HLA Matching at the Eplet Level Protects Against Chronic Lung Allograft Dysfunction.

Donor selection in lung transplantation (LTx) is historically based upon clinical urgency, ABO compatibility, and donor size. HLA matching is not routinely considered; however, the presence or later development of anti-HLA antibodies is associated with poorer outcomes, particularly chronic lung allograft dysfunction (CLAD). Using eplet mismatches, we aimed to determine whether donor/recipient HLA incompatibility was a significant predictor of CLAD. One hundred seventy-five LTx undertaken at the Alfred Hospital between 2008 and 2012 met criteria. Post-LTx monitoring was continued for at least 12 months, or until patient death. HLA typing was performed by sequence-based typing and Luminex sequence-specific oligonucleotide. Using HLAMatchmaker, eplet mismatches between each donor/recipient pairing were analyzed and correlated against incidences of CLAD. HLA-DRB1/3/4/5+DQA/B eplet mismatch was a significant predictor of CLAD (hazard ratio [HR] 3.77, 95% confidence interval [CI]: 1.71-8.29 p < 0.001). When bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) were analyzed independently, HLA-DRB1/3/4/5 + DQA/B eplet mismatch was shown to significantly predict RAS (HR 8.3, 95% CI: 2.46-27.97 p < 0.001) but not BOS (HR 1.92, 95% CI: 0.64-5.72, p = 0.237). HLA-A/B eplet mismatch was shown not to be a significant predictor when analyzed independently but did provide additional stratification of results. This study illustrates the importance of epitope immunogenicity in defining donor-recipient immune compatibility in LTx.

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves.

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.

Effects of cycloheximide on abscisic Acid biosynthesis and stomatal aperture in bean leaves.

Cycloheximide was shown to block abscisic acid (ABA) biosynthesis in nonstressed as well as in stressed Phaseolus vulgaris leaves. Leaf wilting caused by cycloheximide resulted from increased stomatal opening as judged by a decreased stomatal diffusion resistance. The inhibition of ABA biosynthesis by cycloheximide was at least partially responsible for the increase in stomatal opening as suggested by the cooccurrence of inhibition of ABA biosynthesis and increased stomatal opening, and the partial reversal of stomatal opening in cycloheximide-treated leaves by exogenous ABA. Dark treatment failed to close stomatal in cycloheximide-treated leaves, suggesting that stomatal closure in response to darkness may normally be mediated by ABA.

Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves.

The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9'-cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an (18)O(2)-containing atmosphere resulted in the synthesis of ABA with levels of (18)O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA.

Seasonal patterns of juglone in soil beneathJuglans nigra (black walnut) and influence ofJ. nigra on understory vegetation.

The allelopathic nature ofJ. nigra L. was investigated in several planted mixed hardwood stands located near Syracuse, New York. Concentrations of chloroform-extracted juglone from soil collected beneathJ. nigra was determined by thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). Soil juglone concentrations were corrected based on recovery of synthetic juglone added to soil. Soil juglone levels were high in the spring, decreased during the summer, and were high again in the fall. The quantification of juglone from soil by HPLC was found to be more accurate than by TLC. Regression analysis indicated that individual tree variation in soil juglone levels could not be explained by differences in soil moisture, pH, organic matter content, and texture. The results of juglone recovery experiments suggest that chloroform-extractable juglone does not persist in soil. Juglone degradation by microorganisms could only explain a portion of the juglone decline. Ordinations revealed that the herbaceous and woody vegetation beneathJ. nigra, in comparison to vegetation beneathAcer saccharum andQuercus rubra, is distinct in only one of the four stands studied. This vegetational difference did not appear to be a consequence of any strong allelopathic influences ofJ. nigra (Scheffe's method of contrast, chi-square analysis). The allelopathic nature of juglone under these field conditions is questionable.

A comparison of the crystal structures of some quaternary trimethylammonium salts related to dopamine and noradrenaline with those of the corresponding amines: a comment on their nicotine-like biological activities.

The crystal structures of seven substituted phenethylammonium salts and one (phenylpropyl)-ammonium salt have been determined. (I) Trimethyl(phenethyl)ammonium iodide, C11H18N+.I-, Mr = 291.2, orthorhombic, P2(1)2(1)2(1) (No. 19), a = 6.040 (2), b = 7.689 (2), c = 26.528 (9) A, V = 1232 (1) A3, Z = 4, Dx = 1.56 g cm-3, Mo K alpha radiation (lambda = 0.71073 A), mu = 25.33 cm-1, F(000) = 576, T = 298 K, R (wR) = 0.0302 (0.0305) for 1991 reflections with I greater than 3 sigma (I). (II) (p-Hydroxyphenethyl)trimethylammonium iodide, C11H18NO+.I-, Mr = 307.2, triclinic, P1 (No. 2), a = 9.619 (1), b = 9.926 (1), c = 14.179 (2) A, alpha = 95.24 (1), beta = 97.50 (1), gamma = 98.97 (1) degrees, V = 1317.2 (3) A3, Z = 4, Dx = 1.55 g cm-3, Mo K alpha radiation (lambda = 0.71073 A), mu = 23.79 cm-1, F(000) = 608, T = 298 K, R (wR) = 0.0351 (0.0373) for 4320 reflections with I greater than 3 sigma (I). (III) (m-Hydroxyphenethyl)trimethylammonium iodide hemihydrate, C11H18NO+.I-.1/2H2O, Mr = 316.2, monoclinic, P2(1)/n (non-standard, No. 14), a = 8.048 (2), b = 9.782 (3), c = 17.447 (7) A, beta = 90.15 (1) degrees, V = 1374 (2) A3, Z = 4, Dx = 1.53 g cm-3, Mo K alpha radiation (lambda = 0.71073 A), mu = 22.86 cm-1, F(000) = 628, T = 298 K, R (wR) = 0.0719 (0.0655) for 1006 reflections with I greater than 1 sigma (I).(ABSTRACT TRUNCATED AT 250 WORDS)

Xanthoxin Metabolism in Cell-free Preparations from Wild Type and Wilty Mutants of Tomato.

Extracts prepared from the turgid and water-stressed leaves of wild-type tomato (Lycopersicon esculentum Mill cv Ailsa Craig) and the wilty mutants sitiens, notabilis, and flacca were tested for their ability to metabolize xanthoxin to ABA. Extracts from wild type and notabilis converted xanthoxin at similar rates, while extracts from sitiens and flacca showed little or no activity. We also observed no activity when extracts of sitiens and flacca were mixed. Similar results were obtained when ABA aldehyde was used as a substrate, in that extracts from wild type and notabilis were equally active, but extracts from flacca and sitiens showed little activity. None of the tomato extracts showed significant activity with xanthoxin acid, xanthoxin alcohol, or ABA-1',4-'Trans-diol as substrates. Extracts from bean leaves (Phaseolus vulgaris L. cv Blue Lake) were similar to the wild-type tomato extracts in their ability to convert the various substrates to ABA, although excised bean leaves did convert ABA-1',4'-trans-diol and xanthoxin alcohol to ABA when these substances were taken up through the petiole. These results are consistent with a role for xanthoxin as a normal intermediate on the ABA biosynthetic pathway, and they suggest that ABA aldehyde is the final ABA precursor.

Xanthophylls and abscisic Acid biosynthesis in water-stressed bean leaves.

Experiments were designed to obtain evidence about the possible role of xanthophylls as abscisic acid (ABA) precursors in water-stressed leaves of Phaseolus vularis L. Leaves were exposed to (14)CO(2) and the specific activities of several major leaf xanthophylls and stress-induced ABA were determined after a chase in (12)CO(2) for varying periods of time. The ABA specific radioactivities were about 30 to 70% of that of lutein and violaxanthin regardless of the chase period. The specific activity of neoxanthin, however, was only about 15% of that of ABA. The effects of fluridone on xanthophyll and ABA levels and the extent of labeling of both from (14)CO(2) were determined. Fluridone did not inhibit the accumulation of ABA when leaves were stressed once, although subsequent stresses in the presence of fluridone did lead to a reduced ABA accumulation. The incorporation of (14)C from (14)CO(2) into ABA and the xanthophylls was inhibited by fluridone and to about the same extent. The incorporation of (18)O into ABA from violaxanthin which had been labeled in situ by means of the violaxanthin cycle was measured. The results indicated that a portion of the ABA accumulated during stress was formed from violaxanthin which had been labeled with (18)O. The results of these experiments are consistent with a preformed xanthophyll(s) as the major ABA precursor in water-stressed bean leaves.

Conversion of xanthoxin to abscisic Acid by cell-free preparations from bean leaves.

Cell-free extracts from the leaves of Phaseolus vulgaris L. convert xanthoxin to abscisic acid. The enzyme activity in dialyzed or acetone-precipitated extracts shows a strong dependence on either NAD or NADP. The enzyme activity appears to be cytosolic with no significant activity observed in chloroplasts. The activity was observed in extracts from roots of Phaseolus vulgaris, and also in extracts prepared from the leaves of Pisum sativum L., Zea mays L., Cucurbita maxima Duchesne, and Vigna radiata L. Neither water stress nor cycloheximide appear to significantly affect the level of enzyme activity in leaves. No intermediates between xanthoxin and abscisic acid were detected.

Biosynthesis of abscissic acid.

The fungus Cercospora rosicola has been studied as a model system for abscissic acid biosynthesis. 1'-dDeoxyabscissic acid and 4'-hydroxy-a-ionylidene acetic acid have been identified as endogenous compounds in this fungus. The results of feeding these and other putative intermediates suggest that abscissic acid biosynthesis proceeds via the successive oxidations of a 3-methyl-5-(2',6',6'-trimethylcyclohex-2'-en-1'-yl)-2,4-pentadienyl intermediate. Preliminary results suggest that a similar pathway may operate in plants.

Abscisic Acid localization and metabolism in barley aleurone layers.

Aleurone layers of Hordeum vulgare, cv. ;Himalaya' took up [(14)C]-abscisic acid (ABA) when incubated for various times. Radioactivity accumulated with time in a low speed, DNA-containing pellet accounting for 1.6 to 2.3% of the radioactivity recovered in subcellular fractions at 18 hours. Thin layer chromatography of ethanolic or methanolic extracts of the cytosol, which contained greater than 95% of the radioactivity taken up by layers, revealed that labeled ABA was metabolized to phaseic acid (PA) and 4'-dihydrophaseic acid (DPA) and three polar metabolites Mx(1), Mx(2), and Mx(3). ABA was not metabolized by endosperm, incubated under conditions used for layers, indicating that metabolism was tissue-specific. Layers metabolized [(3)H]DPA to Mx(1) and Mx(2). ABA, PA, and DPA-methyl ester and epi-DPA-methyl ester inhibited synthesis of alpha-amylase by layers incubated for either 37 or 48 hours. These layers converted the methyl DPA and epi-methyl-DPA esters to their respective acids. DPA did not inhibit Lactuca sativa germination or root and coleoptile elongation of germinating Hordeum vulgare seeds, or coleoptile elongation of germinating Zea mays seeds.

Abscisic acid levels and metabolism in the leaf epidermal tissue of Tulipa gesneriana L. and Commelina communis L.

We have shown the presence of abscisic acid (ABA) in abaxial epidermal strips taken from Tulipa gesneriana and Commelina communis and that the ABA level rises in the epidermis when leaves are water stressed. ABA levels had risen 50% in the abaxial epidermis of C. communis 30 min after the leaves lost 10% of their fresh weight. Epidermis from both T. gesneriana and C. communis metabolize [(14)C]ABA to several products probably including phaseic acid (PA) and dihydrophaseic acid (DPA).

The relationship between stomatal resistance and abscisic-acid levels in leaves of water-stressed bean plants.

Leaf water potentials of Phaseolus vulgaris L. plants exposed to a -3.0 bar root medium were reduced to between -7 and -9 bars within 25 min and remained constant for the next several hours. This treatment led to considerable variation between leaves in both abscisic-acid (ABA) content and Rs, although the two were well correlated after a 5-h treatment. There was an apparent 7-fold increase in leaf ABA levels necessary to initiate stomatal closure when plants were exposed to a -3.0 bar treatment, but when plants were exposed to a -5.0 bar stress Rs values increased prior to any detectable rise in ABA levels. To explain these seemingly contradictory results, we suggest that the rate of ABA synthesis in the leaf, rather than the total ABA content, determines the status of the stomatal aperture.

Abscisic Acid Metabolism by a Cell-free Preparation from Echinocystis lobata Liquid Endoserum.

A cell-free enzyme system capable of metabolizing abscisic acid has been obtained from Eastern Wild Cucumber (Echinocystis lobata Michx.) liquid endosperm. The reaction products were determined to be phaseic acid (PA) and dihydrophaseic acid (DPA) by co-chromatography on thin layer chromatograms as the free acids, methyl esters, and their respective oxidation or reduction products. The crude enzyme preparation was separated by centrifugation into a particulate abscisic acid (ABA)-hydroxylating activity and a soluble PA-reducing activity. The particulate ABA-hydroxylating enzyme showed a requirement for O(2) and NADPH, inhibition by CO, and high substrate specificity for (+)-ABA. Acetylation of short term incubation mixtures gave evidence for the presence of 6'-hydroxymethyl-ABA as an intermediate in PA formation. Determinations of endogenous ABA and DPA concentrations suggest that the ABA-hydroxylating and PA-reducing enzymes are extensively metabolizing ABA in the intact E. lobata seed.

The effects of water stress on abscisic-acid levels and metabolism in roots of Phaseolus vulgaris L. and other plants.

Abscisic-acid (ABA) levels in roots of bean plants exposed to a-4 bar stress in the root medium increased ca. 10fold within 1 h and 16fold by the end of the 2nd h. Several types of experiments indicated that there is no transport requirement from the shoot for the increase to occur. ABA levels in roots from pea (Pisum sativum L.) and sunflower (Helianthus annuus L.) also increased in response to a-4 bar stress, although not as dramatically as in bean. When (S)-[2-(14)C]-ABA was fed to excised bean roots dihydrophaseic acid (DPA) was the major metabolite formed. The levels of endogenous DPA and phaseic acid increased markedly during a 27-h stress period. These results are consistent with a possible role for ABA in roots of water-stressed plants.

Abscisic Acid Metabolism in Water-stressed Bean Leaves.

Phaseic acid (PA) and dihydrophaseic acid (DPA) are the major metabolites observed when (S)-2-(14)C-abscisic acid (ABA) is fed to 14-day excised primary bean leaves (Phaseolus vulgaris L. cv. Red Kidney). The distribution of (14)C in leaves which were wilted after feeding ABA appears to be the same as that observed in unwilted leaves. A reduction in the relative specific radioactivities of the two metabolites after wilting, compared with the specific radioactivities measured in unwilted plants, indicated that these metabolites continue to be formed endogenously after wilting. Estimates of the endogenous ABA levels showed that they rose from 0.04 mug to approximately 0.5 mug/g fresh weight within 4 hours after the beginning of a 10% wilt and remained at that level during a subsequent 20 hours of wilt. In unwilted leaves, the levels of PA and DPA were 5 times and 20 times higher than that of ABA, respectively. Both PA and DPA levels rose throughout the wilt period. PA rose from 0.20 mug to 1.0 mug and DPA from 0.8 mug to over 3 mug/g fresh weight. From these data, we calculated the rate of ABA synthesis to be at least 0.15 mug/hr.g fresh weight during this period. We have interpreted these results to mean that in wilted leaves an elevated level of ABA is maintained because the rate of synthesis and metabolism are both elevated and approximately equal.

The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed.

Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-(14)C-abscisic acid (ABA) under a variety of conditions. Both dormant and stratified embryos rapidly metabolize abscisic acid to phaseic acid, dihydrophaseic acid, and an unidentified polar metabolite apparently derived from dihydrophaseic acid. Although the stratified embryos may have an increased capacity to metabolize abscisic acid, our calculations suggest that such an increased capacity would probably not be physiologically significant.Dormant intact seeds also metabolize (S)-2-(14)C-abscisic acid during stratification at 5 C or incubation at 25 C. The metabolites appear to be similar to those observed in excised embryos although by 12 days of stratification a fourth metabolite is observed. More than 90% of the (14)C-abscisic acid was metabolized after 26 days of stratification at 5 C or after 12 days of incubation at 25 C. Stratification at 5 C leads to the breaking of dormancy while incubation at 25 C does not.

Germination of Phaseolus vulgaris: IV. Patterns of Protein Synthesis in Excised Axes.

Soluble proteins from excised Phaseolus vulgaris axes incubated for 1 hour in (3)H or (14)C- amino acid mixtures at different times during the period leading up to initiation of cell elongation were compared by acrylamide gel electrophoresis. Differences in electrophoretic patterns were found when proteins from axes incubated during the 1st hour of imbibition were compared with proteins from axes incubated during the hour when cell elongation was initiated. These differences greatly diminished by the 2nd hour of imbibition which suggests that they were due primarily to incomplete axis imbibition. A 5-hour actinomycin D treatment which reduced amino acid incorporation by 40% in the 5th hour had no apparent effect on the electrophoretic pattern during that hour.

The isolation of an abscisic-acid metabolite, 4'-dihydrophaseic acid, from non-imbibed Phaseolus vulgaris seed.

Naturally occurring 4'-dihydrophaseic acid (DPA) has been isolated from mature, non-imbibed bean seed. The concentrations of abscisic acid (ABA), phaseic acid (PA) and DPA in the seed were estimated to be 0.06, 0.11 and 5.95 mg/kg dry wt., respectively. The results suggest that DPA is a major inactivation product of ABA in this tissue. The possible pathway from ABA to DPA is discussed.

Metabolism of 2-C-(+/-)-abscisic Acid in excised bean axes.

Excised embryonic bean axes (Phaseolus vulgaris, var. White Marrowfat) rapidly metabolize 2-(14)C-(+/-)-abscisic acid to two compounds, M-1 and M-2, which have very low growth-inhibitory activity. Chemical tests indicate the M-1 and M-2 are not previously described abscisic acid metabolites. M-2 accumulates in the axes and evidence is presented for the hypothesis that abscisic acid --> M-1 --> M-2. Zeatin, which partially reverses the abscisic acid-mediated growth inhibition of axes, neither decreases abscisic acid uptake nor causes any major changes in its metabolism. It was observed that axes transferred from abscisic acid-containing solutions to buffer resume control rates of fresh weight increase while still containing considerable quantities of abscisic acid.