bluuebunny/crossref_metadata_2025 huggingface dataset:
Vesico‐ureteral reflux in pediatric kidney transplants: Clinical relevance to graft and patient outcome
Authors: Fontana I. , Ginevri F. , Arcuri V. , Basile G. , Nocera A. , Beatini M. , Bonato L. , Barocci S. , Bertocchi M. , Manolitsi O. , Valente R. , Draghi P. , Gusmano R. , Valente U.
Published: March 2003

Abstract:From June 1985 to December 1998, 173 pediatric renal transplants were carried out in 170 patients at our center. From this pool, 73 patients (34 males and 39 females) with a follow‐up of 48 months were examined. In all patients, ureteroneocystostomy was performed according to the Lich‐Grégoire procedure. All patients were treated with cyclosporin A (CsA)‐based immunosuppression, including prednisone and sometimes azathioprine (AZA). Six months after transplantation, voiding cystography (VCU) was performed in all patients and reflux was classified from Grade I to Grade IV. The patients were divided into two groups: those with reflux (Group A: 25 patients) and those without (Group B: 48 patients). Grade I reflux was found in four patients, Grade II in seven patients, Grade III in seven patients, and Grade IV in seven patients. All the patients with severe reflux (Grade IV) underwent a corrective surgical procedure. Both groups were examined for immunologic and non‐immunologic risk factors and no significant differences were found. Analysis of patient and graft survival rates revealed no statistical differences (NS) between Groups A and B. Mean serum creatinine (mg/dL) was 1.06 ± 0.28 and 1.12 ± 0.41 at 4 yr in Groups A and B, respectively (NS). Mean calculated creatinine clearance (cCrC; ml/min) was 76.74 ± 15.92 and 77.96 ± 15.66 in Groups A and B, respectively (NS). The analysis was further extended by considering the grade of reflux (I to IV). Again, no significant differences in the above parameters emerged between the reflux sub‐groups; only in the Grade IV sub‐group was a slight decrease in cCrC detected, although this difference was not statistically significant when compared with the other sub‐groups. In conclusion, vesico‐ureteral reflux (VUR) does not seem to negatively affect graft function. However, as all severe reflux patients (Grade IV) were surgically corrected, no conclusions can be drawn with regard to the influence of Grade IV reflux on long‐term graft function.
Plasmapheresis, intravenous cytomegalovirus‐specific immunoglobulin and reversal of antibody‐mediated rejection in a pediatric renal transplant recipient: A case report
Authors: Furth S. , Neu A. M. , Hart J. , Zachary A. , Colombani P. , Fivush B. A.
Published: March 2003

Abstract: This is a pediatric case report illustrating the development of antibody (Ab)‐mediated rejection in a patient with low levels of pretransplant anti‐human leucocyte antigen (HLA) panel reactive antibodies (PRA). The clinical course of this patient suggests that aggressive use of a combination of plasmapheresis, monoclonal anti‐T‐lymphocyte antibody therapy, and intravenous immunoglobulin (IVIG) therapy can reverse Ab‐mediated rejection in previously allosensitized pediatric transplant recipients.
Pulmonary capillary leak syndrome with intravenous cyclosporin A in pediatric renal transplantation
Authors: Mackie Fiona E. , Umetsu Dale , Salvatierra Oscar , Sarwal Minnie M.
Published: March 2003

Abstract: Despite frequent use of intravenous (i.v.) cyclosporin A (CsA) in the early post‐operative course of transplant recipients, allergic reactions have been infrequently described. Of 134 transplants, we report four pediatric renal transplant recipients with severe reaction to i.v. CsA with pulmonary capillary leak syndrome. Pulmonary edema developed at a mean time of 3.5 h after commencement of i.v. CsA, with two patients requiring mechanical ventilation. Discontinuation of i.v. CsA and conversion to oral CsA was followed by rapid resolution of pulmonary edema, suggesting that cremaphor, the solubilizing agent in the i.v. formulation, is likely to be responsible for this adverse response. Skin prick testing with cremaphor was negative in all patients and alternative mechanisms for the cremaphor response are proposed. It is likely that inadequate mixing of the i.v. CsA solution triggered this reaction, by delivering a higher concentration of cremaphor at the start of the CsA infusion. Pulmonary edema in the early post‐transplant course in the absence of obvious fluid overload should prompt the diagnosis of an i.v. CsA reaction. This life‐threatening reaction is easily reversible if recognized, and can be managed easily without compromise to the allograft, by discontinuing i.v. CsA and switching early to an oral CsA formulation.
Increased tacrolimus levels in a pediatric renal transplant patient attributed to chronic diarrhea
Authors: Eades Shannan K. , Boineau Frank G. , Christensen Michael L.
Published: March 2003

Abstract: Tacrolimus is an immunosuppressant used to prevent rejection of transplanted organs. It is metabolized in both the gut and the liver by the cytochrome P450 (CYP) 3A4 enzyme system and is a substrate for the P‐glycoprotein (P‐gp) drug efflux pump. As CYP3A4 enzymes and P‐gp are present at differing concentrations throughout the gastrointestinal tract, the bioavailability of tacrolimus may be influenced by changes in gastrointestinal transit time in addition to changes in hepatic metabolism. We report the case of a pediatric renal transplant patient who experienced a three‐fold increase in serum tacrolimus concentrations during an episode of gastroenteritis with chronic diarrhea.
Survival after acute graft failure in pediatric thoracic organ transplant recipients
Authors: Hoffman Timothy M. , Spray Thomas L. , Gaynor James W. , Clark Bernard J. , Bridges Nancy D.
Published: March 2003

Abstract: Survival among recipients of repeat thoracic organ transplantation, particularly in the setting of acute graft failure (AGF), is lower than survival after a primary transplant. This has created controversy over the fair allocation of scarce organs. We reviewed our experience to assess the effectiveness of aggressive therapy and retransplantation in pediatric patients with AGF. Between November 1994 and March 1998, 52 patients aged 49 days to 16.9 years (median age 4.7 years) underwent thoracic organ transplantation (32 primary and 4 repeat heart, 16 primary and 4 repeat lung, and 3 primary heart–lung transplants). Acute graft failure occurred in nine (4 heart, 3 lung, 2 heart–lung transplants), six of whom were supported with extracorporeal membrane oxygenation (ECMO), and four of whom underwent repeat transplant. Six of the nine survived, including all of those who were retransplanted, and five of the nine were alive 1 year later. The average postoperative hospital stay after receiving a second organ was 46.5 days vs. a postoperative 22‐day stay in recipients without AGF (p = 0.07). We conclude that the decision to allocate institutional and professional resources to the aggressive support of patients with AGF must be made at the level of the individual transplant center. However, we feel that the outcome of aggressive support and retransplantation justifies the allocation of organs to these patients and suggests that the current policies governing organ allocation for patients with early graft failure should be re‐examined.
Risk factors for bone mineral density loss in pediatric renal transplant patients
Authors: Ellis Eileen N. , Floyd‐gimon Donna , Berry Phillip , Wells Thomas , Seibert Joanna , Belsha Craig
Published: March 2003

Abstract:Bone mineral density (BMD) is decreased in both adult and pediatric renal transplant recipients. To investigate the risk factors associated with this decrease in BMD post‐renal transplant, we studied 33 children, aged 7–22 yr, who had received a renal transplant from 0.3 to 10 yr prior to this study. BMD analysis of the total body, spine, and femur was carried out by using dual‐energy X‐ray absorptiometry (DEXA). Age, weight, Tanner stage, time on dialysis prior to transplantation, cumulative corticosteroid dosage, and cyclosporin A (CsA) dosage since transplantation, and use of corticosteroid therapy prior to transplantation, were recorded. Spine, femur, and total body BMDZ‐scores were greater than two standard deviations (2 SD) below the mean in 45%, 42%, and 17% of patients, respectively. Age correlated inversely with total body and spine BMDZ‐scores (p = 0.001 and p = 0.008); no child under 14 yr of age had a total body or spine BMDZ‐score greater than 2 SD below the mean for age. Patients at a Tanner stage of 4 or 5 had lower total body and spine BMDZ‐scores than did patients at Tanner stages 1–3 (p = 0.043). Time post‐transplant correlated inversely with both spine and total body BMDZ‐score (p = 0.013 and p = 0.023). Only total body BMDZ‐score correlated inversely with cumulative corticosteroid dose (in g, p = 0.045). BMD did not correlate with cumulative CsA dose. Black patients tended to have decreased total body BMD compared with Caucasian patients. In pediatric renal transplant patients, decreases in BMD start in adolescence. Risk factors for BMD loss in these patients include increasing age, time post‐transplant, increasing Tanner stage, and ethnicity. Longitudinal studies in these patients and strategies to improve BMD are needed.
Conversion from cyclosporin A to tacrolimus in pediatric liver transplantation
Authors: Furlan Valérie , Debray Dominique , Fourre Catherine , Taburet Anne‐Marie
Published: March 2003

Abstract: The dosing regimen for conversion from cyclosporin A (CsA) to tacrolimus immunosuppression was studied in 12 pediatric liver allograft recipients. Patients were stratified according to their age. Tacrolimus was started orally at a dosage of 0.05 mg/kg b.i.d., 12 h after stopping CsA administration and increased thereafter if needed. Tacrolimus and CsA concentrations were assayed by immunoassay using, respectively, an IMx and a TDx autoanalyzer (Abbott‐France). Mean CsA concentration was in the therapeutic range 12 h prior to and at the time of introduction of tacrolimus. After 72 h of tacrolimus therapy, CsA concentrations were undetectable whereas mean tacrolimus concentration was 10.4 ng/mL with a mean dose of 0.09 mg/kg b.i.d. The decline of CsA concentration was clearly biphasic. A slower decline in CsA concentration was detected after initiation of tacrolimus therapy, suggesting an inhibition of CsA metabolism by tacrolimus. No nephrotoxicity was observed. This dosage regimen allowed effective immunosuppression while avoiding additive nephrotoxicity.
Demographic and medical predictors of medication compliance among ethnically different pediatric renal transplant patients
Authors: Fennell Robert S. , Tucker Carolyn , Pedersen Tyler
Published: March 2003

Abstract: Medication adherence in African‐American and European‐American pediatric renal transplant recipients was evaluated by four separate measures. Demographic and medical factors were analyzed. Based on pill count/refill history, European‐American females were more compliant than their male counterparts. Based on self‐ratings of compliance, African‐American recipients were more compliant if they had vs. had not had dialysis experience prior to their transplant. These recipients also had higher self‐ratings of compliance if their donors were cadaveric rather than living related.
Predictors of cytomegalovirus disease among pediatric transplant recipients within one year of renal transplantation
Authors: Robinson Lisa‐Gaye , Hilinski Joseph , Graham Fitzroy , Hymes Leonard , Beck‐Sague Consuelo M. , Hsia Jason , Nesheim Steven R.
Published: March 2003

Abstract:Cytomegalovirus (CMV) is the most important opportunistic infection in renal transplant recipients and is associated with an increased risk of rejection. Infection can be acquired post‐operatively (from the transplanted organ) or from re‐activation of latent disease. To identify risk factors for CMV disease in a pediatric population within 1 yr of renal transplant, and to generate hypotheses for the pathogenesis of CMV disease in this population, a review of all recipients from 1992 to 1998 in a children's hospital in Atlanta, Georgia, was undertaken. Medical records of 73 transplants performed on 72 patients were reviewed: nine (12.7%) of 72 individuals, after 73 procedures developed CMV disease. Median time to onset of CMV disease was 52 days post‐transplant (range = 15–95 days). Receipts of mycophenolate mofetil (MMF), demographic factors, and use of cadaveric kidneys were not associated with a significantly elevated risk of CMV disease. Positive donor CMV serostatus was associated with CMV disease (uni‐variate relative risk [RR] = 8.52, Fisher's Exact Test [FET] p = 0.010). Patients with transplants in October or November had a higher risk of developing CMV disease (four of 13; 30.8%) than patients transplanted in other months (five of 60, 8.3%); RR = 3.69; p = 0.047, FET). Most transplants of patients who did not develop CMV disease were performed in January through August (48/64; 75.0%); only 25.0% were performed in September through December. In contrast, six of nine (66.7%) transplants in patients who subsequently developed CMV disease were performed in September through December (p = 0.018, FET). Donor CMV‐positive serostatus and transplant in October and November continued to be independently associated with an increased risk of CMV disease when controlled for other factors. The association of transplant in October and November with CMV disease in November–January may be related to an increased risk of seasonal community CMV exposure and primary CMV infection during the peak season for CMV circulation, with subsequent immune suppression promoting progression to disease. Alternatively, co‐infection with seasonal pathogens after exposure from an infected donor during the period of immune suppression may promote progression from CMV infection to CMV disease. Further studies should be undertaken to explore these and other hypotheses, which may have implications for determination of a need for anti‐viral prophylaxis.
Single‐dose pharmacokinetics and tolerability of everolimus in stable pediatric renal transplant patients
Authors: VanDamme‐Lombaerts R. , Webb N. , Hoyer P. F. , Mahan J. , Lemire J. , Ettenger R. , McMahon L. , Cambon N. , Boger R. , Kovarik J. M. ,   for the Everolimus Study Group
Published: March 2003

Abstract: Everolimus (Certican; RAD), a novel macrolide with potent immunosuppressive and anti‐proliferative activities, prevents acute rejection in adult recipients of renal transplantation. This phase I trial conducted in stable pediatric renal transplant patients examined the single‐dose pharmacokinetics, safety, and tolerability of everolimus in combination with cyclosporin A (CsA; Neoral®) and corticosteroids, with or without azathioprine. Nineteen pediatric patients were enrolled and received a single 1.2 mg/m2 dose of everolimus. Everolimus was safe and well tolerated, with a low incidence of adverse events reported and none judged to be related to the study medication. Everolimus administration did not increase infection rates or produce clinically significant changes in vital signs or changes in electrocardiograms. Apparent clearance and volume of distribution of everolimus increased with age, weight, and body surface area in a generally linear manner across the pediatric demographic ranges. Compared with adults from a previous study, apparent clearance (L/h) and distribution volume (L) were lower in pediatric patients, whereas the elimination half‐life was similar. Single‐dose everolimus co‐administration did not affect the steady‐state pharmacokinetics of CsA. Based on this information, pediatric patients will need a dose scaled down for body size, but can probably maintain the same twice‐daily dosing schedule used in adults.
Cardiac troponin I: A marker of acute heart rejection in infant and child heart recipients?
Authors: Siaplaouras J. , Thul J. , Krämer U. , Bauer J. , Schranz D.
Published: March 2003

Abstract: Acute rejection of the donor heart is a major cause of mortality in infant heart transplant recipients. The early diagnosis of acute cardiac rejection (ACR) is crucial. Non‐invasive methods have shown poor sensitivity in detecting rejection when compared to endomyocardial biopsies (EMB). We assessed troponin I as a new marker to diagnose cardiac rejection. Serum cardiac troponin I (cTNI) levels were retrospectively analysed in 25 heart transplant patients (ages, 2 wk to 13 yr; mean age, 3 months) presenting 36 acute rejections. In early post‐operative rejection and initially elevated cTNI levels, rejection was associated with a second increase of serum cTNI concentrations in 21% of the patients (p = 0.15). If cTNI levels were in normal range before ACR an elevation was monitored in 59% of the rejection periods (p < 0.05). In 25% of the cases (n = 9) cTNI levels remained in normal range during the rejection episode (<0.6 ng/mL), in 22% (n = 8) cTNI levels did not exceed pathological values from 0.6 to 1.5 ng/mL and in 53% (n = 19) the measured levels went beyond 1.5 ng/mL. Maximum concentrations of cTNI were measured mostly 12 d from the moment rejection was suspected (day 1) in patients (median day 3). However, cTNI levels were elevated for 2–43 d after ACR was diagnosed (median 10 d). Twenty per cent of the patients with grade 3 rejection (ISHLT) and 75% of the patients with grade 4 rejection had a corresponding elevated cTNI level (p = 0.013). No false‐positive elevations of cTNI were documented. The present data demonstrate that cTNI is a not a sensitive but a specific marker of ACR in children.
Early changes of Cl− efflux and H+ extrusion induced by osmotic stress in Arabidopsis thaliana cells
Authors: Teodoro Anna Elisabetta , Zingarelli Luisa , Lado Piera
Published: March 2003

In various plant materials changes in turgor pressure, following hyper‐ or hypo‐osmotic stress, were associated with the activation or inactivation of the plasma membrane H+‐ATPase, respectively. To see if the turgor changes might indirectly influence H+‐ATPase activity by regulating ion fluxes through plasma membrane, we investigated, in cultured cells of Arabidopsis thaliana (L.) Heynh., the early effects of hyper‐ and hypo‐osmotic stress on Cl− fluxes in comparison, in the case of hyper‐osmotic treatment, with its effect on net H+ extrusion. The results obtained showed that hyper‐osmotic stress (200 mM mannitol) quickly reduced Cl− efflux (−70%) from cells preloaded with 36C1− for 18 h. This inhibiting effect was independent of the simultaneous mannitol‐induced stimulation of Cl− influx and rapidly reversible after removal of the hyper‐osmotic treatment. The inhibition of Cl− efflux was associated with a stimulation of net H+ extrusion, and these two effects showed the same dependence on the external mannitol concentration. Fusicoccin (FC, 20 µM), which stimulated H+ extrusion to about the same extent as 200 mM mannitol, did not affect Cl− efflux. When cells preloaded with 36C1− for 18 h in the presence of mannitol (from 25 up to 200 mM) were eluted in a mannitol‐free medium an early and strong increase in Cl− efflux was found. The increase of Cl‐ efflux was already detectable for a small hypo‐osmotic jump (25 mM), and was reduced (−50%) by the anion channel inhibitor A9C (300 µM). These results lead to exclude a direct causal relationship mediated by Em changes between the effects of osmoticum on Cl− efflux and net H+ extrusion, and favour the view that the changes in turgor pressure induced by hyper/hypo‐osmotic stress may respectively induce an early inactivation/activation of stretch‐sensitive anion channels.
Nitrogen‐induced reduction in leaf phenolic level is not accompanied by increased rust frequency in a compatible willow (Salix myrsinifolia)‐Melampsora rust interaction
Authors: Hakulinen Johanna
Published: March 2003

Phenolic compounds are potential antimicrobial agents in willows (Salix spp.). However, little is known about their role in willow‐pathogen interactions. Earlier studies have shown that phenolic levels may vary depending on genotype and environmental factors (such as nutrient availability). In the present study, the combined effects of nitrogen fertilization and infection by pathogenic rust (Melampsora sp.) on phenolics and growth in willow (salix myrsinifolia) clones were investigated. Cutting‐propagated plants of eight willow clones were subjected to low and optimum levels of nitrogen fertilization and inoculated with rust urediniospores or mock‐inoculated with water. The plants were harvested 20 days after the inoculations and the dry weight of the leaves, stems and roots determined. Phenolics were analysed from leaf material using HPLC. The willow clones differed significantly in their phenolic content, rust frequency and phytomass production. Plants under optimum nitrogen availability showed increased shoot phytomass production and reduced root production. Rust infection increased the root phytomass of willow under both fertilization treatments, but had no consistent effect on the shoot growth of the clones. Low nitrogen availability increased the phenolic levels in willow leaves, but the rust frequency did not differ significantly between plants grown at low and optimum levels of nitrogen fertilization. In several clones, rust infection increased the concentrations of individual phenolic compounds, but there were no significant correlations between phenolic concentrations and the level of rust infection. The results indicate that resistance in compatible willow‐rust interactions may not be directly governed by phenolic compounds.
Effect of stimulating maize germination on cell cycle proteins
Authors: Cruz‐García Felipe , Zúñiga‐Aguilar José Juan , Vázquez‐Ramos Jorge M.
Published: March 2003

The germination process can be accelerated if seeds are stimulated either by adding cytokinins or by osmopriming. Under these conditions, cells in maize (Zea mays) embryo axes shorten the time at which the first round of DNA replication and mitosis takes place, thus advancing the cell cycle. Using heterologous antibodies against different cell cycle proteins, we have followed the behaviour of several markers for G1 phase (cyclin D, E2F and p53) and a marker of G2 phase (cyclin B) under either control or “accelerated” germination conditions. The results showed two classes of behaviour: either there was no variation in the amount of the protein present under control or accelerated germination conditions, represented by cyclin Band E2F‐type proteins, or the amount of the proteins was drastically reduced, more rapidly under accelerated germination, as was the case for cyclin D‐ and p53‐type proteins. Although the cyclin D‐type protein was synthesized de novo during germination, the balance was towards degradation so that there was no cyclin D detected 15 h after germination in benzyladenine‐treated and osmoprimed seeds. A Cdk4‐type protein seemed to be present in cyclin D immunoprecipitates and its kinase activity paralleled the fluctuations of the cyclin amount during germination. These data are discussed in the context of early seed germination.
Precociously germinating somatic embryos of Vitis vinifera have lower ABA and IAA levels than their germinating zygotic counterparts
Authors: Faure Olivier , Dewitte Walter , Nougarède Arlette , Van Onckelen Henri
Published: March 2003

Somatic embryos of Vitis vinifera (cv. Grenache noir) develop normally up to the torpedo stage, but they germinate precociously and form viable plantlets with very low frequency. Because a peak in abscisic acid (ABA) in mid‐embryogenesis could be one factor preventing precocious germination during normal seed development, we followed the development of ABA content concurrent with that of the somatic embryos. Additionally, we measured changes in indoleacetic acid (IAA) levels. We also compared the levels of both hormones during precocious germination of somatic embryos and during normal germination of their zygotic counterparts. Somatic embryos were able to accumulate ABA and IAA throughout their development but no peak in ABA concentration was detected during embryogenesis. This suggests that the switch from mid‐ to late‐embryogenesis is not triggered. Furthermore, during precocious germination, i.e. from the torpedo stage onwards, the concentrations of ABA and IAA in somatic embryos were much lower than during normal germination of zygotic embryos. Thus, it is likely that when precocious germination occurs, grape somatic embryos do not accumulate ABA and/or IAA in sufficient concentrations to support normal plantlet development. Therefore, for grape somatic embryos we propose that prevention of precocious germination, i.e. triggering late‐embryogenesis, is attainable by an ABA treatment followed by slow desiccation, as already shown for conifer somatic embryos. Our results also suggest that the role of ABA and IAA for improving normal germination after imposed quiescence should be investigated.
Defence response of pepper (Capsicum annuum) suspension cells to Phytophthora capsici
Authors: García‐Pérez M. Dolores , Egea Catalina , Candela M. Emilia
Published: March 2003

Cell suspension cultures of three cultivars of Capsicum annuum L., with different degrees of sensibility to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate. They showed conductivity changes, browning, production of the phytoalexin capsidiol and synthesis or accumulation of pathogenesis‐related (PR) proteins with glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) activities. The cultivation medium was optimised for growth of both the plant and the fungus in order to avoid any stress during their combination. The resistant cv. Smith‐5, showed a more rapid and intense response to the elicitor preparations than the sensitive cvs Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate, when differences became clearly visible after only 6 h incubation. The greatest rate of capsidiol accumulation occurred after 18 h in the mycelium‐elicited cells and after 12 h in those elicited with the filtrate. These times are the optimal for capsidiol accumulation, and the phytoalexin is produced much more rapidly than it can be excreted into the extracellular medium. The inhibition threshold of fungal growth (300 µg capsidiol [g dry weight]−1) was reached only in the resistant cultivar. The induction of an intracellular glucanase (pI 8.9 and Rf 0.18) and an extracellular chitinase (pI 5.4 and Rf 0.70) only in the resistant cultivar 24 h after elicitation suggests that these enzymes are involved in the resistance to Phytophthora capsici, while other hydrolases common to all three cultivars form part of a more general defence. The results indicate that elicitation of pepper cell suspension cultures by signal molecules from P. capsici exhibits properties of a multicomponent dynamic system in which different protective mechanisms play complementary roles in the overall expression of the defence reaction. We confirm that the differential responses of resistant and susceptible pepper cultivars to P. capsici previously seen in plant stem sections are retained in suspension culture.
Nitric oxide decreases superoxide anion generation by microsomes from soybean embryonic axes
Authors: Caro Andrés , Puntarulo Susana
Published: March 2003

Experiments were carried out to evaluate the effects of exposure to nitric oxide on the ability by NADPH‐dependent microsomal electron transfer to generate oxygen radicals. Such interactions could play a role in the potential antioxidant action of nitric oxide (NO). Isolated microsomes from soybean (Glycine max [L.] Merr. cv. Hood) embryonic axes were exposed to an exogenously added source of nitric oxide (NO) (S‐nitrosoglutathione + dithiothreitol). The O2− generation rate by microsomes exposed to NO decreased significantly as compared to the rate measured in microsomes incubated in the absence of NO. The exposure of the microsomes to the NO donor did not alter the microsomal rate of hydroxyl radical generation. Preincubation of the microsomes with the NO donor affected neither iron reduction rate nor activity of cytochrome c reductase. However, cytochrome P450 activity was significantly inhibited after exposure to NO. This inhibition was completely prevented by hemoglobin. The data are consistent with the hypothesis that NO exhibits a potential antioxidant role in the plant cell by decreasing the rate of generation of superoxide anion. Since endogenous NO was detected in homogenates of soybean embryonic axes by EPR studies, this interaction between NO and cytochrome P450 in soybean embryonic axes could be a factor of relevance for the control of oxidative stress in vivo.
Modulation by weak bases or weak acids of the pH of cell sap and phosphoenolpyruvate carboxylase activity in leaf discs of C4 plants
Authors: Rajagopalan A. V. , Gayathri J. , Raghavendra A. S.
Published: March 2003

When leaf discs of a C4 species, Alternanthera pungens (L.) H.B. and K. or Amaranthus hypochondriacus L., were preincubated in 7.5 mM NH4Cl, the pH of the cell sap increased by nearly 0.3 unit, while the activity of phosphoenolpyruvate carboxylase (PEPC) about doubled compared to the cell sap from control leaf discs (preincubated in 5 mM Tricine‐KOH, pH 8.5). The sensitivity of PEPC to L‐malate (a feedback inhibitor) decreased marginally as a result of cytosolic alkalization. The pH of the cell sap and PEPC activity decreased by nearly 0.4 unit and 50%, respectively, when leaf discs were incubated in weak organic acids such as propionic, butyric or salicylic acid. Thus, our results demonstrate a marked modulation in vivo of cell sap pH and PEPC activity in leaf discs from C4 plants by external alkalizing or acidifying reagents. The rise in PEPC activity due to alkalization of leaf discs was not sensitive to cycloheximide, implying that cytosolic protein synthesis was not involved in the activation of PEPC. Despite the marked increase in the PEPC activity due to the base‐loading of leaf discs, the change in malate sensitivity of the enzyme was only marginal, indicating that there was no significant increase in the extent of PEPC‐phosphorylation. Besides the physiological significance, the technique of acid/ base‐loading may be an important tool for studying the regulation of PEPC in leaf discs of C4 species, since the activity of PEPC could be enhanced apparently without phosphorylation of the enzyme.
Ultrastructural and biophysical changes in developing embryos of Aesculus hippocastanum in relation to the acquisition of tolerance to drying
Authors: Farrant Jill M. , Walters Christina
Published: March 2003

Changes in ultrastructural, biochemical and biophysical characteristics of embryonic axes of Aesculus hippocastanum during development are related to changing levels of desiccation tolerance. Histodifferentiation was complete 30 days after flowering (DAF) and fruits were shed about 120 DAF. During this period, the dry mass of embryonic axes increased from about 0.5 to 4 mg and the water content decreased from 10.2 to 2.0 g H2O g−1 dry mass (g g−1). In spite of the large changes in water content, water potentials of freshly harvested material declined slightly during development from −0.65 to −2.0 MPa. Tolerance of desiccation increased as embryos matured. If evaluated on the basis of critical water contents for survival, tolerance appeared to increase continuously, maximum tolerance being achieved at 120 DAF when embryos survived water contents as low as 0.30 g g−1. When evaluated from critical water potentials, three distinct levels of desiccation tolerance could be recognized at −1.8 MPa (30‐40 DAF), −4 M Pa (48‐90 DAF) and −12 MPs (100‐120 DAF). During development, total dry matter increased while sugar content (g g−1' dry mass) and osmotically active material (mmol g−1 dry mass) decreased. The subcellular organisation of axes was always typical of metabolically active tissues. Maximum tolerance (−12 MPa) was associated with a reduced amount of monosaccharides and the appearance of water with unusual calorimetric behaviour. Our data are consistent with several of the current hypotheses regarding the mechanisms of desiccation tolerance. Accumulation of dry matter reserves, reduced levels of monosaccharides, presence of dehydrin‐like proteins and ability to form glasses appear to be associated with the changes in desiccation tolerance. However, none of these factors allow A. hippocastanum embryos to achieve the extreme level of desiccation tolerance typical of orthodox seeds. This may be because A. hippocastanum embryos do not reach physiological maturity and remain metabolically active even after they are shed from the parent plant. Also, embryos may acquire incompetent protectants or lack as yet unidentified protective mechanisms.
Cysteine is the presumed catalytic residue of Citrus sinensis phospholipid hydroperoxide glutathione peroxidase over‐expressed under salt stress
Authors: Faltin Zehava , Camoin Luc , Ben‐Hayyim Gozal , Perl Avi , Beeor‐Tzahar Tal , Strosberg Arthur Donny , Holland Doron , Eshdat Yuval
Published: March 2003

A citrus salt‐stress associated protein (Cit‐SAP), partially purified from citrus cultured cells, was previously identified as the first plant phospholipid hydroperoxide glutathione peroxidase (PHGPx). The nucleotide sequence of its isolated gene (csa) revealed that a TGT, known as codon for Cys, encodes the presumed catalytic residue 41 in the polypeptide chain of Cit‐SAP. In animals, a TGA encodes the rare amino acid selenocysteine (Sec) as the catalytic residue of the analogous enzyme. It is of interest to establish whether the TGT codon for this catalytic residue in the plant enzyme is indeed translated to Cys and not to Sec, and to demonstrate the effect of such a change, if it exists, on the nature of the enzymatic activity of the plant enzyme as compared to that of the animal. In the present study, we have purified for the first time, by affinity chromatography, enzymatically active citrus PHGPx from recombinant Escherichia coli bearing the csa gene. Tryptic digestion of the purified enzyme followed by HPLC afforded the isolation of a peptide which contains residue 41, and its sequence analysis revealed that this residue is indeed a Cys, and not Sec. The enzymatic activity and specificity of the recombinant Cit‐SAP was found to be similar to that observed before for the partially purified plant enzyme. However, the rate of this activity was much lower towards phospholipid hydroperoxides, and none towards hydrogen peroxide, as compared to that of the animal analogue. It is therefore suggested that the presence of a Cys, and not Sec, as the catalytic residue in the plant enzyme, affects its enzymatic activity and may determine a different biological role for the plant PHGPx from that of the animal.
Properties of thiamin‐binding proteins from sesame seed 2S albumins
Authors: Watanabe Katsumi , Chikushi Kumi , Adachi Takashi , Shimizu Mayumi , Yoshida Toyokazu , Mitsunaga Toshio
Published: March 2003

Three thiamin‐binding proteins (TBPs) from sesame (Sesamum indicum L.) seeds (STBP‐I, ‐II and ‐III) were characterized. Binding of thiamin to the three STBPs was inhibited by pyrithiamin, which did not inhibit the binding of thiamin to TBPs from other plant seeds. STBP‐I alone bound 2‐northiamin and hydroxyethylthiamin. Isoelectric points (pIs) of STBP‐I and ‐II both were 7.5. The pI of STBP‐III was 6.5. STBPs did not have immunological homology with TBPs from rice seeds and buckwheat seeds. On the other hand, the amino acid compositions of the small and large polypeptides isolated from STBP‐I, ‐II and ‐III resembled each other. Both the polypeptides contained large amounts of Glu (or Gln) and Arg. The small polypeptides contained more Ser than the large polypeptides. The N‐terminal amino acid sequences (the first 29 residues) of the small polypeptides were identified. The N‐terminal amino acid sequences of the three STBPs were the same. The small polypeptides had homology to castor bean 2S albumin small subunit. These results showed that STBPs were part of a plant protein superfamily and that STBPs differed from the TBPs of other plant seeds as to the binding to thiamin‐related compounds and immunological properties, and further, that STBP‐I, ‐II and ‐III differed in the affinity for thiamin‐related compounds and pI, indicating that STBP‐I, ‐II and ‐III are isomers.
Tissue localization of β‐glucosidase in rye, maize and wheat seedlings
Authors: Nikus Jeanette , Jonsson Lisbeth M. V.
Published: March 2003

The localization of β‐glucosidase was determined at the tissue level in roots and shoots of rye, wheat and maize seedlings, using an immunohistochemical approach with antibodies directed against purified maize β‐glucosidase as the primary antibody. In the roots, the β‐glucosidase was found in the epidermis and the underlying cell layer. In the leaves, staining was seen in the epidermis (rye and wheat) and nearby vascular tissue (rye, wheat and maize). In all 3 species, β‐glucosidase activity was highest in the coleoptile. Here the enzyme was restricted to the epidermis in wheat and to cells near the vascular tissue in maize, but was found in the whole tissue, except the vascular tissue, in rye. Maize, wheat and rye all contain hydroxamic acid glucosides and results are discussed in relation to a proposed role of β‐glucosidase as part of a defense system releasing hydroxamic acid aglucone upon herbivore attack, pathogen penetration or aphid infestation.
Temporal and spatial patterns of GAPDHc mRNA accumulation during an incompatible potato‐Phytophthora infestans interaction. Comparison with a compatible interaction
Authors: Beligni María Verónica , Laxalt Ana María , Lamattina Lorenzo
Published: March 2003

Most inducible defense responses of plants to pathogens are the result of transcriptional activation of specific genes. The cytosolic isoform of the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDHc, EC 1.2.1.12) increases under environmental constraints, including biological stress. This work was directed to investigate the temporal and spatial distribution of GAPDHc mRNA during an incompatible potato (Solanum tuberosum L. cv. Pampeana)‐Phytophthora infestans interaction. GAPDHc mRNA accumulated earlier during an incompatible interaction (24 h upon infection) than during a compatible one (48 h) and remained over basal levels until late stages of infection (96 h). This different pattern for temporal accumulation in whole potato plants could be part of the rapid defense responses observed when plants are resistant to infection. During the incompatible interaction, the most important GAPDHc mRNA induction (20‐fold increase) relative to basal levels, at 12 h post‐inoculation, occurred at the site of infection, whereas a 13‐fold increase occurred in the uninfected surrounding tissue of inoculated points. At later stages (24 h), the highest induction was observed in uninoculated leaflets (15‐fold), whereas the levels in the inoculated leaflets (both the inoculated points and the surrounding areas) remained above the levels present at time zero of infection (8‐ and 11‐fold, respectively). These results suggest that the signal responsible for GAPDHc mRNA accumulation in plants during infection is first activated in infected tissues, but is then transmitted to uninfected parts. Furthermore, biochemical responses in plants upon pathogen attack include components of primary metabolism, and this could be involved in physiological host adaptations against pathogen spreading.
Flavonoid biosynthesis in tomato fruit cuticles after in vivo incorporation of H‐phenylalanine precursor
Authors: Laguna Lidia , Casado Carolina G. , Heredia Antonio
Published: March 2003

Radiolabelling of epicuticular waxes and cutin of isolated tomato fruit cuticles were determined after fruit surface application of H‐phenylalanine precursor. During fruit ripening, the precursor is incorporated in different phenolic components: the flavanone naringenin was found to be the major compound in the epicuticular waxes, while the amount of the labelled flavonoid in the cutin matrix was progressively increased throughout fruit ripening. Confocal microscopy, together with experimental estimation of the mobility (diffusion coefficient, D) and affinity (partition coefficient, K) of the flavonoids naringenin and chalconaringenin for the different cuticular components, indicate that these compounds are extruded to the outer surface of tomato fruits, forming molecular clusters.
Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators
Authors: Lelu Marie‐Anne , Bastien Catherine , Drugeault Aurélie , Gouez Marie‐Laure , Klimaszewska Krystyna
Published: March 2003

To initiate somatic embryogenesis in Pinus sylvestris and Pinus pinaster, immature seeds were collected from June to August and the developmental stage of the zygotic embryos was determined. Four developmental stages were distinguished and the response of the zygotic embryos at each of the four developmental stages was compared intra‐ and inter‐species. For this study, modified Litvay's medium (LM), with or without growth regulators, was chosen. Somatic embryogenesis was initiated and maintained on both media but the two species displayed different propensities. In P. sylvestris, the highest initiation frequency was obtained with intact megagametophytes containing embryos at the four‐cell stage to the stage of cleavage polyembryony (up to 22 and 9%, respectively). The culture medium had no significant effect on the initiation and proliferation of embryogenic cultures. In P. pinaster, however, the best response occurred from excised zygotic embryos at the stage prior to elongation of cotyledon primordia (up to 40% explants responded), on medium with growth regulators. Another characteristic distinguishing the two species in culture was that in some embryogenic cell lines of P. sylvestris, somatic embryos matured spontaneously when initiated and maintained on medium without growth regulators. Some of these embryos developed into plantlets on the same medium at the frequency of 40%. Therefore, in P. sylvestris all the stages of somatic embryogenesis were achieved on the medium without growth regulators. However, in both species, maturation of a large number of somatic embryos was greatly improved on medium containing high concentration of gellan gum (Gelrite 10 g l−1) and abscisic acid (60 μM). Cotyledonary somatic embryos subsequently germinated (72 and 80% for P. sylvestris and P. pinaster, respectively) and developed into plantlets (48 and 29%, for P. sylvestris and P. pinaster, respectively). This represents a significant improvement in plantlet recovery from somatic embryos of both species.
Carbohydrate metabolism during early fruit development of sweet melon (Cucumis melo)
Authors: Gao Zhifang , Petreikov Marina , Zamski Eli , Schaffer Arthur A.
Published: March 2003

In sink tissues of cucurbits, including sweet melon fruits, the galactosyl‐sucrose oligosaccharides, stachyose and raffinose, together with sucrose, are the major translocated carbohydrates. In the present study we investigated the carbohydrate metabolism of young melon (Cucumis melo L. cv. C‐8) fruit during the period of initial fruit set and development, from 3 days prior to anthesis until 20 days after anthesis (DAA), prior to the onset of sucrose accumulation. The enzymes assayed could be classified into two categories according to developmental patterns. Two of the enzymes, alkaline α‐galactosidase I [EC 3.2.1.22], which hydrolyzes both raffinose and stachyose, and acid invertase [EC 3.2.1.26] either increased or remained stable during the first 10 DAA. The remaining measured enzymes (the stachyose‐specific alkaline α‐galactosidase form II, acid α‐galactosidase, alkaline invertase, sucrose synthase [EC 2.4.1.13], galactokinase [EC 2.7.1.6], UDP‐Gal PPase [EC 2.7.7.10], UDP‐Glc‐4 epimerase [EC 5.1.3.2], UDP‐Glc PPase [EC 2.7.7.9], phosphoglucomutase [EC 5.4.2.2] and phosphoglucoisomerase [EC 5.3.1.9]) all showed a similar developmental pattern of steady decrease in activity following anthesis. We also compared the saccharide metabolism of pollinated and non‐pollinated ovaries during the initial days following anthesis. In the absence of pollination, ovary growth dramatically decreased by the first DAA and was accompanied by a sharp decrease in the activity of UDP‐Glc PPase. Other enzymes in the pathway, including the enzymes of stachyose and raffinose hydrolysis, did not decrease in activity until 2 or 4 DAA, after ovary growth was affected. These results provide information to assess the possible regulating enzymes in cucurbit ovary development and fruit set.
Characterization of a spinach chloroplast sequence‐specific DNA‐binding factor for photosystem I psaA operon promoter
Authors: Wu Sang‐Pin , Cheng Ming‐Chih , Grace Chen Shu‐Chen
Published: March 2003

The plastid genes psaA, psaB and rps14, encoding the photosystem I reaction center chlorophyll proteins and ribosomal protein CS14, are organized into an operon on the circular plastid genome. To elucidate the regulatory mechanism of platid transcription during plant development, a DNA‐binding protein has been characterized and partially purified from spinach (Spinacia oleracea L. var. spinosa Ashers et Graeden) chloroplasts. The protein interacts specifically with promoter sequences from −51 to −17 (named region U) of the operon. The purification procedures include high‐salt extraction of chloroplasts, ammonium sulfate fractionation (25–65% saturation), heparin agarose chromatography, heat treatment at 65°C and sequence‐specific DNA‐affinity chromatography. Gel filtration in combination with Southwestern blot analysis conclude that this DNA‐binding protein is dimeric and composed of two 34‐kDa subunits. A 28‐kDa protein without region U‐binding activity was observed to copurify with the DNA‐binding protein using DNA‐affinity chromatography.
Characterization of an 80‐kDa dehydrin‐like protein in barley responsive to cold acclimation
Authors: Bravo León A. , Close Timothy J. , Corcuera Luis J. , Guy Charles L.
Published: March 2003

Barley (Hordeum vulgare L.) exposed to low temperature increases its freezing tolerance. This increase has been associated with several metabolic changes caused by low temperature, including expression of dehydrins (DHN), a family of proteins induced by dehydration and cold acclimation. DHNs play an undetermined role in dehydration responses during freezing. We have studied the accumulation of an 80‐kDa DHN‐like protein (P‐80) in barley under cold acclimation 6/4°C (day/night), postulating that it is localized in tissues where primary ice nucleation occurs. P‐80 was absent in nonacclimated plants and was detectable after 48 h of cold acclimation, reaching a stable level after 6 days. P‐80 decreased when plants were returned to 20–25°C. Drought, ABA and high temperature did not increase the levels of P‐80, suggesting that its expression could be specifically regulated by cold. Immunolocalization by tissue printing and fresh cross sections of leaves showed the protein to be associated with vascular tissues and epidermis. The localization of P‐80 is consistent with our hypothesis because vascular tissue and the epidermis are preferential ice nucleation zones during the onset of freezing. The differential accumulation of P‐80 may have an adaptive value by participating in tolerance mechanisms during freeze‐induced dehydration.