Profile of anemia in acute lymphoblastic leukemia patients on maintenance therapy and the effect of micronutrient supplementation.

BACKGROUND: Anemia is a common finding and important cause of morbidity in patients with acute lymphoblastic leukemia (ALL) at diagnosis or during the course of its protracted treatment. We studied profile of anemia in ALL patients on maintenance therapy and evaluated specific micronutrients as cause of this anemia. PATIENTS AND METHODS: ALL patients who were on maintenance therapy and had grade ≥ 2 anemia were recruited for the study. Serum iron studies, folate, and vitamin B12 were done to identify micronutrient deficiency and to initiate supplementation with specific components if found to be deficient. Toxicities, improvement of anemia, micronutrient levels, and disease outcome were studied after 3 months. RESULTS: From March 2015 to September 2016, 105 ALL patients were found to be on maintenance fulfilling the inclusion criteria. Overall, the proportion of anemia was 80%(N = 84). Majority had normocytic normochromic anemia (71%). Macrocytic anemia was seen in 18% and microcytic hypochromic in 9.5%. In patients with anemia of grade ≥ 2 (N = 84), 38 patients (45%) had biochemical deficiency of serum folate, and 7 (8%) had vitamin B12 deficiency. No biochemical evidence of iron deficiency was found. Supplementation of deficient micronutrients improved anemia: mean hemoglobin significantly increased from 8.06 ± 1.63 to 10.78 ± 1.53 (p < 0.001) at 3 months; and reduced treatment toxicities, mean number of febrile neutropenia episodes (p = 0.007), and treatment interruptions of > 2 weeks (p = 0.002) were lowered. Patients with anemia had significantly more relapses (N = 14,64%) compared to patients without anemia (N = 8,36%), (p = 0.040). CONCLUSION: Timely identification and correction of micronutrient deficiencies causing anemia in ALL patients on maintenance can enhance treatment outcomes.

Late Effects of Breast Cancer Treatment and Outcome after Corrective Interventions.

Purpose: To study the late toxicities of treatment and its impact on Breast cancer survivors among Indian patients. Materials and Methods: Our study recruited 152 curatively treated non metastatic carcinoma breast patients. The baseline demographic details, disease related and treatment related information were collected. The late effects included breast cancer related lymphedema, shoulder dysfunction, treatment induced bone loss, hypothyroidism, cardiac dysfunction, and chemotherapy induced cognitive dysfunction and Quality of life. Results: The median age was 47 years (range 27 -72 years). The cumulative frequency of BCRL and shoulder dysfunction was 31.57% and 34.86% respectively. The improvement in BCRL with corrective intervention was not statistically significant. The BCRL was significantly associated with shoulder dysfunction. The frequency of loss of bone mineral density was 38.15%. There was statistically significant improvement in bone mineral density with interventions. The cumulative rate of hypothyroidism and cardiac dysfunction was 14.47 % and 2.17% respectively which improved after corrective therapy. We did not find any delayed cognitive dysfunction. There was improvement in global health, physical function, role function, fatigue, Nausea, vomiting, pain scores, insomnia, Loss of appetite, diarrhea and arm symptoms over time with intervention. Conclusion: Our study has shown that nearly half of the survivors were suffering from at least one of the late effects. The intervention helped in improving the loss of bone mineral density, hypothyroidism, cardiac dysfunction and quality of life in Breast cancer survivors.

At a 10-Year Follow-up, Tendon Repair Is Superior to Physiotherapy in the Treatment of Small and Medium-Sized Rotator Cuff Tears.

BACKGROUND: Tendon repair and physiotherapy are frequently used treatment methods for small and medium-sized rotator cuff tears. In 2 previous publications of the 1 and 5-year results of this study, we reported significant but small between-group differences in favor of tendon repair. Long-term results are needed to assess whether the results in both groups remain stable over time. METHODS: In this study, 103 patients with a rotator cuff tear not exceeding 3 cm were randomly assigned to primary tendon repair or physiotherapy with optional secondary repair. Blinded follow-up was performed after 6 months and 1, 2, 5, and 10 years. Outcome measures included the Constant score; the self-report section of the American Shoulder and Elbow Surgeons score; the measurement of shoulder pain, motion, and strength; and patient satisfaction. Magnetic resonance imaging (MRI) was performed on surgically treated shoulders after 1 year, and ultrasound was performed on all shoulders after 5 and 10 years. The main analysis was by 1-way analysis of covariance and by intention to treat. RESULTS: Ninety-one of 103 patients attended the last follow-up. After 10 years, the results were better for primary tendon repair, by 9.6 points on the Constant score (p = 0.002), 15.7 points on the American Shoulder and Elbow Surgeons score (p < 0.001), 1.8 cm on a 10-cm visual analog scale for pain (p < 0.001), 19.6° for pain-free abduction (p = 0.007), and 14.3° for pain-free flexion (p = 0.01). Fourteen patients had crossed over from physiotherapy to secondary surgery and had an outcome on the Constant score that was 10.0 points inferior compared with that of the primary tendon repair group (p = 0.03). CONCLUSIONS: At 10 years, the differences in outcome between primary tendon repair and physiotherapy for small and medium-sized rotator cuff tears had increased, with better results for primary tendon repair. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Cefepime vs. cefoperazone/sulbactam in combination with amikacin as empirical antibiotic therapy in febrile neutropenia.

PURPOSE: Beta lactams are standard empirical therapy for febrile neutropenia (FN). The aim of this study was to evaluate the efficacy and safety of cefepime monotherapy compared with cefoperazone/sulbactam plus amikacin (CS + A) for empirical treatment of high risk FN. METHODS: One hundred seventy-five patients with 336 FN episodes were randomized to receive either cefepime (2 g q8h for adults and 50 mg/kg q8h for children) or CS (2 g q8h for adults and 50 mg/kg q8h for children) plus amikacin (15 mg/kg once a day). Positive response was defined as afebrile within 72 h of starting antibiotics, persistent afebrile status more than 48 h and no requirement of second-line antibiotics and antifungal agents. RESULTS: Three hundred thirty-six episodes were assessable for efficacy (168 cefepime, 168 CS + A). The positive response to antibiotics was identical for cefepime (53%) and CS + A (53%). Positive response was similar in MDI (microbiologically documented infection), 50 vs. 35% (p = 0.248), CDI (clinically documented infection), 50 vs. 35% (p = 0.259), combination CDI + MDI, 25 vs. 15% (p = 0.400), FUO (fever of unknown origin), 68 vs. 72% (p = 0.577) respectively in the two groups. The successful discontinuation of antibiotics at 72 h in FUO was similar in both groups (60 vs. 59%, p = 0.544). Total drug-related adverse events were similar in both groups (8 vs. 6%) except renal dysfunction was high in CS + A (1 vs. 7 events). Mortality was the same between two groups (8 vs 7%). CONCLUSIONS: Cefepime monotherapy and CS + A had similar efficacy as first-line therapy for FN. Discontinuation of empirical antibiotics is safe and feasible approach in selected group of FUO patients.

The effect of pharmacogenetic profiling with a clinical decision support tool on healthcare resource utilization and estimated costs in the elderly exposed to polypharmacy.

OBJECTIVE: To compare healthcare resource utilization (HRU) and clinical decision-making for elderly patients based on cytochrome P450 (CYP) pharmacogenetic testing and the use of a comprehensive medication management clinical decision support tool (CDST), to a cohort of similar non-tested patients. METHODS: An observational study compared a prospective cohort of patients ≥65 years subjected to pharmacogenetic testing to a propensity score (PS) matched historical cohort of untested patients in a claims database. Patients had a prescribed medication or dose change of at least one of 61 oral drugs or combinations of ≥3 drugs at enrollment. Four-month HRU outcomes examined included hospitalizations, emergency department (ED) and outpatient visits and provider acceptance of test recommendations. Costs were estimated using national data sources. RESULTS: There were 205 tested patients PS matched to 820 untested patients. Hospitalization rate was 9.8% in the tested group vs. 16.1% in the untested group (RR = 0.61, 95% CI = 0.39-0.95, p = 0.027), ED visit rate was 4.4% in the tested group vs. 15.4% in the untested group (RR = 0.29, 95% CI = 0.15-0.55, p = 0.0002) and outpatient visit rate was 71.7% in the tested group vs. 36.5% in the untested group (RR = 1.97, 95% CI = 1.74-2.23, p < 0.0001). The rate of overall HRU was 72.2% in the tested group vs. 49.0% in the untested group (RR = 1.47, 95% CI = 1.32-1.64, p < 0.0001). Potential cost savings were estimated at $218 (mean) in the tested group. The provider majority (95%) considered the test helpful and 46% followed CDST provided recommendations. CONCLUSION: Patients CYP DNA tested and treated according to the personalized prescribing system had a significant decrease in hospitalizations and emergency department visits, resulting in potential cost savings. Providers had a high satisfaction rate with the clinical utility of the system and followed recommendations when appropriate.

An evaluation of clinical risk factors for estimating fracture risk in postmenopausal osteoporosis using an electronic medical record database.

SUMMARY: Many of the clinical risk factors used in fracture risk assessment (FRAX) calculator are available in electronic medical record (EMR) databases and are good sources of osteoporosis risk factor information. The EPIC EMR database showed a lower prevalence of FRAX risk factors and, consequently, proportion of patients who would be deemed "high risk." INTRODUCTION: The FRAX tool is underutilized for osteoporosis screening. Many of the clinical risk factors for FRAX may be available in EMR databases and may enable health systems to perform fracture risk assessments. We intended to identify variables in an EMR database for calculating FRAX score in a cohort of postmenopausal women, to estimate absolute fracture risk, and to determine the proportions of women whose absolute fracture risks exceed the National Osteoporosis Foundation (NOF) thresholds. METHODS: Our cohort was selected using an EMR database with demographic, inpatient, outpatient, and clinical information for female patients age≥50 in a family practice, internal medicine, or obstetrics/gynecology clinic in 2007-2008. The latest physician encounter was the index date. Variables, problem and medication lists, diagnosis codes, and histories from the EMR were used to populate the 11 clinical risk factor variables used in the FRAX. These risk factor prevalence and treatment-eligible proportions were compared to those of published epidemiology studies. RESULTS: The study included 345 patients. Mean (SD) 10-year risk for any major fracture was 11.1% (6.8) when bone mineral density (BMD) was used and 11.2% (6.5) when BMI was used. About 10.1% of the cohort exceeded the NOF's 20% major fracture risk threshold and 32.5% exceeded the NOF's 3% hip fracture risk threshold when BMD was used. Overall, the number of treatment-eligible patients was slightly lower when FRAX was calculated using BMD versus BMI (13.6 and 36.8%). CONCLUSION: Our cohort using EMR data most likely underestimated the mean 10-year probability of any major fracture compared to other cohorts in published literature. The difference may be in the nature of EMRs for supporting only passive data collection of risk factor information.

Tendon repair compared with physiotherapy in the treatment of rotator cuff tears: a randomized controlled study in 103 cases with a five-year follow-up.

BACKGROUND: There is limited Level-I evidence that compares operative and nonoperative treatment of rotator cuff tears. We compared outcomes of patients treated with primary tendon repair with outcomes of those treated with physiotherapy and optional secondary tendon repair if needed. METHODS: A single-center, pragmatic, randomized controlled study with follow-ups after six months and one, two, and five years was conducted in a secondary-care institution. One hundred and three patients with a rotator cuff tear not exceeding 3 cm were randomized to primary tendon repair (n = 52) or physiotherapy (n = 51). The primary outcome measure was the Constant score. Secondary outcome measures included the self-report section of the American Shoulder and Elbow Surgeons score; the physical component summary measure of the Short Form 36 Health Survey; the measurement of pain, strength, and shoulder motion; patient satisfaction; and findings from magnetic resonance imaging and sonography. Analysis was by intention to treat. RESULTS: The five-year follow-up rate was 98%. Twelve of the fifty-one patients in the physiotherapy group were treated with secondary tendon repair. The results from primary tendon repair were superior to those from physiotherapy plus secondary repair, with between-group mean differences of 5.3 points on the Constant score (p = 0.05), 9.0 points on the American Shoulder and Elbow Surgeons score (p < 0.001), 1.1 cm on a 10-cm visual analog scale for pain (p < 0.001), and 1.0 cm on a 10-cm visual analog scale for patient satisfaction (p = 0.03). In 37% of tears treated with physiotherapy only, there were increasing tear sizes on ultrasound of >5 mm, over five years, associated with an inferior outcome. CONCLUSIONS: Although primary repair of small and medium-sized rotator cuff tears was associated with better outcome than physiotherapy treatment, the differences were small and may be below clinical importance. In the physiotherapy treatment group, there were increasing tear sizes and inferior outcomes in one-third of patients who did not undergo repair.

Determinants of glycaemic control in a practice setting: the role of weight loss and treatment adherence (The DELTA Study).

AIMS: Examine the association between weight loss and adherence with glycaemic goal attainment in patients with inadequately controlled T2DM. MATERIALS AND METHODS: Patients ≥ 18 years with T2DM from a US integrated health system starting a new class of diabetes medication between 11/1/10 and 4/30/11 (index date) with baseline HbA1c ≥ 7.0% were included in this cohort study. Target HbA1c and weight change were defined at 6-months as HbA1c < 7.0% and ≥ 3% loss in body weight. Patient-reported medication adherence was assessed per the Medication Adherence Reporting Scale. Structural equation modelling was used to describe simultaneous associations between adherence, weight loss and HbA1c goal attainment. RESULTS: Inclusion criteria were met by 477 patients; mean (SD) age 59.1 (11.6) years; 50.9% were female; 30.4% were treatment naïve; baseline HbA1c 8.6% (1.6); weight 102.0 kg (23.0). Most patients (67.9%) reported being adherent to the index diabetes medication. At 6 months mean weight change was -1.3 (5.1) kg (p = 0.39); 28.1% had weight loss of ≥ 3%. Mean HbA1c change was -1.2% (1.8) (p< 0.001); 42.8% attained HbA1c goal. Adherent patients (OR 1.70; p = 0.02) and diabetes therapies that lead to weight loss (metformin, GLP-1) were associated with weight loss ≥ 3% (OR 2.96; p< 0.001). Weight loss (OR 3.60; p < 0.001) and adherence (OR 1.59; p < 0.001) were associated with HbA1c goal attainment. CONCLUSIONS: Weight loss ≥ 3% and medication adherence were associated with HbA1c goal attainment in T2DM; weight loss was a stronger predictor of goal attainment than medication adherence in this study population. It is important to consider weight-effect properties, in addition to patient-centric adherence counselling, when prescribing diabetes therapy.

Measuring the evolution and output of cross-disciplinary collaborations within the NCI Physical Sciences-Oncology Centers Network.

Development of effective quantitative indicators and methodologies to assess the outcomes of cross-disciplinary collaborative initiatives has the potential to improve scientific program management and scientific output. This article highlights an example of a prospective evaluation that has been developed to monitor and improve progress of the National Cancer Institute Physical Sciences-Oncology Centers (PS-OC) program. Study data, including collaboration information, was captured through progress reports and compiled using the web-based analytic database: Interdisciplinary Team Reporting, Analysis, and Query Resource. Analysis of collaborations was further supported by data from the Thomson Reuters Web of Science database, MEDLINE database, and a web-based survey. Integration of novel and standard data sources was augmented by the development of automated methods to mine investigator pre-award publications, assign investigator disciplines, and distinguish cross-disciplinary publication content. The results highlight increases in cross-disciplinary authorship collaborations from pre- to post-award years among the primary investigators and confirm that a majority of cross-disciplinary collaborations have resulted in publications with cross-disciplinary content that rank in the top third of their field. With these evaluation data, PS-OC Program officials have provided ongoing feedback to participating investigators to improve center productivity and thereby facilitate a more successful initiative. Future analysis will continue to expand these methods and metrics to adapt to new advances in research evaluation and changes in the program.

Impact of elevated intact parathyroid hormone on mortality and renal disease progression in patients with chronic kidney disease stages 3 and 4.

OBJECTIVE: To estimate the impact of elevated intact parathyroid hormone levels on time to death and renal replacement therapy in patients with chronic kidney disease stages 3 and 4. METHODS: A retrospective cohort analysis from 01/1996 to 09/2007 was conducted in 11,092 patients with chronic kidney disease stages 3 and 4 patients using Cockroft-Gault and Modification of Diet in Renal Disease equations to estimate their glomerular filtration rates. Patients' highest parathyroid hormone levels were used to define the index date and cohort (followed for 1 year). Mortality and renal replacement therapy events were evaluated among cohorts at pre-defined parathyroid hormone levels. RESULTS: As the intact parathyroid hormone levels increased, the mean age, number of females and estimated glomerular filtration rates decreased. Patients with an intact parathyroid hormone level<50 pg/mL were defined as the reference group. Similar results were found using the Modification of Diet in Renal Disease equation for calculating estimated glomerular filtration rate, which was possible in 48% of the patients where race could be identified. Combined mortality and renal replacement therapy adjusted hazard ratio using Cox regression for intact parathyroid hormone level 51-110 pg/mL was 1.12 (0.82-1.54), intact parathyroid hormone level 111-199 pg/mL was 2.42 (1.78-3.29), intact parathyroid hormone level 200-299 pg/mL was 3.01 (2.14-4.27), intact parathyroid hormone level 300-399 pg/mL was 3.12 (2.09-4.60), intact parathyroid hormone level 400-499 pg/mL was 3.91 (2.61-5.85) and intact parathyroid hormone level>500 pg/mL was 2.67 (1.84-3.84). CONCLUSION: Intact parathyroid hormone levels>50 pg/mL in patients with chronic kidney disease stages 3 and 4 are associated with an escalating combined risk of death or RRT.

Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies.

The flavonoid pathway is known to be up-regulated by different environmental stress factors. Down-regulation of the pathway is much less studied and is emphasized in the present work. Flavonoid accumulation was induced by exposing plants for 1 week to nitrogen depletion at 10 degrees C, giving high levels of anthocyanins and 3-glucoside-7-rhamnosides, 3,7-di-rhamnosides and 3-rutinoside-7-rhamnosides of kaempferol and quercetin. Flavonol accumulation as influenced by temperatures and nitrogen supply was not related to the glycosylation patterns but to the classification as quercetin and kaempferol. When nitrogen was re-supplied, transcripts for main regulators of the pathway, PAP1/GL3 and PAP2/MYB12, fell to less than 1 and 0.1% of initial values, respectively, during 24 h in the 15-30 degrees C temperature range. Anthocyanins showed a half-life of approximately 1 d, while the degradation of flavonols was much slower. Interestingly, the initial fluxes of anthocyanin and flavonol degradations were found to be temperature-independent. A kinetic model for the flavonoid pathway was constructed. In order to get the observed concentration-temperature profiles as well as the temperature compensation in the flavonoid degradation flux, the model predicts that the flavonoid pathway shows an increased temperature sensitivity at the end of the pathway, where the up-regulation by PAP/GL3 has been found to be largest.

Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the first step in the phenylpropanoid pathway, and is considered an important regulation point between primary and secondary metabolism. In the present work we analyzed expression of the PAL genes in leaves of Arabidopsis thaliana rosette-stage plants in response to nitrogen depletion at temperatures ranging from 5 to 30 degrees C. Only PAL1 and PAL2 responded strongly to both environmental factors, nitrogen and temperature. Regardless of nitrogen treatments, PAL1 and 2 transcript levels increased at 5 and 10 degrees C. Averaged across all temperatures, nitrogen depletion led to a two-fold increase in PAL1 and PAL2 transcripts. PAL activity was correlated with PAL transcript levels (R=0.94). Accumulation of major soluble phenylpropanoids, sinapic acid esters and flavonoids, increased in response to lowering temperature. The flavonoids, kaempferols, quercetins and anthocyanins, showed significantly increased levels as a result of nitrogen depletion (two-, five- and six-fold increases, respectively) when averaged across all temperatures. PAL1, PAL2 and PAL4 have previously been shown to be related with tissue-specific lignin synthesis, and the present work shows that PAL1 and PAL2 also have functional specialization in abiotic environmental-triggered flavonoid synthesis.

HY5 and HYH are positive regulators of nitrate reductase in seedlings and rosette stage plants.

HY5 and HYH are bZIP transcription factors well known to be involved in photomorphogenesis and light signalling. Loss-of-function mutants of HY5 and HYH revealed that these genes are essential for induction of a key enzyme in nitrogen assimilation, nitrate reductase (EC 1.7.1.1). In Arabidopsis thaliana seedlings nitrate reductase was expressed under low irradiance far-red or red light at the same level as under higher irradiance, photosynthetic active, white light. However, high NR expression at low light levels occurred only in the presence of sucrose in the growth medium. Sucrose did not promote expression in darkness. Whereas HY5 was necessary for high nitrate reductase expression in far-red light, HYH was important in red light. COP1 is known to promote degradation of HY5 and HYH, and in the cop1 mutant, nitrate reductase activity was relatively high also in darkness. PhyA and PhyB mutants were tested, and confirmed the phytochrome dependency for far-red and red light induction of nitrate reductase in seedlings. In rosette leaves of 3-week-old green plants the daily increase in nitrate reductase expression in response to light-on was abolished in the hyh and hy5 hyh double mutant. The hy5 hyh double mutant had lower nitrate reductase activity than any of the single mutants in photosynthetic active light in both seedlings and rosette leaves.

Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway.

The content of flavonoids increases in response to nitrogen and phosphorus depletion in plants. Manipulation of these macronutrients may therefore be used to control the levels of desirable compounds and improve plant quality. Key enzymes in the shikimate pathway, which feeds precursors into the flavonoid pathway, are regulated post-translationally by feedback from aromatic amino acids, and possibly by redox control through photosynthesis. Use of microarrays for global transcript analysis in Arabidopsis has revealed that transcript levels are less influenced by mineral nutrients in the shikimate pathway compared with the flavonoid pathway. The responses in the shikimate pathway appear complex, whereas in the flavonoid pathway, a single gene often responds similarly to mineral depletion, high light intensity and sucrose. MYB [production of anthocyanin pigment 1 (PAP1)/production of anthocyanin pigment 2 (PAP2)] and bHLH [GLABRA3 (GL3)] transcription factors are important for the nutrient depletion response. PAP1/2 stimulate gross activation of the flavonoid pathway, and different investigations support merging signal transduction chains for various abiotic treatments on PAP1/2. Flavonol synthase is not part of the PAP1/2 regulon, and expression is mainly enhanced by high light intensity and sucrose, not mineral depletion. Nevertheless, both cyanidin and flavonol derivatives increase in response to nitrogen depletion. Kaempferols are the dominating flavonols in Arabidopsis leaves under normal cultivation conditions, but quercetin accumulation can be triggered by nitrogen depletion in combination with other abiotic factors.

Nitrogen deficiency enhances expression of specific MYB and bHLH transcription factors and accumulation of end products in the flavonoid pathway.

Expression of regulators of the flavonoid pathway was examined in Arabidopsis thaliana wild type and pap1D plants, the latter being a T-DNA activation-tagged line over-expressing the PAP1/MYB75 gene which is a positive regulator of the pathway. Anthocyanin accumulation was induced in plants grown in soil, on agar plates, and hydroponics by withdrawing nitrogen from the growth medium. The agar-grown seedlings and rosette stage plants in hydroponics were further explored, and showed that nitrogen deficiency resulted in the accumulation of not only anthocyanins, but also flavonols. The examination of transcript levels showed that the general flavonoid pathway regulators PAP1 and PAP2 were up-regulated in response to nitrogen deficiency in wild type as well as pap1D plants. Interestingly, PAP2 responded much stronger to nitrogen deficiency than PAP1, 200- and 6-fold increase in transcript levels, respectively, for wild-type seedlings. In rosette leaves the increase was 900-fold for PAP2 and 6-fold for PAP1. At least three different bHLH domain transcription factors promote anthocyanin synthesis, and transcripts for one of these, i.e. GL3 were found to be sixfold enhanced by nitrogen deficiency in rosette leaves. The MYB12 transcription factor, known to regulate flavonol synthesis, was slightly induced by nitrogen deficiency in seedlings. In conclusion, four out of eight regulators involved in the flavonoid pathway showed an enhanced expression from 2 to 1,000 times in response to nitrogen deficiency. Together with MYB factors, especially PAP2, GL3 appears to be the BHLH partner for anthocyanin accumulation in response to nitrogen deficiency.

Posttranslational regulation of nitrate reductase strongly affects the levels of free amino acids and nitrate, whereas transcriptional regulation has only minor influence.

Diurnal variations in nitrate reductase (NR) activity and nitrogen metabolites were examined in wild-type Nicotiana plumbaginifolia and transformants with various degrees of NR deregulation. In the C1 line, NR was only deregulated at the transcriptional level by placing the NR gene under the control of the cauliflower mosaic virus 35S RNA promoter. In the Del8 and S521D lines, NR was additionally deregulated at the posttranslational level either by a deletion mutation in the N-terminal domain or by a mutation of the regulatory phosphorylation site (serine-521). Posttranslational regulation was essential for pronounced diurnal variations in NR activity. Low nitrate content was related to deregulation of NR, whereas the level of total free amino acids was much higher in plants with fully deregulated NR. Abolishing transcriptional and posttranslational regulation (S521D plants) resulted in an increase of glutamine and asparagine by a factor of 9 and 14, respectively, compared with wild type, whereas abolishing transcriptional regulation (C1 plants) only resulted in increases of glutamine and asparagine by factors <2. Among the minor amino acids, isoleucine and threonine, in particular, showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and wild type as determined with (15)N feeding. Deregulation of NR appears to set the level of certain amino acids, whereas diurnal variations were still determined by light/dark. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the posttranslational level resulted in profound changes of nitrogen metabolite levels.

Is nitrate reductase a major player in the plant NO (nitric oxide) game?

Nitric oxide (NO) is a diffusible, very reactive gas that is involved in the regulation of many processes in plants. Several enzymatic sources of NO production have been identified in recent years. Nitrate reductase (NR) is one of them and it has been shown that this well-known plant protein, apart from its role in nitrate reduction and assimilation, can also catalyse the reduction of nitrite to NO. This reaction can produce large amounts of NO, or at least more than is needed for signalling, as some escape of NO to the outside medium can be detected after NR activation. A role for NO and NR in stomata functioning in response to abscisic acid has also been proposed. The question that remains is whether this NR-derived NO is a signalling molecule or the mere product of an enzymatic side reaction like the products generated by the oxygenase activity of RuBisCO.

Mechanism and importance of post-translational regulation of nitrate reductase.

In higher plants, nitrate reductase (NR) is inactivated by the phosphorylation of a conserved Ser residue and binding of 14-3-3 proteins in the presence of divalent cations or polyamines. A transgenic Nicotiana plumbaginifolia line (S521) has been constructed where the regulatory, conserved Ser 521 of tobacco NR (corresponding to Ser 534 in Arabidopsis) was mutated into Asp. This mutation resulted in the complete abolition of activation/inactivation in response to light/dark transitions or other treatments known to regulate the activation state of NR. Analysis of the transgenic plants showed that, under certain conditions, when whole plants or cut tissues are exposed to high nitrate supply, post-translational regulation is necessary to avoid nitrite accumulation. Abolition of the post-translational regulation of NR also results in an increased flux of nitric oxide from the leaves and roots. In view of the results obtained from examining the different transgenic N. plumbaginifolia lines, compartmentation of nitrate into an active metabolic pool and a large storage pool appears to be an important factor for regulating nitrate reduction. The complex regulation of nitrate reduction is likely to have evolved not only to optimize nitrogen assimilation, but also to prevent and control the formation of toxic, and possibly regulatory, products of NR activities. Phos phorylation of NR has previously been found to influence the degradation of NR in spinach leaves and Arabidopsis cell cultures. However, experiments with whole plants of N. plumbaginifolia, Arabidopsis, or squash are in favour of NR degradation being the same in light and darkness and independent of phosphorylation at the regulatory Ser.

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue.

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme. When cut leaves or roots of this line (S(521)) were placed in darkness in a buffer containing 50 mM KNO(3), nitrite was excreted from the tissue at rates of 0.08-0.2 micromol (g FW)(-1) h(-1) for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1-3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S(521), although 20-40 micromol (g FW)(-1) nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S(521) also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S(521) was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation.

In wild-type Nicotiana plumbaginifolia and other higher plants, nitrate reductase (NR) is rapidly inactivated/activated in response to dark/light transitions. Inactivation of NR is believed to be caused by phosphorylation at a special conserved regulatory Ser residue, Ser 521, and interactions with divalent cations and inhibitory 14-3-3 proteins. A transgenic N. plumbaginifolia line (S(521)) was constructed where the Ser 521 had been changed by site-directed mutagenesis into Asp. This mutation resulted in complete abolishment of inactivation in response to light/dark transitions or other treatments known to inactivate NR. During prolonged darkness, NR in wild-type plants is in the inactivated form, whereas NR in the S(521) line is always in the active form. Differences in degradation rate between NR from S(521) and lines with non-mutated NR were not found. Kinetic constants like Km values for NADH and NO3(-) were not changed, but a slightly different pH profile was observed for mutated NR as opposed to non-mutated NR. Under optimal growth conditions, the phenotype of the S(521) plants was not different from the wild type (WT). However, when plants were irrigated with high nitrate concentration, 150 mM, the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.