GROWTH PATTERNS IN NUTATING AND NONNUTATING SUNFLOWER (HELIANTHUS ANNUUS) HYPOCOTYLS

Dark-grown sunflower (Helianthus annuus L.) seedling hypocotyls (15–30 mm) were marked with two rows of lanolin-coated resin beads, and the events of the following 24 hr, in physiological darkness, were recorded on time-lapse video. Nutational movement of the hypocotyl, followed for 20–24 hr for each of 21 seedlings, was found to have a mean period of 153 ± 26 min (ca. 24 C). Displacement of each bead, with time, was measured with a microcomputer-controlled video analyzer, and relative elemental elongation rate and relative growth rate analyses were carried out to determine the spatial and temporal distribution of growth. Relative elemental elongation rates were plotted against distance and time to produce “growth landscapes.” A strongly nutating seedling showed periodic fluctuations in local growth rates that alternated between values of 0.0 hr^−-1 and >0.12 hr^−-1 near the hypocotyl hook. Nearer the base, maximum growth rates were lower but local periodic changes still were evident. Seedlings, in which nutation appeared during the time period analyzed, showed non-synchronous pulses of growth along the axis. With nutational development, these local growth fluctuations became synchronized along each side and phased (usually 180° out of phase) with the coordinated growth fluctuations along the opposite side. In some seedlings the changes from low to high local growth rates occur nearly simultaneously over two-thirds of the active region. In others, basipetally traveling waves of growth are suggested by the growth landscapes.     

Variations in the amount of glucose phosphate isomerase in lymphocytes and erythrocytes from A/J and C57BL/6J mice

In a comparative study of A/J (Gpi-1a) and C57BL/6J (Gpi-1b) mice, we observed that erythrocytes of A/J mice exhibited significantly higher glucose phosphate isomerase (GPI) activity compared to erythrocytes of C57BL/6J mice on a per cell, per gram of protein, or per gram of hemoglobin basis. Higher GPI activity per cell was detected for peripheral blood lymphocytes of A/J compared to C57BL/6J mice. (A/J X C57BL/6J)F1 mice expressed erythrocyte and peripheral blood lymphocyte GPI activities intermediate to those of the parental mouse strains. The GPI activities of spleen lymphocytes from A/J, C57BL/6J, or (A/J X C57BL/6J)F1 mice were not significantly different from each other. The higher activity in the A/J mice could be due to GPI of a higher catalytic rate or to the presence of more GPI molecules. In order to distinguish these two possibilities, GPI was purified to homogeneity from both strains of mice. The specific activities (activity per milligram of protein) of the purified enzymes from the two strains were found to be similar, indicating that GPI from the A/J strain was not a more active enzyme. Antibody to the purified enzymes was prepared and used in an enzyme-linked immunosorbent assay (ELISA) to compare the relative amounts of enzyme molecules in cells of A/J and C57BL/6J mice. Results of the ELISA tests on peripheral blood lymphocytes indicated that A/J mice contain more molecules of GPI per cell and, therefore, have a higher GPI activity than C57BL/6J mice.     

Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major

Mitchell G. F., Handman E. and Spithill T. W. 1985. Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major. International Journal for Parasitology15: 677–684. In vaccination experiments not involving adjuvants, genetically-susceptible mice were injected with living avirulent cloned promastigotes of Leishmania major or killed promastigotes prior to cutaneous challenge with virulent cloned promastigotes. Emphasis was placed on aspects that may contribute to marked variability between experiments and between laboratories in vaccination of mice against cutaneous leishmaniasis. One variable, the challenge promastigotes, was shown to be important in that cloned virulent parasites (V121) were less pathogenic in terms of rate of cutaneous lesion development, than the parental isolate LRC-L137 when low doses of promastigotes were used and particularly when harvested from the log phase of culture. It is likely that avirulent parasites in mixed isolates can increase the rate of lesion development after cutaneous deposition. As reported previously, intraperitoneal, and more particularly intravenous injections of living avirulent cloned parasites (A12) increase resistance in mice. Most importantly, a difference has been demonstrated in the vaccinating efficacy of killed promastigotes of various isolates injected intravenously. This implies that certain isolates of L. major (e.g. the “Moshkovsky strain”) express “host-protective antigens” at higher levels, or in a qualitatively different manner, than other isolates (e.g. LRC-L137). The finding will greatly facilitate the identification of vaccine antigens in this system using immunochemical and gene cloning approaches.     

Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis

The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).     

Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases

Restriction endonuclease-digested DNAs from several isolates of phase I and phase II Coxiella burnetii were compared using agarose gel electrophoresis and soft-laser scanning densitometry. Our results demonstrate that the two phases are, as previously assumed, alternative phases of the same organism. Although the restriction endonuclease digestion revealed genetic differences between clonal isolates of phase I and phase II C. burnetii Nine Mile strain, these differences do not appear to be related to antigenic phase variation. However, analyses of the fragment patterns generated by restriction enzyme digestion suggest potential grouping of the different isolates.     

Lowering extracellular sodium or pH raises intracellular calcium in gastric cells

Summary The dependence of cytoplasmic free [Ca] (Ca _i ) on [Na] and pH was assessed in individual parietal cells of intact rabbit gastric glands by microfluorimetry of fura-2. Lowering extracellular [Na] (Na _o ) to 20mm or below caused a biphasic Ca _i increase which consisted of both release of intracellular Ca stores and Ca entry across the plasma membrane. The Ca increase was not blocked by antagonists of Ca-mobilizing receptors (atropine or cimetidine) and was independent of the replacement cation. Experiments in Ca-free media and in Na-depleted cells indicated that neither phase was due to reversal of Na/Ca exchange. The steep dependence of the Ca _i increase on Na _o suggested that the response was not due to lowering intracellular [Na] (Na _i ). The effects of low Na _o on Ca _i were also completely independent of changes in intracellular pH (pH _i ). Ca _i was remarkably stable during changes of pH _i of up to 2 pH units, indicating that H and Ca do not share a cytoplasmic buffer system. Such large pH excursions required determination of the pH dependence of fura-2. Because fura-2 was found to decrease its affinity for Ca as pH decreased below 6.7, corrections were applied to experiments in which large pH _i changes were observed. In contrast to the relative insensitivity of Ca _i to changes in pH _i , decreasing extracellular pH (pH _o ) to 6.0 or below was found to stimulate release of intracellular Ca stores. Increased Ca entry was not observed in this case. The ability of decreases in Na _o and pH _o to stimulate release of intracellular Ca stores suggest interactions between Na and H with extracellular receptors.     

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

Microorganisms for degrading simmondsin and related cyanogenic toxins in jojoba

Summary Four microorganisms that metabolize simmondsin (S) and related cyanogenic toxins from jojoba (Simmondsia chinensis) were isolated by enrichment: Pseudallescheria boydii, a fungus which specifically degrades simmondsin ferulate but not S; Fusarium moniliforme; Flavobacterium aurantiacum; and Pseudomonas maltophilia. The latter three organisms grow on S as a sole carbon and nitrogen source in culture media, but only F. moniliforme attacks S in the complete jojoba meal. Combinations of the four microorganisms at two temperatures, and with free air or limited air exchange for up to 20 days, were tested on jojoba meal to determine an optimum detoxification method. Degradation of toxins was most rapid and complete when Pseudallescheria boydii and Fusarium moniliforme together were incubated on jojoba meal at 25°C with free air exchange for 20 days. Mice were fed fermented meals at 0, 5, 10 and 20% substitution levels to determine detoxification and nutritional quality. Average daily gains during rapid growth of weanling (1–3 weeks) and mature (4–8 weeks) mice did not differ significantly from controls for mice on all diets containing fermented meal. Diets containing fungally detoxified jojoba meal were more efficient for maintaenance of mature weight than jojoba meal detoxified with enzymes naturally present in the meal. Meal can be detoxified by ensilage for 20 days at 80% water content. Detoxification is attributed to as yet unidentified enzymes inherent in the jojoba seed.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentionedOffprint requests to: Thomas P. Abbott     

Changes in Thylakoid Galactolipids and Proteins during Iron Nutrition-Mediated Chloroplast Development

Changes in the amounts of thylakoid galactolipids and proteins were monitored for 96 hours following iron resupply to iron-deficient sugar beet (Beta vulgaris L. cv F58-554H1) plants. During this period of iron nutrition-mediated chloroplast development, the amount of galactolipid per leaf area increased linearly with time. Assuming galactolipids are an index for the amount of thylakoids, then there was a linear synthesis of thylakoid membranes during regreening. Total thylakoid protein synthesis, however, lagged behind galactolipid synthesis, suggesting that proteins are inserted secondarily into the galactolipid matrix of the thylakoid membrane during development.

Iron deficiency caused an increase in the free chlorophyll band under the conditions of gel electrophoresis used. Of the chlorophyll proteins resolved, the chlorophyll protein associated with photosystem I was most diminished in iron-deficient tissue, and appeared to recover most rapidly. Changes in the light-harvesting chlorophyll proteins are also discussed.

The number of polypeptides resolved by lithium dodecyl sulfate-polyacrylamide gel electrophoresis was higher in iron-deficient thylakoids. During regreening, the number of resolved polypeptides decreased.