Changes in phosphatidylinositol metabolism in response to hyperosmotic stress in Daucus carota L. cells grown in suspension culture.

Carrot (Daucus carota L.) cells plasmolyzed within 30 s after adding sorbitol to increase the osmotic strength of the medium from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant change in the polyphosphorylated inositol phospholipids or inositol phosphates or in inositol phospholipid metabolism within 30 s of imposing the hyperosmotic stress. Maximum changes in phosphatidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in osmoticum. There was a 30% decrease in [3H]inositol-labeled PIP. The specific activity of enzymes involved in the metabolism of the inositol phospholipids also changed. The plasma membrane phosphatidylinositol (PI) kinase decreased 50% and PIP-phospholipase C (PIP-PLC) increased 60% compared with the control values after 5 min of hyperosmotic stress. The PIP-PLC activity recovered to control levels by 10 min; however, the PI kinase activity remained below the control value, suggesting that the cells had reached a new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A inhibitor, the differences in enzyme activity resulting from the hyperosmotic stress were no longer evident, suggesting that an okadaic acid-sensitive phosphatase was activated in response to hyperosmotic stress. Our work suggests that, in this system, PIP is not involved in the initial response to hyperosmotic stress but may be involved in the recovery phase.     

Localization of the Functional p34cdc2 Homolog of Maize in Root Tip and Stomatal Complex Cells: Association with Predicted Division Sites

We have used an antibody against the functional homolog of the cdc2 kinase of maize to localize the p34cdc2 protein within dividing cells of the root apex and the stomatal complex of leaf epidermis. The microtubule cytoskeletal structure of plant cells was visualized concomitantly with a monoclonal antibody specific for [alpha]-tubulin. We found that the cdc2 protein is localized mainly to the nucleus in plant cells at interphase and early prophase. This finding contrasts markedly with the predominantly cytoplasmic staining obtained using antibody to the PSTAIRE motif, which is common to cdc2 and numerous cdc2-like proteins. In a subpopulation of root cells at early prophase, the p34cdc2 protein is also distributed in a band bisecting the nucleus. Double labeling with the maize p34cdc2Zm antibody and tubulin antibody revealed that this band colocalizes with the preprophase band (PPB) of microtubules, which predicts the future division site. Root cells in which microtubules had been disrupted with oryzalin did not contain this band of p34cdc2 protein, suggesting that formation of the microtubule PPB is necessary for localization of the p34cdc2 kinase to the plane of the PPB. The p34cdc2 protein is also localized to the nucleus and PPB in cells that give rise to the stomatal complex, including those cells preparing for the highly asymmetrical divisions that produce subsidiary cells. Association of the p34cdc2 protein with the PPB suggests that the cdc2 kinase has a role in establishing the division site of plant cells and, therefore, a role in plant morphogenesis.     

Fine-scale mapping of physicochemical and microbial landscapes of the coral skeleton

The coral skeleton harbours a diverse community of bacteria and microeukaryotes exposed to light, O₂ and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O₂ and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef-building corals Porites lutea and Paragoniastrea benhami. The physicochemical environment was more stable in the deep skeleton, and the diversity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O₂ habitat, whereas pH varied from pH 6–9 with depth. Physicochemical gradients of O₂ and pH of the coral skeleton explained the β-diversity of the bacterial communities, and skeletal layers that showed O₂ peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.     

Influence of root density on the critical soil water potential

Estimation of root water uptake in crops is important for making many other agricultural predictions. This estimation often involves two assumptions: (1) that a critical soil water potential exists which is constant for a given combination of soil and crop and which does not depend on root length density, and (2) that the local root water uptake at given soil water potential is proportional to root length density. Recent results of both mathematical modeling and computer tomography show that these assumptions may not be valid when the soil water potential is averaged over a volume of soil containing roots. We tested these assumptions for plants with distinctly different root systems. Root water uptake rates and the critical soil water potential values were determined in several adjacent soil layers for horse bean (Vicia faba) and oat (Avena sativa) grown in lysimeters, and for field-grown cotton (Gossypium L.), maize (Zea mays) and alfalfa (Medicago sativa L.) crops. Root water uptake was calculated from the water balance of each layer in lysimeters. Water uptake rate was proportional to root length density at high soil water potentials, for both horse bean and oat plants, but root water uptake did not depend on root density for horse bean at potentials lower than −25 kPa. We observed a linear dependency of a critical soil water potential on the logarithm of root length density for all plants studied. Soil texture modified the critical water potential values, but not the linearity of the relationship. B E Clothier Section editor     

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff,Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1intratracheally. J. Appl. Physiol.82(1): 226-232, 1997.Interleukin-1 (IL-1) is increased in lunglavages from patients with the acute respiratory distress syndrome, andadministering IL-1 intratracheally causes neutrophil accumulation and aneutrophil-dependent oxidative leak in lungs of rats. In the presentstudy, we found that rats pretreated intraperitoneally with lisofylline[(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species,did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1.However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbersof lung lavage neutrophils as rats given IL-1 intratracheally. Lungs ofrats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lungleak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

     

Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean

Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean. Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed.     

Properties of acetylcholinesterase reconstituted in liposomes of a different charge

Acetylcholinesterase (AChE) purified from mouse brain was reconstituted in liposomes of a different charge, and the properties of liposome-associated AChE were investigated. Relative to the K_m value (38.5 M) of AChE bound to a neutral liposome, the value of AChE reconstituted in a negatively-charged liposome decreased to 23.3 M, whereas that of AChE in a positively-charged liposome increased to 90.9 M. Additionally, AChE bound to a positively-charged liposome expressed a wider range of optimum pH than the enzyme in a negatively-charged liposome. In a stability study, it was found that soluble AChE was unstable at pH 5.5 and 7.4, while it was relatively stable at pH 10. Noteworthy, the immobilization of AChE to liposome enhanced the stability of soluble enzyme at acidic and neutral pH. Moreover, in the stabilization of the enzyme, a neutral liposome was more effective than charged liposomes, of which a positively-charged liposome was more effective than a negatively-charged liposome at acidic pH. Based on these results, it is proposed that while the K_m value and the pH dependence of AChE activity are affected by the charge of liposome, the stability of AChE is determined mainly by a hydrophobic binding to a phospholipid membrane.This work was supported in part by Agency for Defense Development.     

Reexamination of paternal age effect in Down's syndrome

Summary Paternal age distribution for 1279 cases of Down's syndrome born in 1952–1968 was compared with the corresponding distribution for the general population, corrected for the maternal age as well as for the year of birth of the patients. Although there was no difference in the mean paternal age, the two distributions differed significantly, largely due to the excess of fathers aged 55 years and over and to the deficit of those aged 40–44 years in the patients born to mothers aged 30 years and over. The overall pattern of the relative incidence of Down's syndrome with advancing paternal age, with maternal age controlled, seems consistent with the hypothesis proposed by Stene et al. (1977). It increased from 0.8 for fathers aged 20–24 years slowly up to 1.2 for those aged 45–49 years, though with an intermediate drop to 0.8 at the age of 40–44 years, and then sharply to 2.4 for those aged 55 years and over. This rising pattern of the relative incidence with paternal age was essentially the same for the patients born in 1952–1960 and for those born in 1961–1968, although the slope was less steep in the latter than in the former group.This paper is dedicated to Professor Heinrich Schade in honor of his 70th birthday     

Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat-killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat-killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat-killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae-suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148-immunized mice, but the mice did not respond to heat-killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross-reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148-immunized mice into syngeneic recipients. These results suggest that the cross-reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.