IDENTIFICATION OF PROCAMBIUM IN THE PRIMARY ROOT OF TRIFOLIUM PRATENSE (FABACEAE)

Histochemical and morphometric analyses were used to identify and define an early stage of procambial differentiation in 1.5–2.0-cm-long (48 hr after germination) primary roots of Trifolium pratense L. Esterase activity was used as a histochemical marker for early differentiation of procambium. Morphometric analysis of cell length and width as a function of distance from the root cap junction was performed on the same tissue using brightfield and Nomarski DIC optics. This combination of techniques allowed the identification of esterase activity in both the cell wall and cytoplasm and permitted the determination of the exact location, size, and shape of the histochemically stained cells within the apex. Esterase activity identified the proendodermis and procambial cylinder (six to seven cells in diameter) two to three cells proximal to the root cap junction. In this system, esterase activity proved to be an earlier marker for procambial differentiation than morphometric or cytological changes. It is suggested that these techniques will be useful in characterizing procambial pattern development in more complex shoot systems.     

NADPH oxidase of neutrophils elevates o,o'-dityrosine cross-links in proteins and urine during inflammation.

Reactive intermediates generated by phagocytic white blood cells are of central importance in destroying microorganisms, but they may also damage normal tissue at sites of inflammation. To investigate the potential role of such oxidants in tissue injury, we used gas chromatography/mass spectrometry to quantify levels of o,o'-dityrosine in mouse peritoneal neutrophils and urine. In wild-type animals, neutrophils markedly increased their content of protein-bound dityrosine when they were activated in vivo. This increase failed to occur in mice that were deficient in the phagocyte NADPH oxidase. Levels of o,o'-dityrosine in urine mirrored those in neutrophil proteins. When o,o'-[(14)C]dityrosine was injected intravenously into mice, the radiolabel was not metabolized or incorporated into tissue proteins: instead, it was recovered in urine with near-quantitative yield. Patients with sepsis markedly increased their output of o,o'-dityrosine into urine, suggesting that systemic inflammation also may be a potent source of oxidative stress in humans. These observations demonstrate that activated neutrophils produce o,o'-dityrosine cross-links in tissue proteins, which may subsequently be degraded into free amino acids and excreted into urine. Our results indicate that mouse phagocytes use oxidants produced by the NADPH oxidase to create o,o'-dityrosine cross-links in vivo and raise the possibility that reactive intermediates produced by this pathway promote inflammatory tissue damage in humans.     

Immunomodulatory mediators from pollen enhance the migratory capacity of dendritic cells and license them for Th2 attraction

The immune response of atopic individuals against allergens is characterized by increased levels of Th2 cytokines and chemokines. However, the way in which the cytokine/chemokine profile is matched to the type of invading allergen, and why these profiles sometimes derail and lead to disease, is not well understood. We recently demonstrated that pollen modulates dendritic cell (DC) function in a way that results in an enhanced capacity to initiate Th2 responses in vitro. Here, we examined the effects of aqueous birch pollen extracts (Bet.-APE) on chemokine receptor expression and chemokine production by human monocyte-derived DCs. Bet.-APE strongly induced expression and function of CXCR4 and reduced CCR1 and CCR5 expression on immature DCs. In addition, DC treatment with Bet.-APE significantly reduced LPS-induced production of CXCL10/IP-10, CCL5/RANTES; induced CCL22/macrophage-derived chemokine; and did not significantly change release of CCL17/thymus and activation-regulated chemokine. At a functional level, Bet.-APE increased the capacity of LPS-stimulated DCs to attract Th2 cells, whereas the capacity to recruit Th1 cells was reduced. Bet.-APE significantly and dose-dependently enhanced intracellular cAMP, suggesting that water-soluble factors from pollen grains bind a G(alphas)-protein-coupled receptor. E(1)-Phytoprostanes were identified to be one player in the Th2-polarizing potential of aqueous pollen extracts. In summary, our results demonstrate that pollen itself releases regulatory mediators which generate a Th2-promoting micromilieu with preferential recruitment of Th2 cells to the site of pollen exposure.     

Vibrio cholerae strains possess multiple strategies for abiotic and biotic surface colonization

Despite its notoriety as a human pathogen, Vibrio cholerae is an aquatic microbe suited to live in freshwater, estuarine, and marine environments where biofilm formation may provide a selective advantage. Here we report characterization of biofilms formed on abiotic and biotic surfaces by two non-O1/O139 V. cholerae strains, TP and SIO, and by the O1 V. cholerae strain N16961 in addition to the isolation of 44 transposon mutants of SIO and TP impaired in biofilm formation. During the course of characterizing the mutants, 30 loci which have not previously been associated with V. cholerae biofilms were identified. These loci code for proteins which perform a wide variety of functions, including amino acid metabolism, ion transport, and gene regulation. Also, when the plankton colonization abilities of strains N16961, SIO, and TP were examined, each strain showed increased colonization of dead plankton compared with colonization of live plankton (the dinoflagellate Lingulodinium polyedrum and the copepod Tigriopus californicus). Surprisingly, most of the biofilm mutants were not impaired in plankton colonization. Only mutants impaired in motility or chemotaxis showed reduced colonization. These results indicate the presence of both conserved and variable genes which influence the surface colonization properties of different V. cholerae subspecies.     

Mitochondrial genome integrity mutations uncouple the yeast Saccharomyces cerevisiae ATP synthase

The mitochondrial ATP synthase is a molecular motor, which couples the flow of protons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the alpha-, beta-, and gamma-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the gamma-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk.     

Effect of peak pressure and pressure gradient on subsurface shear stresses in the neuropathic foot

The pressure distribution on the plantar surface of the foot may provide insights into the stresses within the subsurface tissues of patients with diabetes mellitus and peripheral neuropathy (PN) who are at risk for skin breakdown. The purposes of this study were to (1) estimate the stress distribution in the subsurface soft tissue from a measured surface pressure distribution and determine any differences between values in the forefoot and rearfoot, and (2) determine the relationship between maximum shear stress (MSS) (magnitude and depth) and characteristics of the pressure distribution. The measured in-shoe pressure distributions during walking characterized by the peak plantar pressure and maximum pressure gradient on the plantar surface of the feet for 20 subjects with diabetes, PN and history of a mid foot or forefoot plantar ulcer were analyzed. The effects of peak pressure and maximum pressure gradient at the peak pressure location on the stress components in the subsurface soft tissue were studied using a potential function method to estimate the subsurface tissue stress. The calculated MSSs are larger in magnitude and located closer to the surface in the forefoot, where most skin breakdown occurs, compared to the rearfoot. In addition, the MSS (magnitude and depth) is highly correlated with the pressure gradient (r=-0.77 & 0.61) and the peak pressure (r=-0.61 & 0.91). The peak pressure and the maximum pressure gradient obtained from the surface pressure distribution appear to be important variables to identify where MSSs are located in the subsurface tissues on the plantar foot that may lead to skin break down.     

Transformation and segregation of GFP fluorescence and glyphosate resistance in horseweed (<Emphasis Type="Italic">Conyza canadensis</Emphasis>) hybrids

The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker allowed for the simple and accurate determination of GR hybrid plants by visual observation. GR plants were shown to be transgenic via the green fluorescence under UV light, and resistant to glyphosate when sprayed with the field-use-rate 0.84 kg acid equivalent ha⁻¹ of glyphosate (i.e. Roundup^TM) herbicide. An in vitro screen for glyphosate resistance in seedlings was developed, and a 5 μM glyphosate concentration was found to reduce dry weight in GS seedlings but not in GR seedlings. The GR plants containing GFP were then hand-crossed with GS plants from eastern Tennessee under greenhouse conditions, with GS plants acting as the pollen acceptor. Resulting seed was collected and germinated for GFP fluorescence screening. Seedlings that exhibited the transgenic GFP phenotype were selected as F₁ hybrids between GR and GS horseweed. Thirty GS×GR hybrids were produced on the basis of a green-fluorescent GFP phenotype of GR plants. GS×GFP/GR F₁ hybrids produced F₂ seeds, and F₂ plants were shown to segregate for GFP fluorescence and glyphosate resistance independently. Both traits segregated at a Mendelian 3:1 ratio, indicating a single gene is responsible for each phenotype.