bluuebunny/crossref_metadata_2025 huggingface dataset:
Irradiation‐induced oral candidiasis in an experimental murine model
Authors: Farah C. S. , Hong S. , Wanasaengsakul S. , Elahi S. , Pang G. , Gotjamanos T. , Seymour G. J. , Clancy R. L. , Ashman R. B.
Published: March 2003

The aim of this experiment was to establish a mouse model of irradiation‐induced oral candidiasis and to explore the cellular populations and mechanisms by which the infection is cleared from the oral mucosa. BALB/c mice received irradiation to the head and neck equivalent to 800 Rad using a Cobalt 60 gamma source. Both irradiated and non‐irradiated mice were infected orally with 1×108Candida albicans yeasts. Compared with untreated controls, irradiated animals developed a more severe infection of longer duration, with hyphae penetrating the oral mucosa. Monoclonal antibody depletion of CD4+ but not CD8+ T cells from the systemic circulation prolonged the infection in irradiated mice, but not in controls. Supernatants of submandibular and superficial cervical lymph node cultures from irradiated animals demonstrated significantly higher titers of interleukin‐12, but similar levels of interferon‐γ compared with controls. Screening for cytokine production by an RNase protection assay detected only macrophage migration inhibition factor in irradiated and non‐irradiated oral tissues from day 8 onwards. The results of this study demonstrate a requirement for CD4+ T cells in the recovery from oral candidiasis induced by head and neck irradiation in mice, and are consistent with a role for Th1‐type cytokines in host resistance.
Testosterone stimulates proliferation and inhibits interleukin‐6 production of normal and hereditary gingival fibromatosis fibroblasts
Authors: Coletta R. D. , Reynolds M. A. , Martelli‐Junior H. , Graner E. , Almeida O. P. , Sauk J. J.
Published: March 2003

Hereditary gingival fibromatosis (HGF) is a rare oral condition characterized by a slow and progressive enlargement of the gingiva, involving both the maxilla and mandible. In vitro, HGF fibroblasts demonstrate a proliferative index significantly higher than fibroblasts from normal gingiva (NG). The objective of this study was to determine the effect of dihydrotestosterone on the proliferation of gingival fibroblasts derived from patients with HGF (n = 4) and from four healthy individuals. Additionally, we analyzed the effect of dihydrotestosterone on interleukin‐6 (IL‐6) production and determined the expression levels of androgen receptors in NG and HGF fibroblasts. Gingival fibroblasts from NG and HGF were incubated with increasing concentrations of dihydrotestosterone with or without androgen blockers, and cultured for 24 h, and the proliferation index was determined by automated cell counter. IL‐6 production, in this system, was quantified using a ‘capture’ enzyme‐linked immunosorbent assay (ELISA). Semi‐quantitative reverse transcriptase–polymerase chain reaction (RT‐PCR) was performed to measure the mRNA expression of androgen receptors. The results indicated that dihydrotestosterone simultaneously downregulates the production of IL‐6 and upregulates the cell proliferation. Finasteride and cyprosterone acetate, two anti‐androgens, partially reversed these effects. Androgen receptor mRNA expression was identified in both NG and HGF fibroblasts; however, the levels in NG were higher than those observed in HGF. These results show that testosterone coordinates the proliferation and production of IL‐6 of normal and HGF fibroblasts.
An essential amino acid residue for catalytic activity of the dextranase of Streptococcus mutans
Authors: Igarashi T. , Morisaki H. , Yamamoto A. , Goto N.
Published: March 2003

Dextranase (Dex) is an enzyme that hydrolyzes glucan, a polymer of glucose synthesized from sucrose by glucosyltransferases (GTFs). By comparing amino acid sequences of Dexs and GTFs, we found that the Dex enzymes of Streptococcus mutans, Streptococcus sobrinus, Streptococcus downei and Streptococcus salivarius had similar amino acid sequences to those of the catalytic sites of GTFs of mutans streptococci. We therefore examined the amino acid essential in Dex catalysis by molecular genetic approaches in this study. Site‐directed mutagenesis was used to convert the Asp‐385 of the Dex molecule of S. mutans Ingbritt to Glu, Asn, Thr or Val. Replacement of Asp‐385 with any of the amino acids resulted in complete disappearance of Dex activity. However, replacement of other Asp residues did not affect the enzyme activity. The inactive enzymes still retained dextran‐binding ability. These results suggest that Asp‐385 of the Dex of S. mutans Ingbritt was essential for enzyme activity and the catalytic and substrate‐binding sites were located at different sites within the Dex molecule.
Streptococcus mutans binding to solid phase dextran mediated by the glucan‐binding protein C
Authors: Sato Y. , Senpuku H. , Okamoto K. , Hanada N. , Kizaki H.
Published: March 2003

Streptococcus mutans GbpC is a wall‐anchored surface protein which is involved in dextran‐dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N‐acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti‐GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(α‐1,6 glucan)‐dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.
The Environment and Childhood Asthma (ECA) Study in Oslo: ECA‐1 and ECA‐2
Authors: Lødrup Carlsen Karin C.
Published: March 2003

An observed increase in asthma admissions in Oslo during the 1980s prompted a prospective birth cohort study to ask the following question: was air pollution (outdoor and indoor) (in a broad sense) associated with asthma development in young children? During 12 months from 1 January 1992, 3,754 children (birth weight ≥2000 g) in Oslo were enrolled at birth into the Environment and Childhood Asthma (ECA) study and followed to 2 years of age (ECA‐part I). Cord blood, a detailed questionnaire (family and pregnancy history of disease, environmental exposures, socio‐economic status) completed by the mother and lung function measurements (n = 803) were collated at birth. Detailed questionnaires completed every 6 months for 2 years included the child's disease history, feeding habits and environmental exposures. A nested case‐control study comprised 306 children with confirmed minimum two episodes of bronchial obstruction (rBO) and 306 controls (without lower respiratory tract disease) with clinical investigations (including tidal breathing lung function, beta‐2 responsiveness and allergy assessment) and environmental exposure assessments (indoor and outdoor). Home dampness and low ventilation, as well as maternal smoking in pregnancy, but not outdoor air pollution increased the risk of rBO. Lung function at birth was decreased among newborns whose mother smoked during pregnancy. To understand better the early risk factors for asthma and allergy development, a follow‐up study started (in 2001; ECA‐part II) of all cases and controls, and those with lung function measured at birth (total 1,230 invited) (9–10 years of age). This involved clinical investigation, allergy assessments, lung function, airway hyper responsiveness measures, exhaled nitric oxide and immunological as well as allergen exposure investigations.
Development of immunoglobulin G and immunoglobulin E antibodies to cow’s milk proteins and ovalbumin after a temporary neonatal exposure to hydrolyzed and whole cow’s milk proteins
Authors: Juvonen Pekka , Månsson Majvi , Kjellman N. ‐I. Max , Björkstén Bengt , Jakobsson Iréne
Published: March 2003

The ingestion of food antigens usually results in the induction of oral tolerance, but the clinical and immunologic consequences of brief exposure to cow’s milk proteins during the neonatal period are not well‐documented. The aim of this work was to study immunoglobulin (Ig)E and IgG responses to cow’s milk proteins and ovalbumin after exposure during the first three days of life in infants who were otherwise exclusively breast‐fed. A group of 129 infants was randomly assigned at birth to one of three feeding regimens: human milk (HM), cow’s milk formula (CMF), or a casein hydrolysate formula (CHF), during the first three days of life. They were then all exclusively breast‐fed for a varying period of time and followed for two years. Serum IgG and IgE antibodies to cow’s milk proteins and ovalbumin (OVA) were analyzed in blood samples obtained at birth, at 4 days and at 2, 4, 8, 12 and 24 months of age. The levels of IgG antibodies to β‐lactoglobulin (IgG‐BLG) and bovine serum albumin (IgG‐BSA) were higher in the CMF and the HM groups than in the CHF group for up to two years. This was particularly obvious for IgG‐BLG in infants who started weaning before two months. The levels of IgG antibodies to casein (IgG‐CAS) were higher in the CMF goup, as compared with the CHF group at 8 and 12 months. The levels of IgG antibodies to OVA were similar in all three feeding groups. The levels of IgE antibodies to CAS or OVA were similar in the three feeding groups. Exposure to cow’s milk during the first three days of life stimulated IgG antibody production to cow’s milk proteins and this was still obvious at 2 years of age, while feeding with a casein hydrolysate during the first three days of life was associated with low levels of IgG antibodies to cow’s milk proteins.
Primary sensitization to inhalant allergens
Authors: Macaubas Claudia , Prescott Susan L. , Venaille Thierry J. , Holt Barbara J. , Smallacombe Troy B. , Sly Peter D. , Holt Patrick G.
Published: March 2003

The neonatal T‐cell system is capable of responding to allergens at birth, indicating the occurrence of prenatal sensitization, and the cytokine profile of these responses is skewed towards the Th‐2 type. This response is further modified by postnatal exposure to different types of allergens. In relation to inhalant allergen (employed by HDM) the low level fetal Th‐2 responses in non‐atopics appear to be down‐regulated rapidly after birth, parallel to an increase in allergen‐specific IFN‐γ production. In contrast, atopics appear to consolidate their initial Th‐2 responses, and around the age of 6 exhibit a cytokine response profile similar to the adult pattern. A pre‐existing deficiency in IFN‐γ production may be one of the key factors determining the postnatal persistence of Th‐2 responses in atopics.
Altered eosinophil levels as a result of viral infection in asthma exacerbation in childhood
Authors: Zhao Jing , Takamura Mayumi , Yamaoka Akiko , Odajima Yasuhei , Iikura Yoji
Published: March 2003

Respiratory viral infection is known to be a significant cause of asthma exacerbation. Eosinophils have been considered to play an important role in the pathogenesis of virus‐induced asthma exacerbations. To determine how often asthma exacerbation is caused by virus infections and to examine the relationship between eosinophilia and asthma episode, we investigated 64 children who experienced asthma attacks between October 1999 and March 2000. We used rapid enzyme immunoassays to detect antigens of respiratory syncytial virus (RSV), influenza A virus, and adenovirus in nasopharyngeal secretions (NPS) of these children, and enumerated eosinophils in the blood and NPS. We detected RSV in 27% and influenza A virus in 17% of the patients. No adenovirus infection or RSV/influenza A co‐infection was detected. RSV‐infected children were younger (3.85 ± 0.83 years old) than influenza A virus‐infected patients (5.23 ± 1.34 years old). Eighty‐two per cent of patients in the RSV group and 36% of patients in the influenza A virus group had moderate‐to‐severe asthma episodes (p < 0.05). In RSV‐infected children, the eosinophil counts in NPS were higher in the ‘severe’ group, and younger patients had a greater number of eosinophils in their NPS than older patients (p < 0.05). These trends were not found in influenza A virus patients. In conclusion, our results indicate that, compared with influenza A virus‐induced asthma attacks, RSV infection had a higher probability of being associated with asthma exacerbation in infants and younger children and induced attacks of greater severity. The increase in the number of eosinophils in the NPS of RSV‐infected children may be responsible, in part, for these differences.
A study of the association between exercise‐induced wheeze and exercise versus methacholine‐induced bronchoconstriction in adolescents
Authors: Henriksen Anne Hildur , Tveit Kjerst Hafstad , Holmen Turid Lingaas , Sue‐Chu Malcolm , Bjermer Leif
Published: March 2003

Among asthmatics, exercise‐induced wheeze (EIW) is a frequent symptom, and 40–77% of asthmatics demonstrate exercise‐induced bronchoconstriction (EIB). In the North‐Trøndelag population‐based survey of 8,571 adolescents (YOUNG‐HUNT), 26% reported wheeze during the previous 12 months (current wheeze). Of those subjects, 50% reported EIW. The aim of the present study was to investigate the association between EIW and EIB in randomly selected adolescents with EIW as the only or predominant asthma‐like symptom, and to relate our findings to results from methacholine bronchoprovocation tests (MT) and measurements of exhaled nitric oxide (ENO). Sixty‐three subjects with current wheeze induced by exercise, but not by allergen exposure, were investigated using a treadmill exercise test (ET) and measurements of ENO. Fifty‐eight subjects completed a MT on a separate study day. EIB was defined as a fall of ≥ 10% in the forced expiratory volume in 1 second (FEV1) after exercise (ΔFEV1%ex). Twenty‐one subjects (33%) had EIB and 33 (57%) had a positive MT. The degree of reported dyspnea during the ET was not correlated to the ΔFEV1%ex. The correlation between EIB and methacholine‐induced bronchoconstriction (MIB) was poor, and the ΔFEV1%ex was more pronounced in smokers than in non‐smokers. Moreover, ENO was not increased in subjects with positive vs. negative ET. Hence, EIW, when reported as the only or predominant asthma‐like symptom, was linked to EIB in only one‐third of the patients. We conclude that EIW is a poor predictor of EIB in epidemiological studies. The poor correlation between EIB and MIB indicates that these two tests measure different mechanisms of bronchial hyper‐responsiveness.
Inclusion of entire pancreas in the composite liver and intestinal graft in pediatric intestinal transplantation
Authors: Kato Tomoaki , Romero Rene , Verzaro Roberto , Misiakos Evangelos , Khan Farrukh A. , Pinna Antonio D. , Nery Jose R. , Ruiz Phillip , Tzakis Andreas G.
Published: March 2003

Abstract: An entire pancreatico–duodenal complex was included in the liver and intestinal graft in eight children who received small‐size grafts. This method showed several advantages compared to the traditional approach. They included reducing time for graft preparation by eliminating donor pancreas resection, no necessity of biliary reconstruction and leaving natural tissue support for blood vessels. The method was not associated with an increased risk of complications such as pancreatitis or rejection. It should be considered in pediatric liver and intestinal transplant recipients who require small‐size grafts.
Prospects for xenotransplantation
Authors: Platt Jeffrey L.
Published: March 2003

Abstract:
The major limitation on the application of transplantation for the treatment of human disease is a severe shortage of human donor organs and tissues. One approach to overcoming this problem is xenotransplantation, that is the transplantation of animal organs into humans. The major hurdle to xenotransplantation is the immune response of the recipient against the graft. Recent years have brought new information concerning this hurdle and insights of strategies for overcoming it. Other hurdles include the physiological function of the graft in the foreign environment including the possibility of molecular incompatibilities between the donor and recipient and the possibility of transferring infectious diseases from the graft to the recipient. The current perspective on these issues will be presented in the review that follows.
Leukocyte‐reduction to prevent transfusion‐transmitted cytomegalovirus infections
Authors: Strauss Ronald G.
Published: March 2003

Abstract: Certain infectious organisms, including cytomegalovirus, are associated ‘exclusively’ with blood leukocytes (WBC), and their transmission by transfusion is strikingly diminished by marked WBC‐reduction of cellular blood components. Based on several reports of WBC‐reduction, it is clear that the risk of CMV is nearly eliminated by consistently removing WBC to a level < 1–5 × 106 WBCs/unit (≤ 1 × 106 preferred in Europe; ≤ 5 × 106 in the United States). Alternatively, the rate of CMV infections is reduced by transfusing blood components collected from donors negative for CMV antibody. However, neither technique is perfect, with a failure rate of 1–4%. Although WBC‐reduction is favored by many experts, practitioners must choose the method that they believe to be most efficacious – being mindful that data do not exist to establish additive protection by combining WBC‐reduction and transfusion of blood components collected from antibody negative donors.
Continuing improvement in cadaver donor graft survival in North American children: The 1998 Annual Report of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) *
Authors: Elshihabi Ihsan , Chavers Blanche , Donaldson Lynn , Emmett Lea , Tejani Amir
Published: March 2003

Abstract: This report of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) covers the years 1987–1997, and analyses data on 3133 cadaver donor (CD) transplants performed in 2736 patients. There has been a steady decline in the number of CD transplants in children since 1996. Kidneys recovered from donors under 10 years of age accounted for 35% of all transplants in 1987, whereas by 1996 they comprised less than 20%. Caucasian children received 54% of CD transplants, whereas African–American children received 21%. Children under 6 years of age received 17% of CD transplants. Approximately half (46%) of the patients were induced with a T‐cell antibody, and at 7 years post‐transplant triple therapy is used in 70% of those with a functioning graft. Cyclosporin A is the primary immunosuppressant, with 92% of the patients being maintained on it at 5 years post‐transplant. Among patients receiving a transplant in 1997, 11% were initiated with another calcineurin inhibitor, tacrolimus. At 15 days post‐transplant 20% of the patients have had a rejection episode and by day 45, 46% have had an acute rejection. The probability of developing a rejection within the first year was reduced from 71% in 1987–1988 to 47% in 1995–1996.For infants (0–1 years) the first episode of rejection ends in graft failure or patient death in 18% of patients, compared with 8% for older children. In more recent years irreversible graft failure has been reduced significantly. Estimated graft survival probability for primary transplants at 1 year is 81%, at 3 years 72%, and at 5 years 64%. Recipient age < 2 years (relative risk (RR) = 1.95), donor age < 10 years (RR = 1.26), absence of induction therapy (RR = 1.29), African–American race (RR = 1.34), and HLA‐AB/DR mismatches (RR = 1.25) are relative risk factors for graft failure.One‐year graft survival for primary grafts has shown a consistent improvement from 72% in the 1987–1988 cohort to 88% in the 1995–1996 cohort. Patient survival for primary grafts at 1 year is 96.3%, at 3 years 94.1%, and at 5 years 91.4%. For infants however, the three‐year patient survival is 77%. Mean height deficit at baseline (n = 2648) is − 2.03 and at 5 years (n = 517) it is − 2.33. Steady improvement continues in short‐term graft survival of CD transplants, however, at 7 years post‐transplant CD graft survival is 16% less than that of living donor (LD) transplants. Measures to prevent acute rejection and thereby delay the onset of chronic rejection are needed. Additional areas of concern include the high mortality of infants and the continuing growth retardation for the majority of children over 6 years of age at the time of transplantation.
Serum C‐reactive protein in pediatric kidney and liver transplant patients
Authors: Their Maria , Rönnholm Kai , Sairanen Heikki , Holmberg Christer , Jalanko Hannu
Published: March 2003

Abstract: The clinical manifestations of different complications after organ transplantation (Tx) are often vague, and simple laboratory tests for early diagnosis would be valuable. In this work we retrospectively analyzed our data on the daily measurements of serum C‐reactive protein (CRP) in 132 children after 63 liver and 83 kidney Txs. A total of 3,886 CRP measurements were performed and 353 episodes of elevated CRP were recorded. One‐third of these were regarded as a response to surgery. The CRP level normalized within 5–10 days post‐operatively in patients who had a favorable outcome, but in patients with a poor outcome CRP tended to remain elevated. Half of the CRP elevations were associated with complications such as acute rejection, infection or thrombotic events. An elevated serum CRP level was found in 68% of the rejection episodes, in 88% of the bacterial infections, and in 73% of the viral infections. The most significant elevations were associated with bacterial infection. In nine of 11 vascular complications, an elevation of CRP was also recorded. Serum CRP responded to rejection therapy in 86% of the episodes. The initial CRP level did not predict steroid‐resistant rejection. CRP seemed to be a more sensitive marker than fever or white blood cell (WBC) count in all complications. We conclude that the daily measurement of serum CRP is a simple and fairly sensitive, but non‐specific, method for detecting rejection and infectious complications after pediatric liver and kidney Tx.
Glomerular filtration rate as a putative ‘surrogate end‐point’ for renal transplant clinical trials in children
Authors: Filler Guido , Browne Richard , Seikaly Mouin G.
Published: March 2003

Abstract: Only with prospective randomized controlled trials is it possible to evaluate the several immunosuppressive regimens available to renal allograft recipients. Commonly used surrogate markers of clinical outcome, such as patient and graft survival, are constantly improving. Current immunosuppressive protocols have improved 1‐yr graft survival to over 90%. The small differences in graft survival among the various immunosuppressive regimes require large patient cohorts in order to establish statistical significance. Such studies are often difficult to conduct in a timely manner, particularly in children. This necessitates the search for better surrogate markers sensitive enough to detect differences in smaller cohorts and in a shorter period of time. While the degree of fibrosis in transplant biopsies might well predict long‐term graft survival, protocol biopsies are expensive, invasive, and unpopular among clinicians. In native kidneys, glomerular filtration rate (GFR) closely correlates with disease progression and interstitial fibrosis and appears to be well positioned as a less invasive surrogate marker for long‐term outcome. Nonetheless, the ideal marker for GFR remains obscure. Serum creatinine has several major drawbacks, making it a poor predictor of GFR. This review discusses the several methods used to estimate or measure GFR with emphasis on 125I‐iothalamate clearance and serum cystatin C (cys‐C). Of all the serum markers, cys‐C is the most reliable and the most promising. However, cys‐C and other endogenous markers cannot replace the diagnostic sensitivity and reliability of radiolabeled markers of GFR such as 125I‐iothalamate in renal transplant clinical trials. Unfortunately, clearance of most radiolabeled markers of GFR including 125I‐iothalamate remain costly and time consuming.
Ethylene may be involved in abortion of the maize caryopsis
Authors: Cheng Chi‐Yuang , Lur Huu‐Sheng
Published: March 2003

The involvement of ethylene and 1‐aminocyclopropane‐1‐carboxylic acid (ACC) in shade‐induced kernel abortion at an early kernel growth stage was investigated. Changes in levels of glucose, sucrose, starch, ACC and ethylene were measured during the first 6 days after pollination (DAP) at sampling intervals of 12 or 24 h. In response to shading treatment, dry weight accumulation of apical kernels ceased within 96 h after pollination (HAP). Glucose levels in shaded kernels 12 HAP were significantly higher than in control kernels, while sucrose and starch levels were similar until 48 HAP. In the shade treatment ACC and ethylene levels increased rapidly around 24 HAP in both apical and basal kernels. When ACC was applied to ears at 32 HAP and 5 DAP, dry weight accumulation was significantly reduced only in apical kernels of 32 HAP. Significant reduction in endosperm nuclei number was also found in both shaded and ACC‐treated kernels. The results of the present study suggest that ethylene may be involved in the processes leading to kernel abortion in maize. Kernels at the very early developmental stages seem to be most sensitive to ethylene.
Species‐ and age‐dependent sensitivity to ozone in young plants of pea, wheat and spinach: Effects on acyl lipid and pigment content and metabolism
Authors: Carlsson Anders S. , Wallin Göran , Stina Sandelius Anna
Published: March 2003

Acyl lipids and pigments were analyzed in young plants of garden pea, spring wheat and spinach exposed to < 5 or 65 nl l−1 ozone 12 h per day for 6 days. In one set of experiments, the plants were exposed to 14CO2 for 2 h 3 days prior to ozone exposure. The plants responded differently to the moderately enhanced level of ozone used Spinach was not at all sensitive while in both pea and wheat, leaves of different ages differed in ozone sensitivity. In pea, ozone sensitivity increased with leaf age. In the second and third oldest leaves, the amounts of galactolipids per leaf area and the proportions of 18:3 of the total lipid extract and of phosphatidylglycerol decreased. In the second oldest leaf, ozone also caused a decreased proportion of 18:3 of monogalactosyldiacylglycerol. In the fourth oldest leaf, lipid composition and galactolipid unsaturation was unaffected, but ozone caused decreased leaf expansion resulting in increased acyl lipid content per leaf area. In both the first and second leaves of wheat, ozone fumigation caused a marked decrease in the content of monogalactosyldiacylglycerol and in the first leaf, the contents of phosphatidylcholine and phosphatidylethanolamine increased. The proportion of 18:3 in phosphatidylcholine was larger in ozone‐fumigated than in control plants, while the reverse applied for phosphatidylglycerol. In the oldest sampled leaves of pea and wheat, ozone caused an increase in the radioactivity associated with β‐carotene, indicating increased turnover. Thus, while spinach was unaffected, in both pea and wheat ozone caused a decrease in the proportion of chloroplast membrane lipids to non‐chloroplast membrane lipids in older leaves while younger leaves were less sensitive.
Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants
Authors: Wang Aiming , Fan Hanli , Singsit Chong , Ozias‐Akins Peggy
Published: March 2003

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut (Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R1 seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.
Regulation of proline biosynthesis in kiwifruit buds with and without hydrogen cyanamide treatment
Authors: Walton Eric F. , Podivinsky Ellen , Wu Rong‐Mei , Reynolds Paul H. S. , Young Lester W.
Published: March 2003

Proline accumulates in kiwifruit (Actinidia deliciosa[A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) buds in spring during budbreak and flower differentiation. However, proline accumulation occurred earlier, and to a level approximately 6.5 times greater, in buds treated with the dormancy breaking chemical hydrogen cyanamide (HC). Peak proline accumulation coincided with peak pyrroline‐5‐carboxylate reductase (P5CR, EC 1.5.1.2) activities in both HC‐treated and control (untreated) vines. Peak levels of P5CR activity were the same in treated and control vines, suggesting that the activity of P5CR does not regulate proline biosynthesis. Ornithine‐δ‐aminotransferase (OAT, EC 2.6.1.13) activity declined over the sampling period and, consequently, was not correlated with proline accumulation in buds from either treated or control vines. This suggests that ornithine does not make a significant contribution to the proline pool in this system. Pyrroline‐5‐carboxylate synthetase (P5CS, EC number not assigned) and P5CR have been cloned from a kiwifruit bud cDNA library. Northern hybridisation analyses indicate that both P5CS and P5CR are transcriptionally regulated. The P5CS northern hybridisation data, together with the P5CR and OAT enzyme data, suggest that glutamate provides the carbon skeletons for proline synthesis and that P5CS transcription has a significant role in the regulation of proline biosynthesis in kiwifruit buds. This role for P5CS is consistent with those reported in other plants and demonstrates a conservation of the pathway, and its overall regulation in quite diverse plants. The only difference in the regulation of proline biosynthesis between kiwifruit buds from control and HC‐treated vines would appear to be temporal.
A study of the relation between CP29 phosphorylation, zeaxanthin content and fluorescence quenching parameters in Zea mays leaves
Authors: Buffoni Mauro , Testi Maria G. , Pesaresi Paolo , Garlaschi Flavio M. , Jennings Robert C.
Published: March 2003

The hypothesis that phosphorylation of the minor photosystem II antenna complex CP29 (CP34 formation) in Zea mays (cv. Dekalb DK300), under conditions of illumination and low temperature stress, may constitute a protective mechanism against photoinhibition, has been investigated. It is demonstrated that illumination at low temperature induces a marked increase in reversible non‐photochemical quenching yield of chlorophyll fluorescence, together with CP34 formation. These two parameters, however, are not related as CP34 dephosphorylates to CP29 in the dark, with a half‐time of about 10 min, while the enhanced non‐photochemical quenching yield is stable for many hours. The enhanced non‐photochemical quenching yield seems to correlate with zeaxanthin formation. The influence of CP34 formation on photoinhibition was also directly investigated. No measurable effect on this parameter could be observed after treatment with high light. It is concluded that CP34 is probably not directly involved in photoprotective processes.
Evidence for the activation of a MAP kinase upon phosphate‐induced cell cycle re‐entry in tobacco cells
Authors: Wilson Cathal , Pfosser Martin , Jonak Claudia , Hirt Heribert , Heberle‐Bors Erwin , Vicente Oscar
Published: March 2003

Mitogen‐activated‐protein (MAP) kinases are components of signal transduction pathways which respond to a variety of stimuli in different organisms. In quiescent mammalian cells, the reactivation of cell division induced by different mitogenic signals is mediated by the rapid phosphorylation and activation of MAP kinases. We have investigated whether a similar situation occurs in plants, arresting tobacco (Nicotiana tabacum L.) cells in the G1 phase of the cell cycle by phosphate starvation, and then inducing them to re‐enter the cell cycle by refeeding with phosphate. The transient activation of a kinase activity with the characteristics of a MAP kinase was observed during the first hour after refeeding, when the cells were still in G1. Using myelin basic protein (MBP) as substrate, an increase in this phosphorylating activity, with a molecular mass of approximately 45 kDa, was detected in cell extracts between 35 and 55 min after induction, in in‐gel phosphorylation assays and after immunoprecipitation with anti‐MAP kinase antibodies. The specificity of the antibodies against recombinant tobacco MAP kinases suggested that the MAP kinase p45ntf4 was responsible for the observed activity. These data provide experimental evidence for the activation in vivo of a plant MAP kinase, possibly mediating the reactivation of cell division in G1‐arrested cells.
Relationship between tissue sugar content, phloem import and lateral root initiation in wheat
Authors: Bingham I. J. , Blackwood J. M. , Stevenson E. A.
Published: March 2003

Split‐root experiments were conducted to test the hypothesis that adjustments in lateral root initiation, as might occur in response to localized soil conditions, are determined by the sugar content of the root and do not depend on changes in the import of phloem‐translocated phytohormones. Wheat (Triticum aesticum L. cv. Alexandria) seedlings were grown in hydroponics with their seminal roots divided between two compartments within the culture vessel. Two seminal roots of treated plants were supplied with standard nutrient solution supplemented with 50 mM glucose, whilst the remaining three roots received nutrient solution without glucose. Control plants had their roots divided in the same ratio, but both ‘halves’ received nutrient solution without glucose. Feeding glucose to one ‘half’ of the root system increased the frequency (number per unit length) of lateral root primordia in the fed axes. The increase was first observed 15 h after the start of treatment and was located within the apical 30 mm of root. At this time there was no significant treatment effect on the frequency of primordia in non‐fed axes. The enhanced initiation of lateral roots in glucose‐fed root tips was associated with an increase in their concentration of glucose and sucrose plus low molecular mass fructans. In contrast, there was a reduction in partitioning of 14C‐photosynthate to these root tips compared to the non‐fed roots of treated plants and controls. The results indicate that lateral root initiation can be stimulated by sugars in the absence of an increase in phloem translocation. It is proposed that proliferation of lateral roots in response to localized soil conditions, such as nutrient patches, may be signalled by an increase in sugar content of the tissue, rather than an altered flux of phytohormones or other material co‐transported with sucrose in the phloem.
Dynamics of nitrogen remobilization in defoliated Phleum pratense and Festuca pratensis under short and long photoperiods
Authors: Bakken Anne Kjersti , Macduff James H. , Collison Mike
Published: March 2003

The impact of photoperiod on the rate and magnitude of N remobilization relative to uptake of inorganic N during the recovery of shoot growth after a severe defoliation was compared over 18 days in two temperate grass species, timothy (Phleum pratense L. cv. Bodin) and meadow fescue (Festuca pratensis Huds. cv. Salten). Plants were grown in flowing solution culture with N supplied as 20 mM NH4NO3 and pre‐treated by extending the 11 h photosynthetically significant light period either by 1 h (short‐day or SD plants) or 7 h (long‐day or LD plants) of very low light intensity, during the 10 days prior to defoliation. Following a single severe defoliation, 15N‐labelled NH4+ or NH4++ NO3− was supplied over a 20‐day recovery period under the same SD and LD conditions. Changes in the relative contributions of remobilized N and newly acquired mineral N to shoot regrowth were assessed by sequential harvests. Both absolute and relative rates of N remobilization from root and stubble fractions were higher in LD than SD plants of both species, with the enhancement more acute but of shorter duration in timothy than fescue. Remobilized N was the predominant source of N for shoot regrowth in all treatments between days 0 and 8 after cutting; on average more so for fescue than timothy, because the presence of NO3− reduced the proportional contribution of remobilized N to the regrowth of timothy but not of fescue. Net uptake of mineral N began to recover between days 4 and 6 after cutting, with NO3− uptake restarting 1 or 2 days earlier than NH4+ uptake, even when NH4+ was the only form of N supply. LD timothy plants supplied solely with NH4+ were slowest to resume uptake of mineral N. Supplying NO3− in addition to NH4+ after defoliation promoted shoot regrowth rate but not remobilization of N. Rates of regrowth (shoot dry weight production per plant) were not correlated with rates of N remobilization from stubble either over the short‐term (days 0–8) or longer term (days 0–18), interpreted as evidence against a causal dependence of regrowth rate on N remobilization under these conditions. The results are discussed in relation to hypotheses for source/sink‐driven rates of N remobilization and their interactions with mineral N uptake following defoliation.
Proline accumulation inside symbiosomes of faba bean nodules under salt stress
Authors: Trinchant Jean‐Charles , Yang Yu‐Suo , Rigaud Jean
Published: March 2003

A two‐week salt treatment (NaCl, 100 mM) induced a 50% inhibition of acetylene reduction activity (ARA) of faba bean (Vicia faba L. var. minor cv. Soravi) nodules, associated with a large increase in the nodule pool of amino acids. The concentration of proline in the different nodule compartments was determined after calculating their respective volumes from their areas on electron micrographs. The proline concentration exhibited a large increase, especially in the cytosol where its amount was 8‐fold enhanced under salt stress, whereas the low proline content of bacteroids was less affected. Increase of proline concentration in faba bean nodules subjected to salt stress was correlated with an enhancement of the cytosolic Δ1‐pyrroline‐5‐carboxylate synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS) activity. Experiments with purified symbiosome preparations showed that the greatest proline content occurred in the peribacteroid space (PBS), where proline was the most abundant amino acid, with a concentration reaching 15.3 mM under salt stress. Proline accumulation in the PBS resulted both from a diffusive transport from the host cell to the symbiosomes through the peribacteroid membrane (PBM) and from the very low rate of uptake by faba bean bacteroids. This accumulation could be partly responsible for the 1.7‐fold enlargement of the symbiosome volume observed in salt‐stressed nodules. In incubations of bacteroids, isolated from salt‐stressed or unstressed plants and supplied with O2 by purified oxyleghemoglobin, addition of proline stimulated neither O2 consumption nor ARA. These results were consistent with proline playing a role as osmoticum, rather than energy source for bacteroid N2 fixation in amide‐exporting legumes such as faba bean.
The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt‐tolerant relative Lycopersicon pennellii
Authors: Shalata Abed , Tal Moshe
Published: March 2003

The possible involvement of the antioxidative system in the tolerance to salt stress was studied in the cultivated tomato Lycopersicon esculentum Mill. cv. M82 (M82) and its wild salt‐tolerant relative L. pennellii (Corn) D'Arcy accession Atico (Lpa). All analyses, except that of monodehydroascorbate reductase (MDHAR), were performed of the youngest fully‐expanded leaf of control and salt (100 mM NaCl) stressed plants, 4, 7, 10, 14, 18 and 22 days after completing the stress treatment. In Lpa, constitutive level of lipid peroxidation and activities of catalase (CAT) and glutathione reductase (GR) were lower while the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were inherently higher than in M82. Relative to M82, lipid peroxidation was much lower and the activities of SOD, CAT and APX were higher in Lpa at 100 mM NaCl. The activity of DHAR decreased more in Lpa than in M82 under salt stress, and the activity of MDHAR, which was lower in Lpa than in M82 under control conditions, increased much more and to a higher level in salt‐treated Lpa plants. GR activity decreased similarly in the two species under salt stress. The results of these analyses suggest that the wild salt‐tolerant Lpa plants are better protected against active oxygen species (AOS), inherently and under salt stress, than the relatively sensitive plants of the cultivated species.
Protein kinase activities in ripening mango (Mangifera indica) fruit tissue. II. Purification and characterization of a casein kinase I
Authors: Frylinck Lorinda , Dubery Ian A.
Published: March 2003

A protein kinase (PK‐II), phosphorylating casein, was purified from ripening mango, Mangifera indica L., fruit tissue. The purification procedure consisted of ammonium sulphate fractionation and sequential anion exchange‐, dye‐ligand, and gel filtration chromatography. The enzyme was purified over 500‐fold to near homogeneity with a recovery of 4%. The purified enzyme had a specific activity of ca 1 µmol mg−1 protein min−1 with ATP as phosphoryl donor. SDS‐PAGE results indicated a monomeric enzyme with molecular mass of 35 kDa. The protein kinase phosphorylated the acidic substrates casein and phosvitin, but had a very low activity with histones and protamine sulphate. The optimum pH and temperature for catalysis were determined to be 9.6 and 35°C, respectively. Mn2+ could not substitute for the Mg2+ needed for activity and Ca2+ had a slight stimulatory effect. Phospholipids, cAMP, calmodulin and the calmodulin inhibitor, calmidazolium, did not have any significant effect on activity, but the enzyme was inhibited by heparin and the specific inhibitor, CKI‐7, (N‐[2‐aminoethyl]‐5‐chloroisoquinoline‐8‐sulphonamide). Autoradiographic studies revealed the ability of the protein kinase to autophosphorylate as well as the presence of endogenous protein substrates in the crude extract. Initial velocity studies with casein as substrate and product inhibition studies with ADP indicated a Km (ATP) and Km (casein) of 14 µ M and 0.18 mg ml−1, respectively, with a Ki (ADP) of 3.2 µM. The enzyme can be classified as a casein kinase I type of protein kinase (EC 2.7.10).
Developmental timing of transgene expression is dosage dependent
Authors: Bucherna Nándor , Thyge Okkels Finn , Palmgren Gorm
Published: March 2003

Expression of a β‐glucuronidase (GUS) gene (uidA) driven by the cauliflower mosaic virus 35S promoter was studied during development in progeny from a primary transformant of tobacco carrying 3 independently segregating inserts. In the R1S2‐generation, plants could be grouped to 4 GUS expression phenotypes, showing either continuous GUS activity [S(‐) plant] or silencing of GUS after 13, 5 or 2 weeks [S(13), S(5) and S(2) plants, respectively]. In S(13), S(5) and S(2) plants, GUS activity was reduced 7‐, 17‐ and 50‐fold, respectively, as compared to the S(‐) plant. Southern blot and segregation analysis showed that S(2) plants contained all 3 inserts of which at least 1 was homozygous. All S(5) plants contained 2 inserts, 1 of which was homozygous and the other hemizygous. The S(13) plant contained 2 hemizygous inserts, while the S(‐) plant contained 1 hemizygous insert. Analysis of the R1S3‐generation showed that the timing and degree of silencing was directly linked to copy number and that each of the 3 inserts seemed to contribute equally to gene silencing. The physically linked neomycin phosphotransferase II gene driven by the weak nopaline synthase promoter was also subject to dosage‐dependent gene but this occurred later and was less pronounced.
Ca2+, calmodulin, and protein dephosphorylation are required for GA‐induced gene expression in petunia corolla
Authors: Leitner‐Dagan Yael , Weiss David
Published: March 2003

Gibberellic acid (GA3) induces the expression of different genes, including chalcone synthase (chs) and gip, in detached petunia corollas. To initiate a study on gibberellin (GA)‐signal transduction in this tissue, we examined the effect of agents that inhibit or promote specific steps in signal‐transduction pathways. The calcium chelator 1,2‐bis(o‐aminophenoxy)ethane N,N,N′,N′‐tetraacetic acid (BAPTA) had no effect on GA‐induced gene expression, while the calcium‐channel blocker, ruthenium red (RR), inhibited the activation of the genes. The calmodulin antagonist N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide hydrochloride (W‐7) inhibited the induction of chs and gip by the hormone, and its analog, N‐(6‐aminohexyl)‐1‐naphthalenesulfonamide hydrochloride (W‐5), had lower effect. The activation of chs and gip by GA3 was completely blocked by the protein phosphatase inhibitor, okadaic acid (OA), and partially inhibited by the protein kinase inhibitor, 1‐(5‐isoquinoline‐sulfonyl)‐ 2‐methylpiperazine dihydrochloride (H‐7). We suggest that Ca2+ from intracellular sources, calmodulin and protein dephosphorylation and phosphorylation are involved in GA‐induced gene expression in petunia corollas.
Purification and binding features of a pea fructose‐1,6‐bisphosphatase domain involved in the interaction with thioredoxin f
Authors: Hermoso Rosario , Lázaro Juan José , Chueca Ana , Sahrawy Mariam , López‐Gorgé Julio
Published: March 2003

We previously demonstrated that a cluster in the available Asn‐170Glu region of pea chloroplast fructose‐1,6‐bisphosphatase (FBPase) could be involved in its interaction with the physiological modulator thioredoxin (Trx). Using as template a cDNA coding for pea chloroplast FBPase, a DNA insert coding for a 19 amino acid fragment (

Pro‐167Gly) was amplified by PCR. After insertion in the pGEX‐4T vector‐1, it was expressed in Escherichia coli as a fusion protein (GST‐19) with the vector‐coded glutathione transferase (GST). This protein appears in the supernatant of cell lysates, and was purified to homogeneity. After thrombin digestion, the 19 amino acid insert was isolated as a polypeptide which displayed a positive reaction against pea chloroplast FBPase antibodies. GST‐19 linked to glutathione‐Sepharose beads, but not the GST, strongly interacts with pea Trx f, suggesting that this binding depends on the 19 amino acid insert. ELISA and Western blot experiments also demonstrate the existence of a GST‐19‐Trx f interaction, as well as a negligible quantity of Trx f bound by the vector‐coded GST. Putative competitive inhibition assays of FBPase activity carried out in the presence of increasing concentrations of the 19 amino acid insert do not demonstrate any enzyme inhibition. On the contrary, this protein fragment enhances the enzyme activity proportionally to its concentration in the assay mixture. This indicates that the FBPase‐Trx f binding promotes some type of structural modification of the Trx molecule, or of the FBPase‐Trx docking site, thus facilitating the reductive modulation of FBPase.