Injection of the Arterial System of the Mouse

Cannulation of the aorta of the mouse through an incision in the left ventricle has permitted the introduction of various injection media into the arterial tree. Heparinized mice, sacrificed by ether, were exsanguinated and injected with latex, latex-thorotrast or vinyl acetate. The apparatus consists of a special cannula made by combining a 1 inch segment of a 23 gauge hypodermic needle and fine polyethylene tubing (PE 50, intramedic). Nearly all of the vascular tree was injected with this technic. Latex-thorotrast injections have the decided advantage of permitting combined X-ray and histologic study of the material. The procedure was devised in the course of an attempt to evaluate the relationship between circulatory changes and experimentally produced bone absorption and new bone formation in mice.     

Allocation of nitrogen to an inducible defense and seed production in Nicotiana attenuata

Resource-based tradeoffs in the allocation of a limiting resource are commonly invoked to explain negative correlations between growth and defense in plants, but critical examinations of these tradeoffs are lacking. To rigorously quantify tradeoffs in a common currency, we grew Nicotiana attenuata plants in individual hydroponic chambers, induced nicotine production by treating roots with methyl jasmonate (MJ) and standardized leaf puncturing, and used ¹⁵N to determine whether nitrogen-based tradeoffs among nicotine production, growth, and seed production could be detected. Plants were treated with a range of MJ quantities (5, 45 or 250 μg plant^?1) to effect a physiologically realistic range of changes in endogenous jasmonic acid levels and increases in nicotine production and accumulation; MJ treatments were applied to the roots to target JA-induced nicotine production, since nicotine biosynthesis is restricted to the roots. Leaf puncturing and 5 μg MJ treatments increased de novo nicotine synthesis and whole-plant (WP) nicotine pools by 93 and 66%, while 250 μg MJ treatments increased these values 3.1 and 2.5-fold. At these high rates of nicotine production, plants incorporated 5.7% of current nitrogen uptake and 6.0% of their WP nitrogen pools into nicotine. The ¹⁵N-labeled nicotine pools were stable or increased for the duration of vegetative growth, indicating that the N-nicotine was not metabolized and re-used for growth. Plants with elevated nicotine production grew more slowly and the differences in plant biomass gain between MJ-treated plants and controls were linearly related to the differences in nicotine accumulation. Despite the reductions in rosette-stage growth associated with nicotine production, estimates of lifetime fitness (cumulative lifetime seed production, mass/seed, seed viability) were not affected by any treatment. Only two treatments (leaf puncturing and 250 μg MJ) increased the allocations of ¹⁵N acquired at the time of induction to seed production. On average, plants used only 14.9% of their WP nitrogen pool for seed production, indicating that either the nitrogen requirements for seed production or the reproductive effort of these hydroponically-grown plants are low. To determine if seed production is strongly influenced by the amount of vegetative biomass attained before reproduction, the experiment was repeated with plants that had 44% of their leaf area (or 29% of their WP biomass) removed before MJ treatments with a removal technique that minimized the nicotine response. MJ treatments of these plants dramatically increased nicotine production and accumulation, but these plants also suffered no measurable fitness consequences from either the leaf removal or MJ treatments. We conclude that when N. attenuata plants are grown in these individual hydroponic chambers, their allocation to reproduction is sufficiently buffered to obscure the large increases in nitrogen allocations to an inducible defense. To determine whether soil-grown plants are similarly buffered, we grew two genotypes of plants in the high-nutrient soil from a 1-year-old burn in a piñyon-juniper forest (the plants' natural habitat) and in low-nutrient soil from an adjacent unburned area, and induced nicotine production in half of the plants with a 500 μg root MJ treatment. Plants grown in burned soils had an estimated lifetime fitness that was on average 2.8-fold greater than that of plants grown in unburned soils. MJ treatment reduced fitness estimates by 43% and 71% in the burned and unburned soils, respectively. We conclude that while hydroponic culture allows one to rigorously quantitate nitrogen allocation to growth, reproduction and defense, the allocation patterns of plants grown in hydroponic culture differ from those of plants grown in soil. Under hydroponic conditions, plants have low reproductive allocations and reproductive-defense tradeoffs are not detected. Reproductive-defense tradeoffs are readily discernible in soil-grown plants, but under these growing conditions, the nitrogen-basis for the tradeoff is difficult to quantify.     

Some mechanisms of salt tolerance in crop plants

Summary In the first part of this review the main features of salt tolerance in higher plants are discussed. The hypothesis of intracellular compartmentation of solutes is used as a basis for models of tolerance mechanisms operating in roots and in leaves. Consideration is given to the implications of the various mechanisms for the yield potential of salt-tolerant crop plants.Some work on the more salt-tolerant members of the Triticeae is then described. The perennial speciesElytrigia juncea andLeymus sabulosus survive prolonged exposure to 250 mol m^–3 NaCl, whereas the annual Triticum species are severely affected at only 100 mol m^–3 NaCl. In the perennial species the tissue ion levels are controlled within narrow limits. In contrast, the more susceptible wheats accumulate far more sodium and chloride than is needed for osmotic adjustment, and the effects of salt stress increase with time of exposure.Two different types of salt tolerance are exhibited in plants capable of growing at high salinities. In succulent Chenopodiaceae, for example, osmotic adjustment is achieved mainly by accumulation of high levels of sodium and chloride in the shoots, accompanied by synthesis of substantial amounts of the compatible solute glycinebetaine. This combination of mechanisms allows high growth rates, in terms of both fresh and dry weight. At the opposite end of the spectrum of salt tolerance responses are the halophytic grasses, which strictly limit the influx of salts into the shoots, but suffer from very much reduced growth rates under saline conditions. Another variation is shown in those species that possess salt glands. The development and exploitation of crop plants for use on saline soils is discussed in relation to the implications of these various mechanisms.     

Purification and properties of the S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B

The S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B, was separated from other alkylsulphohydrolases and purified to homogeneity. Under the experimental conditions used the enzyme completely hydrolysed d-octan-2-yl sulphate (d-1-methylheptyl sulphate), but showed no activity towards the corresponding l-isomer. Additional evidence has been obtained to indicate that it is probably optically stereospecific for d-secondary alkyl sulphate esters with the ester sulphate group at C-2 and with a chain length of at least seven carbon atoms. Enzyme activity towards racemic samples of heptan-2-yl sulphate (1-methylhexyl sulphate), octan-2-yl sulphate and decan-2-yl sulphate (1-methylnonyl sulphate) increased with increasing chain length. l-Octan-2-yl sulphate is a competitive inhibitor of the enzyme, as are certain primary alkyl sulphates and primary alkanesulphonates. Inhibition by each of the last two types of compounds is characteristic of the behaviour of an homologous series. Inhibition increases with increasing chain length and plots of log K(i) values against the number of carbon atoms in each alkyl chain show the expected linear relationship. A crude preparation of the S2 secondary alkylsulphohydrolase was used to show that this particular enzyme hydrolyses l-octan-2-yl sulphate, but is probably inactive towards the corresponding d-isomer. The similarity of the S1 and S2 enzymes to the CS2 and CS1 enzymes respectively of Comamonas terrigena was established, and some comments have been made on the possible roles of these and other alkylsulphohydrolases in the biodegradation of detergents.     

Effects of calcium on growth and leaf ion concentrations of Gossypium hirsutum grown in saline hydroponic culture

The optimum Ca²⁺ concentration for growth of cotton (Gossypium hirsutum cv. Acala SJ-2) was in the range 1 to 15 mol m^–3 for plants growing in hydroponic culture with 100–150 mol m^–3 NaCl. Most saline (but not sodic) soils contain higher Ca²⁺ concentrations. CaCl₂ was inhibitory to the growth of cotton above 20–50 mol m^–3. Increasing concentrations of Ca²⁺ in the range 0–2 mol m^–2 drastically reduced Na⁺ accumulation in the leaves. As CaCl₂ concentrations were increased above the optimum for growth there was a further reduction in leaf Na⁺ accumulation, but this was more than offset by increased leaf Ca²⁺ and Cl^– concentrations. Leaf K⁺ concentrations were not much affected by changes in external CaCl₂ concentrations. The response of Mg²⁺ varied from an increase to a decrease with increasing external CaCl₂ and was influenced by nutritional status. There was no evidence that high Ca²⁺ caused a deficiency of Mg²⁺ in cotton. Except for Cl^–, whose concentrations tended to decrease initially and then increase as the CaCl₂ concentration increased, the anions were largely unaffected by changes in external CaCl₂.     

TGF-beta 1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4+ T cells at priming and at recall: differential involvement of Stat4 and T-bet

TGF-beta1 plays a critical role in restraining pathogenic Th1 autoimmune responses in vivo, but the mechanisms that mediate TGF-beta1's suppressive effects on CD4(+) T cell expression of IFN-gamma expression remain incompletely understood. To evaluate mechanisms by which TGF-beta1 inhibits IFN-gamma expression in CD4(+) T cells, we primed naive wild-type murine BALB/c CD4(+) T cells in vitro under Th1 development conditions in the presence or the absence of added TGF-beta1. We found that the presence of TGF-beta1 during priming of CD4(+) T cells suppressed both IFN-gamma expression during priming as well as the development of Th1 effector cells expressing IFN-gamma at a recall stimulation. TGF-beta1 inhibited the development of IFN-gamma-expressing cells in a dose-dependent fashion and in the absence of APC, indicating that TGF-beta1 can inhibit Th1 development by acting directly on the CD4(+) T cell. During priming, TGF-beta1 strongly inhibited the expression of both T-bet (T box expressed in T cells) and Stat4. We evaluated the importance of these two molecules in the suppression of IFN-gamma expression at the two phases of Th1 responses. Enforced expression of T-bet by retrovirus prevented TGF-beta1's inhibition of Th1 development, but did not prevent TGF-beta1's inhibition of IFN-gamma expression at priming. Conversely, enforced expression of Stat4 partly prevented TGF-beta1's inhibition of IFN-gamma expression during priming, but did not prevent TGF-beta1's inhibition of Th1 development. These data show that TGF-beta1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4(+) T cells at priming and at recall.     

Genetic regulation of autoimmune disease: BALB/c background TGF-beta 1-deficient mice develop necroinflammatory IFN-gamma-dependent hepatitis

Autoimmune hepatitis (AIH) in humans arises spontaneously in genetically susceptible individuals and is associated with the presence of Th1 cells in the liver. The understanding of AIH has advanced more slowly than that of other organ-specific autoimmune diseases, however, largely because of the lack of an appropriate animal model. We now describe a new mouse model characterized by spontaneous development of necroinflammatory hepatitis that is restricted by genetic background. Mice deficient in the immunomodulatory cytokine TGF-beta1 were extensively back-bred to the BALB/c background. The BALB/c background dramatically modified the phenotype of TGF-beta1(-/-) mice: specifically, BALB/c-TGF-beta1(-/-) mice developed a lethal necroinflammatory hepatitis that was not observed in TGF-beta1(-/-) mice on a different genetic background. BALB/c background TGF-beta1(-/-) livers contained large numbers of activated CD4(+) T cells that produced large quantities of IFN-gamma, but little IL-4, identifying them as Th1 cells. BALB/c background TGF-beta1(-/-)/IFN-gamma(-/-) double knockout mice, generated by cross-breeding, did not develop necroinflammatory hepatitis, demonstrating that IFN-gamma is mechanistically required for its pathogenesis. This represents the first murine model of hepatitis that develops spontaneously, is restricted by genetic background, and is dependent upon the Th1 cytokine IFN-gamma, and that thus recapitulates these important aspects of AIH.     

Betaine distribution in the Malvaceae

Aerial parts of 26 taxa, distributed in 18 genera and all 5 tribes of the Malvaceae have been examined for the presence of betaines. Glycinebetaine was obtained in high yield (0.5-4.6%, dry weight) from all the plants studied, except Abelmoschus moschatus, in extracts of which glycinebetaine was not detected. Trigonelline was recorded for 16 of the plants tested, but the yields were low (0.005-0.07%, dry weight). Roots and flowers of a few of the species were also examined for betaines. The same compounds as those found in the aerial parts were usually detected, but the glycinebetaine contents of the roots and flowers were considerably lower.