bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethylene: A regulator of stress‐induced ACC synthase activity in nonclimacteric fruit
Authors: Mullins Ewen D. , McCollum T. G. , McDonald Roy E.
Published: March 2003

In spite of precautions, harvesting, packing and shimpping of grapefruit (Citrus paradisi) can often result in injury to the peel, which in turn leads to an increased evolution of the ripening hormone ethylene by the fruit. The present study is an attempt to determine how the stress‐induced ethylene pathway is regulated using the 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase (EC 4.4.1.14) action inhibitor aminoethoxyvinylglycine (AVG). Incubation of mature grapefruit flavedo discs in the presence of AVG created a super‐induction of ACC synthase (ACS) activity. Maximum enzyme activity was achieved following an incubation period of between 24 and 36 h. From AVG‐treated flavedo discs, cDNA encoding ACS was isolated, employing reverse transcriptase‐polymerase chain reaction (RT‐PCR), and the accumulation of ACS transcripts, which peaked after 36 h incubation, was found to complement the recorded enzyme activity. Saturation of ACS activity was achieved with 100 μM AVG. Incubation of flavedo with AVG significantly decreased both the conversion of S‐adenosyl‐l‐methionine (AdoMet) to ACC and the evolution of ethylene from the treated tissue, and in vitro incubation of extracted ACS enzyme with AVG led to repression of the action of the enzyme. Treatment of flavedo discs with ACC and ethylene inhibited the AVG‐induced ACS activity. Northern blot analysis showed that ethylene sumppressed the accumulation of ACS mRNA. It is suggested that in nonclimacteric fruit, the synthesis of ethylene can be regulated through a process of autoinhibition working at the level of gene expression.
Influence of soil fertilization, plant spacing, and cokppicing on growth, stomatal conductance, abscisic acid, and camptothecin levels in Camptotheca acuminata seedlings
Authors: Liu Zhijun , Adams John C. , Viator H. P. , Constantin Roysell J. , Carpenter Stanley B.
Published: March 2003

Changes in amount of the plant alkaloid, camptothecin (CPT), were investigated in field‐grown Camptotheca acuminata seedlings in response to fertilization, variation in plant density, and cokppicing. The relationship between CPT and growth, and abscisic acid (ABA) levels were also examined. Fertilization caused positive responses in growth of both the slow‐ and the fast‐growing C. acuminata plants. It lowered stomatal conductance (g), reduced leaf CPT level in slow‐growing but not fast‐growing seedlings in mid‐summer (July), and increased leaf ABA level in dense plantings in early fall (September). Shoots induced to develop as a result of cokppicing were significantly higher in leaf CPT level than the original non‐cokppiced shoots. These results suggest that in the C. acuminata plantation management, cokppicing should be employed to produce high concentrations of CPT, but that soil fertilization should be used cautiously so that greater growth is not achieved at the cost of lower CPT biosynthesis.
Involvement of phytochrome(s), Ca2+ and phosphorylation in light‐dependent control of transcript levels for plastid genes (psbA, psaA and rbcL) in rice (Oryza sativa)
Authors: Grover Monendra , Dhingra Amit , Sharma Arun K. , Maheshwari Satish C. , Tyagi Akhilesh K.
Published: March 2003

In the present investigation, an attempt has been made to understand the mechanism of light signal transduction in regulation of steady‐state transcript levels of the plastid genes psbA, psaA and rbcL in rice (Oryza sativa L. ssp. indica cv. Pusa 169) seedlings. Red light irradiation of 5‐day‐old etiolated seedlings for 5 min upregulates the transcript levels of the above‐mentioned plastid genes, while far‐red light exposure subsequent to red light reverses this effect, indicating the involvement of phytochrome(s). Ca2+ and the Ca2+ ionophore, A23187 increase the level of transcripts in the dark‐grown seedlings. Ca2+ chelators (EGTA and BAPTA) as well as Ca2+ channel blockers (nifedipine and verapamil) suppress the accumulation of all transcripts in light. This suggests the involvement of Ca2+ in the photoregulation of plastid genes. Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A (PP1 and PP2A), and sodium fluoride (NaF), a general inhibitor of protein phosphatases, significantly prevent the light‐induced increase in transcript levels of the plastid genes. Staurosporine, a protein kinase inhibitor, causes an increase in the transcript levels in darkness and a further enhancement in conjunction with light. The results suggest that light signal transduction downstream to phytochrome(s) involves Ca2+ and phosphorylation for controlling the pattern of plastid gene expression. Phosphorylation inhibits light‐activated gene expression in chloroplasts.
Effects of adaptation to winter stress on biomass production, growth and morphology of three contrasting white clover cultivars
Authors: Frankow‐Lindberg Bodil E.
Published: March 2003

Changes in biomass production and partitioning, together with morphology and growth of three white clover (Trifolium repens L.) cultivars after exposure to winter stress were recorded in a controlled environment study. The cultivars used were Grasslands Huia (H, New Zealand), AberHerald (AH, bred in UK from material of Swiss origin) and Sandra (S, Sweden), sub‐populations of these (survival populations), and a wild ecotype from the Uppsala area. The sub‐populations were collected after 2 (with the exception of S) and 4 years of winter stress, respectively, at Uppsala (59°49′ N, 17°39′ E). In the controlled environment study the photoperiod was 12 h and there were two temperature regimes, namely 24/18 and 12/9°C (day/night), respectively. The experimental plants were cloned from eight different plants of each population. There were few interactions between populations and temperature regimes. Biomass production was lowest in the Swedish populations and in the surviving populations of the other cultivars collected after 4 years of exposure to winter stress. However, biomass partitioning, with the exception of allocation to branches, remained unaffected. Morphology differed significantly between cultivars, but, with the exception of internode length, was unaffected by exposure to winter stress. Internode length was significantly reduced in surviving populations of both H and AH after exposure to four winters, when its value was similar to that of the Swedish populations. The reduced internode length was a result of a greater reduction in the stolon extension rate than in the leaf appearance rate of survival populations compared with the original populations. It is hypothesised that reduced internode length would have implications for the carbon economy of the plant.
Properties of plant plasma membrane lipid models – bilayer permeability and monolayer behaviour of glucosylceramide and phosphatidic acid in phospholipid mixtures
Authors: Berglund Anna H. , Norberg Peter , Quartacci Mike F. , Nilsson Ralf , Liljenberg Conny
Published: March 2003

Phosphatidic acid (PA) and glucosylceramide (Cer), constituents of plant plasma membranes, were used in interaction studies with the major plasma membrane lipid components, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). With molecular species combinations, representative for plant plasma membranes, packing conditions during compression of monolayers of PC/PE mixtures with different amounts of PA or Cer added, were investigated. In contrast to the behaviour of single PA or single Cer, which exhibited condensed compression curves, as compared with curves representative for phosphoglycerides, the triple mixtures of PC/PE with PA or Cer showed markedly expanded monolayer films. These data were evaluated as a spontaneous heterogeneous dispersion of PA and Cer in the PC/PE mixture. Membrane vesicles produced with different amounts of PA added to a PC/PE mixture of 1:1 (mol/mol) had an almost linear increase in permeability for glucose (chosen as a common polar low‐molecular mass metabolite) with increasing percentage PA. The presence of PA in plasma membranes and its possible function are discussed in relation to recent reports on anionic protein‐lipid interactions. PC/PE vesicles with different amounts of Cer added did not influence the permeability for glucose at 2.5 and 5 mol%, but did so, significantly, at 7.5 and 9 mol%.
An in vivo N NMR study of agropine synthesis in transformed root cultures of Nicotiana tabacum
Authors: Ford Y.‐Y. , Ratcliffe R. G. , Robins R. J.
Published: March 2003

The biosynthesis of the opine agropine in transformed Nicotiana tabacum L. root cultures was studied using in vivo

N nuclear magnetic resonance (NMR) spectroscopy. Roots were incubated with [

N]ammonium and/or [

N]nitrate, and the incorporation of the label into agropine, conjugated polyamines, Γ‐aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln) and nicotine was monitored by NMR. The largest labelled pool was agropine in cells grown on a

N‐enriched medium and its synthesis was maintained, to the detriment of the Gln pool, under conditions of nitrogen (N) starvation. These observations indicate that the synthesis of agropine is not tightly regulated and that it represents a significant sink for carbon (C) and N in the plant. The addition of α‐naphthaleneacetic acid (NAA) and kinetin to the growth medium caused de‐differentiation of the root cultures and perturbation of secondary N metabolism. The amount of agropine relative to Gln increased but the total accumulation of agropine decreased, in part because of the increase in the synthesis of conjugated polyamines. These results show that agropine biosynthesis perturbs both primary and secondary N metabolism, and that the perturbation differs according to the culture conditions and the imposed stress.
Activities of SOD and the ascorbate‐glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt‐tolerant relative Lycopersicon pennellii
Authors: Mittova Valentina , Volokita Micha , Guy Micha , Tal Moshe
Published: March 2003

The activities of the ascorbate‐glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and SOD were studied in cell organelles of the cultivated tomato Lycopersicon esculentum (M82) and its wild salt‐tolerant related species Lycopersicon pennellii (Lpa). All four enzymes of the ascorbate‐glutathione cycle were present in chloroplasts/plastids, mitochondria and peroxisomes of leaf and root cells of both tomato species. In all leaf and root organelles of both species, the activity of MDHAR was similar to, or higher than, that of APX, while the activity of DHAR was one order of magnitude lower than that of MDHAR. Based on these results, it is suggested that in the organelles of both tomato species, ascorbate is regenerated mainly by MDHAR. In both tomato species, GR activity, and to a lesser extent DHAR activity, was found to reside in the soluble fraction of all leaf and root cell organelles, while APX and MDHAR activities were distributed between the membrane and soluble fractions. A higher SOD to APX activity ratio in all Lpa organelles was the major difference between the two tomato species. It is possible that this higher ratio contributes to the inherently better protection of Lpa from salt stress, as was previously reported.
Fruit size: Towards an understanding of the metabolic control of fruit growth using avocado as a model system
Authors: Cowan A. Keith , Cripps Ryan F. , Richings Evelyn W. , Taylor Nicky J.
Published: March 2003

Final fruit size is the consequence of complex metabolic events that occur between fruit set and maturation. Disruption of these biochemical and molecular processes at any stage during fruit growth will impact on final fruit size. Because fruit size is a function of cell number rather than cell size, factors affecting cell division cycle activity assume importance. In this paper, we focus attention on the metabolic control of fruit growth using avocado as a model system. Three areas of current interest are highlighted, viz. the contribution by isoprenoid metabolism in the control of cell proliferation, the role played by carbohydrate content and composition in signalling changes in metabolite status and gene expression and maintenance of plant hormone homeostasis. Central to the process of fruit growth and control of final fruit size by cell division is 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR) and activity of the sucrose non‐fermenting 1‐related protein kinase (SnRK1) complex. It is argued that sugar content and composition of sink cells impact on SnRK1 (and hexokinase) to modulate expression of sugar‐metabolizing enzymes, HMGR and molybdenum cofactor (MoCo)‐containing enzymes. These changes, in turn, impact on hormone metabolism by affecting allocation of the purine‐derived MoCo to aldehyde oxidase and thus the endogenous concentration of indole‐3‐acetic acid, abscisic acid and cytokinin (CK) to alter plant hormone homeostasis. These aspects are integrated into a model to explain the metabolic control of avocado fruit growth and final fruit size.
Partial characterization of H+‐translocating inorganic pyrophosphatase from 3 citrus varieties differing in vacuolar pH
Authors: Marsh Kenneth , González Pedro , Echeverría Ed
Published: March 2003

Vacuolar pyrophosphatase (V‐PPase) from juice cells of 3 citrus varieties (differing in their vacuolar pH) were partially characterized using purified tonoplast vesicles. Total V‐PPase activity was highest in vesicle samples from sweet limes with vacuolar pH of 5.0, while samples from acid limes (with lowest vacuolar pH of 2.0) had the minimal total V‐PPase activity. Samples from ‘Valencia’ orange had intermediate V‐PPase levels. When assayed at equal V‐PPase activity (measured as Pi production), V‐PPase was not able to generate a pH gradient (ΔpH) in vesicles from acid lime, despite its capacity to form a ΔpH in the presence of ATP. Vesicles from sweet lime and ‘Valencia’ orange were able to form similar ΔpHs in the presence of PPi and ATP supplied together or separately. Antibodies raised against a peptide corresponding to the catalytic site of mung bean V‐PPase reacted with samples from all varieties, coinciding with their capacity to hydrolyze PPi. However, antibodies raised against the entire V‐PPase polypeptide from mung bean recognized V‐PPase from sweet lime and ‘Valencia’ orange, but did not recognize acid lime samples even at elevated protein concentrations. The structural differences highlighted by antibody recognition, substrate affinity and proton‐pumping reactions of V‐PPase presented here may reflect evolutionary adaptations related to its reduced function under in vivo conditions and are in agreement with our understanding of acid, sugar accumulation and vacuolar pH changes during the development and maturation of citrus fruits.
The inhibition of the carbon concentrating mechanism of the green alga Chlorella saccharophila by acetazolamide
Authors: Pollock Steve Vincent , Colman Brian
Published: March 2003

The effects of the carbonic anhydrase (CA) inhibitors acetazolamide (AZ) and dextran‐bound sulfonamide (DBS) on HCO3−‐dependent O2 evolution in Chlorella saccharophila were evaluated. Addition of 4 μM AZ or 0.4 mg ml−1 DBS to photosynthesizing cells reduced the O2 evolution rate at low dissolved inorganic carbon (DIC) concentration, decreased the size of the intracellular acid‐labile carbon pool, and decreased the apparent affinity of the cells for DIC. Measurement of the whole‐cell affinity of cells for CO2 and HCO3− in the presence and absence of inhibitors indicated that active HCO3− transport was inhibited by AZ and DBS. The inhibition of HCO3− transport was independent of the inhibition of external and internal CA. These results suggest that the active uptake of HCO3− occurs initially by the interaction of HCO3− and a CA‐like transporter.
Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation
Authors: Minocha Rakesh , McQuattie Carolyn , Fagerberg Wayne , Long Stephanie , Woon Noh Eun
Published: March 2003

The effects of Al on red spruce (Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereological and microscopic methods. Exposure to Al for 24–48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al. Using energy‐dispersive X‐ray microanalysis on sections of freeze‐substituted cells that had no obvious disruption in cytoplasmic or cell wall structure, Al (always in the presence of P) was detected in dense regions in cell walls, cytoplasm, plastids and vacuoles after 48 h exposure to Al. Stereological quantification of spruce cell structure showed that, after 24 h of Al treatment, intact cells had increased vacuolar and total cell volume, but the nuclear volume did not change. In addition, Al treatment resulted in increased surface area of Golgi membranes and endoplasmic reticulum. The biochemical and ultrastructural alterations in red spruce cells, in combination with the presence of Al in cellular organelles of visually intact cells, suggest that Al movement occurred across the plasma membrane without major cellular disruption. Detailed short‐term time course studies are needed to determine if intracellular Al in these cells results from its passage into cells through submicroscopic lesions in the plasma membrane or it is taken up into the symplast through the intact membrane by an active, but slow, process.
Studies on water transport through the sweet cherry fruit surface: III. Conductance of the cuticle in relation to fruit size
Authors: Knoche Moritz , Peschel Stefanie , Hinz Matthias
Published: March 2003

Rain‐cracking of sweet cherry fruit has been related to water absorption through the fruit surface and large fruit has been reported to be more susceptible to cracking than small fruit. Therefore, the effect of fruit size on water conductance of the cuticular membrane (CM) of exocarp segments excised from cheek, suture or stylar end region of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated. Segments consisting of epidermis, hypodermis and several layers of mesocarp cells were mounted in diffusion cells filled with deionized water. Mass loss due to transpiration was monitored gravimetrically during an 8‐h incubation period (25 ± 2°C) over dry silica in the dark. Conductance was calculated from the amount of water transpired per unit surface area and time divided by the difference in water vapour concentration across the segment. For an average size cv. Sam sweet cherry CM conductance was 1.06 × 10−4, 0.91 × 10−4 and 2.09 × 10−4 m s−1 in cheek, suture and stylar end region, respectively. Fruit size had no significant effect on conductance in cheek or suture regions, but for the stylar end region conductance was positively related to fruit size. Stomatal density in the cheek, but not the suture or stylar end region increased as fruit size increased. The area of the stylar scar was positively related to fruit size. Conductance of the stylar scar averaged 37.6 ± 4.0 × 10−4 m s−1 and was 54‐fold higher than that of the CM between stomata in the cheek region (mean 0.69 × 10−4 m s−1). Conductance calculated on a whole fruit basis is estimated to increase by 108% as fruit size increases from 6 to 12 g. Increased conductance on a whole fruit basis may be attributed to increased fruit surface area and increased conductance per unit fruit surface area, particularly in the stylar end region.
Limited acclimation of photosynthesis to blue light in the seaweed Gracilaria tenuistipitata
Authors: Mercado Jesús M. , Sánchez Pilar , Carmona Raquel , Niell F. Xavier
Published: March 2003

Changes in photosynthetic capacity of the seaweed Gracilaria tenuistipitata Zhang et Xia acclimated to monochromatic blue light were studied. For this purpose, affinity for external inorganic carbon, light use efficiency, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli acclimated to 45 µmol m−2 s−1 of blue light. Thalli cultured in white light of the same photon fluence rate were used as a control. Lower maximal photosynthetic rates (i.e. at light and carbon saturation) were obtained in the thalli cultured in blue light. Apparently, this lower photosynthetic capacity was not due to differences in affinity and/or capacity for use of external dissolved inorganic carbon (DIC) since (1) CA activity did not change significantly and (2) similar values of photosynthetic conductance for DIC at alkaline pH were obtained (0.95 × 10−6 m s−1). In addition, the pool size of Rubisco was not modified by the blue light treatment since there were no significant differences in Rubisco content between white (12.14% of soluble proteins) and blue light (12.13% of soluble proteins) treatments. In contrast, Fv/Fm was increased by 11% and photosynthetic efficiency for oxygen production was reduced by 50% in blue light. This absence of correlation between quantum yields for maximum stable charge separation of photosystem II and oxygen evolution suggests that blue light promote changes in rates of photosynthetic electron flow.
Biomonitoring air pollution with the desert lichen Ramalina maciformis
Authors: Garty Jacob , Levin Tal , Cohen Yehudit , Lehr Haya
Published: March 2003

To investigate the environmental impact of anthropogenic activity in the Negev Desert, we monitored the following in order to determine the physiological integrity of the epilithic fruticose lichen Ramalina maciformis: (1) production of ethylene; (2) potential quantum yield of photosystem II expressed as chlorophyll fluorescence ratio Fv/Fm; and (3) electrolyte leakage of cell membranes in terms of electric conductivity. The degree of pollution was assessed by measuring the amounts of B, Cd, Co, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, Sr and Zn in the lichen thallus. Some of the lichen‐carrying stones collected in the relatively unpolluted control site were relocated on the same hill, to test the possible impact of relocation. An additional amount of lichen‐carrying stones was marked as controls. The greater part of the lichen‐carrying stones was transferred to 10 biomonitoring sites in and around Ramat Hovav, an industrial area in the Negev Desert, Israel. Transplanted lichen in polluted sites contained higher amounts of mineral elements than lichens in the control site after an exposure period of 7 months. Statistical analysis revealed positive correlation of ethylene production and Ni content, negative correlation of Fv/Fm ratios and B, Cu, Mn, Na, Ni, Sr and Zn, and positive correlation of electric conductivity and B, Mn, Ni and Sr. Both elemental content and physiological alterations in transferred samples of R. maciformis point to a high degree of contamination in Ramat Hovav. The present study suggests that the lichen R. maciformis should be classified as a sensitive species endangered by anthropogenic activity in the desert. Furthermore, this lichen species is likely to be suitable as a bioindicator of pollution in other parts of the Negev Desert. Ethylene production and electric conductivity as parameters of lichen‐vitality, indicated stress in 5 of the 12 biomonitoring sites (42%) and may therefore be considered as more sensitive than the Fv/Fm ratio, which indicated stress in 3 of the 12 sites (25%).
Networking of phospholipases in plant signal transduction
Authors: Wang Xuemin , Wang Cunxi , Sang Yongming , Qin Chunbo , Welti Ruth
Published: March 2003

Phospholipases are activated in response to various cellular and environmental cues. Their activation can affect many cellular processes through their roles in signal transduction. Recent advances in the biochemical and molecular understanding of phospholipase D (PLD) have provided insights into potential networks of PLDs and other phospholipases in plants. PLDs are a family of heterogeneous enzymes, and the activities of the multiple types of PLDs are regulated in distinctly different manners. Phosphoinositides, free fatty acids, lysophospholipids, and calcium are differential modulators of PLDs. Since these modulators are substrates, products, or downstream targets of phospholipase As and phospholipase Cs, there are many potential regulatory and metabolic interrelationships among the various PLDs and other phospholipases.
Abscisic acid, phaseic acid and gibberellin contents associated with dormancy and germination in barley
Authors: Jacobsen John V. , Pearce David W. , Poole Andrew T. , Pharis Richard P. , Mander Lewis N.
Published: March 2003

Analyses of abscisic acid (ABA), ent‐kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after‐ripened barley (Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after‐ripened dry grain contained similar amounts of ABA, of ent‐kaurenoids and of GAs, determined by gas chromatography‐mass spectrometry‐selected ion monitoring. In embryos of after‐ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent‐kaurenoids and GAs, including GA1, the first biologically active GA in the early 13‐hydroxylation biosynthetic pathway, occurred to a much greater extent in after‐ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent‐kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent‐kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after‐ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.
Two subtilisin‐like proteases from soybean
Authors: Beilinson Vadim , Moskalenko Oleksandr V. , Livingstone Donald S. , Reverdatto Sergei V. , Jung Rudolf , Nielsen Niels C.
Published: March 2003

Two subtilisin‐like proteases (SLP) were identified in soybean (Glycine max[L.] Merr.). The first, SLP‐1, was localized in seed coats early in seed development, but became undetectable with anti‐SLP‐1 antibodies as seed fill progressed. A partial purification of SLP‐1 was achieved using a two step chromatographic procedure. NH2‐terminal sequence analysis of the partially purified enzyme permitted primers to be designed that were used to amplify cDNA encoding SLP‐1. A genomic clone encoding SLP‐1 was also obtained. Characterization of the cDNA and partially purified SLP‐1 revealed the initial translation product was an 82 694 MW precursor. After removal of a signal peptide, the mature protein was formed by removal of an NH2‐terminal propeptide. A COOH‐terminal peptide also appeared to be removed from some of the protease molecules. DNA blot analysis suggested that at least one additional SLP gene was present in soybean. The second gene, SLP‐2, was subsequently cloned and characterized. Although the coding regions for SLP‐1 and SLP‐2 were homologous, their promoters were quite divergent. RT‐PCR revealed that SLP‐2 message was found in the mature plant and in cotyledons of germinating seeds. Although SLP‐2 mRNA could be identified in developing seeds, the message was at least an order of magnitude less abundant than that for SLP‐1, and it was mis‐spliced such that a chain termination event would preclude obtaining a product. As with SLPs from other organisms, the functions of the soybean proteases are unknown. However, SLP‐1 is one of only a few proteins from soybean seed coats that have been described.
Carbon and nitrogen metabolism in barley plants exposed to UV‐B radiation
Authors: Ghisi Rossella , Trentin Anna Rita , Masi Antonio , Ferretti Massimo
Published: March 2003

The effect of UV‐B radiation on FW, leaf and stem length, photosynthetic O2 evolution, levels of carbohydrates and nitrates, and extractable activities of some of the enzymes involved in C and N metabolism was evaluated in barley (Hordeum vulgare L. cv. Express) seedlings during the 9 days following transfer to an UV‐B enriched environment. The results show that under our experimental conditions UV‐B radiation scarcely affects the photosynthetic competence of barley leaves, expressed as RuBP carboxylase (EC 4.1.1.39) activity, O2 evolution rate and chlorophyll content. Nevertheless, this treatment induced significant alterations of the enzyme activity of nitrate reductase (EC 1.6.6.1) and glutamine synthetase (EC 6.3.1.2), although only after a few days of treatment. The effects were not confined to the exposed tissue, but were detectable also at the root level. In fact, nitrate reductase decreased in response to UV‐B in both leaf and root tissue, whereas glutamine synthetase was affected only in the root. In contrast, nitrate content was not influenced by the treatment, neither in root nor in leaf tissue, whilst leaf sucrose diminished in exposed plants only on the last day of treatment.
Isolation and expression of Lea gene in desiccation‐tolerant microspore‐derived embryos in Brassica spp.
Authors: Wakui Kenji , Takahata Yoshihito
Published: March 2003

Desiccation tolerance can be induced in microspore‐derived embryos of Brassica spp. by application of abscisic acid (ABA). As part of our effort to elucidate the molecular mechanism in induction of desiccation tolerance of embryos, we have isolated Lea genes and examined their expression in microspore‐derived embryos of B. napus and B. campestris. The cDNA clones of Lea genes were isolated from desiccation‐tolerant microspore‐derived embryos of both species. Two cDNA clones of B. napus and B. campestris shared a similar homology of 92.9% identity between the two deduced proteins. These proteins also had high homology with Lea 76 which was isolated from zygotic embryos of B. napus, but were 54 amino acids shorter than Lea 76. Acquisition of desiccation tolerance of the microspore‐derived embryos and accumulation of mRNA and its transcripts of the Lea genes began within 12 h of ABA treatment. The embryos treated with ABA for 48–168 h showed a high desiccation tolerance and accumulation of Lea proteins at high levels. The close relationships between the induction of desiccation tolerance of embryos and the expression level of Lea genes suggest an important function of Lea proteins for desiccation tolerance in embryos.
Glycolate metabolism in algal chloroplasts: inhibition by salicylhydroxamic acid (SHAM)
Authors: Goyal Arun
Published: March 2003

Unicellular green algae such as Chlamydomonas and Dunaliella excrete small amounts of glycolate during active photosynthesis. This phenomenon has been explained by the fact that these algae do not have leaf‐type peroxisomes and glycolate oxidase; instead, they have a limited capacity to metabolise glycolate in their mitochondria by a membrane‐associated glycolate dehydrogenase. Salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidase in plant and algal mitochondria, stimulates glycolate excretion by the algae or their isolated chloroplasts 5‐fold. In the presence of SHAM, cells of Chlamydomonas or Dunaliella grown with high‐CO2 (5% CO2 in air, v/v) or adapted with air levels of CO2 excreted glycolate at a rate of about 14 µmol glycolate mg−1 Chl h−1. Aminooxyacetate (AOA), an inhibitor of aminotransferases, also increases glycolate excretion by the algal cells or chloroplasts but at a lower rate (about 50%) than SHAM. The algal, light dependent, SHAM‐sensitive glycolate oxidizing system in the chloroplasts appears to be the primary site for glycolate oxidation, and it is different and more active then the minor mitochondrial glycolate dehydrogenase.
Lipid metabolism in the moss Dicranum scoparium: effect of light conditions and heavy metals on the accumulation of acetylenic triacylglycerols
Authors: Guschina Irina A. , Dobson Gary , Harwood John L.
Published: March 2003

Lipid metabolism in the moss Dicranum scoparium Hedw. was studied using radiolabelling from [1‐14C]acetate. The effect of two environmental parameters, light and heavy metals, on such metabolism was examined. Radiolabelling was approximately linear for 48 h after which the radioactivity in the total and polar lipid fractions remained constant in the light, whereas it declined in the dark. Pulse‐chase experiments confirmed that lipid labelling was influenced by light exposure. Light exposure altered the pattern of polar lipid labelling, especially of those lipids associated with chloroplast membranes. Within the neutral lipids, diacylglycerols and triacylglycerols (TAGs) were the major classes labelled. D. scoparium contained up to 45% of total acyl moieties as 9,12,15‐octadecatrien‐6‐ynoic (18:3A) acid and this was found in a TAG subfraction which could be separated by TLC. Although TAGs always represented 65–75% of total neutral lipid labelling, the proportion of TAGs containing 18:3A was increased with time and by light but reduced by exposure to environmentally relevant levels of Cu2+ and Pb2+. The data suggest that 18:3A is produced in D. scoparium by the action of a bi‐functional Δ6‐desaturase on α‐linolenate. Furthermore, important environmental factors (such as light and heavy metals) significantly change the metabolism of TAGs containing 18:3A.
High‐dose acyclovir prophylaxis reduces cytomegalovirus disease in liver transplant patients
Authors: Barkholt L. , Lewensohn‐Fuchs I. , Ericzon B.‐G. , Tydén G. , Andersson J.
Published: March 2003

Cytomegalovirus (CMV) is still a major pathogen in liver transplantation (LTX). The clinical efficacy of prophylactic high‐dose acyclovir therapy (800 mg qid) was assessed for the prevention of CMV infection and disease in liver recipients. Fifty‐five patients were enrolled in a prospective, randomised, double‐blind and placebo‐controlled trial; 28 on acyclovir vs. 27 on placebo. The therapy was given for 12 weeks. The patients were followed for 24 weeks.

CMV infection was diagnosed in 60% (16 on acyclovir, 17 on placebo) and CMV disease developed in 38% (7 on acyclovir, 14 on placebo) of the patients. The total mortality was 27% (6 on acyclovir, 10 on placebo). Acyclovir delayed 32% of the CMV infections and prevented 59% of the CMV disease cases which occurred in the placebo cohort. The time to CMV disease was significantly prolonged in patients on acyclovir as compared to patients on placebo (P=0.013).

Adverse events included neurotoxicity which occurred in 5 cases in the acyclovir, but none in the placebo arm, and nephrotoxicity which was detected in 6 patients in the acyclovir and 5 in the placebo arm, respectively.

We conclude that acyclovir prophylaxis significantly reduced the incidence of CMV disease, and delayed the onset of CMV infection in liver transplant patients.
Visual symptoms after lung transplantation: a case of progressive multifocal leukoencephalopathy
Authors: Ouwens J.P. , Haaxma‐Reiche H. , Verschuuren E.A.M. , Timens W. , Steenhuis L.H. , De Boer W.J. , Van Der Bij W.
Published: March 2003

After solid organ transplantation, signs and symptoms of the central nervous system may present a diagnostic challenge. A 43‐year‐old patient developed a decrease in vision 15 months after bilateral lung transplantation. The initial diagnosis was a left posterior cataract, but left eye cataract extraction did not improve his vision. Seizures led to investigation of a broader differential diagnosis (cyclosporine intoxication, post‐transplant lymphoproliferative disorder, infectious disease, chronic lymphatic leukemia). The clinical diagnosis of progressive multifocal leukoencephalopathy (PML) was confirmed by demonstration of JC virus in the cerebrospinal fluid and by autopsy findings. Modulation of the immunosuppressive regimen was unsuccessful. This case illustrates that decreased vision in immunocompromised patients may be the first manifestation of PML.
Epstein–Barr virus post‐transplant lymphoproliferative disease and virus‐specific therapy: pharmacological re‐activation of viral target genes with arginine butyrate
Authors: Mentzer S.J. , Perrine S.P. , Faller D.V.
Published: March 2003

Abstract: Lymphoproliferative disorders associated with the Epstein–Barr virus (EBV) include non‐Hodgkin’s lymphoma, Hodgkin’s lymphoma, and “post‐transplant lymphoproliferative disorders” (PTLD), which occur with immunosuppression after marrow and organ transplantation. PTLD is characterized by actively proliferating, latently infected EBV(+) B‐lymphocytes, and often manifests a rapidly progressive fatal clinical course if the immunosuppression cannot be reversed. Lung transplant recipients are a subset of patients at special risk for developing PTLD. The incidence of PTLD development in these patients has been estimated at 5–10%. Whereas immunologic and antiviral therapy have been moderately effective for treating EBV‐associated infections in the lytic phase, they have been less useful in the more common latent phase of the disease. One common treatment for herpesvirus infections has targeted the virus‐specific enzyme thymidine kinase (TK). The lack of viral TK expression in EBV(+) tumor cells, due to viral latency, makes anti‐viral therapy alone ineffective as an anti‐neoplastic therapy, however. We have developed a strategy for the treatment of EBV‐associated lymphomas/PTLD using pharmacologic induction of the latent viral TK gene and enzyme in the tumor cells, followed by treatment with ganciclovir. Arginine butyrate selectively activates the EBV TK gene in latently EBV‐infected human lymphoid cells and tumor cells. A Phase I/II trial has been initiated, employing an intra‐patient dose escalation of arginine butyrate combined with ganciclovir. In six patients with EBV‐associated lymphomas or PTLD, all of which were resistant to conventional radiation and/or chemotherapy, this combination produced complete clinical responses in four of six patients, with a partial response occurring in a fifth patient. Pathologic examination in two of three patients demonstrated complete necrosis of the EBV lymphoma, with no residual disease, following a single three‐week course of the combination therapy. Possible side‐effects of the therapy included nausea and reversible lethargy at the highest doses. One patient suffered acute liver failure, thought to be secondary to release of FasL from the necrotic tumor. Analysis of patient‐derived tumor cells in culture demonstrated that arginine butyrate produced selective induction of the EBV TK gene, which then conferred sensitivity to ganciclovir, resulting in tumor apoptosis. Additional patient accrual is sought for further evaluation of this therapy.
Cross‐species reactivity of a panel of antibodies with monkey and porcine tissue
Authors: Carter Anthony S. , Cerundolo Lucia , Koo Dicken D. H. , Rust Nigel A. , Morris, Peter J. , Fuggle Susan V.
Published: March 2003

The continuing shortage of organs available for transplantation limits the number of patients able to benefit from this highly successful form of therapy. Interest in alternative sources of organs has now turned towards the pig because of its physiological similarity to human. There is a requirement therefore for reagents not only for research purposes but possibly for studying xenotransplants in the clinical situation in the future. In this study, we have concentrated on determining the cross‐species reactivity of a large panel of antibodies directed against human leukocyte markers, testing peripheral blood leukocytes and also including renal tissue to determine non‐leukocyte cross‐reactivity. A total of 63 out of 127 antibodies cross‐reacted with cynomolgus monkey cells. Twenty of these antibodies stained similar populations of leukocytes to human, whereas the remaining 43 reacted with clearly different populations. The majority of antibodies (108/127) were unreactive with porcine leukocytes, reflecting the evolutionary differences between pig and man. Of the 19 antibodies cross‐reactive with porcine cells, seven reacted with similar proportions of leukocytes to human, whereas the remaining 12 antibodies stained entirely different populations. The most interesting, and potentially most useful, antibodies were four that reacted with human, cynomolgus monkey and porcine tissue in a similar manner, suggesting that the epitopes recognized are present on similar molecules. These antibodies were directed against CD29 (MEM101A, K20) and CD18 (BU87, 7E4), the common β1‐ and β2‐integrin subunits respectively. This study demonstrates that there are antigens common to cynomolgus monkey, pig and man that react with currently available antibodies. Nevertheless, when determining cross‐species reactivity of human antibodies, it is important to consider the possibility that there may be additional non‐leukocyte reactivity in other tissues.
IgY antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding
Authors: Fryer Jonathan , Firca Joseph , Leventhal Joseph , Blondie Beth , Malcolm Andrew , Ivancic David , Gandhi Ripal , Shah Ateet , Pao Winnie , Abecassis Michael , Kaufman Dixon , Stuart Frank , Anderson Byron
Published: March 2003

Avian IgY antibodies are structurally different from mammalian IgGs and do not fix mammalian complement components or bind human Fc receptors. As these antibody‐mediated interactions are believed to play significant roles in both hyperacute rejection (HAR) and acute vascular xenograft rejection (AVXR), IgY antibodies to xenoantigen target epitopes may inhibit these rejection processes. In this report, we show that chicken IgY antibodies to α‐Gal antigen epitopes and to other porcine aortic endothelial cell (PAEC) antigens block human xenoreactive natural antibody binding to both porcine and rat cardiac tissues and porcine kidney tissues. Chicken IgY antibodies blocked complement‐mediated lysis of PAECs by human serum, and inhibited antibody‐dependent cell‐mediated lysis of PAECs by heat‐inactivated human serum plus peripheral blood leukocytes. Binding of IgY to porcine endothelial cells did not affect cell morphology nor expression of E‐selectin. These results suggest that avian IgYs could be of potential use in inhibiting pig‐to‐human xenograft rejection.
Characterization of a monoclonal anti‐porcine CD3 antibody
Authors: Huang Christene A. , Lorf Thomas , Arn J. Scott , Koo Gloria C. , Blake Tom , Sachs David H.
Published: March 2003

Huang CA, Lorf T, Arn JS, Koo GC, Blake T, Sachs DH. Characterization of a monoclonal anti‐porcine CD3 antibody. Xenotransplantation 1999; 6: 201‐212. © Munksgaard, Copenhagen

Abstract: Partially inbred miniature swine have been developed in this laboratory as a large animal model for studies related to transplantation tolerance and as a source of hematopoietic cells and organs for xenotransplantation. The identification of swine CD3 specific mAbs capable of activating or depleting T cells in vitro and inducing an immunosuppressive state in vivo greatly facilitates studies of the swine immune system, transplantation tolerance and xenotransplantation research. Flow cytometry was used to determine the phenotypic profile of the swine specific mAb 898H2‐6‐15 (2‐6‐15). The specificity of 2‐6‐15 was further defined biochemically by surface labeling and immunoprecipitation. The ability of this mAb to activate pig T cells in vitro was examined by several criteria including proliferation assays, calcium flux analysis and detection of surface CD25 upregulation by fluorescence activated cell sorter (FACS) analysis. Monoclonal antibody 898H2‐6‐15 is specific for swine CD3 and is capable of inducing proliferation and CD25 upregulation in cultured swine peripheral blood lymphocytes. In addition, it induces calcium flux in purified pig T cells. Surprisingly, in contrast to described antibodies to CD3 in swine and other species, the binding of this antibody to porcine CD3 is dependent on the presence of extracellular calcium. Thus calcium was required in order to immunoprecipitate labeled surface molecules for biochemical analysis and to stain cell surfaces for FACS analysis of swine lymphocytes. In this paper, we describe a new swine CD3 specific mAb, 898H2‐6‐15 (2‐6‐15) the characteristics of which make it an extremely useful tool for in vitro and in vivo studies of the swine immune system and xenotransplantation. The availability of swine T cell specific reagents should facilitate the monitoring of swine T cell engraftment and/or release amongst xenogeneic mixed chimeras and thymic transplant recipients as well as provide a means to treat potential GvHD across xenogeneic barriers. We are currently evaluating the in vivo effects of 2‐6‐15 in the pig.
Human serum induces apoptosis of xenogeneic cardiomyocytes in vivo and in vitro
Authors: Lucien Jamie , Shimada Munehiko , Watzka Stefan , Ogiwara Masanori , Brockhausen Inka , Sandhu Jasbir , Coles John G.
Published: March 2003

Abstract: Discordant xenotransplantation is complicated by delayed xenograft rejection (DXR). Previous studies have demonstrated that anti‐apoptotic genes are protective against DXR. This study examines the hypothesis that apoptosis plays a role in human anti‐xenograft responses. C57BL/6 mice and NOD SCID mice were given a single intravenous injection of either a lethal dose (LD, survival < 30 min) or a sublethal dose (SLD) of human serum, and isolated pig and mouse rod‐shaped cardiomyocytes were exposed to human serum in vitro. In situ detection of apoptotic cells in mouse hearts was assessed using a terminal deoxynucleotidyl transferase‐mediated dUTP nicked‐end labeling assay. Mice transfused with human serum had approximately a 10‐fold increased percentage of apoptotic cells after SLD 18 h post‐injection compared with animals given saline, and a fourfold increase over LD. Administration of cobra venom factor (CVF) decomplemented SLD 18 h did not significantly (P > 0.05) alter the percentage apoptosis. The addition of 20 mM Gal‐α‐1,3‐Gal to SLD 18 h significantly (P < 0.05) reduced percentage apoptosis to levels comparable to saline treated control animals. In vitro using mouse and pig cardiomyocytes demonstrated parallel results as in vivo experiments.

Human serum induces apoptosis of cardiomyocytes in immunocompetent and immunoincompetent mice in vivo, as well as mouse and pig cardiomyocytes in vitro. Further, this apoptotic response can be inhibited by the addition of Gal‐α‐1,3‐Gal without affecting the capacity of the serum to cause HAR. These results demonstrate that a putative human serum factor induces a delayed apoptotic injury of xenograft tissues, and supports the hypothesis that apoptosis may be an important mediator of DXR.