ligand binding site within the V domain of both molecules suggests that these receptors likely recognize the fish homologues of the B7 family. The mRNA expression pattern of rbtCD28 and rbtCTLA4 in naive trout is reminiscent to that reported in humans and mice, because rbtCTLA4 expression within trout leukocytes was quickly up-regulated following PHA stimulation and virus infection. The cytoplasmic tail of rbtCD28 possesses a typical motif that is conserved in mammalian costimulatory receptors for signaling purposes. A chimeric receptor made of the extracellular domain of human CD28 fused to the cytoplasmic tail of rbtCD28 promoted TCR-induced IL-2 production in a human T cell line, indicating that rbtCD28 is indeed a positive costimulator. The cytoplasmic tail of rbtCTLA4 lacked obvious signaling motifs and accordingly failed to signal when fused to the huCD28 extracellular domain. Interestingly, rbtCTLA4 and rbtCD28 are not positioned on the same chromosome and thus do not belong to a unique costimulatory cluster as in mammals. Finally, our results raise questions about the origin and evolution of positive and negative costimulation in vertebrate immune systems. T cell activation is initiated through complex cell-to-cell interactions. TCRs expressed on the surface of T cells first recognize antigenic peptides presented by MHC molecules on the surface of APCs. TCRs and other surface receptors and ligands stabilize the contact between T cells and APCs, which triggers signal transduction pathways resulting in T cell activation. According to the two-signal model ( 1 , 2 ), T cell activation by Ag requires both specific recognition of the peptide by the TCR (signal 1) and additional signals delivered by other costimulatory receptors (signal 2). Among the known costimulatory receptors, CD28 and CTLA4 (also known as CD152), which are expressed on T cells and bind their corresponding ligands B7-1/B7-2 (CD80/CD86) on APCs, represent a well-studied system in mammals ( 3 ). CD28 is a glycosylated homodimeric protein expressed on the surface of double-positive thymocytes, mature CD4 + T cells, CD11b − CD8 + T cells, and γδ CD3 + T cells ( 4 , 5 ). CD28 is expressed at higher levels on activated T cells than on resting cells ( 6 ). In mammals, CD28 binds to B7-1 and B7-2, which are expressed on the surface of APCs, and delivers a critical costimulatory signal to T lymphocytes. In the absence of CD28 ligation, TCR binding either induces apoptosis or anergy in T cells ( 5 ). Engagement of CD28 alone cannot activate T cells, even if synergetic effects are provided via stimulation by T cell mitogens or anti-CD3 treatment ( 7 ). A tyrosine-based motif in cytoplasmic tail of CD28 functions as a binding site for the p85 PI3K subunit when it is phosphorylated ( 8 , 9 ). Other CD28 intracytoplasmic motifs, which associates with IL-2-inducible tyrosine kinase, lymphocyte-specific tyrosine kinase, and the adaptor growth-factor receptor bound protein-2 are responsible for signal transduction ( 10 , 11 , 12 ). The binding of growth-factor receptor bound protein-2 to CD28 also activates the GTPase RAS ( 13 ). CD28 signaling is also thought to contribute to the mobilization of lipid rafts to the immunological synapse ( 14 ), the region of contact between T cell and APC, which lowers the overall threshold of TCR engagement required for effective cytokine production and proliferation ( 15 ). In contrast, CTLA4 is a powerful negative regulator of T cell activation and was first cloned via differential screening of a cytotoxic T cell cDNA library ( 16 ). CD28 and CTLA4 belong to the same family and share high sequence similarity to each other ( 16 , 17 ). Both receptors interact with B7-1 and/or B7-2 but induce different signals: CD28 amplifies signaling triggered by the TCR-CD3 complex, whereas CTLA4 generates negative signals that inhibit T cell activation ( 18 ). CTLA4 has higher affinity for B7-1 and B7-2 compared with CD28, and its expression is also induced by TCR engagement ( 19 ). The mechanisms of CTLA4-mediated suppression likely involve both competition with CD28 for B7-1/B7-2 binding and potent inhibitory signals delivered by CTLA4 ( 20 ). The inhibitory signaling mechanisms triggered by CTLA4 are not clear. The CTLA4 cytoplasmic tail lacks typical ITIM motifs and shares with CD28 a p85 binding site ( 21 ). Thus, the extent of an immune response is likely controlled through the finely tuned expression of costimulatory receptors from the CD28/CTLA4 family on the surface of activated T cells ( 22 , 23 ). Very little is known about T cell responses and activation in bony fish. TCR-αβ cDNAs ( 24 , 25 , 26 , 27 ) and polymorphic class IA and class IIA/B MHC molecules ( 28 , 29 , 30 ) have been reported in several species, suggesting that the fish TCR recognizes antigenic peptides presented by MHC molecules similar to that of mammals. T cell-mediated responses in fish are supported by several lines of evidence. Allograft rejection provided the first experimental indications suggesting that bony fish possess a functional T cell-mediated immune response ( 31 , 32 ). In vitro assays for allospecific cytotoxicity have also been established using clonal and nonclonal catfish cell lines ( 33 , 34 ) or clonal rainbow trout ( 35 ). Autologous cell-mediated specific lysis of virus-infected syngenic target cells has also been reported in cloned goldfish ( 36 , 37 ). Finally, both public and private T cell-specific expansions have been observed in rainbow trout during secondary immune responses to viral hemorrhagic septicemia virus (VHSV), 3 a fish rhabdovirus, using TCR-β CDR3-length spectratyping ( 38 ). TCR-MHC-peptide interactions induce signaling events through CD3 in mammals and most likely in lower vertebrates because a complete set of CD3 genes, including TCR ζ, CD3 ε, and CD3 γδ, has been found in Pufferfish and Xenopus ( 39 , 40 ). CD8α has been identified in rainbow trout ( 41 ); however, the cytoplasmic tail lacks the consensus p56 lck motif, suggesting that TCR/CD3/CD8-mediated signaling events may be different in teleosts. In this study, we have identified and initially characterized two members of the CD28 family in rainbow trout, rbtCD28 and rbtCTLA4. Their sequence features and expression patterns suggest that they are the likely homologues of mammalian CD28 and CTLA4. The potential costimulatory capacities of these fish receptors were investigated using a human T cell line expressing chimeric receptors composed of the extracellular domain of human CD28 (hCD28) fused to the cytoplasmic tail of rbtCD28 or rbtCTLA4. The chimeric hCD28-rbtCD28 receptor mediated enhanced TCR-induced IL-2 production, suggesting a costimulatory function for rbtCD28. In contrast, the divergent cytoplasmic tail from rbtCTLA4 did not mediate similar signaling activities. This study therefore provides the first characterization of costimulatory receptors in lower vertebrates.
Journal of Immunology↗
Mercury on a landscape scale—Balancing regional export with wildlife health
The Cosumnes River watershed requires a 57–64 percent reduction in loads to meet the new Delta methylmercury (MeHg) total maximum daily load allocation, established by the Central Valley Regional Water Quality Control Board. Because there are no large point sources of MeHg in the watershed, the focus of MeHg load reductions will fall upon non-point sources, particularly the expansive wetlands considered to be a primary source of MeHg in the region. Few management practices have been implemented and tested in order to meet load reductions in managed wetlands, but recent efforts have shown promise. This project examines a treatment approach to reduce MeHg loads to the Sacramento-San Joaquin River Delta by creating open-water deep cells with a small footprint at the downstream end of wetlands to promote net demethylation of MeHg and to minimize MeHg and Hg loads exiting wetlands at the Cosumnes River Preserve. Specifically, the deep cells were were located immediately up gradient of the wetland’s outflow weir and were deep enough (75–91 centimeter depth) to be vegetation-free. The topographic and hydrologic structure of each treatment wetland was modified to include open-water deep cells so that the removal of aqueous MeHg might be enhanced through (1) particle settling, (2) photo-degradation, and (3) benthic microbial demethylation. These deep cells were, therefore, expected to clean MeHg from surface water prior to its discharge to the Cosumnes River and the downstream Delta. Our goal was to test whether the implementation of the deep cells within wetlands would minimize MeHg and total Hg export. Further, we sought to test whether continuous flow-through hydrology, would lower MeHg concentrations in resident biota, compared to traditional wetland management operations. The dominant practice in seasonal wetlands management is the “fill-and-maintain” approach, in which wetlands are filled with water and the water levels maintained without substantial draining until drawdown. Our approach was to create and characterize replicate treatment wetland complexes, in conjunction with monitoring of hydrologic, biologic, and chemical indicators of MeHg exposure for two full annual cycles within winter-spring flooded seasonal wetlands. In addition to the creation of deep cells within treatment wetlands, hydrology was manipulated so that there was a constant flow-through of water, while the control wetlands utilized the fill-and-maintain approach. Specifically, the treatment wetlands were maintained in a flow-through manner, while the control wetlands were maintained in a fill-and-maintain manner from September through May, to test the hypothesis that the flow of water through the seasonal wetland can lower fish bioaccumulation through dilution of MeHg-concentrated water within the wetland by constant inflows of water into the wetland. The major tasks of this study included: (1) field design and implementation, (2) water and wetland management, (3) hydrologic monitoring and water quality sampling, (4) MeHg export and load estimates, (5) caged fish experiments for examining MeHg bioaccumulation, (6) site and process characterization to improve understanding and transferability of results, (7) adaptive management, transferability, and outreach, and (8) reporting of results and conclusions. This report summarizes the key findings of this study, which focuses on MeHg load estimates from control and treatment wetlands, quantification of three MeHg removal mechanisms (particulate settling, benthic demethylation, and photo-demethylation) in the deep cells within the treatment wetlands, and MeHg bioaccumulation in wetland fishes. Key findings include: Over two years of study, mean whole-water MeHg load decreased 37 percent in deep cells, when comparing inlet of check weir flows to outlet. Of the 37 percent MeHg load removed within the deep cell, photodegradation accounted for 7 percent and particle flux to the benthos accounted for 24 percent of the mass removed, with the remaining 6 percent apparent MeHg loss unexplained. Benthic MeHg degradation did not appear to be a major MeHg removal process in the deep cells, as changes in the ambient MeHg pool over 7-day bottle incubations showed that the surface sediment exhibited net MeHg production in the majority (87 percent) of incubation experiments. In only 13 percent of the incubations (3 out of 24) was net MeHg degradation observed. Estimates of benthic diffusive flux of MeHg across the sediment/water interface were small relative to particulate flux and variable (positive or negative), suggesting this is likely a minor term in the overall MeHg budget within the deep cells. Although the deep cells served as net MeHg sink overall, MeHg export from the flow-through treatment wetlands (shallow and deep combined) exceeded export from the fill-and-maintain managed control wetlands, because of the differences in hydrologic management between the two wetland types. Shallow wetlands under flow-through conditions generated a net export of MeHg. Most of the annual MeHg export from the treatment wetlands occurred within the first 3 months of flood up (September to November), shortly after hydrologic management began. Despite the effectiveness of the deep cell in lowering MeHg export concentrations, total mercury (THg) concentration did not decrease in biosentinel fish ( Gambusia affinis , Mosquitofish) between the deep cell inlet and outlet. Mosquitofish THg concentrations were higher in treatment wetlands than in control wetlands during the first year of study, likely because of an associated increase in MeHg availability immediately following wetland construction activities. Mosquitofish THg concentrations declined in the treatment wetlands during the second year of study, and fish THg concentrations in treatment wetlands were no different from those in the control. Similarly, the increased hydrologic flow rates in the treatment wetlands did not lower fish THg concentrations nor aqueous MeHg concentrations in the shallow cells, suggesting that MeHg flux from the sediment to water column exceeded the flow-through flushing rate in the shallow portion of the treatment wetlands. Reductions in MeHg concentrations of surface water and fish may require higher flow rates than used in the study to achieve the region’s regulatory goals. However, the flow rates necessary may not be feasible for these managed wetlands because of limited water supply and the associated costs for water and pumping. The use of deep cells in seasonal wetlands were effective in lowering MeHg exports under continuous water flow-through hydrology. However, fill-and-maintain hydrology had lower exports overall, because of a single major drainage event at the end of the flood season. Future studies focused on limiting MeHg export should consider combining deep cells with the fill-and-maintain or fill-and-trickle hydrologic management approach.
California↗
Automated derivation of hydrologic basin characteristics from digital elevation model data
Digital elevation model (DEM) data in a raster format can be used to automatically derive the drainage characteristics of an area. A procedure has been designed that is capable of operating on matrices of elevation data having no algorithmically imposed size limit, while performing within the resolution and accuracy tolerances of the DEM data. Each cell is processed as the center of a 3- by 3-cell spatial window in the raster elevation data. If a cell is a local minimum in comparison with two of its non-adjacent neighbors, it is labeled as a drainage cell. The linkages of the drainage cells within user-specified distance and elevation thresholds are established in a separate process. The products of these processing steps are digital masks of the drainage cells and the watershed basins, both in raster format. A drainage cell mask derived using this procedure is useful in computing slope values for a raster data base. Slope has traditionally been calculated for each cell by fitting a plane through the eight nearest cells. However, if the terrain represented by these cells is V-shaped, such as a gully, a plane does not fit well; in fact, the desired slope value is the slope along the bottom of the gully, regardless of the steepness of the gully sides. The automated drainage process will label such a cell as a drainage cell, and its slope can then be computed from the elevation values of neighboring drainage cells.
Conference Paper↗
Simulated changes in water levels caused by potential changes in pumping from shallow aquifers of Virginia Beach, Virginia
A steady-state ground-water flow model of the southern watersheds of Virginia Beach, Virginia, was refined and used to simulate changes in aquifer water levels caused by potential changes in pumping in the Transition Area of Virginia Beach, Va., a 20-square mile planning zone that runs through the middle of the city. Cessation of dewatering at borrow pits, pumping to irrigate a golf course, pumping to irrigate lawns of a hypothetical neighborhood, and pumping to irrigate both the golf course and lawns of the hypothetical neighborhood were simulated. Simulated recoveries from cessation of dewatering of borrow pits were generally restricted to the immediate area of the pits. The simulated recoveries averaged about 20 feet (ft) near the center of the cells representing the active areas of the pits and 2 ft at the cells representing the extent of the pits. At a golf course, 4 hypothetical wells pumping 300,000 gallons per day (gal/d) from the Yorktown sand aquifer resulted in drawdowns averaging 10 ft in the pumping cells and 1 ft at a distance of 1.2 miles (mi) from the center of the pumping cells. The extent of the 1-ft drawdown was virtually the same as that simulated previously and reported in a permit application for the golf course. Simulated pumping of 150,000 gal/d from 4 cells in the confined sand aquifer representing a 40-acre neighborhood resulted in drawdowns averaging 7 ft in the pumping cells and 1 ft at a distance of 0.8 mi from the center of the cells. Simulated pumping of 300,000 gal/d from the same 4 cells resulted in drawdowns averaging 15 ft in the pumping cells and 1 ft at a distance of 1.4 mi from the center of the cells. Simulated pumping of 150,000 gal/d at the golf course and another 150,000 gal/d in the hypothetical neighborhood resulted in drawdowns that averaged 5 ft around the cells representing the golf course wells spaced 1,300 ft apart and 7 ft around the contiguous cells representing the 40-acre neighborhood. A drawdown of 1 ft encompassed most of the eastern half of the Transition Area.
Virginia↗
Clonal foraging in perennial wheatgrasses: A strategy for exploiting patchy soil nutrients
1. Foraging by means of plasticity in placement of tillers in response to low- and high-nutrient patches was examined in the rhizomatous wheatgrass Elymus lanceolatus ssp. lanceolatus . Its ability to exploit soil nutrient patches was compared to that of the closely related but caespitose E. lanceolatu s ssp. wawawaiensis . 2. Clones of 14 genets of each taxon were planted in boxes consisting of two 30 × 30 cm cells: the `origin cell' where clones were planted, and the adjacent `destination cell', with each cell containing soil with either low or high levels of nutrients. 3. The rhizomatous taxon, which can produce intravaginal, short-rhizome and long-rhizome tillers, preferentially produced short-rhizome and intravaginal tillers in high-nutrient destination cells. Effects of nutrient status of the origin cell as well as of the destination cell on total tiller numbers indicated clonal integration, yet tiller placement responded to local conditions. 4. Roots of both taxa accessed nutrients in destination cells (the caespitose subspecies by root growth only), and above-ground biomass of both taxa increased to a similar extent with high-nutrient destination cells. With the patch sizes used in this experiment, root growth was as important as ramet placement in exploiting nutrients in destination cells. 5 There was no relationship between degree of plasticity in ramet placement and biomass of the clone when high-nutrient destination cells were present.
Journal of Ecology↗
An evaluation of methods for measuring algal growth
Algal growth was expressed as milligrams dry weight per litre with use of the electronic particle counter for determining cell number and cell volume. A gravimetric constant in milligrams dry weight per cubic micrometre was determined. This constant was applicable throughout the growth curve since there was a linear relationship between total cell weight and total cell volume. Cell volume must be considered in measuring growth because there is a change in average cell volume in addition to an increase in cell numbers throughout the growth curve. Chlorella vulgaris and Selenastrum capricornutum exhibited similar growth curves when growth was expressed as cells per litre or as milligrams dry weight per litre. Chlamydomonas reinhardtii appeared to have a lower algal growth potential when measured in cells per litre but had the same growth potential as C. vulgaris and S. capricornutum when expressed as milligrams dry weight per litre. The growth rate for Scenedesmus quadricauda was less than the other three, regardless of how the results were expressed. The image analysis system was used to count different types of algae in a mixed culture. C. reinhardtii , C. vulgaris , and Anacystis nidulans were not grossly distorted by the sample preparation method and could be distinguished from one another by variations in morphology. The counts could be separated by computing the ratio of area to length for each particle and by allowing the computer to sort and count each of the three types. Typical ratios ranged from 5 to 7 for G. reinhardtii , 2 to 4 C. vulgaris , and 0.2 to 1.3 for A. nidulans . The number of types to be counted in a mixture was limited only by the ability to apply shape factors without having an overlap between each type. Both the image analysis and electronic particle counter produced equal count and size measurements. Fluorescence was a sensitive measure of growth on a laboratory prepared medium since there was a logarithmic relationship between fluorescence and cells per millilitre for both C. vulgaris and S. capricornutum . Fluorescence measurements were not satisfactory for determining growth in samples of varying chemical composition (which occurs in natural samples) because the chlorophyll-to-cell ratio varied with media composition. This was especially pronounced with C. vulgaris .
Journal of Research of the U.S. Geological Survey↗
Accounting for intracell flow in models with emphasis on water table recharge and stream-aquifer interaction: 2. A procedure
Intercepted intracell flow, especially if cell includes water table recharge and a stream ((sink), can result in significant model error if not accounted for. A procedure utilizing net flow per cell ( F n ) that accounts for intercepted intracell flow can be used for both steady state and transient simulations. Germane to the procedure is the determination of the ratio of area of influence of the interior sink to the area of the cell ( A i / A c ). A i is the area in which water table recharge has the potential to be intercepted by the sink. Determining A i / A c requires either a detailed water table map or observation of stream conditions within the cell. A proportioning parameter M , which is equal to 1 or slightly less and is a function of cell geometry, is used to determine how much of the water that has potential for interception is intercepted by the sink within the cell. Also germane to the procedure is the determination of the flow across the streambed ( F s ), which is not directly a function of cell size, due to difference in head between the water level in the stream and the potentiometric surface of the aquifer underlying the streambed. The use of F n for steady state simulations allows simulation of water levels without utilizing head-dependent or constant head boundary conditions which tend to constrain the model-calculated water levels, an undesirable result if a comparison of measured and calculated water levels is being made. Transient simulations of streams usually utilize a head-dependent boundary condition and a leakance value to model a stream. Leakance values for each model cell can be determined from a steady state simulation, which used the net flow per cell procedure. For transient simulation, F n would not include F s . Also, for transient simulation it is necessary to check F n at different time intervals because M and A i / A c are not constant and change with time. The procedure was used successfully in two different models of the aquifer system in the Ozarks. The use of F n was essential to the two model studies because most model cells in both models contained water table recharge and multiple sinks.
Water Resources Research↗
Green fluorescent protein is lighting up fungal biology
Prasher ( 42 ) cloned a cDNA for the green fluorescent protein (GFP) gene from the jellyfish Aequorea victoria in 1992. Shortly thereafter, to the amazement of many investigators, this gene or derivatives thereof were successfully expressed and conferred fluorescence to bacteria and Caenorhabditis elegans cells in culture ( 10 , 31 ), followed by yeast ( 24 , 39 ), mammals ( 40 ), Drosophila ( 66 ), Dictyostelium ( 23 , 30 ), plants ( 28 , 49 ), and filamentous fungi ( 54 ). The tremendous success of GFP as a reporter can be attributed to unique qualities of this 238-amino-acid, 27-kDa protein which absorbs light at maxima of 395 and 475 nm and emits light at a maximum of 508 nm. The fluorescence of GFP requires only UV or blue light and oxygen, and therefore, unlike the case with other reporters (β-glucuronidase, β-galacturonidase, chloramphenicol acetyltransferase, and firefly luciferase) that rely on cofactors or substrates for activity, in vivo observation of gfp expression is possible with individual cells, with cell populations, or in whole organisms interacting with symbionts or environments in real time. Complications caused by destructive sampling, cell permeablization for substrates, or leakage of products do not occur. Furthermore, the GFP protein is extremely stable in vivo and has been fused to the C or N terminus of many cellular and extracellular proteins without a loss of activity, thereby permitting the tagging of proteins for gene regulation analysis, protein localization, or specific organelle labeling. The mature protein resists many proteases and is stable up to 65°C and at pH 5 to 11, in 1% sodium dodecyl sulfate or 6 M guanidinium chloride (reviewed in references 17 and 67 ), and in tissue fixed with formaldehyde, methanol, or glutaraldehyde. However, GFP loses fluorescence in methanol-acetic acid (3:1) and can be masked by autofluorescent aldehyde groups in tissue fixed with glutaraldehyde. Fluorescence is optimal at pH 7.2 to 8.0 ( 67 ). Limitations on GFP as a reporter for some applications are its low turnover rate, 2-h lag time for autoactivation of its chromophore, improper folding at high temperatures (37°C), which results in nonfluorescent and insoluble forms of the protein, and requirement for oxygen, which is not present in equal concentrations in all subcellular locations or cell types (reviewed in references 17 and 67 ). These characteristics of GFP, however, have not posed a problem for many applications, and mutant forms of GFP that have an ability to fold properly at high temperatures, increased solubility and fluorescence, reduced photobleaching ( 16 , 17 , 51 ), and reduced half-lives ( 1 ) have been developed. Coupled with fluorescence-activated cell sorting, confocal microscopy or quantitative image analysis techniques, GFP technology can be used to isolate transformed cells or specific cell types from populations of cells ( 14 ), to quantify gene expression of individual cells within whole organisms ( 8 ), or to assess the dispersal and biomass of organisms in complex environments, such as in animal or plant hosts ( 38 , 59 ), in biofilms ( 55 ), in fermentors ( 41 ), on leaf surfaces ( 53 , 61 ), or in soils ( 2 ). The vast majority of studies utilizing GFP expression in fungi have been with yeast (reviewed in reference 13 ). Ustilago maydis was the first filamentous fungus for which successful expression of gfp was reported ( 54 ), followed closely by Aspergillus nidulans ( 22 , 57 ) and Aureobasidium pullulans ( 61 ). Presently, gfp expression has been reported for 16 species comprising 12 genera of filamentous fungi, including Colletotrichum ( 21 , 44 ), Mycosphaerella ( 52 ), Magnaporthe ( 32 , 35 ), Cochliobolus ( 38 ), Trichoderma ( 2 , 70 ), Podospora ( 5 ), Sclerotinia ( 63 ), Schizophyllum ( 37 ), Aspergillus ( 20 , 47 , 50 ) and Phytophthora ( 7 , 62 ). In this review we draw on published reports, with the goal of providing an overview of GFP technology as it applies to the biology of filamentous fungi. These reports are not exhaustive of potential applications of GFP technology, as examples of genomic approaches to utilizing GFP in bacterial and yeast systems attest ( 4 , 46 , 60 , 65 ). Expression of gfp in filamentous fungi requires a gfp variant that is efficiently translated in fungi, a transformation system, and a fungal promoter that satisfies the requirements of a given experimental objective. Transformation of fungi has recently been reviewed by Gold et al. ( 26 ). Robinson and Sharon ( 44 ) suggest that GFP can actually be used to optimize transformation protocols. In addition to reporting the construction of a new fungal transformation vector that expresses SGFP under the control of the ToxA gene promoter from Pyrenophora tritici-repentis ( 12 ) and demonstrating its use in plant pathogens belonging to eight different genera of filamentous fungi ( Fusarium, Botrytis, Pyrenophora, Alternaria, Cochliobolus, Sclerotinia, Colletotrichum , and Verticillium ), in this review we also enumerate and describe a comprehensive list of vectors for expressing GFP in fungi.
Applied and Environmental Microbiology↗
Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity
Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.
Diseases of Aquatic Organisms↗
Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway
Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.
Biochemical and Biophysical Research Communication↗
Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos
In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers.
Zygote↗
Rewetting approximation for a simulator of flow in a surficial aquifer overlain by seasonally inundated wetlands
An approximation is presented to rectify situations occurring as part of the three-dimensional simulation of surficial aquifer flows in which surface grid cells become dry (zero saturated thickness) and then cannot readily receive flows that would again partially saturate them. The approximation permits the cells to be rewetted by adjusting dry cell pressures by the same amount as the pressure change computed for the uppermost nondry cells in the vertical column. This is continued in successive timesteps until the dry cells once again become partially or fully saturated. The rewetting procedure was tested in a recent transient simulation of flows in the surficial Biscayne aquifer of Dade County, Florida, which is partly covered by seasonally inundated wetlands. The uppermost layer of the model (layer 1) was used to represent overland sheetflow and grid cells of high equivalent hydraulic conductivity in this layer became dry and were rewetted seasonally, requiring a relatively robust representation of rewetting. The rewetting procedure made possible a simulation of overland sheetflow stages and aquifer water-table altitudes between 1945 and 1989 that was generally correct to within about 1 foot in inundated and noninundated regions. In a 6-year time period (1962–67) in which the behavior of the rewetting procedure was studied in detail, there were 4,723 simulated cell rewettings, of which 3,172 were in the overland sheetflow layer and 1,551 were in the uppermost aquifer layer (layer 2). The average head adjustment in a cell rewetting was 0.10 foot in layer 1 and 0.15 foot in layer 2. This study demonstrates an empirical approach to the problem of cell rewetting that might prove useful in other models of flow in surficial aquifers.
Groundwater↗
Isolation, growth, and metabolism of an obligately anaerobic, selenate- respiring bacterium, strain SES-3
A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO 2 with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se 0 ). Hence, SES-3 can carry out a complete reduction of selenate to Se 0 . Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [ 14 C]lactate to 14 CO 2 coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m -chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se 0 by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [ 75 Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se 0 . This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se 0 may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.
Nevada↗