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Polyribosomes from Peas: II. Polyribosome Metabolism during Normal and Hormone-induced Growth.

Polyribosomes as large as 10-mers (strands of messenger RNA bearing 10 ribosomes) were isolated from etiolated pea (Pisum sativum L. var. Alaska) stem tissue during all stages of development when methods were used which essentially eliminated ribonuclease activity during extraction. Actively growing tissue, harvested from the apical 10 mm, yielded many large polyribosomes and a low (<20%) proportion of monosomes. Similar tissue, allowed to age by applying lanolin to decapitated apices, showed a progressive decrease in number of larger polyribosomes and an increase in the proportion of monosomes. Hormone treatments, which prolonged growth and delayed aging, delayed the loss in large polyribosomes and the increase in proportion of monosomes. Growth-stimulating hormones, added to previously aged tissue, stimulated the production of many large polyribosomes in pre-existing cells.It is suggested that (a) large polyribosomes occur in all regions of the pea stem, (b) changes in polyribosome distribution appear to precede changes in growth rate, (c) loss of larger polyribosomes is closely related to a decrease in mRNA templates followed more gradually by loss of ribosomes, (d) hormone-stimulated continuation of growth is accomplished through maintenance of available mRNA.Methods are described, involving detailed analysis of polysome distribution, which, although they cannot be used to measure changes in initiation of ribosomes on to mRNA, do permit measurement of the amount of polysomal-associated mRNA present in tissues at different stages of growth. These analyses lead to the further suggestion that hormone stimulation of growth of previously nongrowing tissue is accomplished primarily through an increase in available mRNA prior to synthesis of ribosomes.

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Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L.

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.

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Red light enhancement of the phototropic response of etiolated pea stems.

In the subapical third internode of 7-day-old etiolated pea seedlings, the magnitude of phototropic curvature in response to continuous unilateral blue illumination is increased when seedlings are pre-exposed to brief red light. The effect of red light on blue light-induced phototropism becomes manifest maximally 4 or more hours after red illumination, and closely parallels the promotive action of red light on the elongation of the subapical cells. Ethylene inhibits phototropic curvature by an inhibitory action on cell elongation without affecting the lateral transport of auxin. Pretreatment of seedlings with gibberellic acid causes increased phototropic curvature, but experiments using (14)C-gibberellic acid indicate that gibberellic acid itself is not laterally transported under phototropic stimuli. Neither red light nor gibberellic acid treatment has any promotive effect on blue light-induced lateral transport of (3)H-indoleacetic acid. Under conditions where phototropic curvature is increased by red light treatment, low concentrations of indoleacetic acid applied in lanolin paste to the apical cut end of the seedling cause an increased elongation response in subapical tissue. This could explain increased phototropic curvature caused by red light treatment.

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Apical correlative effects in leaf epinasty of tomato.

The influence of the stem apex on leaf curvature was investigated using debudded tomato (Lycopersicon esculentum Mill. cv Anahu) plants and petiole explants, consisting of a section of petiole attached to a section of stem.Decapitation of the main shoot of tomato plants induced hyponasty of petioles in young leaves. Application of auxin in place of the removed apex or fumigation of intact tomato plants with ethylene produced epinastic curvature at the base of the petiole. Simultaneous carbon dioxide treatments prevented the development of petiolar epinasty due to auxin and ethylene treatments. Application of ethylene gas to the decapitated shoot or injection into the stem, induced petiolar epinasty. In a saturating level of ethylene gas, tomato petioles did not respond to indole-3-acetic acid applied to the cut apex. Auxin-induced ethylene production in petiole explants preceded the development of epinasty. Application of indoleacetic acid in lanolin to the entire lower side of the petioles of leaves in situ produced petiole epinasty. Petiolar epinasty due to apically applied indoleacetic acid resulted from differential cell elongation.The auxins indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and naphthalene-1-acetic acid induced epinasty when applied apically to decapitated tomato plants, while gibberellic acid, kinetin, abscisic acid, and auxin or gibberellin antagonists had no effect. When such compounds were applied to petiole explants, only indole-3-acetic acid and kinetin caused an increase in ethylene production and the effect of kinetin was relatively weak.Application of 2,3,5-triiodobenzoic acid around the stem did not change the effect on petiolar epinasty of auxin applied to the decapitated shoot or around the stem. Radioautography showed that the label from (14)C-indoleacetic acid applied apically entered the petiole and midrib tissue; however, extraction showed that only a fraction of the label in these tissues was in the form of indoleacetic acid.Removal of leaflets from leaves induced hyponasty in the midrib region, and application of auxin to the leaflet stubs produced midrib epinasty; carbon dioxide did not block the action of auxin in this type of epinasty. Removal of leaflets from leaves did not alter the effect of apically applied auxin on petiolar epinasty.The data are consistent with the hypothesis that the oblique orientation of leaves in tomato plants is influenced by two epinastic responses. Petiolar epinasty is controlled by the apical region on the stem and is due to the action of auxin-induced ethylene; and midrib epinasty is due to an action of auxin other than through ethylene.

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[C]Ethylene Metabolism during Leaf Abscission in Cotton.

Changes in (14)C(2)H(4) metabolism in the abscission zone were monitored during cotton (cv. Deltapine 16) leaf abscission. Rates of (14)C(2)H(4) oxidation to (14)CO(2) and tissue incorporation in abscission zone segments cut from the second true leaf of nonabscising leaves of intact plants were similar (about 200 disintegrations per minute per 0.1 gram dry weight per 5.5 hours) and relatively constant over a 5-day period. Deblading to induce abscission caused a dramatic rise in (14)C(2)H(4) oxidation, but tissue incorporation was not markedly affected. This rise occurred well before abscission, reaching a peak of 1,375 disintegrations per minute per 0.1 gram dry weight per 5.5 hours 2 days after deblading when abscission was 40%. The rate then gradually declined, but on day 5 when abscission reached completion, it was still nearly three times higher than in segments from nonabscising leaves. Application of 0.1 millimolar abscisic acid in lanolin to the debladed petiole ends increased the per cent abscission slightly and initially stimulated (14)C(2)H(4) oxidation. In contrast, naphthaleneacetic acid applied in a similar manner delayed and markedly inhibited both abscission and (14)C(2)H(4) oxidation.Petiole segments cut 1 centimeter from the abscission zone of intact second true leaves also incorporated and oxidized (14)C(2)H(4) to (14)CO(2) but at rates two and six times higher, respectively, than that of comparable adjacent abscission zone segments. However, in marked contrast to the abscission zone segments, no changes in oxidation were observed when the leaves were debladed to induce abscission.THESE RESULTS DEMONSTRATE THAT: (a) prior to abscission, the ethylene oxidation, but not the tissue incorporation pathway, rapidly increases in the abscission zone; (b) this increase does not occur in adjacent petiole tissue; and (c) changes in the rate of oxidation and per cent abscission brought about by hormone treatments parallel one another. The possible significance of these changes in ethylene metabolism is discussed with respect to the hypothesis that ethylene action and metabolism are directly related.

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Bud Induction with Cytokinin : A LOCAL RESPONSE TO LOCAL APPLICATION.

A portion of the surface of detached Graptopetalum paraquayense E. Walther leaves can be used to assay small amounts of reagents in lanolin for their ability to induce shoots only at the site of application. The cytokinins benzyladenine, kinetin, and 6-(gamma,gamma-dimethylallylamino)purine (DMAAP) were tested, and DMAAP was most effective in bud induction at concentrations below 1%. The higher the hormone concentration, the sooner the appearance of leaf primordia and the higher the ultimate yield of buds. Leaves treated with DMAAP for 2 days developed buds as rapidly as those with longer treatments.

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Ribonucleic Acid and protein metabolism in pea epicotyls : I. The aging process.

Aging of actively growing, etiolated pea Pisum sativum L. var Alaska plants was initiated by removing the plumules of plants in the third internode stage, and applying lanolin to the cut apices of otherwise intact plants. During the subsequent 4-day aging period, several degenerative events occurred in this apical 10-millimeter region. Ribosomal RNA and messenger RNA contents declined, polyribosomes disaggregated, and the protein synthesizing capacity of the polysomes decreased.Two-dimensional, silver-stained protein patterns revealed that aging altered the relative amounts of specific cellular proteins accumulated in vivo. In addition, polypeptide patterns generated by cell-free translation of total and polysomal RNA, isolated from unaged and aged tissues, showed major modifications. More than 200 spots could be resolved by two-dimensional gel fluorography of translation products using RNA from fresh tissues. Of these 200 spots, about eight appeared or increased when total RNA from aged tissue was used, and about 58 disappeared or declined. When polysomal RNA from aged tissue was used as template, about 12 spots appeared or increased, whereas about 64 disappeared or decreased. In general, the products which increased after aging were lower molecular weight and those that decreased were higher molecular weight.

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Gravitropism in Higher Plant Shoots : III. Cell Dimensions during Gravitropic Bending; Perception of Gravity.

Cross and longitudinal sections were prepared for light microscopy from vertical control plants (Xanthium strumarium L. Chicago strain), free-bending horizontal stems, plants restrained 48 hours in a horizontal position, and plants restrained 48 hours and then released, bending immediately about 130 degrees . Top cells of free-bending stems shrink or elongate little; bottom cells continue to elongate. In restrained stems, bottom cells elongate some and increase in diameter; top cells elongate about as much but decrease in diameter. Upon release, bottom cells elongate more and decrease in diameter, while top cells shorten and increase in diameter, accounting for the bend. During restraint, bottom cells take up water while tissue pressures increase; top cells fail to take up water although tissue pressures are decreasing.Settling of amyloplasts was observed in cells of the starch sheath.Removal of different amounts of stem (Xanthium; Lycopersicon esculentum Miller, cv Bonny Best; Ricinus communis L. cv Yolo Wonder) showed that perception of gravity occurs in the bending (elongation) zone, although bending of fourth and fifth internodes from the top was less than in uncut controls. Uniform application of 1% indoleacetic acid in lanolin to cut stem surfaces partially restored bending. Reversing the gradient in tension/compression in horizontal stems (top under compression, bottom under tension) did not affect gravitropic bending.

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Regulation of potato tuber protein accumulation by gibberellic Acid.

Many studies have shown that gibberellic acid (GA(3)) inhibits tuberization in potato (Solanum tuberosum L.). In this study, we have utilized the 40 kilodalton glycoprotein, patatin, as a marker for biochemical events associated with the process of tuberization. To determine the effects of exogenous applications of GA(3) on the induction of the accumulation of this major tuber protein, we measured patatin levels in tubers from treated whole plants, petioles from a single-node cutting system with GA(3) applied in a lanolin paste, and stolon tips cultured in vitro on an inductive medium supplemented with GA(3). In all three systems, GA(3) inhibited the accumulation of patatin and the major 15 and 22 kilodalton tuber proteins. This effect appeared to be selective since most of the other proteins were not affected and, in tubers, at least one protein was stimulated by GA(3). These results suggest that GA(3) can reverse biochemical events of tuberization in tubers as well as prevent the accumulation of the major tuber proteins in other inducible tissues.

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Can lateral redistribution of auxin account for phototropism of maize coleoptiles?

Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip.

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The role of cytokinin in sieve tube regeneration and callose production in wounded coleus internodes.

Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 mug/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 mug/mL and 20 mug/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 mug/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed.

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Tracheid production in response to changes in the internal level of indole-3-acetic Acid in 1-year-old shoots of scots pine.

Different concentrations of indole-3-acetic acid (IAA) were applied in lanolin to 1-year-old shoots of Pinus sylvestris (L.) in a manner known to stimulate cambial activity. The internal concentration of free IAA was measured at a distance below the application point by combined gas chromatography-selected ion monitoring-mass spectrometry using [(13)C(6)]IAA as a quantitative internal standard, and related to the production of tracheids at the same site. The experiment was performed with: (a) debudded cuttings, where the major source of endogenous IAA, the apical buds, were replaced with exogenous IAA, and (b) intact, attached shoots, where endogenous IAA was supplemented by applying IAA around the circumference of the shoot. In both experimental systems, an increase in the internal IAA level was positively related to increased tracheid production. It was also demonstrated that the concentration of internal IAA measured at the sampling site was comparable with endogenous IAA levels found in intact control shoots, and that a wide range of applied IAA concentrations was associated with a relatively small range of internal IAA levels.

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The association between ambient air conditions (temperature and absolute humidity), irritant sodium lauryl sulfate patch test reactions and patch test reactivity to standard allergens.

To support the decision as to whether erythematous patch test reactions to allergens are irritant or allergic, sodium lauryl sulfate (SLS, 0.5% in water) has been added to the standard patch tests since July 1996 in the Dortmund Department of Dermatology. Data on 1600 patients patch tested up until June 2001, as well as standardized data on ambient temperature and humidity obtained by the German Meteorological Service, were included in a logistic regression analysis taking age, sex and atopy as potential confounders into account. The pattern of association was heterogeneous: while doubtful reactions to nickel sulfate were significantly associated with dry/cold weather conditions, but not with SLS reactivity, the opposite was observed for lanolin alcohol, benzocaine and Myroxylon pereirae resin (balsam of Peru). Doubtful reactions to other allergens, namely formaldehyde, fragrance mix or p-phenylenediamine, were associated with both factors. For several other allergens of the standard series, no distinct, significant pattern could be discerned. In conclusion, meteorological conditions and SLS reactivity independently contribute information on individual irritability at the time of patch testing, and both should be considered.

Allergens↗

Allergic and non-allergic periorbital dermatitis: patch test results of the Information Network of the Departments of Dermatology during a 5-year period.

Periorbital dermatitis is common and can be due to the external use of ophthalmic drugs. We evaluated patch test results of the Information Network of the Departments of Dermatology. During a 5-year period (1995-99), of a total 49,256 patch-tested patients, 1053 (2.1%) were eventually diagnosed as allergic periorbital contact dermatitis (APD) and 588 (1.2%) as non-allergic periorbital dermatitis (NAPD). Patient characteristics between APD, NAPD and other cases (OCs) differed with respect to sex (19.7% male in both periorbital groups versus 36.3% in OCs), atopic dermatitis (10.4% in APD versus 60.2% in NAPD versus 16.9% in OCs) and age, APD being substantially more often (68.2%) aged 40 and above than NAPD (52.6%). Several of the top allergens in OCs [such as fragrance mix, Myroxylon pereirae resin (balsam of Peru), lanolin alcohol and potassium dichromate] caused significantly fewer positive test reactions in both periorbital groups. In contrast, thimerosal, phenylmercuric acetate, sodium disulfite, gentamicin sulfate, phenylephrine hydrochloride and benzalkonium chloride tested positively significantly more often in APD but not in NAPD, verifying them as true ophthalmic allergens. Finally, in 42 cases (4%) of APD patients, additional allergens were identified by testing of the patients' own substances (mostly beta-blockers, oxybuprocaine and dexpanthenol), supporting the necessity of testing with ophthalmic drugs as is where individual substances are not readily available.

Adult↗

Reproducibility of patch test results: a concurrent right-versus-left study using TRUE Test.

Wide variations in the reproducibility rate of positive patch test responses have been reported. We hypothesized that a major source of non-reproducibility resides in the methodological pitfalls of routine patch testing. Simultaneous duplicate patch testing on opposite sides of the upper back was performed on 500 consecutive patients, using the TRUE Test system consisting of 2 panels, each one containing 12 standard allergens. A rigorous methodological design was applied and relevance was assessed for all discordant patch test reactions. A total of 435 positive patch test reactions were observed either on one or both sides in 289 patients (58.8%). Of these, 22 (5%) were discordant, i.e. interpreted as positive allergic on one side whilst negative or doubtful on the opposite side. The allergens responsible for discordant reactions were nickel sulfate (4 patients), cobalt chloride (3), lanolin alcohol (3), fragrance mix (2), carba mix (2), thiuram mix (2), colophonium (1), potassium dichromate (1), p-phenylenediamine (1), formaldehyde (1), balsam of Peru (Myroxylon pereirae resin) (1) and thimerosal (1). Of the 19 (4%) patients with discordant patch test reactions, the allergen was deemed to be of definite present or past relevance in 9 patients (1.8% of the total and 3.1% of all patients with positive patch test reactions) and of possible relevance in a further 2 patients. These data suggest that patch testing is a reasonably reproducible procedure as long as methodological error is minimized.

Adolescent↗

Common contact sensitizers in the Czech Republic. Patch test results in 12,058 patients with suspected contact dermatitis*.

The patch test results of 12 058 patients (4416 male and 7642 female) referred to 9 clinics in the Czech Republic between January 1997 and December 2001 were evaluated. Patients were tested with the same series of allergens by using the standardized patch testing method. The current standard tool for diagnosing allergic contact dermatitis (ACD) in the Czech Republic is the Trolab test panel (Hermal, Reinbeck, Germany) which consists of 23 allergens. Only a few data exists on ACD in the Czech Republic. All patients were tested with the 23 allergen European standard series. Of these patients, 7661 (63.5%) had 1 or more positive reactions. On average, there were 2.8 positive reactions per patient. ACD, according to clinical relevance, was diagnosed in 5339 (69.7%) of these patients. The most frequent allergens were metals (22.9%), especially nickel sulfate (13.8%), and followed by Myroxylon pereirae resin (balsam of Peru) (7.3%), fragrance mix (5.8%), formaldehyde (4.2%) and lanolin alcohol (3.0%). Our results were compared with results from other countries. We conclude that the European standard series is suitable for detection of ACD in the Czech Republic.

Adolescent↗

Which children should we patch test?

BACKGROUND: Allergic contact dermatitis (ACD) in childhood was considered rare until recently. However, reports are increasing, which may reflect an increased incidence and/or more frequent patch testing of children. It is also likely that allergen exposure in children has changed with time. AIMS: To determine the most common contact allergens and the rate of positive patch-test reactions among children with suspected contact allergy. METHODS: We carried out a retrospective case study of 114 children (66 girls and 48 boys) aged from 3 to 15 years (median 11.5) patch tested over a 3-year period. Indications for patch testing included uncontrolled or deteriorating atopic dermatitis, localized dermatitis or a history of reacting to a specific allergen. RESULTS: Of 110 children for whom we had notes, 83 (75%) had a history of atopy. Positive reactions that were of current, past or possible relevance were seen in 61 children (54%); in 58 (52%) of 111 tested with the standard series (SS) and in 6 (10%) of 60 tested with the medicament series. None of the children patch tested to the corticosteroid (n = 47), shoe (n = 15), fragrance (n = 12), cosmetic (n = 10) or rubber (n = 5) series had a positive reaction. However, 11 (10%) reacted to rubber allergens within the SS and one of five to their own shoes. The lowest rate of relevant positive reactions was among those with deteriorating atopic dermatitis (22%) and facial (33%) or perioral dermatitis (40%), and the highest rate amongst those with eyelid (86%) or hand (71%) dermatitis. Nickel was the most common allergen (20%) in line with previous reports (82% female), followed by rubber chemicals (10%), fragrance (7.2%), cobalt (5.4%) and lanolin (wool alcohol) (4.5%). CONCLUSIONS: The reported incidence of ACD among children, in particular nickel and rubber allergy, appears to be increasing, which may relate to changing fashions and hobbies. Contact allergy should be considered in all children with dermatitis, particularly with eyelid or hand dermatitis, and patch testing carried out more frequently.

Adolescent↗

Allergy due to topical medications in chronic otitis externa and chronic otitis media.

Thirty-four patients suffering from chronic otorrhoea were tested for delayed type contact allergy. Patch testing showed a relevant positive reaction in 19 patients (56%). The most frequent allergens were aminoglycosides with neomycin and framycetin as major offenders. Other antimicrobial agents (clioquinol, polymyxin B), cream bases (lanolin) and corticosteroids (tixocortol) were less common allergens encountered. These results indicate that it is almost obligatory to perform patch testing in any patient with long-standing otitis which does not respond to local therapy. Scoring of the patch tests has to be extended to 7 days, as notably the aminoglycosides and corticosteroids only become positive after such a long interval. Because of the high risk of sensitization, topical preparations containing neomycin and framycetin should not be used routinely. We recommend the use of either a topical antiseptic or a topical antibiotic with low allergenic potential for the initial treatment of otorrhoea.

Administration, Topical↗