bluuebunny/crossref_metadata_2025 huggingface dataset:
Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation
Authors: Hideg Éva , Ogawa Ken'ichi , Kálai Tamás , Hideg Kálmán
Published: March 2003

Arabidopsis thaliana leaves were infiltrated with DanePy (3‐(N‐diethylaminoethyl)‐N‐dansyl)aminomethyl‐2,5‐dihydro‐2,2,5,5‐tetramethyl‐1H‐pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts. These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.
Three MADS‐box genes similar toAPETALA1andFRUITFULLfrom silver birch (Betula pendula)
Authors: Elo Annakaisa , Lemmetyinen Juha , Turunen Marja‐Leena , Tikka Liisa , Sopanen Tuomas
Published: March 2003

Despite intensive research on genetic regulation of flower development there are still only a few studies on the early phases of this process in perennial plants like trees. The aim of this study has been to identify genes that regulate early stages of inflorescence development in silver birch (Betula pendulaRoth) and to follow the expression of these genes during development of the unisexual birch inflorescences. Here we describe the cloning and characterization of 3 cDNAs representing MADS‐box genes designatedBpMADS3, BpMADS4andBpMADS5,all belonging to theAP1/SQUAgroup of plant MADS‐box genes. According to RNA blot analysis, all 3 genes are active during the development of both male and female inflorescences. However, differences in patterns of expression suggest that they play different roles.BpMADS3is most similar in sequence toAP1andSQUA,but it seems to have the highest expression at late developmental stages.BpMADS4is most similar in sequence to theArabidopsisgeneFRUITFULL, but is expressed, in addition to developing inflorescences, in shoots and roots.BpMADS5is also similar toFRUITFULL;its expression seems to be inflorescence‐specific and continues during fruit development. Ectopic expression of eitherBpMADS3, BpMADS4orBpMADS5with the CaMV 35S promoter in tobacco results in extremely early flowering. All of these birch genes seem to act early during the transition to reproductive phase and might be involved in the determination of the identity of the inflorescence or flower meristem. They could apparently be used to accelerate flowering in various plant species.
Novel cytokinins: The predominant forms in mature buds of Pinus radiata
Authors: Zhang Huaibi , Horgan Kathryn Joan , Stewart Reynolds Paul Hugh , Norris Gill Erna , Jameson Paula Elizabeth
Published: March 2003

To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double‐solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high‐pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine‐9‐(glucopyranosyl riboside), dihydrozeatin‐9‐(glucopyranosyl riboside) and confirmed the presence of zeatin‐9‐(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl‐linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl‐linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O‐ or N‐glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.
Small complex‐type N‐linked glycans are attached to cell‐wall bound exo‐β‐glucanases of both mung bean and barley seedlings
Authors: Kotake Toshihisa , Tonari Akiko , Ohta Masaya , Matsuura Fumito , Sakurai Naoki
Published: March 2003

N‐linked glycans of wall‐bound exo‐β‐glucanases from mung bean and barley seedlings, namely Mung‐ExoI and Barley‐ExoII, were characterized. The N‐linked glycans of Mung‐ExoI and Barley‐ExoII were liberated by gas‐phase hydrazinolysis followed by re‐N‐acetylation. Their structures were determined by two‐dimensional sugar‐mapping analysis and MALDI‐TOF mass spectrometry. N‐glycans from both glucanases were of paucimannosidic‐type (small complex‐type) structures, Manα1‐6(±Manα1‐3)(Xylβ1‐2)Manβ1‐4GlcNAcβ1‐4(±Fucα1‐3) GlcNAc, which are known as typical vacuole‐type N‐glycans. The results suggest that N‐glycans of cell‐wall glucanase were produced by partial trimming of complex‐type N‐glycans by exoglycosidases during its transport from Golgi apparatus to cell walls or in the cell walls.
Seasonal changes of lipid content in the leaves of some ferns
Authors: Rozenstvet O. A. , Saksonov S. V. , Filin V. R. , Dembitsky V. M.
Published: March 2003

Seasonal distribution of total lipids, of total and individual phospholipids as well as of one betaine lipid, diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS), found in the leaves of Dryopteris carthusiana, Dryopteris filix‐mas, Matteuccia struthiopteris and Pteridium aquilinum was studied. Using thin‐layer chromatography it was shown that lipid composition was different in these species and changed substantially throughout the development of scrolls (unrolled young leaves) to vegetatively mature leaves. The maximum concentration of DGTS for all the examined species was found in scrolls. DGTS then decreased to its minimum level or entirely disappeared to reappear at later stages of leaf development. This may indicate a specific role of DGTS in the plant membrane.
Effects of putrescine accumulation in tobacco transgenic plants with different expression levels of oat arginine decarboxylase
Authors: Panicot Mireia , Masgrau Carles , Borrell Antonio , Cordeiro Alexandra , Tiburcio Antonio Fernández , Altabella Teresa
Published: March 2003

Arginine decarboxylase (ADC; EC 4.1.1.19) is a key enzyme in one of the two possible ways to synthesize putrescine (Put) in plants. In previous work (Masgrau et al. 1997), we observed an altered phenotype (growth inhibition, leaf chlorosis and necrosis) in tobacco transgenic plants (Nicotiana tabacum L. var. Wisconsin‐38) containing the oat ADC cDNA under the control of a tetracycline inducible promoter, the severity of which was correlated with Put content. Now we have analysed the T2 generation of a selected transgenic line (line 52), which in previous generations was characterized by presenting a moderate increase in ADC activity and polyamine levels, but no phenotype alterations. Studying two selected individuals, one with a high expression level of the transgene and the other with a moderate expression level, we demonstrate that only the one with increased polyamine content displays the altered (toxic) phenotype. The possible causes of toxicity have been analysed. The results suggest that either Put or its oxidation products, via diamine oxidase (DAO; EC 1.4.3.6), are the responsible factors for the deleterious effects observed in the transgenic plants.
Winter‐drought induced embolism in Norway spruce (Picea abies) at the Alpine timberline
Authors: Mayr Stefan , Wolfschwenger Marion , Bauer Helmut
Published: March 2003

At the timberline in the Central Alps, climatic conditions during winter frequently cause excessive drought stress (frost drought, ‘Frosttrocknis’), which we hypothesized to induce cavitation in trees. We investigated the extent of winter‐embolism in Norway spruce (Picea abies (L.) Karst.) growing near the timberline and analysed adaptations in vulnerability and anatomy. We found conductivity losses of up to 100% at the highest elevation (2020 m) correlated with low water potentials down to − 4.0 MPa. Vulnerability thresholds (50% loss in conductivity) decreased from − 3.39 MPa at 800 m to − 3.88 MPa at 1600 m corresponding to a decrease in tracheid cross‐sectional area as well as pit and pit pore diameters. These thresholds were lower than potentials measured in embolized twigs near the timberline at the sampling dates probably due to lower potentials and/or a role of freeze‐thaw events earlier in winter. Data indicated refilling processes, which may be of particular relevance for trees at the timberline, since adaptations in drought‐induced vulnerability failed to prevent winter‐embolism.
Ethanol sensitivity of rice and oat coleoptiles
Authors: Kato‐Noguchi Hisashi
Published: March 2003

The ability to avoid the ethanol‐induced injury was evaluated in rice (Oryza sativa L.) and oat (Avena sativa L.) coleoptiles. The growth of the rice and oat coleoptiles was inhibited by ethanol exogenously applied at concentrations greater than 200 and 30 mM, respectively. At 300 mM ethanol, oat coleoptiles were brown and flaccid but rice coleoptiles did not show any visible symptoms of toxicity. The acetaldehyde level in rice and oat coleoptiles was increased by exogenously applied ethanol and the increases were greater in oat than in rice coleoptiles under aerobic and anaerobic conditions. At 300 mM ethanol, the acetaldehyde concentrations in the rice and oat coleoptiles were 46 and 87 nmol g−1 FW under aerobic conditions, respectively, and 52 and 124 nmol g−1 FW under anaerobic conditions, respectively. The activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) in the direction of ethanol to acetaldehyde was greater in oat than in rice coleoptiles and ADH protein in oat coleoptiles was more induced by exogenously applied ethanol than that in rice coleoptiles. These results suggest that in vivo conversion rate of ethanol to acetaldehyde by ADH is lower in rice than oat coleoptiles, which may be one of the reasons that ethanol sensitivity of rice is much lower than that of oat coleoptiles. The great ability of rice to avoid the ethanol‐induced injuries may contribute its anoxia tolerance when glycolysis and ethanolic fermentation replace the Krebs cycle as the main source of energy under anaerobic conditions.
Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination
Authors: Stasolla Claudio , Loukanina Natalia , Ashihara Hiroshi , Yeung Edward C. , Thorpe Trevor A.
Published: March 2003

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of 14C‐labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14C‐thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.
Diurnal variation of dimethylallyl diphosphate concentrations in oak (Quercus robur) leaves
Authors: Brüggemann Nicolas , Schnitzler Jörg‐Peter
Published: March 2003

In the present work a rapid and sensitive non‐radioactive assay for the determination of cellular dimethylallyl diphosphate (DMADP) was developed and used for the analysis of the diurnal variation of DMADP levels in oak leaves. The method is based on the acid‐catalysed hydrolysis of DMADP to isoprene, which subsequently is determined by gas chromatography. Diurnal variation of cellular DMADP levels in oak leaves of young saplings was measured on 6 days in 1998 and 1999, showing a 2 to 3‐fold light‐dependent increase from approximately 15 pmol mg−1 DW in the night to 31–75 pmol mg−1 DW around noon. The leaf DMADP contents showed a significant positive correlation with net assimilation and isoprene emission rates, indicating that the availability of cellular DMADP might be an important regulatory factor of leaf isoprene emission.
Gibberellin regulates post‐microsporogenesis processes in petunia anthers
Authors: Izhaki Anat , Borochov Amihud , Zamski Eliezer , Weiss David
Published: March 2003

Previous studies have suggested that gibberellins (GAs) are produced in petunia anthers and transported to the corolla to induce growth and pigmentation. In this work, we studied the role of GA in the regulation of anther development. When petunia plants were treated with the GA‐biosynthesis inhibitor paclobutrazol, anther development was arrested. Microscopic analysis of these anthers revealed that paclobutrazol inhibits post‐meiotic developmental processes. The treated anthers contained pollen grains but the connective tissue and tapetum cells were degenerated. A similar phenotype was obtained when the Arabidopsis GA‐signal repressor, SPY, was over‐expressed in transgenic petunia plants, i.e. anther development was arrested following microsporogenesis. The expression of the GA‐induced gene, GIP, can be used in petunia as a molecular marker to study GA responses. GA3 treatment of young anthers promoted, and paclobutrazol inhibited, GIP expression, suggesting that the hormone controls the natural activation of the gene in the anthers. Analyses of GIP expression during anther development revealed that the gene is induced only after microsporogenesis. This observation further suggests a role for GA in the regulation of post‐meiotic processes during petunia anther development.
Purification and characterization of secreted acid phosphatase in phosphorus‐deficient Arabidopsis thaliana
Authors: Coello Patricia
Published: March 2003

Arabidopsis roots responded to the absence of an exogenous phosphate source with an increase in the specific activities of secreted acid phosphatases. Increases in enzyme activity were revealed beginning 2 days after P‐withdrawal, reaching a maximum at 6 days. We characterized the secreted acid phosphatase. Two proteins, migrating at 52 and 63 kDa in SDS‐PAGE, co‐purified with the activity. Purified enzyme had a pH optimum of 5 and a pI of 5.9. In addition to the general phosphatase substrate, p‐nitrophenyl‐phosphate, the enzyme readily hydrolysed pyrophosphate, polyphosphate, ATP and PEP. Low or negligible activity was observed with glucose‐1P, fructose‐1P and phytic acid. The activity of the purified secreted acid phosphatase was stimulated by calcium and inhibited by molybdate, phosphate, fluoride, vanadate and nitrate. Activity was not inhibited by tartrate. The substrate profile and the biochemical properties suggest that Arabidopsis secreted acid phosphatase may have a role in mobilizing organic phosphate in the soil.
Expression and characterization of recombinant cytosolicNAD+‐dependent malate dehydrogenase from Mesembyanthemum crystallinum
Authors: Trípodi Karina E. J. , Casati María E. Gómez , Uttaro Antonio D. , Podestá Florencio E.
Published: March 2003

An NAD+‐dependent cytosolic malate dehydrogenase (MDH, EC 1.1.1.37) from leaves of Mesembryanthemum crystallinum in the Crassulacean Acid Metabolism (CAM) mode was cloned, expressed in E. coli and characterized. The recombinant enzyme had a subunit molecular mass of 39.5 kDa and was recognized by antibodies raised against the cytosolic MDH from Ananas comosus. Its activity showed a maximum in the pH range of 7.5–9.5. The purified MDH is highly but not completely specific for oxaloacetate, as indicated by a low activity using various other α‐ketoacids as substrates. The sequence data, subunit mass and immunoreactivity suggest that the MDH that has been cloned and characterized corresponds to the cytosolic isoform. Yet, the biochemistry of this enzyme comparative with the only other CAM plant cytosolic MDH characterized so far (that of pineapple) hints at a distinct isoform being expressed in M. crystallinum leaves.
The zygotic embryogenesis and ripening of Brassica rapa seeds provokes important alterations in the levels of free and conjugated abscisic acid and polyamines
Authors: Puga‐Hermida María Isabel , Gallardo Mercedes , Matilla Angel Jesús
Published: March 2003

Prior to the embryogenic study of Brassica rapa (turnip tops), the PA (polyamine) changes during flower development were determined. Whereas the flower was closed, no notable changes were found. However, pollination strongly stimulated the synthesis of the F (free) and ASC (acid soluble conjugated) forms, the pistil and not the petals being the main responsible. During embryogenesis, major alterations appeared in the synthesis of free and conjugated PA and ABA. Seeds, and not the silique wall, contributed most to the total content of the different F‐PA (free polyamines) in the silique, this contribution involving all the PA studied. During seed desiccation, F‐Put remained almost undetectable whereas F‐Spd and F‐Spm were very high. This is also applicable to ASC‐Spd and ASC‐Spm, this not being noted in the silique wall. During the first 5 stages of silique embryogenesis, the wall had higher levels of AIC‐PA (acid insoluble conjugated polyamines) than in the seeds, primarily due to AIC‐Spd and AIC‐Spm. By contrast, during the middle and final phases AIC‐PA was found more in the seeds than in the silique wall. The free and conjugated ABA showed the presence of 3 and 2 peaks in the seeds and the silique wall, respectively. The first corresponded to the onset of development, the second appeared when the highest moisture content was reached, and the third coincided with the triggering of carotenoid synthesis when the seed and the silique wall had lost more than 70–75% of their moisture. Finally, it is shown that the dry seeds of turnip tops were highly heterogeneous, not only in their germinative capacity, but also in the levels of free and conjugated ABA, which increased inversely to the germinative capacity.
Neutrophil respiratory burst following liver transplantation: in vitro effects of granulocyte colony‐stimulating factor
Authors: Jaeger K. , Ruschulte H. , Heine J. , Scheinichen D. , Leuwer M. , Winkler M. , Kuse E.R.
Published: March 2003

Early postoperative infections and septic complications are predominant causes of morbidity and mortality in patients following orthotopic liver transplantation (OLTx). Prophylactic granulocyte colony‐stimulating factor (G‐CSF) administration after OLTx was found to decrease the number of sepsis episodes and sepsis‐related mortality. Since polymorphonuclear neutrophils (PMNs) are one of the major determinants of antimicrobial defense, alteration of their functions may influence the development of sepsis in these patients. Therefore, we investigated in vitro whether or not priming with G‐CSF affects the neutrophils’ respiratory burst (RB) in immunosuppressed liver‐transplanted patients. Venous blood was drawn from liver allograft recipients (n=12) between the 5th and 15th day postoperatively. Patients without clinical signs of infection or rejection were included in this study. Leukocytes were obtained as supernatant following sedimentation and incubated with 1000 IE ml−1 G‐CSF. The RB was measured by the intracellular oxidation of non‐fluorescent dihydrorhodamine to the fluorescent rhodamine by flow cytometry. The results were expressed as a percentage of increasing stimulation compared to the control responses, which are made up of the percentage of cells with RB reaction after stimulation with phorbol ester (PMA), bacteria (E. coli), or the combination of a cytokine (TNF‐α) and a bacterial peptide (FMLP) in the absence of G‐CSF. In vitro priming with G‐CSF resulted in significantly increased activity of the RB after PMA (from 71.7% to 85.6%) and TNF‐α/FMLP (from 58.4% to 72.7%) stimulation. These data demonstrate that G‐CSF in vitro augments the RB of PMNs, thereby suggesting a possible therapeutic role for G‐CSF as immunomodulating agent during bacterial and fungal infections following OLTx.
Cerebral toxoplasmosis and Guillain‐Barré syndrome after allogeneic peripheral stem cell transplantation
Authors: González M.I. , Caballero D. , López C. , Alburquerque T. , Hernández R. , De La Loma A. , Cañizo C. , Vázquez L. , San Miguel J.F.
Published: March 2003

We report an unusual case of cerebral toxoplasmosis associated with Guillain‐Barré syndrome ( GBS) in a 25‐year‐old patient diagnosed with chronic myelogenous leukaemia ( CML), who underwent a mismatched allogeneic peripheral stem cell transplantation ( PSCT). On day +83 he started with fever, and 7 days later tremor, muscular weakness, diplopia, dysarthria, respiratory difficulty, and universal arreflexia appeared, compatible with GBS. As the patient had a positive cytomegalovirus ( CMV) antigenemia, this was the aetiology suspected for his neurologic findings, but specific treatment failed to improve his clinical situation, and he died on day +123. Necropsy demonstrated cerebral toxoplasmosis and axonal degeneration of nerve roots compatible with the axonal form of GBS. Interestingly, the polymerase chain reaction ( PCR) signal for Toxoplasma gondii in two different cerebrospinal fluid (
CSF) samples had been negative. In addition, this case showed unique magnetic resonance imaging (
MRI) abnormalities. We conclude that a negative
PCR on
CSF cannot exclude toxoplasmosis in a transplant patient, and we emphasise the importance of considering Toxoplasma as an aetiology of fever and neurological symptoms in the transplant setting.
Disseminated Dactylaria constricta infection in a renal transplant recipient
Authors: Malani P.N. , Bleicher J.J. , Kauffman C.A. , Davenport D.S.
Published: March 2003

Abstract: We report the case of a 32‐year‐old renal transplant recipient who developed disseminated Dactylaria constricta infection. The patient died despite treatment with amphotericin B, itraconazole, and fluconazole.
Intracranial Nocardia dissemination during minocycline therapy
Authors: Weber L. , Yium J. , Hawkins S. , Weber L. , Yium J. , Hawkins S.
Published: March 2003

Abstract: Nocardia species is a well‐known pathogen in immunocompromised hosts, including renal transplant recipients. Primary pulmonary infection can disseminate to other organs and recommended first‐line therapy is high‐dose trimethoprim/sulfamethoxazole (TMP/SMX). We report two cases of primary pulmonary Nocardia sp. in immunosuppressed patients who were treated with minocycline, a second‐line drug. During treatment with minocycline, both patients developed central nervous system (CNS) lesions of Nocardia sp. and were then treated with TMP/SMX with resolution of disease. The literature on Nocardia and treatment with minocycline is reviewed. Treatment of pulmonary Nocardia sp. with 200 mg minocycline daily is not adequate to prevent disseminated CNS disease.
CMV retinitis after allogeneic bone marrow transplantation: a report of five cases
Authors: Larsson K. , Lönnqvist B , Ringdén O , Hedquist B , Ljungman P , Larsson K. , Lönnqvist B. , Ringdén O. , Hedquist B. , Ljungman P.
Published: March 2003

Four cases of cytomegalovirus (CMV) retinitis (CMVR) after allogeneic blood stem cell transplant (SCT) were documented in Huddinge University Hospital between 1994 and 1999. Prior to 1994, only one case was documented. All five patients were transplanted due to malignant disease, two with sibling donors and three with matched unrelated donors. Despite adequate antiviral treatment against CMV retinitis, the result has been almost total unilateral blindness in three patients. However rare, the complication seems to have become more common since we began doing more matched unrelated donor transplants, which leads to a more pronounced T‐cell defect and to a delayed immune reconstitution compared to sibling transplants. We conclude that CMV retinitis is a rare but important complication to allogeneic blood stem cell transplantation.
Persistent lack of human herpesvirus‐6 specific T‐helper cell response in liver transplant recipients
Authors: Singh N. , Bentlejewski C. , Carrigan D.R. , Gayowski T. , Knox K.K. , Zeevi A. , Singh N. , Bentlejewski C. , Carrigan D.R. , Gayowski T. , Zeevi A. , Knox K.K.
Published: March 2003

Background. Specific immunologic defects predisposing to human herpesvirus‐6 (HHV‐6), e.g. the role of HHV‐6 specific T‐helper cell memory response in liver transplant recipients, have not been assessed.

Methods. T‐helper function (mitogen ConA response) as a marker of overall immunocompetence and T‐helper response (memory response) specific to HHV‐6 and cytomegalovirus (CMV) were assessed in 15 liver transplant recipients and compared with 25 healthy subjects. Samples were tested pretransplant, at 2 weeks, 1 month, 2–3 months, and 1 year posttransplantation. Stimulation index (SI) >3 was considered a positive response.

Results. Seven percent (1/15) of the transplant recipients at any time posttransplantation, as compared to 64% (16/25) of the healthy subjects, had a positive HHV‐6 memory response (P = 0.00065). HHV‐6‐specific memory response in transplant recipients at 2 weeks (SI 1.43), 1 month (SI 1.1), and 2–3 months (SI 1.3) was significantly more suppressed than in healthy subjects (SI 17.5, P = 0.0001). Although transplant recipients as compared to healthy subjects also had a lower CMV‐specific memory response posttransplant (P = 0.0439), CMV‐specific memory response recovered significantly at 1 month (P = 0.03) and at 2–3 months (P = 0.027) as compared to that at 2 weeks. However, HHV‐6 memory response was persistently absent up to 2–3 months with partial recovery at 1 year; 7% of the patients at 2–3 months, but 25% at 1 year had a positive HHV‐6 specific memory response. Forty percent (6/15) of the patients developed HHV‐6 viremia a mean of 4 weeks posttransplant. Patients with HHV‐6 viremia had greater suppression of HHV‐6 memory response at 1 month than those without viremia (mean SI, 0.96 vs. 1.3, P = 0.08). All but one of the patients had a positive ConA response.

Conclusion. Prolonged suppression of HHV‐6 memory response, but not overall T‐helper cell function was documented and may play a role in the pathogenesis of HHV‐6 infection in liver transplant recipients. Memory response to CMV after liver transplantation was significantly more robust than to HHV‐6.
IFN‐γ but not IL‐4 is important for mouse CD4+ T cell‐mediated macrophage activation following their exposure to pig cells in vitro
Authors: Yi Shounan , O'Connell Philip J.
Published: March 2003

In order to investigate the mechanism by which CD4+ T cells and macrophages interact in the xenogeneic immune response, murine CD4+ T cells and macrophages were used as responder cells in culture with irradiated fetal pig spleen cells (FPSC) as pig xenogeneic stimulators. In this in vitro model, murine CD4+ T cells and macrophages were cultured individually, or together with FPSC. In addition, mouse CD4+ T cells were also cultured with autologous macrophages which were previously stimulated by FPSC. The cultured murine cells were analyzed for expression of CD4+ T cell and macrophage activation markers (cell surface markers and cytokines) as well as cytokine production. CD4+ T cells and macrophages cultured alone or together without FPSC showed unchanged low levels of expression of activation markers. Coculture of macrophages with FPSC and in the absence of CD4+ T cells induced increased expression levels of all the activation markers examined except B7.2 and ICAM‐1. Addition of CD4+ T cells to the coculture further enhanced this up‐regulation. Coculture of CD4+ T cells with FPSC‐stimulated macrophages, but not naive macrophages, or FPSC alone, resulted in significantly increased numbers of CD4+ T cells coexpressing their activation markers, especially IFN‐γ and CD40L, and this expression was enhanced further by including FPSC in the coculture. The activation of both CD4+ T cells and macrophages in their coculture with FPSC was suppressed by neutralizing IFN‐γ but not IL‐4. Our results demonstrated that interaction of CD4+ T cells and autologous macrophages was required for their optimal activation in response to pig xenogeneic stimulation. The mechanisms involved included cell–cell and/or cytokine interactions, and in particular IFN‐γ mediated communication was involved. Macrophages activated by pig cells in the absence of CD4+ T cells were able to activate naive CD4+ T cells, thus providing an important communication pathway between innate immune activation and a T cell mediated response in xenograft rejection.
Hyperacute lung rejection in the pig‐to‐human model. 2. Synergy between soluble and membrane complement inhibition
Authors: Azimzadeh A. , Zorn G. L. , Blair K. S. A. , Zhang J. P. , Pfeiffer S. , Harrison R. A. , Cozzi E. , White D. J. G. , Pierson R. N.
Published: March 2003

Azimzadeh A, Zorn GL III, Blair KSA, Zhang JP, Pfeiffer S, Harrison RA, Cozzi E, White DJG, Pierson RN III. Hyperacute lung rejection in the pig‐to‐human model. 2. Synergy between soluble and membrane complement inhibition. Xenotransplantation 2003; 10: 120–131. © Blackwell Munksgaard, 2003

Background. The role of complement in hyperacute lung xenograft rejection has not been elucidated. The present study evaluates the effect of complement (C) C3/C5 convertase inhibition on hyperacute rejection of pig lung by human blood. Methods. In an established ex‐vivo model, lungs from pigs heterozygous for human decay accelerating factor (hDAF), non‐transgenic littermate control pigs, or farm‐bred pigs were perfused with fresh human blood that was either unmodified or treated with soluble complement receptor type 1 (sCR1: TP10, 100 μg/ml). Results. Non‐transgenic lungs from littermate controls had a median survival time of 35 min (range 5 to 210; P=0.25 vs. farm‐bred piglets: median 5 min, range 5 to 10). Lungs expressing hDAF survived for a median of 90 min (range 10 to 161; P=0.5 and 0.01 vs. littermate and farm‐bred controls, respectively), with sCR1, whereas hDAF (–) lungs failed by 35 min (range 6 to 307), hDAF (+) lungs survived for 330 min (range 39 to 577) [P=0.002 vs. farm‐bred; P=0.08 vs. hDAF (–); P=0.17 vs. sCR1/hDAF (–)]. The rise in pulmonary vascular resistance (PVR) at 5 min was blunted only by hDAF (+) with sCR1 (0.26 ± 0.2 vs. 0.5 to 0.7 mmHg/ml/min for other groups). Plasma C3a and sC5b‐9 and tissue deposition of C5b‐9 were dramatically diminished using sCR1, and further decreased in association with hDAF. Histamine and thromboxane were produced rapidly in all groups. Conclusion. Complement plays an important role in lung HAR. However, even potent inhibition of C3/C5 convertase, both membrane bound in lung and by a soluble‐phase inhibitor in the blood, does not prevent activation of inflammatory responses known to be particularly injurious to the lung. Our findings implicate a role for innate immune pathways resistant to efficient complement regulation. The role of anti‐species antibody, coagulation pathway dysregulation, and additional environmental or genetic influences remain to be defined.
Swim speed of free‐ranging Adélie penguins Pygoscelis adeliae and its relation to the maximum depth of dives
Authors: Ropert‐Coudert Y. , Kato A. , Sato K. , Naito Y. , Baudat J. , Bost A. , Le Maho Y.
Published: March 2003

Swim speed and depth utilization were recorded at a sampling rate of 1 Hz in 14 free‐ranging Adélie penguins in Adélie Land, Antarctica during the austral summers of 1996/1997 and 1998/1999. The average swim speeds during the descent, bottom and ascent phases of dives were independent of the maximum depth, while the variability in swim speed decreased with increasing maximum depth, reflecting the physiological constraints of diving. Descent speed, which varied less with maximum depth than speeds measured during other parts of dives, was significantly different among birds. In addition to the speed analysis, a new category of dive profiles with a flat bottom phase and an extremely reduced swim speed is reported. The probable benthic nature of such dives is discussed.
Mounting frequency and number of cloacal contacts increase with age in common terns Sterna hirundo
Authors: González‐Solís Jacob , Becker Peter H.
Published: March 2003

We investigated courtship feeding and copulatory behaviour of common terns Sterna hirundo in relation to age. We found a significant positive correlation between the number of courtship feedings delivered to females and the number of mountings. Moreover, both courtship feeding and mounting rates were positively correlated with female and male age. Also, the number of cloacal contacts per mounting increased significantly with male or female age, which may indicate an increase in copulatory ability with age. The number of mountings for older birds was up to three times that for younger birds. Presuming that transfer of sperm is proportional to the number of cloacal contacts, older birds may have transferred up to six times more sperm than younger birds during the pre‐laying and laying period. These results may account for the preference of young females for older mates as well as for older extra‐pair mates.
Targeting complement in therapy
Authors: Kirschfink Michael
Published: March 2003

With increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases, strategies that interfere with its deleterious action have become a major focus in pharmacological research. Endogenous soluble complement inhibitors (C1 inhibitor, recombinant soluble complement receptor 1, antibodies) blocking key proteins of the cascade reaction, neutralizing the action of the complement‐derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)‐mediated adhesion of inflammatory cells to the vascular endothelium have successfully been tested in various animal models over the past years. Promising results consequently led to clinical trials. Furthermore, incorporation of membrane‐bound complement regulators (decay‐accelerating factor (CD55), membrane co‐factor protein (CD46), CD59) in transgenic animals has provided a major step forward in protecting xenografts from hyperacute rejection. At the same time, the poor contribution of complement to the antitumor response, which is caused by multiple resistance mechanisms that hamper the efficacy of antibody‐based tumor therapy, is increasingly recognized and requires pharmacologic intervention. First attempts have now been made to interfere with the resistance mechanisms, thereby improving complement‐mediated tumor cell destruction.
Human natural killer cell receptors and co‐receptors
Authors: Biassoni Roberto , Cantoni Claudia , Pende Daniela , Sivori Simona , Parolini Silvia , Vitale Massimo , Bottino Cristina , Moretta Alessandro
Published: March 2003

Summary: In the absence of sufficient signaling by their HLA class I‐specific inhibitory receptors, human natural killer (NK) cells become activated and display potent cytotoxicity against cells that are either HLA class I negative or deficient. This indicates that the NK receptors responsible for the induction of cytotoxicity recognize ligands on target cells different from HLA class I molecules. On this basis, the process of NK‐cell triggering can be considered as a mainly non‐MHC‐restricted mechanism. The recent identification of a group of NK‐specific triggering surface molecules has allowed a first series of pioneering studies on the functional/molecular characteristics of such receptors. The first three members of a receptor family that has been termed natural cytotoxicity receptors (NCR) are represented by NKp46, NKp44 and NKp30. These receptors are strictly confined to NK cells, and their engagement induces a strong activation of NK‐mediated cytolysis. A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various target cells. Importantly, mAb‐mediated blocking of these receptors has been shown to suppress cytotoxicity against most NK‐susceptible target cells. However, the process of NK‐cell triggering during target cell lysis may also depend on the concerted action of NCR and other triggering receptors, such as NKG2D, or surface molecules, including 2B4 and NKp80, that appear to function as co‐receptors rather than as true receptors. Notably, a dysfunction of 2B4 has been associated with a severe form of immunodeficiency termed X‐linked lymphoproliferative disease. Future studies will clarify whether also the altered expression and/or function of other NK‐triggering molecules may represent a possible cause of immunological disorders.

This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanità (I.S.S.), Ministero della Sanità, and Ministero dell’Università e della Ricerca Scientifica e Tecnologica (M.U.R.S.T.) and Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. The financial support of Telethon‐Italy (grant no. E.0892) is gratefully acknowledged.
Determining the immune mechanisms of protection from AIDS: correlates of immunity and the development of syngeneic macaques
Authors: Kent Stephen J. , Ada Gordon L. , Hayes Eric , Lewis Ian M.
Published: March 2003

Summary: The AIDS pandemic is a global emergency and a preventive vaccine is urgently needed. CD4 and CD8 T‐cell responses appear important in controlling human immunodeficiency virus (HIV)‐1 in humans and simian immunodeficiency virus (SIV) in macaques. The utility of vaccines that induce high levels of SIV‐ or HIV‐specific T cells has recently become clearer. Since T cells recognize virus‐infected cells rather than free virus, T‐cell‐based vaccines only have the capacity to control infections (non‐sterilizing immunity) and to prevent continuing or persisting infection. An HIV/SIV infection of macaques that is partially controlled by vaccine‐induced T‐cell responses permits a critical window of opportunity for the efficient generation and recruitment of additional T‐ and B‐cell immune responses to the incoming viral inoculum. Although CD8‐depletion experiments in macaques have defined the utility of CD8 T responses in control of SIV infections in macaques, direct evidence on the utility of either CD4 or CD8 T‐cell responses in protective immunity to SIV following vaccination is lacking. The availability of genetically identical macaques would allow cell transfer studies and help define with more certainty the role of cellular immune responses in protection from AIDS. The review also focuses on the development of syngeneic macaques by twinning and cloning technologies.
Monoclonal antibodies against rat leukocyte surface antigens
Authors: Van Den Berg Timo K. , Puklavec Michael J. , Barclay A. Neil , Dijkstra Christine D.
Published: March 2003

Summary: Monoclonal antibodies have proven to be powerful tools for studying the properties of leukocyte surface antigens and the cells that express them. In the past decades many monoclonal antibodies (mAb) for identifying the different rat leukocyte surface antigens have been described. A list of mAb is provided in Table 1 below. The rat leukocyte surface antigens are divided into different sections, including rat CD antigens (a), rat leukocyte surface antigens without CD designation (b), rat major histocompatibility complex (MHC) antigens (c), rat T‐cell receptors (d) and rat immunoglobulins (e). The molecular and functional characteristics of rat leukocyte surface antigens are discussed in more detail in some of the other chapters of this issue (e.g. Van den Berg et al., p. 45). A more extensive overview of the properties of leukocyte surface antigens is provided by Barclay et al. (1).
Role of NK cells in adoptive immunotherapy of metastatic colorectal cancer in a syngeneic rat model
Authors: Kuppen Peter J. K. , Gorter Arko , Hagenaars Martin , Jonges Liesbeth E. , Giezeman‐Smits Katinka M. , Nagelkerke J. Fred , Fleuren Gertjan , Van De Velde Cornelis J. H.
Published: March 2003

Summary: This article reviews our immunotherapy research with natural killer (NK) cells in a syngeneic rat colorectal cancer liver and lung metastasis model. Using adoptive transfer of interleukin (IL)‐2‐activated NK cells, NK cells were shown to selectively infiltrate the tumors. More NK cells were found in tumors when the NK cells were directly injected into tumor‐draining blood vessels than when the cells were injected in systemic blood vessels. Under optimal conditions, a limited, though significant, effect of adoptively transferred NK cells on tumor growth was shown. We observed that both endogenous and adoptively transferred NK cells were predominantly present in the stroma surrounding the tumor cell nodules. It is possible that they did not penetrate the nodules containing the tumor cells because of the presence of a basal membrane‐like structure around these nodules. Adoptively transferred NK cells may initiate elimination of tumor cells by activating other effector cells, whereas some may eliminate tumor cells by direct cell–cell contact. A diverse array of molecules was shown to be involved in this process. CD45 on NK cells was found to be important in initiating the lysis‐inhibitory signal upon binding of ‘self’ major histocompatibility complex (MHC) class I on potential target cells. Our results indicate that NK‐cell cancer therapy is still promising and needs improvement.
The role of Th2 cytokines in mast cell homeostasis
Authors: Shelburne C. P. , Ryan J. J.
Published: March 2003

Summary: Homeostatic mechanisms regulating mast cell numbers and function in peripheral tissues have largely focused on cytokines, such as stem cell factor, interleukin (IL)‐3, IL‐4, and IL‐10, which regulate mast cell maintenance and proliferation. Despite these advances, little attention has been paid to the mechanisms that mediate mature mast cell turnover, and control of mast cell hyperplasia generated during Th2‐mediated responses. These are important issues, as mast cells are now known to be multi‐functional effector cells, that have the capacity to mediate both innate and Th2‐induced immune responses. Numerous secretagogues may elicit mast cells to release a large number of important mediators that can cause chronic inflammation. Therefore, how mast cell homeostasis is regulated may have significant effects on normal physiology, and contribute to the genesis of inflammatory disease. Our laboratory has characterized an in vitro model of mast cell homeostasis, by which long‐term exposure of murine bone‐marrow‐derived mast cells to the Th2‐derived cytokines IL‐3, IL‐4, and IL‐10, will induce downregulation of critical mast cell effector proteins such as Kit and FcεRI, followed by mast cell apoptosis. These data offer a novel role for Th2 cytokines, acting to both initiate and resolve mast cell activation and proliferation. Loss of these signals may contribute to a multitude of diseases, such as mastocytosis and allergy.
Exploring lymphocyte differentiation pathways
Authors: Cooper Max D.
Published: March 2003

Summary: Highlights in a 4‐decade exploration of lymphocyte differentiation begin with comparative studies in birds and mammals leading to recognition of the separate T‐ and B‐cell differentiation pathways and their cooperative interaction. The global effects of aborting IgM B‐cell development with anti‐µ antibodies indicated that B cells can undergo immunoglobulin isotype switching. A search for the mammalian bursa equivalent that began with an extended excursion through the gut‐associated lymphoepithelial tissues ultimately led to the hematopoietic tissue origin of mammalian B cells. The identification of the precursors of B cells in hematopoietic tissues provided an expanded view of the life history of B cells. A recurring theme in this essay is the interplay between understanding normal lymphocyte differentiation and the defects that underlie immunodeficiency diseases and lymphoid malignancies.
The distal pole complex: a novel membrane domain distal to the immunological synapse
Authors: Cullinan Patrick , Sperling Anne I. , Burkhardt Janis K.
Published: March 2003

Summary: While much interest has focused on the finding that T cell–antigen presenting cell (APC) interaction induces the recruitment of proteins to the immunological synapse (IS), we have recently discovered that APC binding induces the formation of a novel protein complex distal to the site of T‐cell receptor ligation. This ‘distal pole complex’ (DPC) is important for appropriate T‐cell activation, functioning either to remove proteins from the synapse or as a signaling complex in its own right. The first component of the DPC to be identified was CD43, a cell‐surface mucin that has been proposed to function as a negative regulator of T‐cell signaling. CD43 movement was found to depend on ezrin and moesin, members of the ERM family, which serve to link CD43 and other cargo molecules to the actin cytoskeleton. ERM proteins interact with several other important surface receptors and cytoplasmic signaling molecules, some of which we have identified as additional components of the DPC. Disruption of the DPC leaves early T‐cell activation events intact but affects cytokine expression. Here, we review what is currently known about the formation and function of the DPC and speculate on how this novel protein complex serves to facilitate T‐cell activation.
Visualization of signaling pathways and cortical cytoskeleton in cytolytic and noncytolytic natural killer cell immune synapses
Authors: Vyas Yatin M. , Maniar Hina , Dupont Bo
Published: March 2003

Summary:  Recent applications of imaging approaches and other methods of cell biology have provided high‐resolution visualization of the location of fluorescent proteins in living and fixed cells during cell–cell interactions between lymphocytes, antigen presenting cells and target cells. We review the composition and dynamics of molecular and cytoskeletal events occurring during natural killer cell interactions with susceptible and nonsusceptible target cells. The natural killer cell immune synapse and the concomitant changes in cytoskeletal components and cytoplasmic organelles are described. The findings are compared with the observations made in T helper cells and cytotoxic T cells. It is concluded that the cytolytic immune synapses display spatial–temporal dynamics that are accelerated as compared with T helper cells. In addition, the cytolytic conjugates have unique characteristics relating to their effector function. Furthermore, the natural killer cell immune synapses in cytolytic and noncytolytic interactions are distinctly different and display patterns consistent with characteristic signaling pathways identified in biochemical studies of disrupted cells. The precise relationship between different stages of the natural killer cell immune synapse formation and progression in signal transduction pathways is yet to be established.