Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K⁺ concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H⁺ fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.     

Photosynthesis of cotton plants exposed to elevated levels of carbon dioxide in the field

The cotton (Gossypium hirsutum L.) plant responds to a doubling of atmospheric CO₂ with almost doubled yield. Gas exchange of leaves was monitored to discover the photosynthetic basis of this large response. Plants were grown in the field in open-top chambers with ambient (nominally 350 l/l) or enriched (nominally either 500 or 650 l/l) concentrations of atmospheric CO₂. During most of the season, in fully-irrigated plants the relationship between assimilation (A) and intercellular CO₂ concentration (c_i) was almost linear over an extremely wide range of c_i. CO₂ enrichment did not alter this relationship or diminish photosynthetic capacity (despite accumulation of starch to very high levels) until very late in the season, when temperature was somewhat lower than at midseason. Stomatal conductance at midseason was very high and insensitive to CO₂, leading to estimates of c_i above 85% of atmospheric CO₂ concentration in both ambient and enriched chambers. Water stress caused A to show a saturation response with respect to c_i, and it increased stomatal closure in response to CO₂ enrichment. In fully-irrigated plants CO₂ enrichment to 650 l/l increased A more than 70%, but in water-stressed plants enrichment increased A only about 52%. The non-saturating response of A to c_i, the failure of CO₂ enrichment to decrease photosynthetic capacity for most of the season, and the ability of the leaves to maintain very high c_i, form in part the basis for the very large response to CO₂ enrichment.Abbreviations c_a- atmospheric CO₂ concentration - c_i- intercellular CO₂ concentration - A- rate of assimilation of CO₂ - g_s- stomatal conductance to water vapor - g_b- boundary layer conductance to water vapor - g_m- mesophyll conductance to CO₂ - VPD- vapor pressure deficit - _w leaf water potential - L- stomatal limitation to CO₂ uptake     

Localization of a DNA-binding determinant in the bacteriophage P22 Erf protein

Four amber fragments of the recombination-promoting P22 Erf protein were characterized. The intact Erf monomer contains 204 amino acids. The amber mutations produce fragments of 190, 149, 130 and 95 amino acid residues, all of which are inactive in vivo. The 190 residue fragment is more susceptible to proteolysis in cell extracts than is intact Erf. It breaks down to a stable remnant that is slightly larger than the 149 residue fragment. The 149 and 130 residue fragments are stable; electron microscopy of the purified fragments reveals that they have similar morphologies, retaining the ring-like oligomeric structure, but lacking the tooth-like protruding portions of intact Erf. Intact Erf and the 149 residue fragment have similar affinities for single-stranded DNA; the affinity of the 130 residue fragment is 40-fold lower in low salt at pH 6.0. The 95 residue fragment is unstable in vivo. These observations, combined with previous observations, are interpreted as suggesting that the boundary of the amino-terminal domain of the protein lies between residues 96 and 130, that certain residues between 131 and 149 form part of an interdomain DNA-binding segment of the protein, that the boundary of the carboxy-terminal domain lies to the C-terminal side of residue 149, and that the carboxy-terminal domain is not necessary for assembly of the ring oligomer, although it is essential for Erf activity in vivo.     

Nitrogen dynamics in conventional and no-tillage agroecosystems with inorganic fertilizer or legume nitrogen inputs

Summary Nitrogen (N) dynamics were studied in conventional and no-tillage agroecosystems on the Georgia Piedmont. Nitrogen inputs in the form of ammonium nitrate (95 kg-N/ha) and winter legume (crimson clover) residues were compared. The legume provided adequate N for summer crop (grain sorghum) production, but water use by the legume reduced the ability of sorghum to utilize the N. Legume N inputs became availabe to plants more gradually than fertilizer inputs. Weed growth and potential denitrification activity were higher in legume-N treatments than in fertilizer-N treatments. Tillage affected the timing of N avaialbility more than the total amount of available N. Summer crops took up more N under conventional tillage while winter crops took up more N under no-tillage. Denitrification activity was higher under no-tillage but was not a significant output from the agroecosystems. Leaching losses of N differed seasonally between treatments but were not significant in any of the agroecosystems.     

Examination of sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals for antibodies reactive with peptides corresponding to the principal neutralizing determinant of HIV-1 gp120 and for in vitro neutralizing activity.

Sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals from the United States and Tanzania were examined for antibody reactivity to four synthetic peptides which corresponded to the principal neutralizing determinant from the V3 region of HIV-1 gp120. We observed that the majority of sera from both countries contained antibodies reactive with a V3 peptide whose sequence is based on that of the HIV-1 MN isolate. We were unable to establish a relationship between the presence of V3-reactive antibodies, as measured by enzyme-linked immunosorbent assay and neutralization of homologous HIV-1 isolates, in sera from either the United States or Tanzania. We observed that some sera which contained high antibody titers to the V3 peptides failed to neutralize HIV-1, while others with no antibody reactivity to the panel of V3 peptides exhibited in vitro neutralizing activity. These results suggest that neutralizing epitopes exist outside the V3 loop and that the presence of V3-reactive antibodies in sera does not imply in vitro neutralization of the homologous HIV-1 isolate. In addition, it appears that the V3 loop may consist of both neutralizing and nonneutralizing epitopes. The identification of neutralizing as well as nonneutralizing epitopes will be important for the design of potential HIV-1 vaccines.     

Topographical EEG maps of human responses to odors

The physiological response to seven odors (birch tar, galbanum,heliotropine, jasmine, lavender, lemon and peppermint) was assessedby EEG recordings from 19 scalp loci from 16 young adult females.Topographic maps were constructed from the amplitude spectrain four frequency bands: delta (1–4 Hz), theta (4–8Hz), alpha (8–13 Hz) and beta (13–30 Hz). Eightseconds of representative and artifact-free EEG were selectedfor FFT analysis before onset of odor delivery, and at threetimes after stimulus onset. EEG was also quantified at 30 safter stimulus termination. Subjects differed in their subjective responses to the odors,with the most consistently arousing and strong odors being galbanum,lavender, lemon and peppermint. Heliotropine was notably weak.The most pleasant odors were lemon and peppermint, while birchtar, galbanum and lavender were consistently unpleasant. EEGmap changes occurred in one or more frequency bands in eachsubject in response to one or more of the odors. EEG map changessometimes occurred even with weak odors and even when the subjectseemed unaware of the odor's presence. This was most notablewith heliotropine. Across subjects, the most consistent responses to odors werein the theta band. Analysis of variance confirmed that certainodors caused statistically significant theta increases overthe left anterior group of electrodes. Both right hemispheregroups tended to have significant theta increases. The odorsthat caused the greatest increase in theta were birch tar, jasmine,lavender and lemon. On the other hand, during blank controltrials, theta activity diminished. There was also a significantodor by epoch interaction over the right posterior set of electrodes.Several of the odors caused increased theta at one or more epochs.Lemon caused an immediate increase in theta that abated withtime. Birch tar induced a delayed response that persisted afterthe stimulus was turned off. Jasmine and lavender tended toinduce theta sooner than birch tar, but the effect did not outlastthe stimulus. Increased theta was not associated with EEG signsof drowsiness. We conclude that all odors affected the EEG in at least somesubjects, and all subjects responded to at least some odors.Widespread increase in theta occurred in most subjects duringstimulation with such odors as birch tar, jasmine, lavenderand lemon.     

Isolation and Expression of an Arrestin cDNA from the Horseshoe Crab Lateral Eye

Abstract: Electrophysiological studies of photoreceptors from the horseshoe crab Limulus polyphemus continue to provide fundamental new knowledge of the photoresponse in invertebrates. Therefore, it is of particular interest to characterize the molecular components of the photoresponse in this system. Here we describe an arrestin cloned from a cDNA library constructed using poly(A)⁺ RNA isolated from Limulus lateral eyes. The protein, deduced from the arrestin cDNA, is most similar to arrestin from locust antennae (56% identity) and Drosophila phosrestin I (53% identity). Limulus arrestin was expressed in a heterologous system, and its properties were compared with those of a 46-kDa light-regulated phosprotein (pp46A) in Limulus photoreceptors described in previous studies from this laboratory. Arrestin and pp46A (a) have the same apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) have an isoelectric point in the basic pH range, (c) require calmodulin and elevated Ca²⁺ levels for phosphorylation, (d) are immunoreactive with monoclonal antibody C10C10 directed against a sequence in bovine arrestin (S-antigen) that is perfectly conserved in the deduced arrestin protein, and (e) are associated with photoreceptors. We conclude that the arrestin described here and pp46A are the same protein. The results of this and previous studies show that in Limulus photoreceptors, light regulates the phosphorylation of arrestin in complex ways.     

Further characterization of a clinically relevant model of melanoma metastasis and an effective vaccine

A major problem in evaluating the effectiveness of tumor cell vaccination and other biological therapies is the variability of experimental models. In this study we have further developed and characterized a model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination: stage I-growth of the primary (local) tumor, stage II-dissemination to regional lymph nodes, and stage III-metastasis to distant organs (lungs). C57BL/6 mice were challenged subcutaneously with B16 F10 murine melanoma cells in the midtail, and within 3 weeks 100% of the mice had local tumors growing in their tails. By 5–7 weeks after challenge, most of the mice had developed metastases to the inguinal lymph nodes and subsequently had metastatic colonies in the lungs and in the bone marrow. Preimmunization of mice with a formalinized extracellular antigen vaccine, derived from B16F10 melanoma cells, provided partial inhibition of the growth of the primary melanoma tumors, as well as reducing the number of metastases to the regional (inguinal) lymph nodes and lungs along with concomitantly increasing survival time. This model for melanoma metastasis provides a reasonable and reproducible test system for the study of anti-melanoma immunity and the different cellular and humoral mechanisms involved.This work was supported in part by National Institutes of Health grants R37 CA45148 and R30 CA13943     

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

Structural studies of bacteriophage lambda heads and proheads by small angle X-ray diffraction.

We have examined a series of lambda proheads and mature structures by small angle X-ray diffraction. This technique yields spherically averaged density distributions and some information about surface organization of particles in solution.We find that gpE ² of proheads and heads forms shells with one of two radii; A^?, B^?, groE^?, and Nu3^? proheads have shells of radius 246 Å, while mature heads, urea-treated A^? proheads and C^? proheads have a radius of 300 Å. The expansion of proheads to mature heads is accompanied by a corresponding decrease in the thickness of the shell. groE^? proheads contain a core. This core is lost spontaneously from the structure and is only observed if the structures are fixed with glutaraldehyde prior to examination by X-ray diffraction or electron microscopy.C^? proheads expand to mature head size spontaneously. A preparation of C^? proheads which was fixed with glutaraldehyde at an early stage of the purification had the smaller, prohead radius. Unfixed particles from this preparation expanded to the mature head size after further purification and standing in the cold for several days. This result suggests that gpC may be involved in regulating head expansion.The radii of the protein shells of mature heads are identical for a series of phages that contain between 78% and 105% of the wild-type complement of DNA, and this radius is the same as that of proheads expanded in the absence of DNA. These results with phage lambda indicate that assembly of a double shell structure composed of coat and scaffolding protein, followed by expansion to a larger shell containing only coat protein is a general feature of the morphogenesis of dsDNA phages.