The VP7 outer capsid protein of rotavirus induces polyclonal B-cell activation

The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection.     

TIMP-2 is released as an intact molecule following binding to MT1-MMP on the cell surface

Binding of tissue inhibitor of metalloproteinase-2 (TIMP-2) to pro-MMP-2 and mature membrane type-1 MMP (MT1-MMP) on the cell surface is required for activation of MMP-2. It has been reported that following binding to cell surface receptors, TIMP-2 undergoes endocytosis and extensive degradation in lysosomes. The purpose of this study was to reexamine the fate of TIMP-2 following binding to transfected HT1080 cell surface MT1-MMP at 4 degrees C. Following 37 degrees C incubation, 125I-TIMP-2 release, endocytosis, and degradation were characterized under varying conditions. More than 85% of the total 125I-TIMP-2 bound to cells was released as intact functional molecules; <15% was degraded. Transfection of HT1080 cells with dominant negative mutant dynamin cDNA resulted in delayed endocytosis and release of 125I-TIMP-2 from cells. Pharmacologic agents that induce clustering of cell surface receptors (concanavalin A) and interfere with endosomal/lysosomal function (bafilomycin A(1)) resulted in enhanced binding of 125I-TIMP-2 to cell surface receptors. Abrogation of activation of proMT1-MMP with a furin inhibitor prevented binding and endocytosis of 125I-TIMP-2. Biotinylation of cell surface MT1-MMP followed by Western blotting confirmed the presence of mature MT1-MMP on the cell surface and degraded MT1-MMP in the intracellular compartment. In conclusion, these studies demonstrate that TIMP-2 is released from cells primarily as an intact functional molecule following binding to MT1-MMP on the cell surface.     

Vasoconstriction during venous congestion: effects of venoarteriolar response, myogenic reflexes, and hemodynamics of changing perfusion pressure

We dissected the relative contribution of arteriovenous hemodynamics, the venoarteriolar response (VAR), and the myogenic reflex toward a decrease in local blood flow induced by venous congestion. Skin blood flow (SkBF) was measured in 12 supine subjects via laser-Doppler flowmetry 1) over areas of forearm and calf skin, in which the VAR was blocked by using eutectic mixture of local anesthetics (EMLA sites) and 2) over the contralateral forearm or calf skin (control sites), using two different techniques: limb dependency of 23-37 cm below the heart and cuff inflation to 40 mmHg. During limb dependency, SkBF decreased at the control sites, whereas it remained unchanged at the EMLA sites. In contrast, during cuff inflation, SkBF decreased at the control sites and also decreased at the EMLA sites. The percent change in SkBF from baseline was greater during cuff inflation than limb dependency at both the control sites and the EMLA sites. Estimated skin vascular resistance remained unchanged at the EMLA sites during cuff inflation, as well as limb dependency. Thus the decrease in SkBF during venous congestion with cuff inflation is not solely due to the cutaneous VAR but also to a reduction in local perfusion pressure. The VAR is therefore most specifically quantified by venous congestion induced by limb dependency, rather than cuff inflation. Finally, from both techniques, we calculated that during venous congestion induced by limb dependency (calf), approximately 45% of the nonbaroreflex vasoconstriction is induced by the VAR and approximately 55% by the myogenic reflex.     

Carnoy fixation: practical and theoretical considerations

Summary The Carnoy-fixation, paraffin-embedding procedure was modified to minimize shrinkage and to suit the schedule of general histology laboratories. Blocks of tissues were fixed in Carnoy's fluid overnight, transferred to absolute alcohol in the morning and washed in three changes of alcohol for a total of 6–8 hours. Tissues were then left in methyl benzoate overnight, transferred to an Autotechnicon in the morning, cleared in xylene and xylene-paraffin 1 hour each and infiltrated in 2–3 changes of paraffin for a total of 4–5 hours. This procedure has worked well in our hands for nine years.A review of the chemical literature showed that the components of Carnoy's fluid-ethanol, chloroform and acetic acid-can interact by hydrogen bond formation with each other and with various groups in tissues. These association compounds apparently stabilize tissue structures and prevent or minimize shrinkage. Ethanol alone causes collapse of protein structures; exposure must be limited to eight hours or less. Addition of water to an ethanol-acetic acid solution causes considerable swelling of proteins and subsequent shrinkage in absolute alcohol; hence, the ratio fixative: tissue should be not less than 201. In our experience, Carnoy's fluid is the fixative of choice for studies of fibrous proteins and associated carbohydrates by histochemical and special staining technics.     

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.     

Flooding and salinity effects on growth and survival of four common forested wetland species

The survival, growth, and biomass of baldcypress (Taxodium distichum (L.) Rich.), water tupelo (Nyssa aquatica L.), Chinese tallow (Sapium sebiferum (L.) Roxb.), and green ash (Fraxinus pennsylvanica Marsh.) seedlings were examined in an experiment varying water levels (watered, flooded) and salinity levels (0, 2, and 10 ppt, plus a simulated storm surge with 32 ppt saltwater). All seedlings, except for those flooded with 10 ppt saltwater, survived to the end of the experiment. In 10 ppt saltwater, flooded baldcypress, water tupelo, and green ash survived two weeks whereas Chinese tallow survived for 6 weeks. However, a second set of slightly older baldcypress, water tupelo, and Chinese tallow seedlings survived eight weeks of flooding with 10 ppt saltwater. When carried through the winter to the beginning of the second growing season, flooded baldcypress and Chinese tallow seedlings from the 0 and 2 ppt treatments leafed out, but only Chinese tallow recovered from the saltwater surge treatment. The diameter and growth (height) of each species was not affected when watered with 2 ppt saltwater, except for the effects of the height growth of baldcypress. Growth was reduced for all species when watered with 10 ppt saltwater. The diameter growth of green ash was reduced by freshwater flooding. The diameter growth of baldcypress and water tupelo was greater when flooded with fresh water. Flooding with 2 ppt saltwater caused a significant reduction in diameter growth in water tupelo, green ash, and Chinese tallow, but not in baldcypress. Root and stem biomass values were not significantly different for any species between the 0 and 2 ppt salinity watering treatments. However, seedlings watered with 10 ppt saltwater had significantly lower root and stem biomass values, except for baldcypress roots and green ash stems. Baldcypress was least affected by flooding with 0 and 2 ppt saltwater, although there were slight reductions in root biomass with increasing salinity. Because of the susceptibility of the seedlings of these four species to increases in flooding and salinity, their regeneration may be limited in the future, thereby causing shifts in species composition.     

Effects of flooding and salinity on photosynthesis and water relations of four Southeastern Coastal Plain forest species

The influence of flooding and salinity on photosynthesis and water relations was examined for four common coastal tree species [green ash (Fraxinus pennsylvanica Marshall), water tupelo (Nyssa aquatica L.). Chinese tallow (Sapium sebiferum (L.) Roxb.), and baldcypress (Taxodium distichum (L.) Richard)]. Both chronic (as might be associated with sea level rise) and acute (similar to hurricane storm surges) exposures to these stresses were examined. Chronic freshwater flooding of green ash, water tupelo, and Chinese tallow seedlings reduced photosynthesis (A) relative to that of watered seedlings, while baldcypress was unaffected. Chinese tallow A declined with increasing length of flooding. A salinity increase of the floodwater to 2 ppt decreased A of baldcypress and water tupelo, but not A of green ash and Chinese tallow, which was already severely reduced by freshwater flooding. All seedlings of the four species died within 2 to 6 weeks when flooded with 10 ppt saltwater. Photosynthesis of all four species did not differ between 0 and 2 ppt watering. Watering with 10 ppt salinity initially reduced A of all four species, but the seedlings recovered over time. Photosynthesis was severely decreased for all species when flooded with 21 ppt salinity for 48 hours. Reduced A continued following the treatment. Photosynthesis of only green ash and water tupelo was reduced by watering with 21 ppt salinity for 48 hours. Flooding of low-lying areas with increased salinity would lead to shifts in species composition of coastal forests due to these differential tolerances.     

Growth and survival of Escherichia coli O157:H7 under acidic conditions.

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.     

pBINPLUS: An improved plant transformation vector based on pBIN19

We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number inE. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.