Molecular Studies on the Role of a Root Surface Agglutinin in Adherence and Colonization by Pseudomonas putida

Pseudomonas putida aggressively colonizes root surfaces and is agglutinated by a root surface glycoprotein. Mutants of P. putida derived chemically or by Tn5 insertion demonstrated enhanced or decreased agglutinability. Two nonagglutinable Tn5 mutants (Agg⁻) and two mutants with enhanced agglutinability (Agg^s) possessed Tn5 in unique restriction sites. Agg⁻ mutants colonized root surfaces of seedlings grown from inoculated seeds, but at levels lower than those observed with the Agg⁺ parent. In short-term binding studies, Agg⁻ cells adhered at levels that were 20- to 30-fold less than those for Agg⁺ parental cells. These data suggest that the agglutination interaction plays a role in the attachment of P. putida to root surfaces.     

Greater airway narrowing in immature than in mature rabbits during methacholine challenge

Shen, X., V. Bhargava, G. R. Wodicka, C. M. Doerschuk, S. J. Gunst, and R. S. Tepper. Greater airway narrowing in immature thanin mature rabbits during methacholine challenge. J. Appl. Physiol. 81(6): 2637-2643, 1996.It hasbeen demonstrated that methacholine (MCh) challenge produces a greaterincrease in lung resistance in immature than in mature rabbits (R. S. Tepper, X. Shen, E. Bakan, and S. J. Gunst.J. Appl. Physiol. 79: 1190-1198, 1995). To determine whether this maturational difference in the response to MCh was primarily related to changes in airway resistance (Raw) or changes in tissue resistance, we assessed airway narrowing in1-, 2-, and 6-mo-old rabbits during intravenous MCh challenge (0.01-5.0 mg/kg). Airway narrowing was determined frommeasurements of Raw in vivo and from morphometric measurements on lungsections obtained after rapidly freezing the lung after the MChchallenge. The fold increase in Raw was significantly greater for 1- and 2-mo-old animals than for 6-mo-old animals. Similarly, the degree of airway narrowing assessed morphometrically was significantly greaterfor 1- and 2-mo-old animals than for 6-mo-old animals. The foldincrease in Raw was highly correlated with the degree of airwaynarrowing assessed morphometrically(r² = 0.82, P < 0.001). We conclude that thematurational difference in the effect of MCh on lung resistance isprimarily caused by greater airway narrowing in the immature rabbits.

     

Effect of shoot tip and leaf removal on gravitropism in pea

Intact, light-grown pea (Pisum sativum L. cv. Alaska) seedlings were subjected to continuous horizontal gravistimulation and their growth and bending response compared with seedlings whose shoot tip and youngest leaf had been excised and with seedlings to which a counterweight to replace the mass of the decapitated tissue was added. While all seedlings achieved vertical orientation in 2 to 3 h, seedlings that were counterweighted bent upward at a significantly slower rate than the non-counterweighted, decapitated plants. In addition to this effect of mass on the rate of bending, decapitation also removed a major supply of auxin to cells in the bending zone which resulted in the slower bending of treated plants. Thus when using decapitation both the loss of mass and the time course of the response must be considered to understand its effect on gravitropism.     

Mechanisms for the mechanical response of airway smooth muscle to length oscillation

Shen, X., M. F. Wu, R. S. Tepper, and S. J. Gunst. Mechanisms for the mechanical response ofairway smooth muscle to length oscillation. J. Appl.Physiol. 83(3): 731-738, 1997.Airway smoothmuscle tone in vitro is profoundly affected by oscillations in musclelength, suggesting that the effects of lung volume changes on airwaytone result from direct effects of stretch on the airway smooth muscle.We analyzed the effect of length oscillation on active force andlength-force hysteresis in canine tracheal smooth muscle at differentoscillation rates and amplitudes during contraction with acetylcholine.During the shortening phase of the length oscillation cycle, the activeforce generated by the smooth muscle decreased markedly below theisometric force but returned to isometric force as the muscle waslengthened. Results indicate that at rates comparable to those duringtidal breathing, active shortening and yielding of contractile elementscontributes to the modulation of force during length oscillation;however, the depression of force during shortening cannot be accountedfor by cross-bridge properties, shortening-induced cross-bridgedeactivation, or active relaxation. We conclude that the depression ofcontractility may be a function of the plasticity of the cellularorganization of contractile filaments, which enables contractileelement length to be reset in relation to smooth muscle cell length asa result of smooth muscle stretch.

     

A simple rapid photometric estimation of the growth response of immobilized cells to fusaric acid and gamma radiation

A simple and rapid photometric method was developed in order to evaluate the growth responses of cells to various factors in vitro. Cells of Asparagus officinalis L. were mixed with autoclaved agar at 40°C and 90 l drops were dispensed into several Petri dishes. After the drops solidified, they were covered with a liquid growth medium and incubated on a gyratory shaker. Growth was measured every 2 or 3 days by two methods. In the first, the agar drop was placed under a stereomicroscope with substage illumination and the light passing through the embedded cells was measured by a light-meter. Growth, expressed by the increase in cell density, was inversely proportional to the intensity of transmitted light. In the second method, the agar drop was melted in a microwave oven and the packed cell volume was measured. The correlation between the two methods showed that the photometric method can be used to assess growth response of immobilized cells, during the first two weeks of culture. This method was used to evaluate growth responses to the toxin fusaric acid and to gamma radiation. The photometric method requires a small amount of inoculum, standard microscopic equipment and can be used to determine the effect of various factors on the growth of intact plant cells in vitro without disruption.Abbreviations FA fusaric acid - NAA naphthyl-1-acetic acid - CH casein hydrolysate - 2-i,P N⁶-(2-isopentyl) adenine - 2,4-D 2,4-dichloro-phenoxy acetic acid - PCV packed cell volume - gy grey     

Reduction of neutrophil influx diminishes lung injury and mortality following phosgene inhalation.

Phosgene inhalation causes a severe noncardiogenic pulmonary edema characterized by an influx of neutrophils into the lung. To study the role of neutrophils in lung injury and mortality after phosgene, we investigated the effects of leukocyte depletion with cyclophosphamide, inhibiting the generation of the chemotaxin leukotriene B4 with the 5-lipoxygenase inhibitor AA861 and impairing neutrophil migration with the microtubular poison colchicine. Cyclophosphamide, AA861, and colchicine injected before exposure significantly reduced percent neutrophils, protein, and thiobarbituric acid-reactive products in bronchoalveolar lavage fluid of rats exposed to phosgene (0.5 ppm X 60 min). Cyclophosphamide, AA861, and colchicine also significantly decreased mortality from phosgene (2.0 ppm X 90 min) in mice. Colchicine significantly reduced neutrophil influx, lung injury, and mortality even when given 30 min after phosgene exposure. We conclude that lung injury and mortality after phosgene exposure are associated with an influx of neutrophils into the lung. Prevention of neutrophil migration with colchicine may hold therapeutic potential in phosgene poisoning.     

ONTOGENETIC AND HISTOCHEMICAL CHANGES IN THE VEGETATIVE SHOOT TIP OF CHENOPODIUM ALBUM

Gifford , Ernest M., Jr ., and Herbert B. Tepper . (U. California, Davis.) Ontogenetic and histochemical changes in the vegetative shoot tip of Chenopodium album. Amer. Jour. Bot. 49(8): 902–911. Illus. 1962.—A distinct central zone is present in apices of young, rapidly growing seedlings of Chenopodium album. This zone is clearly evident when the plane of section includes developing leaf buttresses and/or newly formed leaf primordia, but is not visible if the cells associated with these developing foliar organs are not in the plane of section. Cells in the central zone contain markedly lower concentrations of RNA than those of the peripheral zone. Differences in DNA, SH-protein, total protein, and histone concentration of cells in the apex parallel the zonal distribution of RNA, but are much less obvious. As the plant ages and the rate of leaf production decreases, the zonal distribution of the above-mentioned compounds becomes less and less obvious. Cells at sites of leaf initiation in older seedlings still have slightly higher RNA concentrations, but a central zone such as is visible in the young seedlings is no longer evident. The axial tunica cells do, however, contain noticeably lower DNA and histone concentrations than the remaining cells of the shoot apex. Changes occur in the size of nucleoli during ontogeny and the possible relationship between nucleolar size and modifying factors is discussed.     

Computational design of auxotrophy-dependent microbial biosensors for combinatorial metabolic engineering experiments

Combinatorial approaches in metabolic engineering work by generating genetic diversity in a microbial population followed by screening for strains with improved phenotypes. One of the most common goals in this field is the generation of a high rate chemical producing strain. A major hurdle with this approach is that many chemicals do not have easy to recognize attributes, making their screening expensive and time consuming. To address this problem, it was previously suggested to use microbial biosensors to facilitate the detection and quantification of chemicals of interest. Here, we present novel computational methods to: (i) rationally design microbial biosensors for chemicals of interest based on substrate auxotrophy that would enable their high-throughput screening; (ii) predict engineering strategies for coupling the synthesis of a chemical of interest with the production of a proxy metabolite for which high-throughput screening is possible via a designed bio-sensor. The biosensor design method is validated based on known genetic modifications in an array of E. coli strains auxotrophic to various amino-acids. Predicted chemical production rates achievable via the biosensor-based approach are shown to potentially improve upon those predicted by current rational strain design approaches. (A Matlab implementation of the biosensor design method is available via http://www.cs.technion.ac.il/~tomersh/tools).