bluuebunny/crossref_metadata_2025 huggingface dataset:
LeGRP1: A new member of glycine‐rich proteins from tomato (Lycopersicon esculentum)
Authors: Uthappa Mukatira , Muchhal Umesh S. , Baldwin James C. , Raghothama K. G.
Published: March 2003

Glycine‐rich proteins (GRPs) are members of a family of proteins with unique repeats of glycine‐containing motifs. This feature facilitates their association with plant structural components such as cell wall and vascular tissues. A transcript encoding a new member (LeGRP1) of the GRP family was identified from roots of tomato by the method of differential display of mRNA species. The cDNA is 1.15 kb in size and has an open reading frame encoding a peptide of 284 amino acids. Glycine residues make up 55% of the deduced peptide and the sequence is characterized by repeating (Gly‐X)n domains. The presence of a hydrophobic N‐terminus with a putative signal peptide indicates a probable association of the protein with the cell wall. Southern analysis shows that several LeGRP1 homologues are present in the genome. The LeGRP1 transcript accumulates predominantly in the roots, stem and petioles of tomato. A relatively lower level of LeGRP1 message was observed in the leaf mid‐rib, while the message is undetectable in the leaf lamina. Abiotic stresses like cold, heat, salt, desiccation and phosphate starvation did not significantly affect the expression of LeGRP1, whereas wounding resulted in a significant repression of the gene expression. Temporal regulation of LeGRP1 was observed during fruit development.
Control of the glutathione S‐transferase and mas1′ promoter‐driven GUS activity in auxin heterotrophic and autotrophic tobacco calli by exogenous 2,4‐d‐induced ethylene
Authors: Csiszár Jolán , Szabó Margit , Tari Irma , Erdei László
Published: March 2003

Auxin autotrophic and heterotrophic lines of tobacco calli may differ not only in their indoleacetic acid (IAA) synthetizing abilities and sensitivities to exogenous auxins, but also in their gene expression patterns. Auxin autotrophic callus tissues from the leaf protoplasts of transgenic Nicotiana tabacum SR1 plants involving mas1′::GUS gene fusion were generated and the growth of cultures was compared with that of the heterotrophic lines of the same transgenic tissues on MS medium containing different concentrations of IAA or 2,4‐d. The mas1′::GUS gene fusion expression was investigated, together with the glutathione S‐transferase activities (GST, EC 2.5.1.18) in auxin autotrophic and heterotrophic tobacco calli. Both the mas1′ promoter and GST gene promoters contain ocs or ocs‐like elements, responsible for both auxin and ethylene/wound inducibility. The mas1′ promoter exhibited a much higher expression activity in the heterotrophic cultures growing on IAA than in the autotrophic ones, but in contrast with the natural auxin, the mas1′::GUS activity decreased at elevated 2,4‐d concentrations in the heterotrophic tissues and increased with increasing 2,4‐d concentrations in the autotrophic lines. The induction of GST activity by different exogenous auxin concentrations was much higher in the autotrophic lines, especially in the case of 2,4‐d. Higher concentrations of external 2,4‐d resulted in increased ethylene production, which displayed different kinetics in the two types of calli. The ethylene‐inducing 2,4‐d concentrations increased the growth of the heterotrophic, but decreased that of the autotrophic lines. Blocking the ethylene receptors and hence the signal perception by 2,5‐norbornadiene (NBD) in the heterotrophic tissues increased the 2,4‐d‐induced mas1′ promoter and GST activities, suggesting that the gaseous hormone counteracted the auxin response pathway. This was not found in the autotrophic tissues, where NBD decreased the mas1′‐driven GUS activity. The GST activities were slightly decreased, or almost independent of the action of ethylene. It is suggested that the cross‐talk between the auxin‐ and ethylene‐induced signal transduction pathways may differ in the auxin autotrophic and heterotrophic lines.
Hormone physiology of pea mutants prevented from flowering by mutations gi or veg1
Authors: Beveridge Christine A. , Batge Shona L. , Ross John J. , Murfet Ian C.
Published: March 2003

The veg1 (vegetative) mutant in pea (Pisum sativum L.) does not flower under any circumstances and gi (gigas) mutants remain vegetative under certain conditions. gi plants are deficient in production of floral stimulus, whereas veg1 plants lack a response to floral stimulus. During long days in particular, these non‐flowering mutant plants eventually enter a stable compact phase characterised by a large reduction in internode length, small leaves and growth of lateral shoots from the upper‐stem (aerial) nodes. The first‐order laterals in turn produce second‐order laterals and so on in a reiterative pattern. The apical bud is reduced in size but continues active growth. Endogenous hormone measurements and gibberellin application studies with gi‐1, gi‐2 and veg1 plants indicate that a reduction in gibberellin and perhaps indole‐3‐acetic acid level may account, at least partially, for the compact aerial shoot phenotype. In the gi‐1 mutant, the compact phenotype is rescued by transfer from a 24‐ to an 8‐h photoperiod. We propose that in plants where flowering is prevented by a lack of floral stimulus or an inability to respond, the large reduction in photoperiod gene activity during long days may lead to a reduction in apical sink strength that is manifest in an altered hormone profile and weak apical dominance.
Pectin‐bound β‐galactosidase present in cell walls of carrot cells under the different calcium status
Authors: Konno Haruyoshi , Nakashima Susumu , Nakato Takako , Katoh Kenji
Published: March 2003

Glycosyl‐hydrolytic enzymes from suspension‐cultured carrot (Daucus carota L. cv. Kintoki) cells grown in calcium (Ca2+)‐deficient and normal liquid media were studied after extraction successively by K‐phosphate (pH 7.0) and Na‐acetate (pH 5.2) containing 3 M LiCl. The same activities were detected in two protein fractions from control and Ca2+‐deprived cells. The specific activities of α‐galactosidase and polygalacturonase decreased under Ca2+ deprivation, but β‐galactosidase activity in the buffer‐soluble protein from Ca2+‐deprived cells increased 1.7‐fold compared to control cells. Upon ion exchange and size‐exclusion chromatography the fraction (Ca‐Ia‐I) in the buffer‐soluble protein from Ca2+‐deprived cells represented β‐galactosidase activity associated with a galacturonic acid‐rich polysaccharide peak, whereas the corresponding fraction could hardly be detected in the buffer‐soluble protein from control cells. Several of the same glycosidase activities were detected in the extract solubilized with cyclohexane‐trans‐1,2‐diaminetetra‐acetate (CDTA) from active cell walls of Ca2+‐deprived cells as in the extract of control cells, but the β‐galactosidase activity was considerably reduced under Ca2+ deprivation. Following the same chromatography the fraction (CDTA‐Ca‐1) of β‐galactosidase activity in the extract solubilized with CDTA from active cell walls of Ca2+‐deprived cells was also completely overlapping with the peak of galacturonic acid‐rich polysaccharide. The molecular mass of fractions Ca‐Ia‐I and CDTA‐Ca‐1 was 300 kDa, and the polysaccharides in these two fractions were composed of approximately equal amounts of rhamnosyl and galacturonosyl residues. These results suggest that the increase of β‐galactosidase in the buffer‐soluble protein fraction from Ca2+‐deprived cells is the result of solubilization of a part of the acidic pectic polymer‐bound β‐galactosidase due to the structural changes in the cell walls that occur during Ca2+ deprivation.
Carbon dioxide and ethylene interactions in tulip bulbs
Authors: De Wild Hans P.J. , Gude Henk , Peppelenbos Herman W.
Published: March 2003

The effect of CO2 on ethylene‐induced gummosis (secretion of polysaccharides), weight loss and respiration in tulip bulbs (Tulipa gesneriana L.) was investigated. A pretreatment with 1‐MCP prevented these ethylene‐induced effects, indicating that ethylene action must have been directed via the ethylene receptor. Treatment with 0.3 Pa ethylene for 2 days caused gummosis on 50% of the total number of bulbs of cultivar Apeldoorn, known to be sensitive for gummosis. Addition of CO2 (10 kPa) reduced the ethylene‐induced gummosis to 18%. In a second experiment the influence of ethylene and CO2 on respiration and FW loss of bulbs of the cultivar Leen van der Mark was studied. A range of ethylene partial pressures (0.003–0.3 Pa) was applied continuously for 29 days. Ethylene caused a transient peak in O2 consumption rate during the first days after the start of application. The relation between O2 consumption rate and ethylene partial pressure could be described by Michaelis‐Menten kinetics. Respiratory peaks were reduced by CO2. This inhibition by CO2 could not totally be due to competition with ethylene at the receptor binding‐site, as was indicated by the use of an O2 consumption model. Pre‐treatment of bulbs with 1‐MCP and subsequent exposure to CO2 showed that CO2 could influence respiration irrespective of any interaction with ethylene. Ethylene and CO2 both stimulated weight loss. The effect of combined treatments of ethylene and CO2 on weight loss was at least as strong as the sum of the separate effects, which implies that competition between ethylene and CO2 at the receptor binding‐site was unlikely.
Salt stress induces up‐regulation of an efficient chloroplast antioxidant system in the salt‐tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species
Authors: Mittova Valentina , Tal Moshe , Volokita Micha , Guy Micha
Published: March 2003

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt‐tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H2O2 level and membrane lipid peroxidation was observed in chloroplasts of salt‐stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt‐stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione‐S‐transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non‐specific peroxidases (POD) were found in chloroplasts of salt‐treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt‐stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt‐treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt‐treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt‐dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up‐regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre‐exposure of Lpa plants to salt‐treatment conferred cross‐tolerance to paraquat‐induced oxidative stress while increased oxidative damage by paraquat‐treatment was found in salt‐stressed Lem plants.
A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves
Authors: Nakamura Kousuke , Shiraishi Naomasa , Hosoo Shingo , Sueyoshi Kuni , Sugimoto Toshio , Nanmori Takashi , Nakagawa Hiroki , Oji Yoshikiyo
Published: March 2003

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark‐activated or dark‐induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark‐activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark‐treated leaves by means of successive chromatographies on Q‐Sepharose, Blue Sepharose, FPLC Q‐Sepharose, and ATP‐γ‐Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+‐independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14‐3‐3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3‐hydroxy‐3‐methylglutaryl‐Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark‐activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark‐activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.
Cloning of a vacuolar invertase from Belgian endive leaves (Cichorium intybus)
Authors: Van den Ende Wim , Michiels An , Le Roy Katrien , Van Laere André
Published: March 2003

Although a lot of vacuolar invertase (EC 3.2.1.26) cDNAs are available from a diversity of plant species, up to now no sequence information is available on invertases from any dicot fructan‐containing species. Therefore, we describe the cloning of vacuolar acid invertase cDNA from etiolated Belgian endive leaves (Cichorium intybus L. var. foliosum cv. Flash), formed throughout the forcing process of the witloof chicory roots. Full‐length cDNA was obtained by a combination of RT‐PCR, PCR and 5′‐ and 3′ RACE RT‐PCR, starting with primers based on conserved amino acid sequences. The cloned chicory acid invertase groups together with vacuolar type invertases and fructan biosynthetic enzymes. A putative role for vacuolar type invertases in fructan synthesizing plants is discussed.
Oxygen‐mediated cold‐acclimation in cucumber (Cucumis sativus) seedlings
Authors: Erez Amnon , Cohen Eliahou , Frenkel Chaim
Published: March 2003

Cold acclimation of etiolated cucumber seedlings, consisting of cooling at 12°C for 48 h followed by a warming period at 25°C, led to tolerance to subsequent chilling at 2°C. Tolerance, as evidenced by freedom from chilling injury and continued growth, developed during the warming period in a time‐course manner for 12 h but decreased with prolonged warming. A similar increase and subsequent decrease was also observed in the content of palmitic, linoleic and linolenic acids in total lipid fraction from cucumber hypocotyl tissue. During the warming period supra‐ambient oxygen stimulated, whereas subambient oxygen inhibited, the increase in fatty acid content as well as development of chilling tolerance. A strong correlation between oxygen‐mediated changes in fatty acid content and associated development of cold tolerance suggests that both these processes are interrelated. Cold acclimation, but not cold stress, led to an increase followed by a decrease in CO2 evolution suggesting that a respiratory upsurge is yet another feature of cold acclimation in cucumbers.
A gene encoding stellacyanin is induced in Capsicum annuum by pathogens, methyl jasmonate, abscisic acid, wounding, drought and salt stress
Authors: Kong Hye Young , Jung Ho Won , Lee Sung Chul , Choi Doil , Hwang Byung Kook
Published: March 2003

A stellacyanin cDNA clone (CASLP1) was isolated from a pepper cDNA library from hypersensitive response (HR) lesions of leaves infected with an avirulent strain of Xanthomonas campestris pv. vesicatoria. The deduced amino acid sequences of CASLP1 are homologous to those of stellacyanins from cucumber, maize, pea and Arabidopsis. The CASLP1 mRNA was not constitutively expressed in all organs of pepper, but strongly induced and accumulated in pepper tissues infected with X. campestris pv. vesicatoria, Colletotrichum coccodes, Phytophthora capsici or C. gloeosporioides. In situ hybridization results revealed that CASLP1 transcripts were strongly localized in the phloem areas of vascular bundles in infected tissues of pepper stems and fruits. CASLP1 mRNA accumulation was found in lower pepper leaves infected by either virulent or avirulent strains of X. campestris pv. vesicatoria and non‐pathogenic Pseudomonas fluorescens, but not in uninoculated upper leaves. Induction of this CASLP1 gene occurred in pepper leaves applied with methyl jasmonate (MeJA), but not with ethylene, salicylic acid, dl‐β‐amino‐n‐butyric acid and benzothiadiazole. Accumulation of CASLP1 transcripts was locally or systemically induced in pepper leaves upon mechanical wounding and was activated in a MeJA‐dependent manner. The CASLP1 transcript was also strongly induced in leaf and stem tissues after exposure of pepper plants to abscisic acid, salt and drought.
Differences in growth and nitrogen productivity between a stay‐green genotype and a wild‐type of Lolium perenne under limiting relative addition rates of nitrate supply
Authors: Macduff James , Raistrick Neil , Humphreys Mervyn
Published: March 2003

The stay‐green mutation of the nuclear gene sid inhibits chlorophyll degradation during leaf senescence in grasses. Decreased productivity is expected under conditions of limited external N availability, due to the higher retention of N in senescent tissues. However, this has not been reported when plants are grown at limiting low external concentrations of N. In the present study a different approach was taken, based on the relative addition rate method for defining limiting N supply. Wild‐type and stay‐green genotypes of Lolium perenne L. were grown for 30 days in flowing solution culture and then supplied with NO3– on an hourly basis over 35 days at relative addition rates (RARs) of 0.03, 0.06, 0.09 and 0.12 day−1, ranging from severe N limitation to optimal supply. Plants were steady‐state labelled with 15NO3– prior to RAR treatments, then switched to 14NO3– to allow measurement of the re‐distribution of N absorbed prior to RAR control. Following acclimation, relative growth rates (RGRs) approached the corresponding RAR, but were significantly lower for stay‐green than wild‐type at RARs of 0.03 and 0.06 day−1. Tiller numbers were lower in stay‐green plants after 35 days at all RARs except 0.12 day−1. Concentrations of total N in senescent laminae of stay‐green plants exceeded those in wild‐type plants by a similar margin (4.8–6.8 mg g−1 DW) irrespective of RAR. Maximum nitrogen productivity (Pn) was 3.9 g DW g−1 N day−1 (Nmin = 7.1 mg g−1 DW) in wild‐type plants, and 5.1 g DW g−1 N day−1 (Nmin = 10.7 mg g−1 DW) in stay‐green plants. The higher N productivity of stay‐green plants indicated these plants used a smaller pool of metabolically available N more efficiently in biosynthesis compared with wild‐type plants. The retention of N, absorbed prior to RAR treatments, in senescent laminae was significantly higher in stay‐green plants at RAR of 0.03 day−1 after day 21 (i.e. 20% versus 15% of the total N recovered). However, in terms of the whole N economy of the plant the margin represented only 1.7% of the total N content on day 35.
Cloning and characterization of a glutathione S‐transferase induced by a herbicide safener in barley (Hordeum vulgare)
Authors: Scalla René , Roulet Alain
Published: March 2003

The effect of the herbicide safener mefenpyr‐diethyl on glutathione S‐transferase (GST, EC 2.5.1.18) activities of dark‐grown barley (Hordeum vulgare cv. Alexis) was examined. Mefenpyr‐diethyl treatment increased the GST activity with 1‐chloro‐2,4‐dinitrobenzene (CDNB) and the herbicide fenoxaprop as substrates. Glutathione (GSH) peroxidase activity was markedly increased. GST subunits were analysed by high performance liquid chromatography (HPLC). The quantities of two major subunits were increased by the safener treatment, while the quantities of two other major subunits remained constant. A cDNA encoding the most abundant inducible GST (HvGST6) was cloned and expressed in E. coli. The purified enzyme exhibited a low activity with herbicides as substrates. By contrast, it exhibited a strong GSH peroxidase activity.
The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles
Authors: Koren'kov Victor D. , Shepherd Ryan W. , Wagner Georg J.
Published: March 2003

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca2+/H+ and Cd2+/H+ antiporter activities of tonoplast vesicles from non‐transformed, wild‐type plants. Solubilization and reconstitution of oat‐seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6‐fold loss of protein, about a 6‐fold enhancement of Cd2+/H+ antiport specific activity (at 10 µM Cd2+), and almost complete loss of Ca2+/H+ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd2+ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca2+ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd2+ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd2+/H+ activity and verapamil binding to vesicles, while Ca2+ pre‐treatment was much less protective of Ca2+/H+ activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd2+/H+ and Ca2+/H+. Our observation that solubilization and reconstitution have similar effects on both Cd2+/H+ and Ca2+/H+ activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.
Monoterpene levels in needles of Douglas fir exposed to elevated CO2 and temperature
Authors: Snow Michael D. , Bard Raymond R. , Olszyk David M. , Minster Lynde M. , Hager Angela N. , Tingey David T.
Published: March 2003

Monoterpene levels in current year needles of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings were measured at the end of 4 years of exposure to ambient or elevated CO2 (+179 µmol mol−1), and ambient or elevated temperature (+0.3.5^C). Eleven monoterpenes were identified and quantified using gas chromatography/flame ionization detector/mass spectroscopy, with eight of these compounds regularly occurring in all trees examined. Elevated CO2 exposure significantly reduced the levels for four of the eight main compounds in needles. Total monoterpene production was reduced by 52% (P < 0.05). Elevated temperature also reduced monoterpene levels (P < 0.07). The combination of elevated temperature and elevated CO2 resulted in a 64% reduction in total monoterpenes compared with needles on ambient temperature trees. Two‐way anova showed no significant temperature‐CO2 interaction. It is hypothesized that seasonal reductions in needle monoterpene pools under elevated CO2 and temperature conditions may be due to a combination of competing carbon sinks, including increased carbon flux through the roots.
Synergistic interactions between volicitin, jasmonic acid and ethylene mediate insect‐induced volatile emission in Zea mays
Authors: Schmelz Eric A. , Alborn Hans T. , Tumlinson James H.
Published: March 2003

Plants display differential responses following mechanical damage and insect herbivory. Both caterpillar attack and the application of caterpillar oral secretions (OS) to wounded leaves stimulates volatile emission above mechanical damage alone. Volicitin (N‐17‐hydroxylinolenoyl‐l‐glutamine), present in beet armyworm (BAW, Spodoptera exigua) OS, is a powerful elicitor of volatiles in excised maize seedlings (Zea mays cv. Delprim). We consider some of the mechanistic differences between wounding and insect herbivory in maize by examining the activity of volicitin, changes in jasmonic acid (JA) levels, and volatile emission from both intact plant and excised leaf bioassays. Compared to mechanical damage alone, volicitin stimulated increases in both JA levels and sesquiterpene volatiles when applied to intact plants. In a bioassay comparison, excised leaves were more sensitive and produced far greater volatile responses than intact plants following applications of both volicitin and JA. In the excised leaf bioassay, volicitin applications (10–500 pmol) to wounded leaves resulted in dose dependent JA increases and a direct positive relationship between JA and sesquiterpene volatile emission. Interestingly, volicitin‐induced JA levels did not differ between intact and excised bioassays, suggesting a possible interaction of JA with other regulatory signals in excised plants. In addition to JA, insect herbivory is known to stimulate the production of ethylene. Significant increases in ethylene were induced only by BAW herbivory and not by either wounding or volicitin treatments. Using intact plant bioassays, ethylene (at 1 µl l−1 or less) greatly promoted volatile emission induced by volicitin and JA but not mechanical damage alone. For intact plants, wounding, elicitor‐induced JA and insect‐induced ethylene appear to be important interacting components in the stimulation of insect‐induced volatile emission.
The production of transgenic pigs for potential use in clinical xenotransplantation: microbiological evaluation
Authors: Tucker Alexander , Belcher Carolyn , Moloo Badru , Bell Judith , Mazzulli Tony , Humar Atul , Hughes Allison , McArdle Peter , Talbot Antony
Published: March 2003

Debate over the infection hazards of pig‐to‐human xenotransplantation has focused mainly on the porcine endogenous retroviruses (PERV). However, hazards of exogenous infectious agents possibly associated with the xenograft have also been evaluated (Xenotransplantation 2000; 7: 143). We report the results of a health monitoring program demonstrating the exclusion of more than 80 potential pathogens from nine cohorts of pigs reared in a high welfare bioexclusion facility as potential xenograft source animals. A dynamic bacterial flora of pigs reared under barrier conditions was characterized, emphasizing the significance of monitoring for multiresistant antimicrobial sensitivity patterns. Evidence was found for exclusion of two commonly residual exogenous viruses, porcine cytomegalovirus and porcine lymphotropic herpesviruses, among a proportion of the cohorts tested. Finally, there was histopathological evidence for low grade pneumonitis among sentinel pigs, likely to have been associated with the use of quaternary ammonium disinfectants during the production process, indicating a need for review of toxicology data for disinfectant agents used in such bioexclusion systems. Intensive health monitoring programs, based upon regularly updated recommendations from the microbiological research community, will enable significant reductions in the potential hazards associated with pig‐to‐human xenotransplantation.
Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells
Authors: Tsuyuki Shigeru , Kono Mari , Bloom Eda T.
Published: March 2003

Tsuyuki S, Kono M, Bloom ET. Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells. Xenotransplantation 2002; 9:410–421. © Blackwell Munksgaard, 2002

Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti‐pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas+ human T‐cell line, Jurkat. Anti‐human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose‐dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas+ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS‐7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL‐2 and anti‐CD3, and fresh IL‐2‐activated human (natural killer) NK cells. Moreover, porcine Fas L‐transfected COS‐7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock‐transfected COS‐7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.
Effect of various forms of the C1 esterase inhibitor (C1‐INH) and DAF on complement mediated xenogeneic cell lysis
Authors: Fukuta Daisuke , Miyagawa Shuji , Yamada Mako , Matsunami Katsuyoshi , Kurihara Takashi , Shirasu Akio , Hattori Hiroyuki , Shirakura Ryota
Published: March 2003

Fukuta D, Miyagawa S, Yamada M, Matsunami K, Kurihara T, Shirasu A, Hattori H, Shirakura R. Effect of various forms of the C1 esterase inhibitor (C1‐INH) and DAF on complement mediated xenogeneic cell lysis. Xenotransplantation 2003; 10: 132–141. © Blackwell Munksgaard, 2003

The purpose of the present study was to assess the effect of various forms of the surface‐bound form of the C1 esterase inhibitor (C1‐INH‐PI) and decay accelerating factor (DAF) on xenogenic cells. cDNAs of various deletion mutants of the C1‐INH‐PI, such as delta‐1‐99 amino acid (AA), delta‐108‐183AA loop, delta‐whole loop, delta‐exon5, delta‐exon6 + 7, and delta‐exon5 + 6 + 7, and that of DAF, the delta‐short consensus repeat (SCR) 1‐DAF were established. While all deletion mutants of C1‐INH‐PI except the delta‐1‐99AA were expressed in the cytoplasm but not on the cell surface, the delta‐1‐99AA was clearly expressed on the xenogeneic cell surface. Amelioration of complement‐mediated xenogeneic cell lysis by delta‐1‐99AA was next tested, and compared with delta‐SCR1 DAF. Both molecules blocked human complement‐mediated cell lysis by approximately 57 to 90 and 93 to 98%, respectively, in Chinese hamster ovarian tumor (CHO) cells and pig endothelial cells (PECs). The CHO cell transfectants were incubated with 20% normal human serum, and the amounts of C4 and C3 deposition on the cell surface were analysed by flow cytometry. The DAF transfectant showed a large amount of C4‐deposition and much less C3‐deposition than the controls (approximately 85% suppression), whereas the delta‐1‐99AA showed approximately a 40% suppression in both C4‐ and C3‐deposition. Consequently, both the delta‐1‐99AA C1‐INH‐PI and delta‐SCR1 DAF molecules are quite effective in down‐regulating the xenogeneic cell lysis, but accomplished this in different manners.
The role of intrahepatic lymphocytes in mediating protective immunity induced by attenuated Plasmodium berghei sporozoites
Authors: Krzych Urszula , Schwenk Robert , Guebre‐Xabier Mimi , Sun Peifang , Palmer Dupeh , White Katherine , Chalom Isaac
Published: March 2003

Summary: Exposure to irradiated Plasmodium sporozoites (g‐spz) results in protection against malaria. Like infectious spz, g‐spz colonize hepatocytes to undergo maturation. Disruption of liver stage development prevents the generation of protection, which appears, therefore, to depend on liver stage antigens. Although some mechanisms of protection have been identified, they do not include a role for intrahepatic mononuclear cells (IHMC). We demonstrated that P. berghei g‐spz‐immune murine IHMC adoptively transfer protection to naive recipients. Characterization of intrahepatic CD4+ T cells revealed an immediate, albeit transient, response to g‐spz, while the response of CD8+ T cells is delayed until acquisition of protection. It is presumed that activated CD8+ T cells home to the liver to die; g‐spz‐induced CD8+CD45RBloCD44hi T cells, however, persist in the liver, but not the spleen, during protracted protection. The association between CD8+CD45RBloCD44hi T cells and protection has been verified using MHC class I and CD1 knockout mice and mice with disrupted liver stage parasites. Based on kinetic studies, we propose that interferon‐g, presumably released by intrahepatic effector CD8+ T cells, mediates protection; the persistence of CD8+ T cells is, in turn, linked to Plasmodium antigen depots and cytokines released by CD4+ T cells and/or NK T cells.
A less‐invasive approach with orthodontic treatment in Beckwith–Wiedeman patients
Authors: Giancotti A , Romanini G , Girolamo R Di , Arcuri C
Published: March 2003

The Beckwith–Wiedeman syndrome (BWS) is a rare genetic disorder, linked to an alteration on the short arm of chromosome 11 that comprises multiple congenital anomalies. Macroglossia is the predominant finding, with subsequent protrusion of dentoalveolar structures, which results in a protruding mandible, anterior open bite, abnormally obtuse gonial angle and increased mandibular length. A less‐invasive treatment with orthopaedic appliances in a patient with early tongue reduction is presented. This work summarizes the oral signs linked to macroglossia, and highlights the influence of macroglossia on mandibular growth structures. In our opinion, glossotomy could be carried out in the paediatric patient as a preventive measure in that it curbs the tongue's influence on skeletal growth and dramatically reduces the duration and extensiveness of subsequent treatment.
Structural basis of MHC class I recognition by natural killer cell receptors
Authors: Sawicki Mark W. , Dimasi Nazzareno , Natarajan Kannan , Wang Jian , Margulies David H. , Mariuzza Roy A.
Published: March 2003

Summary: Natural killer (NK)‐cell function is regulated by NK receptors that recognize MHC class I (MHC‐I) molecules on target cells. Two structurally distinct families of NK receptors have been identified, the immunoglobulin‐like family (killer cell immunoglobulin‐like receptors (KIRs), leukocyte immunoglobulin‐like receptors (LIRs)) and the C‐type lectin‐like family (Ly49, CD94/NKG2A, NKG2D, CD69). Recently, the three‐dimensional structures of several NK receptors were determined, in free form or bound to MHC‐I. These include those of unbound KIRs, NKG2D, CD69, LIR‐1 and the CD94 subunit of the CD94/NKG2A heterodimer. Together, these structures define the basic molecular architecture of both the immunoglobulin‐like and C‐type lectin‐like families of NK receptors. In addition, crystal structures have been reported for the complex between Ly49A and H‐2Dd, and for KIR2DL2 bound to HLA‐Cw3. The complex structures provide a framework for understanding MHC‐I recognition by NK receptors from both families and reveal striking differences in the nature of this recognition, despite the receptors’ functional similarity.

This research was supported, in part, by National Institutes of Health grants R01 AI47900 and R37 36900 (RAM) and a fellowship from the Cancer Research Institute (MWS). We are grateful to DW Wolan and IA Wilson for providing coordinates of NKG2D prior to publication, and to members of our laboratories for encouragement.
Novel immune‐type receptor genes
Authors: Litman Gary W. , Hawke Noel A. , Yoder Jeffrey A.
Published: March 2003

Summary: Novel immune‐type receptor (NITR) genes, which initially were identified in the Southern pufferfish (Spheroides nephelus), encode products which consist of an extracellular variable (V) and V‐like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail, which typically possesses an immunoreceptor tyrosine‐based inhibition motif (ITIM). Multiple NITR genes have been identified in close, contiguous chromosomal linkage. The V regions of NITRs resemble prototypic forms defined for immunoglobulin (Ig) and T‐cell antigen receptor (TCR), are present in multiple families and exhibit regionalized variation in sequence, which also occurs in Ig and TCR. Comparisons of exons encoding transmembrane and cytoplasmic regions of multiple NITRs suggest that exon shuffling has factored in the diversification of the NITR gene complex. Zebrafish (Danio rerio) NITRs exhibit many of these characteristics. NITRs that have been identified in additional species of bony fish demonstrate additional variation in the number of extracellular domains as well as in the presence of intramembranous charged residues, cytoplasmic tails and ITIMs. The presence in NITRs of V regions that are related closely to those found in Ig and TCR, as well as regulatory motifs and other structural features that are characteristic of immune inhibitory receptors encoded at the leukocyte receptor cluster, suggests that the NITRs are representative of an integral stage in the evolution of innate and adaptive immune function.

This research was supported by grants AI23338 to GWL and GM20231 to JAY from the National Institutes of Health as well as a grant from The Pediatric Cancer Foundation, Inc. to GWL.
Thymic development and repertoire selection: the rat perspective
Authors: Hünig Thomas , Torres‐Nagel Nora , Mehling Beatrix , Park Hyun‐Jung , Herrmann Thomas
Published: March 2003

Summary: This review summarizes our current knowledge of T‐cell maturation and repertoire selection in the rat thymus. Some unique features of early thymocyte development and of CD4/CD8 lineage decision are described. A detailed analysis of lineage progression through the CD4, CD8 “double positive” compartment and T‐cell receptor‐induced CD8 T‐cell maturation in cell culture is provided. A second emphasis is placed on interactions between germline‐encoded T‐cell receptor elements with MHC molecules in thymic repertoire selection and alloreactivity
Rat macrophages: membrane glycoproteins in differentiation and function
Authors: Van Den Berg Timo K. , Döpp Ed A. , Dijkstra Christine D.
Published: March 2003

Summary: Macrophages (mφ) play a crucial role in the immune system. The rat offers unique advantages for studying the biology of mφ. Firstly, monoclonal antibodies (mAb) against many rat mφ surface glycoproteins have become available. These have not only demonstrated a considerable heterogeneity among mφ, but have also allowed the characterization of various mφ surface molecules in terms of structure, expression regulation and function. Furthermore, substantial numbers of rat mφ can be isolated from various sites (e.g. blood, peritoneal cavity, lung and other tissues), enabling proper molecular and functional analysis of these mφ populations. Finally, a number of (unique) experimental models for human diseases have been developed in the rat, making possible the evaluation of the involvement of mφ in pathogenesis. For this, a method for the selective elimination of mφ from various tissues in vivo has proven very useful. Here, we will review the contribution that the rat has made to understanding the immunobiology of mφ. In particular, we will discuss the surface (glyco)proteins on rat mφ in differentiation and function, and the involvement of mφ in rat models of disease.
Arthritis induced in rats with non‐immunogenic adjuvants as models for rheumatoid arthritis
Authors: Holmdahl Rikard , Lorentzen Johnny C. , Lu Shemin , Olofsson Peter , Wester Lena , Holmberg Jens , Pettersson Ulf
Published: March 2003

Summary:

Rat models are useful for studies of the pathogenesis of rheumatoid arthritis (RA) since rats are extraordinarily sensitive to induction of arthritis with adjuvants. Injection of not only the classical complete Freund's adjuvant but also mineral oil without mycobacteria and pure adjuvants such as pristane and squalene, induce severe arthritis in many rat strains. Models like pristane‐induced arthritis in rats are optimal models for RA since they fulfill the RA criteria including a chronic relapsing disease course. Arthritogenic adjuvants like pristane, avridine, squalene and mineral oil are not immunogenic since they do not contain major histocompatibility complex (MHC) binding peptides. Nevertheless, the diseases are MHC‐associated and dependent on the activation of αβTCR (T‐cell receptor)‐expressing T cells. However, it has not been possible to link the immune response to joint antigens or other endogenous components although immunization with various cartilage proteins induce arthritis but with different pathogeneses. To unravel the mechanisms behind adjuvant‐induced arthritis, a disease‐oriented genetic approach is optimal. Several loci that control onset of arthritis, severity and chronicity of the disease have been identified in genetic crosses and most of these have been confirmed in congenic strains. In addition, many of these loci are found in other autoimmune models in the rat as well as associated with arthritis in mice and humans.
One cell, one antibody: prelude and aftermath
Authors: Nossal G. J. V.
Published: March 2003

Summary: This essay seeks to summarize one scientist's pilgrim's progress through the world of antibody formation, attempting to capture the flavor of problems as encountered within their times. It seeks to portray a world where the direct template hypothesis ruled the day, where the function of lymphocytes was unknown, let alone the difference between T and B cells, and where antibody genes were but a dream. Struggles to establish the credentials of the clonal selection hypothesis are presented in some detail, as are the implications which followed the ‘one cell − one antibody’ discovery. The other two main preoccupations of the author are presented more briefly, namely the essential features of the germinal center and the cellular basis of immunological tolerance within the B lymphocyte compartment. Naturally, the experiments become more sophisticated as both knowledge and technology mature. A continuing thread within the unfolding story is that one must not shy away from developing new techniques when problems demand them.
Fas ligand expressing mononuclear cells around intrahepatic bile ducts co‐express CD68 in primary biliary cirrhosis
Authors: Iwata Mitsuhiro , Harada Kenichi , Hiramatsu Katsushi , Tsuneyama Kouichi , Kaneko Shuichi , Kobayashi Kenichi , Nakanuma Yasuni
Published: March 2003

Abstract: Aims/Background: Apoptosis, including the Fas system, has been implicated in progressive bile duct loss in primary biliary cirrhosis (PBC). In this study, we attempted to analyze Fas ligand (FasL) expressing mononuclear cells infiltrating in the portal tracts of PBC. Methods: We immunohistochemically assessed co‐expression of leukocyte markers and FasL on infiltrating mononuclear cells in 18 patients with PBC. Twenty‐five patients with chronic hepatitis C (CH‐C) were used as controls. Results: In PBC, FasL expressing cells were scattered in the portal tracts,

and some were accentuated around the damaged bile ducts. In addition, these cells co‐expressed CD68 (71%), a marker of monocytes, but not UCHL‐1, CD3 and CD57, markers of activated T cells and natural killer cells. By contrast, in CH‐C, the biliocentric pattern of FasL expression was not evident, and about half of FasL expressing cells (42–56%) co‐expressed UCHL‐1, CD3 or CD57. CD14, a receptor for bacterial products such as lipopolysaccharides, was also detected on a proportion of FasL expressing mononuclear cells around the damaged bile ducts in PBC. Conclusion: The results suggest that in PBC, FasL expressing CD68+ monocytes are at least partly involved in apoptotic bile duct loss mediated by the Fas system, and a surface molecule, CD14, participates in this process.
Role of myofibroblasts in tumour encapsulation of hepatocellular carcinoma in haemochromatosis
Authors: Bridle Kim R. , Crawford Darrell H. G. , Powell Lawrie W. , Ramm Grant A.
Published: March 2003

Abstract: Background/Aims: Hepatocellular carcinoma is a common malignancy and a major complication of untreated haemochromatosis. Encapsulation of liver tumours has been associated with a better prognosis and longer disease‐free periods following resection. This study investigated the source of the tumour capsule in patients with haemochromatosis and coexisting hepatocellular carcinoma and examined potential factors influencing development. Methods: Five haemochromatosis patients with encapsulated hepatocellular carcinoma were studied. Myofibroblasts were identified using combined immunohistochemistry and in situ hybridisation for α‐smooth muscle actin and procollagen α1(I) mRNA, respectively. Immunohistochemistry was also performed for transforming growth factor (TGF)‐β1, platelet‐derived growth factor (PDGF)‐β receptor and malondialdehyde. Results: Procollagen α1(I) mRNA co‐localised to α‐smooth muscle actin positive myofibroblasts. The number of myofibroblasts was maximal within the capsule and decreased away from the tumour. TGF‐β1 protein was expressed in iron‐loaded cells in non‐tumour liver at the interface of tumour capsule. PDGF‐β receptor expression was observed in mesenchymal cells in the tumour capsule and in portal tracts. Malondialdehyde adducts were observed in the tumour, non‐tumour tissue and in the capsule. Conclusions: This study provides evidence that myofibroblasts are the cell type responsible for collagen production within the tumour capsule surrounding hepatocellular carcinoma in haemochromatosis. The production of TGF‐β1 by iron‐loaded hepatic cells at the tumour capsule interface may perpetuate the myofibroblastic phenotype, resulting in the formation of the tumour capsule.