bluuebunny/crossref_metadata_2025 huggingface dataset:
Nebulized budesonide after hospitalization for recurrent bronchial obstruction in children younger than 18 months
Authors: Lødrup Carlsen K. C. , Carlsen K. H. , Nikander K. , Leegaard J. , Havnen J. , Steen‐Johnsen J. , Winsness A.
Published: March 2003

A multi‐center, double‐blind, randomized dose–response study was performed to assess the effect of 3 months of treatment with two different doses of inhaled nebulized budesonide in children with acute recurrent bronchial obstruction (BO) causing hospitalization. Steroid‐naive children younger than 18 months were included when admitted to hospital because of BO for at least the second time, and were followed‐up monthly for 15 months. Forty‐five of 49 subjects (43 boys, 2 girls) (mean age 9.3 months upon inclusion) completed the study. Twenty‐four patients (20 boys, 4 girls) received nebulized budesonide 0.5 mg twice daily for 1 month followed by 0.25 mg daily for the next 2 months, whereas 25 children received 0.1 mg twice daily throughout the 3‐month treatment period. Outcome (number of BO episodes, time to first BO after start of treatment, and use of rescue medication), as well as height/length and weight, were assessed at the start of treatment and monthly for the following 3 months, as well as for 12 months after cessation of treatment (15 months in total). There was an overall tendency towards better symptom control (fewer episodes of acute BO during treatment and follow‐up, fewer hospital visits because of acute BO, lower clinical score during follow‐up, and less use of rescue medication during follow‐up) in the high‐dose treatment group vs. the low‐dose treatment group. However, the differences did not reach statistical significance for any of the outcomes. The only significant difference in effect between the groups was fewer children in the high‐dose group treated openly with nebulized budesonide during follow‐up. Length/height and weight gain did not differ significantly between the two treatment groups throughout the study. There was no significant dose‐dependent beneficial effect of 3 months of treatment with nebulized budesonide in infants and toddlers with at least two hospitalizations for acute bronchial obstruction.
Bee‐venom allergy in children: Long‐term predictive value of standardized challenge tests
Authors: Schuetze Georg E. , Forster Johannes , Hauk Pia J. , Friedl Katrin , Kuehr Joachim
Published: March 2003

Venom immunotherapy (VIT) is able to protect insect venom‐allergic patients against life‐threatening sting reactions. Standardized sting challenges can be used as a diagnostic tool to check whether VIT is required. No data are available on the long‐term predictive value of sting challenge tests. The purpose of this study was to investigate the long‐term predictive value of sequential bee‐sting challenges with respect to the ability to predict future sting reactions in bee‐venom (BV) allergic children. Between 1988 and 1992, 92 BV‐allergic children had been challenged with sequential bee stings at intervals of 2–6 weeks to determine the necessity of VIT. In 1996, all 92 families were followed‐up using standardized telephone interviews. Until the follow‐up, 61 children (66.3%) had experienced at least one natural bee sting. Based on the results of the initial challenge tests, 13 of the 61 patients had been started on VIT. Two of these 13 (15.4%) developed systemic reactions 1 year after VIT of 5 years, of which one was mild and one was severe. Among the 48 re‐stung patients who were not treated with VIT, three children (6.3%) experienced mild systemic reactions, whereas 45 children reported no more than a local reaction. The long‐term predictive value of sequential bee‐sting challenge tests for systemic reactions in children not treated with VIT remained at a level of 93.8% (95% confidence interval: 82.8–98.7%) even over a period of more than 6 years. Based on this data, we conclude that sequential bee‐sting challenges are a powerful tool to determine the necessity for VIT in BV‐allergic children.
Interleukin‐6, interleukin‐8, interleukin‐11, and interferon‐γ levels in nasopharyngeal aspirates from wheezing children with respiratory syncytial virus or influenza A virus infection
Authors: Oh Jae‐Won , Lee Ha‐Baik , Park Il‐Kyu , Kang Jung‐Oak
Published: March 2003

The differences between respiratory syncytial virus (RSV) and influenza A virus (IFAV) in the pathogenesis of wheezing in young children have not been clearly defined. The aim of this study was to assess the contributions of RSV vs IFAV in the pathogenesis of upper airway inflammation in wheezy young children. We compared interleukin (IL)‐6, IL‐8, IL‐11, and interferon‐γ (IFN‐γ) levels in nasopharyngeal aspirates (NPA) from non‐asthmatic children with respiratory virus infections (RSV in 17 children and IFAV in 13 children), asthmatic children with viral infections (RSV in nine children, IFAV in 10 children), and 22 unaffected healthy children (controls). Levels of IL‐11 in NPA from asthmatic children were significantly higher than those from non‐asthmatic children with RSV infection, and RSV infection enhanced the IL‐11 production in NPA significantly compared to IFAV infection. Nasopharyngeal epithelium from children with RSV infection secreted more IL‐6 than that of children with IFAV infection. There was little difference in the IL‐8 and IFN‐γ levels between asthmatic and non‐asthmatic children with RSV or IFAV infection. In conclusion, asthma enhanced IL‐11 production in RSV infection rather than IFAV infection in early childhood. There was a trend towards greater IL‐6 production in RSV infection compared with IFAV infection.
Influence of external biliary drainage on cyclosporin A (Neoral®) absorption in a pediatric liver transplant recipient
Authors: Zylber‐katz Ester , Verstandig Anthony , Granot Esther
Published: March 2003

Abstract: We report on a patient who underwent a liver transplant 8 yr ago at the age of 2. The post‐operative course and further follow‐up were uneventful, maintaining immunosuppression with cyclosporin A (CsA) (Sandimmune®) and prednisone; 1.5 yr ago, the patient was converted to Neoral®. The mean ± SD trough CsA level was 127 ± 37.2 ng/mL, when the patient was maintained on a daily dose of 180 mg. Following an increase in γ‐GTP levels, a biliary‐enteric anastomotic stricture was found. Dilatation was performed and a tube placed for external biliary drainage. Three days later the trough CsA level was at the limit of detection; consequently, the Neoral dose was increased to 480 mg/d. CsA concentration measured 5 days later reached 164 ng/mL. After restoring internal biliary drainage the dose was decreased to 180 mg/d and the CsA level was 142 ng/mL. Later on the CsA dose was further reduced to 160 mg/d (a third of the dose during external biliary drainage) and trough levels were maintained at 90–120 ng/mL. We suggest that CsA dose adjustment and continuous drug monitoring are necessary when bile flow is compromised, in order to prevent rejection of the transplanted liver.
Intensification of therapy using hematopoietic stem‐cell support for high‐risk neuroblastoma
Authors: Matthay Katherine K.
Published: March 2003

Abstract: The use of new strategies for dose intensification using peripheral blood stem cell or autologous purged bone marrow rescue has raised expectations for cure in advanced neuroblastoma, although conflicting reports exist regarding the efficacy of these approaches. Using risk groups based on both biological and clinical staging, the Children’s Cancer Group and the Pediatric Oncology Group have agreed upon common prognostic criteria for treatment stratification. We summarize below the prognostic classification and treatment approaches that have improved the overall outcome for children with advanced neuroblastoma. Intensive induction therapy, myeloablative therapy, hematopoietic stem cell purging, and post‐transplant therapy for minimal residual disease all have an important role in the treatment. Possible future improvements may incorporate more tumor‐specific therapy with targeted radiotherapy, monoclonal antibodies, tumor vaccines, and differentiating agents.
Graft failure secondary to necrotizing enterocolitis in multi‐visceral transplantation recipients: Two case reports
Authors: Khan Farrukh A. , Kato Tomoaki , Berho Marianna , Nery Jose R. , Pinna Antonio D. , Colombani Paul , Tzakis Andreas G.
Published: March 2003

Abstract: We report on two recipients of multi‐visceral grafts who exhibited sudden onset of acute abdomen discomfort 2 weeks post‐transplantation after a fairly uneventful immediate post‐operative course. Both patients were shown to have pneumatosis intestinalis and one had air in the portal vein. Both patients underwent exploration, which showed non‐viable intestine (terminal ileum and colon in the first patient and the entire small intestine distal to the ligament of Treitz in the second patient). There was no vascular thrombosis. The necrotic intestine was resected in both cases. The first patient developed sepsis and died 15 days later despite the rescue efforts. The second patient was re‐transplanted twice and is doing well. The histopathology of the segments involved revealed cryptitis, vasculitis, and features of transmural necrosis. Accordingly, both clinical and pathologic features are diagnostic of necrotizing enterocolitis. To our knowledge this is the first report of this complication following intestinal or multi‐visceral transplantation.
A review of nutritional problems and the cystic fibrosis lung transplant patient
Authors: Dosanjh A.
Published: March 2003

Abstract: Lung transplantation is an option for some cystic fibrosis (CF) patients. CF is associated with a variety of non‐pulmonary problems, which should be managed before and after tansplantation. This commentary discusses some of the nutritional issues affecting CF patients. Theses issues include: the need for nutritional supplements; gastrostomy tube placement; osteoporosis and disabetes.
Domino as a bridge to definitive liver transplantation in a neonate
Authors: Casas‐Melley Adela T. , Thomas Philip G. , Krueger Leslie J. , Falkenstein Kathleen P. , Flynn Louise M. , Conley Susan B. , Dunn Stephen P.
Published: March 2003

Abstract: An interim liver transplant was used to extend survival in a neonate. This was accomplished by the initial transplant of a left‐lateral segment of a metabolically abnormal liver obtained from a 7‐yr‐old patient with primary oxalosis. This bridging strategy was required because our neonatal patient was dying of fulminant hepatic failure caused by hepatic vein thrombosis and a small liver or liver segment could not be found. Although problems with hyperoxaluria were encountered in the neonate post‐transplant, the interim liver transplant enabled the baby to survive and grow until the age of 4 months. At that time, a definitive transplant was performed using the left‐lateral segment of his mother's liver. This case represents the first reported use of a pediatric domino transplant where a metabolically abnormal liver was used to allow sufficient growth to permit a definitive liver transplantation.
Factors influencing growth and final height after renal transplantation
Authors: Ninik A. , McTaggart S. J. , Gulati S. , Powell H. R. , Jones C. L. , Walker R. G.
Published: March 2003

Abstract: Growth retardation occurs commonly in children and adolescents with chronic renal insufficiency. While some children exhibit catch‐up growth following renal transplantation, for many children growth remains sub‐optimal. The aim of the current study was to review the factors influencing growth and final height following renal transplantation. Data from all children who had a renal transplant performed between 1985 and 1998 at the Royal Melbourne and Royal Children's Hospitals, Melbourne (n = 85), were examined retrospectively. Two children who died in the first year post‐transplant and one patient lost to follow‐up within 6 months of their transplant were excluded. Children with multiple grafts had only growth following their most recent graft analyzed. The mean height standard deviation score (Ht‐SDS) at the time of transplantation was −2.11 (range: −5.05 to 0.27), improving to −1.50 (range: −3.67 to 1.27) at 7 yr post‐transplant. On univariate analysis, the dose of cyclosporin at 6 months and at 1 and 3 yr, and the graft function at 1 yr, had a significant positive correlation with the change in Ht‐SDS (ΔHt‐SDS) at each of those time‐points post‐transplant. At all time‐points there was a strong correlation between pretransplant height and subsequent growth. A sub‐group of children who were 16 yr of age or older at December 1999, and who were considered to have reached their final height, were examined to determine predictors of final height. Multiple regression analysis of clinical and laboratory parameters from the sub‐group of patients ≥ 16 yr of age showed that height at the time of transplant, age at the time of transplant, and final glomerular filtration rate, were significant independent predictors of growth (r2 = 0.82, p = 0.01). In addition, the immunosuppressive regimen at 1, 3, and 5 yr post‐transplant had a significant effect on growth. This study confirms the importance of each of these factors for post‐transplant growth.
Liver transplantation in Ellis–van Creveld syndrome: A case report
Authors: Black Dalliah , Reutter Jason , Johnson Mark , Fair Jeffrey , Woosley John , Gerber David
Published: March 2003

Abstract: This case represents a rare association of Ellis–van Creveld (EvC) syndrome, a chondroectodermal disorder, with congenital paucity of bile ducts. Sequential liver biopsies during the patient's childhood demonstrated progressive fibrosis that can occur in other chondrodysplastic malformations. However, this EvC case is the first report to demonstrate paucity of intra‐hepatic bile ducts progressing to cirrhosis and subsequently requiring transplant.
Cholelithiasis in infant and pediatric heart transplant patients
Authors: Sakopoulos Andreas G. , Gundry Steven , Razzouk Anees J. , Andrews H. Gibbs , Bailey Leonard L.
Published: March 2003

Abstract: There have been numerous studies which demonstrate a relatively high incidence of gallstones in adult solid‐organ transplant recipients receiving cyclosporin A (CsA) immunosuppression. The purpose of the present study was to investigate our experience with cholelithiasis in babies and children undergoing heart transplant (HTx). From May 1985 to December 1998, 311 neonatal and pediatric cardiac transplants were performed at our institution. Routine abdominal ultrasound was performed at 3 months, 1 yr, and bi‐annually thereafter on all transplant recipients. Asymptomatic or symptomatic gallstone development was detected during abdominal ultrasound in 10 of 311 patients (3.2%). Eight of these 10 patients (80%) were transplanted when younger than 3 months of age. Eight per cent of all infants transplanted at < 3 months of age developed cholelithiasis (p < 0.05 compared to older age at HTx). Fifty per cent of gallstones were detected and treated within 6 months post‐HTx, while the remaining 50% of patients with gallstones underwent cholecystectomy 3–6 yr later. Only 20% (two of 10) had symptoms of cholelithiasis/cholecystitis. Five patients (50%) underwent laparoscopic cholecystectomy. Only one patient older than 1 yr of age, who was symptomatic, underwent open cholecystectomy. There were no complications from surgery. There were no differences in liver function tests or cholesterol levels in transplant recipients with or without gallstones, and all mean values were within normal limits. Hence, although the incidence of pediatric post‐transplant cholelithiasis in infant and pediatric heart transplant recipients is low, almost all occurrences are associated with HTx during early infancy and, because of this, patients in this group should be routinely screened. Laparoscopic or open cholecystectomy are extremely well tolerated and we recommend that surgery be performed when cholelithiasis is found in pediatric heart treatment patients.
Flower senescence in daylily (Hemerocallis)
Authors: Lay‐Yee Michael , Stead Anthony D. , Reid Michael S.
Published: March 2003

Daylily (Hemerocallis hybrid cv. Cradle Song) flower development and senescence was categorised into seven stages, each separated by one day. As the bud developed (Stages I to III) its fresh and dry weight increased, but during flower opening (Stages III to IV) only the fresh weight increased, reaching a maximum when the flower was fully open (Stage IV). Fresh and dry weight declined as the flower senesced (Stages V to VII), and the petal tissue became water‐soaked. Flower respiration was relatively constant through Stages I to III, but during opening the CO2 production increased, then declined as the flowers senesced. Ethylene production was low throughout flower development, and there was a transient increase, up to 2.5 nl flower−1 h−1, as flowers senesced. Petal protein content per gram fresh weight decreased throughout flower development and senescence either on or off the plant. One‐dimensional SDS‐PAGE revealed a general gradual loss of protein bands during flower senescence and loss of some specific bands (e.g. at M, 30 000 and 40 000). Flowers, detached from the plant at Stage III, placed in deionised (DI) water for 7 h and then transferred to fresh DI water or actinomycin D (25 mg l−1), showed an increase in fresh weight up to a maximum at about 12 h after transfer, followed by a rapid decline. By 48 h the flowers had completely senesced, the tissue becoming watersoaked and free moisture appearing on the tips of collapsed petals. In contrast, senescence of flowers treated during opening with cycloheximide (1 mM) was greatly delayed, with little loss of fresh weight or reduction in flower diameter, even up to 181 h after transfer. When such flowers did finally senesce, they did not show the same pattern of senescence as seen with those in water or actinomycin D. Cycloheximide‐treated flowers wilted gradually and showed no water‐soaking and no free moisture at the petal tips. Cycloheximide treatment retarded the decline in protein content of the petals; and the protein population, as visualised by SDS‐PAGE, was maintained for up to 204 h after transfer to cycloheximide.
Effects of salt stress on H+‐ ATPase and H+‐PPase activities of tonoplast‐enriched vesicles isolated from sunflower roots
Authors: Ballesteros Emilia , Donaire Juan Pedro , Belver Andres
Published: March 2003

The control of ion concentration in the cytosol and the accumulation of ions in vacuoles are thought to be key factors in salt tolerance. These processes depend on the establishment in vacuolar membranes of an electrochemical H+ gradient generated by two distinct H+‐translocating enzymes: a H+‐PPase and a H+‐ATPase. H+‐lrans locating activities were characterized in tonoplast‐enriched membrane fractions isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots exposed for 3 days to different NaCl regimes. The 15/32% sucrose interface was enriched in membrane vesicles possessing a vacuolar‐type H+‐ATPase and a H+‐PPase, as indicated by inhibitor sensitivity, pH optimum, substrate specificity, ion effects kinetic data and immunolabelling with specific antibodies. Mild and severe stress did not alter the pH profile, ion dependence, apparent Km nor the amount of antigenic protein of either enzyme. Saline treatments slightly increased K+‐stimulaied PPase activity with no change in ATPase activity, while both PPi‐dependent and NO3‐sensitive ATP‐dependent H+ transport activities were strongly stimulated. These results are discussed in terms of an adaptative mechanism of the moderately tolerant sunflower plants to salt stress.
Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll b‐deficient mutants
Authors: Falbel Tanya G. , Staehelin L. Andrew
Published: March 2003

We have analyzed precursor pools in the chlorophyll (Chi) synthesis pathway for a set of eighteen well studied Chl b‐defident mutants in monocotyledonous (barley, maize and wheat) and dicotyledonous plants (Antirrhinum, Arabidopsis, soybean, tobacco and tomato) that form abnormal thylakoid membrane systems. All of these mutants have a partial block in Chl synthesis and nearly all of them accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The large number of mutants at several genetic loci affecting this critical branchpoint in tetrapyrrole biosynthesis suggests that the Mg‐chelatase enzyme, catalyzing the first committed step of Chi biosynthesis, is a multimeric complex composed of the products of some of these genetic loci, and perhaps regulated by others. We hypothesize that these mutants are Chi b‐deficient and have reduced amounts of light‐harvesting antenna complexes (LHCs.) and develop abnormal thylakoid membranes as a direct result of limited Chl synthesis. The observed bottleneck in Chl synthesis can also explain the light‐intensity‐dependent and temperature‐dependent expression of the mutant phenotype. This hypothesis offers a simple explanation for the wide variety of pbenotypes that have been reported for the many Chl‐deficient mutants in the literature. Our findings are also consistent with the notion that Chl b is made from “left over” Chl a molecules and suggest that the Chi b‐deficient mutants should be considered more appropriately as leaky Chl‐deficient mutants.
Gamma‐ray‐induced changes in hypodermal mesocarp tissue plasma membrane of pre‐ and post‐storage muskmelon
Authors: Lester Gene E. , Whitaker Bruce D.
Published: March 2003

Gamma irradiation (1.0 kGy) of intact, newly harvested, mature muskmelon fruit (Cucumis melo L. var. reticulatus Naud.) appears to have an immediate deleterious effect, but also a long‐term beneficial effect, on the integrity and function of the plasma membrane (PM) of hypodermal mesocarp tissue. The initial consequences of gamma irradiation included an increase in the free sterol:phospholipid ratio, resulting at least in part from deglycosylation of steryl glycosides, a decrease in the spinasterol:7‐stigmastenol ratio in each of the PM steryl lipids (free sterols, steryl glycosides, and acylated steryl glycosides), and a decrease in H+‐ATPase activity. Irradiation did not increase protein loss, suggesting that the decrease in H+‐ATPase activity resulted from either direct inactivation of the enzyme or altered PM ordering caused by the steryl lipid modifications. The long‐term beneficial effects of irradiation, observed following 10 days of commercial storage, included greater retention of total PM protein, a diminished decline in total PM phospholipids (PL) and in the PL:protein ratio, and maintenance of greater overall H+‐ATPase activity (activity was the same as in controls on a per mg protein basis, but there was > 30% more protein in the PM of stored irradiated fruit). These results indicate that 1 kGy gamma irradiation administered prior to storage slowes the progression of two key parameters of senescence, PM protein loss and PL catabolism.
Photocontrol of chs gene expression in petunia flowers
Authors: Katz A. , Weiss D.
Published: March 2003

In the present work we studied the effect of light and diurnal light‐dark periods on the expression of the anthocyanin biosynthetic gene, chalcone synthase (chs), in petunia corollas. We found that light is essential for chs expression at the early stages of flower development. At these stages chs transcript level fluctuated during the day‐night cycles. Our results also showed that the fluctuation is regulated by the diurnal light signal and not by an endogenous clock. The effect of the light was cumulative and thus chs mRNA reached maximum levels at the end of the diurnal light period. At later stages of flower development, when chs transcript is rapidly accumulated, expression was only slightly affected by light and did not fluctuate during the diurnal light‐dark period. We suggest that the low sensitivity of chs expression to external signals at the later stages of flower development is essential to ensuring normal flower coloration under changing environmental conditions.
The ribosomal protein S7 gene is transcribed and edited in sugar beet mitochondria
Authors: Estiati A. , Kubo T. , Mikami T.
Published: March 2003

We have isolated a mitochondrial rps7 gene from sugar beet (Beta vulgaris L. cv. TK81‐O). It is present in a single genomic environment, with northern blot analysis showing transcription of this gene. Sequence analysis of RT‐PCR‐derived cDNA of rps7 showed that three RNA‐editing events occurred on rps7 mRNAs. As far as we know, this is the first report of the complete identification of the RNA editing of a mitochondrial rps7 in the angiosperms.
Differential reponses to paraquat‐induced oxidative injury in a pea (Pisum sativum) protoplast system
Authors: Doulis Andreas G. , Donahue Janet L. , Alscher Ruth G.
Published: March 2003

Antioxidant responses to varying degrees of paraquat stress in freshly isolated photosynthesizing pea (Pisum sativum L.) protoplasts from cultivars Progress and Nugget were studied. Leaves of comparable maturity were used for protoplast isolation. Nugget protoplasts were more resistant to paraquat in the micromolar range under our conditions. In Nugget, a non‐bleaching paraquat concentration (10 µM) inhibited CO2‐dependent O2 evolution ca 50% during the first 40 min, remaining at that rate (“coping behavior”) for up to 100 min. In contrast, Progress protoplasts treated with the same concentration of paraquat did not exhibit coping behavior. Antioxidant enzyme activities were unaltered throughout the time course of the experiment in treated protoplasts from Nugget and in chloroplasts isolated from them. Thus, the coping behavior of Nugget protoplasts cannot be attributed to changes in activities of the three antioxidant enzymes tested. Paraquat treatment did not affect antioxidant enzyme activities in Progress protoplasts nor in chloroplasts isolated from them. When higher doses of paraquat were used (12 h, 0.1 mM paraquat), protoplasts from both cultivars were rapidly bleached and total protein decreased to ca 30% of pre‐stress levels. Glutathione reductase (GR, EC 1.6.4.2) activity dropped in protoplasts from both cultivars under the severe stress conditions in concert with declines in protein levels. However, superoxide dismutase (SOD, EC 1.15.1.1) activity remained constant over the first 9 h of the time course, increasing to ca 150& of original levels by the final, 12‐h time point. The activity of the plastid Cu,Zn isoform, expressed as a percentage of total SOD activity, declined over the time course of the experiment while that of mitochondrial MnSOD appeared to increase. This change in activity of MnSOD correlated with cell decline, therefore, and was not correlated with protection. These data are in agreement with some earlier reports and are compatible with the hypothesis that SOD activity levels increase in response to reactive oxygen species levels, even under conditions leading to cell death.
Substrates regulate the phosphorylation status of nitrate reductase
Authors: Rouby Marina B. , González Claudio A. , Kenis Juana D.
Published: March 2003

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7‐day‐old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg2+. NR sensitivity to Mg2+ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg2+ inhibition of NR, which was strongly diminished in the presence of 10 mM NO3−+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg2+ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0‐40% saturation fraction (F1).

After incubation of F1 with ATP, the sensitivity of the enzyme to Mg2+ inhibition remained low, but it strongly increased after mixing F1 with a 45‐60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho‐NR, would be required to keep the phosphorylated enzyme inactive in a +Mg2+ medium. Addition of 10 mM NO3−+ 100 µM NADH together with desalted F2 did not prevent Mg2+ inhibition suggesting that NO3− did not interfere with IP binding to phospho‐NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg2+, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO3− at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho‐NR induced by the substrates.

The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase‐ or the kinase‐proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.
NO2‐induced nitrate reductase activity in needles of Norway spruce (Picea abies) under laboratory and field conditions
Authors: Von Ballmoos Peter , Ammann Markus , Egger Alfred , Suter Marianne , Brunold Christian
Published: March 2003

The induction of activity of the enzyme nitrate reductase (NR, EC 1.6.6.1, 1.6.6.2) in needles of Norway spruce (Picea abies[L.] Karst.) by nitrogen dioxide (NO2) was studied under laboratory and field conditions. In fumigation chambers an increase in nitrate reductase activity (NRA) was detected 4 h after the start of the NO2 treatment. During the first 2 days with 100 µg NO2 m−3, NRA reached a constant level and did not change during the following 4 days. At the same level of NO2, NRA was lower in needles from trees grown on NPK‐fertilized soil than on non‐fertilized soil. After the transfer of spruce trees from fertilized soil to NPK‐rich nutrient solution, NRA was transiently increased. This effect was assigned to root injuries causing nitrate transport to the shoot and subsequent induction of NRA. Neither trees on fertilized soil nor trees transferred to NPK‐poor nutrient solution had increased NRA unless NO2 was provided. The NO2 gradient in the vicinity of a highway was used to test the long‐term effect of elevated levels of NO2 on needle NRA of potted and field‐grown spruce trees. Compared with less polluted sites, permanently increased NRAs were detected when NO2 concentrations were above 20 µg m−3. Controls of field measurements some 10 years after the introduction of catalytic converters in cars showed no significant change neither in NO2 levels nor in the decreasing NRA of spruce needles with the distance from the highway.
Influence of phosphorus and formononetin on isozyme expression in the Zea mays‐Glomus intraradices symbiosis
Authors: Fries Leadir L. M. , Pacovsky Raymond S. , Safir Gene R.
Published: March 2003

Maize (Zea mays L. cv. Great Lakes 586) plants were either inoculated with the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith, or grown in the presence of the isoflavone formononetin or were provided with both G. intraradices and formononetin. All plants were grown in soil containing one of five levels of inorganic P (between 8 and 110 µg g−1 soil). By 3 weeks there were significant differences in a number of enzyme activities and in the pattern of isoenzymes in roots colonized by the VAM fungus or treated with formononetin. One NAD‐malate dehydrogenase (MDH) isozyme was expressed only in mycorrhizal roots, whether treated or not with formononetin. Despite differences in the soil P level, the expression of this isozyme was not observed in non‐mycorrhizal roots, indicating specific expression in the mycorrhizae. We suggest that MDH isozyme could serve as a specific, early indicator of the Zea‐Glomus symbiosis. Differences in the esterase (EST) isozyme pattern were not detectable between VAM and non‐VAM roots, suggesting that this enzyme system is not a good parameter for the evaluation of mycorrhizal colonization. As available P in the soil increased, total EST activity appeared to increase as well. Interestingly, total peroxidase (POX) activity increased along with P suggesting that as plant P nutrition improved, both cell wall ramification and the quantity of defense peroxidases increased as well. Total POX activity from mycorrhizal roots was inversely correlated with root colonization, indicating that there was suppression of POX activity by the host under low soil P. Most interestingly, formononetin further decreased POX activity regardless of the level of P or mycorrhizal status. This may suggest one mechanism by which formononetin enhances root VAM colonization. The presence of this isoflavone suppressed POX activity in mycorrhizal roots allowing a rapid penetration and spread of the fungus in the root cortex. The interplay between host root, soil P levels, secondary metabolites and endogenous host enzyme activities and a particular VAM fungus has a profound effect on the efficiency, duration and functioning of an endomycorrhizal symbiosis.
Recovery and characterization of amino acid analog‐resistant carrot and tobacco suspension‐cultured cells after cryostorage for over 10 years
Authors: Kamal Sami , Ethington Sam , Luo Ximing , Widholm Jack
Published: March 2003

Several Daucus carota (carrot) and Nicotiana tabacum (tobacco) cell lines that had been selected as resistant to Pro, Trp, Lys and Met analogs were thawed after over 10 years of cryostorage in liquid nitrogen. Some of these cell lines had been cloned. Viable cells were recovered in all cases, and growing lines were recovered from the carrot lines when placed either on feeder plates or directly into liquid medium. Some tobacco cultures were recovered only in liquid medium, but two Trp analog‐resistant cloned lines could not be recovered after several attempts. Generally the cryopreserved lines retained the original resistance and corresponding free amino acid levels while the same lines maintained as callus with monthly transfers for the same period often lost the resistance. Our study shows that carrot and some, but not all, tobacco cultured cell lines can be cryopreserved for over 10 years and still be recovered with their original characteristics.
Transient freezing regulates expression of extensin‐type genes in winter oilseed rape
Authors: Kozbial Piotr Z. , Jerzmanowski Andrzej , Shirsat Anil H. , Kacperska Alina
Published: March 2003

In order to identify the molecular mechanisms underlying the process of cold acclimation in plants, we examined the expression of extensin‐type genes in Brassica napas L. var. oleifera L. leaves. subjected to various low temperature treatments. Northern analysis showed that two transcripts of 1 260 nt and 1 450 nt, strongly hybridized to the extA probe and accumulated to high levels in leaves subjected to transient freezing treatments, giving rise to ca 20% membrane injury (2 h at −3°C for non‐acclimated or at −12°C for cold‐acclimated leaves). The accumulation of the frost‐induced extensin mRNAs (especially of the 1 450‐nt transcript) was markedly higher than that seen upon mechanical wounding. Frost‐dependent induction of extensin mRNAs was markedly elevated both in extent and duration in cold‐acclimated as compared to non‐acclimated leaves. Post‐incubation of cold‐acclimated and frost‐treated leaves on ice instead of at room temperature prolonged the time course of extensin mRNA accumulation and resulted in a marked increase of the level of the transcripts in the material studied. Factors possibly involved in the induction of extensin gene transcription are discussed, as well as the role of extensin cell wall proteins in plant responses to freezing.
Hydrogen peroxide production by roots and its stimulation by exogenous NADH
Authors: Frahry Gitta , Schopfer Peter
Published: March 2003

H2O2 production by roots of young seedlings was monitored using a non‐destructive in vivo assay at pH 5.0. A particularly high rate of H2O2 production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H2O2 production. In the soybean root H2O2 production can be stimulated 10‐fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase‐catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O2 to H2O2. Comparative measurements showed that the NADH‐induced H2O2 production of the roots resembles the H2O2‐forming activity of horseradish peroxidase with respect to NADH and O2 concentration requirements and sensitivity to inhibition by KCN, NaN3, superoxide dismutase and catalase. NADH‐induced H2O2 production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H2O2 production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH‐induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O2. It is concluded that the basal H2O2 production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O2−‐producing oxidase.
Molecular characterization of the spinach G‐box binding protein family
Authors: Bolle Cordelia , Lübberstedt Thomas , Herranen Mirkka , Herrmann Reinhold G. , Oelmüller Ralf
Published: March 2003

The promoters of the spinach ferredoxin‐NADP+‐oxidoreductase gene and one member of the gene family for the small subunit of ribulose‐1,5‐bisphosphate carboxylase contain ACGT sequences relevant for gene expression. Site‐directed mutagenesis revealed that these sequences operate quantitatively and are not involved in the light response. We have isolated a cDNA for a basic leucine zipper protein (bZIP) from spinach. After transcription and translation in cell‐free systems, the protein binds in vitro to double‐stranded oligonucleotides designed according to both sequences, although with different efficiencies. The genomic DNA segment for this bZIP contains 10 introns. The bZIP gene promoter harbors also an ACGT sequence; however, promoter/uidA gene fusions revealed that these nucleotides are not essential for expression. At least three other genes with high similarities are present in the spinach genome; however, they appear to be either pseudogenes, because they contain in‐frame stop codons in highly conserved epitopes, or must be posttranscriptionally modified in order to code for functional proteins. The high sequence similarities suggest that all four sequences derive from gene duplications.
Ribosomes in incompatible pollen tubes in the Solanaceae
Authors: Walles Björn , Han Shu Ping
Published: March 2003

Some members of the Solanaceae have a self‐incompatibility mechanism preventing self‐fertilization. Stylar ribonucleases (S‐RNases) are responsible for growth inhibition of self‐pollen tubes. A prevalent model postulates that the S‐RNases act as intracellular cytotoxins that degrade ribosomal RNA, and possibly also messenger RNA, in the incompatible pollen tubes. Since ribosomes and polysomes are easily noticed with the electron microscope, it should be possible to confirm disintegration of these structures. However, our inspection by electron microscopy revealed the presence of ribosomes and polysomes in pollen tubes formed after self‐pollination of the self‐sterile species Brugmansia (Datura) suaveolens and Nicotiana alata. There was no decrease over time in the number of bound ribosomes per unit of rough endoplasmic reticulum (RER) membrane. The results indicate that the inhibition of tube growth is not due to a general degradation of ribosomal and messenger RNA. Therefore, the substrate for S‐RNases presumably is very specific.
Multiple forms of ADP‐glucose pyrophosphorylase from tomato leaf
Authors: Chen Bing‐Yuan , Janes Harry W.
Published: March 2003

ADP‐glucose pyrophosphorylase (AGP, EC 2.7.7.27) was partially purified from tomato (Lycopersicon esculentum Mill. cv. Laura) leaf by a procedure previously used for purification of AGP from tomato pericarp. SDS‐PAGE and western blot analysis of the final preparation indicated that the leaf enzyme is composed of two subunits of 50 and 54 kDa. Two‐dimensional PAGE and western blot analysis of the same preparation, however, revealed at least six isoforms of the large subunit and two isoforms of the small subunit. The leaf AGP is very sensitive to regulation by 3‐phosphoglycerate and inorganic phosphate. Its properties are compared to those of AGP from tomato fruit.
Mechanisms of cobalt uptake in plants: 60CO uptake and distribution in Chara
Authors: Liu J. , Reid R. J. , Smith F. A.
Published: March 2003

The mechanism of cobalt uptake was investigated using cells of the giant alga Chara corallina in which it is possible to resolve separately uptake by the cell wall and actual influx across the cell membrane. The absorption of 60Co by Chara cells appeared to saturate within 2 h, but this was mainly due to rapid uptake into the cell wall which accounted for 87–92% of the total activity. Even after prolonged desorption most of the cell‐associated 60Co was found on the cell wall. The intracellular distribution of absorbed 60Co was investigated by fractionating the cell into cytoplasm and vacuole. It was shown that 60Co influx to the vacuole occurs simultaneously with influx to the cytoplasm. The transported species appears to be Co2+ rather than the less charged Co(OH)+ or Co(OH)2. 60Co influx is pH dependent (optimum pH 7–9), and is sensitive to some other divalent metals. Influx from solutions containing 1 µM60Co was inhibited by 5 µM Cd2+, Cu2+, and Zn2+, but Mn2+ and Ni2+ had no significant effect. The sensitivity of Co uptake to N‐ethyl maleimide (NEM) and cysteine suggests that the transport system involves direct binding of CO2+ to ‐SH groups.
‘Multimodal’ kinetics: Cyanobacterial nitrate reductase and other enzyme, transport and binding systems
Authors: Nissen Per , Martín‐Nieto José
Published: March 2003

Concentration‐dependence data for nitrate reductase (EC 1.7.99.4) from heterocystous, nitrogen‐fixing cyanobacteria (J. Martín‐Nieto, E. Flores and A. Herrero, 1992. Plant Physiol. 100: 157–163) have been interpreted most plausibly to reflect the operation of a single enzyme with two independent catalytic sites. However, data from a total of 30 experiments (published as well as unpublished) are, overall, much better (P < 0.0001) represented according to a ‘multimodal’ kinetic model rather than as due to two separate sites. This new term is introduced to refer to enzyme systems displaying multiple concentration‐dependent phases separated by sharp inflections. This phenomenon is taken to reflect the operation of a single catalytic site undergoing discontinuous conformational transitions and thus able to function in distinct kinetic ‘modes’. Moreover, plots of log Km versus log Vmax in these kinetic systems are perfectly linear, as also previously found for multiphasic plant uptake systems. The same multi‐mode kinetic behavior is exhibited by a wide variety of enzyme, uptake and ligand‐binding systems from plants, animals and microorganisms, including monomeric proteins purified to homogeneity. Multimodal kinetics thus constitute a widespread, albeit largely unrecognized, phenomenon in nature.
Antioxidative defence and photoprotection in pine needles under field conditions. A multivariate approach to evaluate patterns of physiological responses at natural sites
Authors: Tausz Michael , Jiménez Maria Soledad , Grille Dieter
Published: March 2003

Measurements of antioxidants and chloroplast pigments have been widely used as markers of stress and vitality of conifers in the field. However, due to the high variability of these data and the multiple environmental influences tress are exposed to, a quantification of physiological stress responses has only scarcely been possible. Physiological stress responses cannot be monitored by single stress markers, but are governed by many different interactions. The objective of this study was to evaluate patterns of biochemical stress markers in an objective and repeatable manner. For this purpose, a data set of 12 stress‐physiological variables (chloroplast pigments, epoxidation state of the xanthophyll cycle, α‐tocopherol, ascorbate and dehydroascorbate, GSH and GSSG) measured on field‐grown Pinus canariensis needles (n = 90) was subjected to explorative statistical techniques. Four principal components (PC), which explained 80% of the variance of the original data, were extracted by principal component analysis. According to stress‐physiological principles, complex responses were assigned to these PCs. Principal component 1 was positively affected by concentrations of a‐tocopherol and total ascorbate, and negatively by the proportion of epoxides in the xanthophyll cycle and by α‐carotene contents. Principal component 2 was composed of chlorophyll, lutein, neoxanthin, and β‐carotene contents, PC 3 contained information about GSH concentrations and the proportions of GSSG and dehydoascorbate; and PC 4 mainly comprised the pool size of the xanthophyll cycle. These components could be ascribed physiological principles such as antioxidative response in chloroplasts (PC 1), pigment content (PC 2), or antioxidant regeneration (PC 3). Via cluster analysis a classification of samples was made based on the patterns of their PC scores. The resulting clusters represented typical physiological response patterns: Cluster 1 was related to initial stages of oxidative damage, cluster 2 to antioxidative responses, whereas cluster 3 represented healthy trees. The spatial distribution of members of these clusters among field plots revealed that different response patterns could be observed at the same plot, a fact that might be ascribed to small scale differences and/or individually differing resistances, and something that is frequently overlooked in the field.