Characterization of the PF-ILD phenotype in patients with advanced pulmonary sarcoidosis.

BACKGROUND: Advanced pulmonary sarcoidosis causes significant morbidity and can lead to death. Large trials demonstrated efficacy of antifibrotics in patients with progressive fibrosing interstitial lung diseases (PF-ILD), including a few with sarcoidosis. To date, little is known about this progressive fibrosing phenotype in sarcoidosis. Diffusion capacity of carbon monoxide (DLCO) may be a useful functional marker to screen for advanced pulmonary sarcoidosis. In this study, we describe a cohort with advanced pulmonary sarcoidosis and we gain insights in the progressive fibrosing phenotype in sarcoidosis. METHODS: Patients with sarcoidosis and a DLCO < 50% predicted were included in this retrospective cohort study. First measurement of DLCO < 50% predicted was the baseline. Lung function data, HRCT, pulmonary hypertension (PH) and mortality were collected. Patients with > 10% fibrosis on HRCT meeting the criteria for ILD-progression within 24 months were labelled as PF-ILD. With Cox-regression analysis predictors of mortality were established. RESULTS: 106 patients with a DLCO < 50% predicted were included. Evolution of forced vital capacity (FVC) varied widely between patients from - 34% to + 45% after 2 years follow-up, whereas change in DLCO varied between - 11% and + 26%. Fourteen patients (15%) met the PF-ILD criteria, of whom 6 (43%) died within 10 years versus 10 (13%) in the non PF-ILD group (p = 0.006). PH was present 12 (11%), 56 (53%) demonstrated > 10% fibrosis on HRCT. Independent predictors of mortality and lung transplantation in the whole cohort are PH, PF-ILD and UIP-like pattern. CONCLUSION: In conclusion, within this group with advanced pulmonary sarcoidosis disease course varied widely from great functional improvement to death. PF-ILD patients had higher mortality rate than the mortality in the overall pulmonary sarcoidosis group. Future research should focus on the addition of antifibrotics in these patients. Trial registration retrospectively registered.

Predicting local tumour progression after ablation for colorectal liver metastases: CT-based radiomics of the ablation zone.

PURPOSE: To assess whether CT-based radiomics of the ablation zone (AZ) can predict local tumour progression (LTP) after thermal ablation for colorectal liver metastases (CRLM). MATERIALS AND METHODS: Eighty-two patients with 127 CRLM were included. Radiomics features (with different filters) were extracted from the AZ and a 10 mm periablational rim (PAR)on portal-venous-phase CT up to 8 weeks after ablation. Multivariable stepwise Cox regression analyses were used to predict LTP based on clinical and radiomics features. Performance (concordance [c]-statistics) of the different models was compared and performance in an 'independent' dataset was approximated with bootstrapped leave-one-out-cross-validation (LOOCV). RESULTS: Thirty-three lesions (26 %) developed LTP. Median follow-up was 21 months (range 6-115). The combined model, a combination of clinical and radiomics features, included chemotherapy (HR 0.50, p = 0.024), cT-stage (HR 10.13, p = 0.016), lesion size (HR 1.11, p = <0.001), AZ_Skewness (HR 1.58, p = 0.016), AZ_Uniformity (HR 0.45, p = 0.002), PAR_Mean (HR 0.52, p = 0.008), PAR_Skewness (HR 1.67, p = 0.019) and PAR_Uniformity (HR 3.35, p < 0.001) as relevant predictors for LTP. The predictive performance of the combined model (after LOOCV) yielded a c-statistic of 0.78 (95 %CI 0.65-0.87), compared to the clinical or radiomics models only (c-statistic 0.74 (95 %CI 0.58-0.84) and 0.65 (95 %CI 0.52-0.83), respectively). CONCLUSION: Combining radiomics features with clinical features yielded a better performing prediction of LTP than radiomics only. CT-based radiomics of the AZ and PAR may have potential to aid in the prediction of LTP during follow-up in patients with CRLM.

Shifting brain inhibitory balance and connectivity of the prefrontal cortex of adults with autism spectrum disorder.

Currently, there are no effective pharmacologic treatments for the core symptoms of autism spectrum disorder (ASD). There is, nevertheless, potential for progress. For example, recent evidence suggests that the excitatory (E) glutamate and inhibitory (I) GABA systems may be altered in ASD. However, no prior studies of ASD have examined the 'responsivity' of the E-I system to pharmacologic challenge; or whether E-I modulation alters abnormalities in functional connectivity of brain regions implicated in the disorder. Therefore, we used magnetic resonance spectroscopy ([1H]MRS) to measure prefrontal E-I flux in response to the glutamate and GABA acting drug riluzole in adult men with and without ASD. We compared the change in prefrontal 'Inhibitory Index'-the GABA fraction within the pool of glutamate plus GABA metabolites-post riluzole challenge; and the impact of riluzole on differences in resting-state functional connectivity. Despite no baseline differences in E-I balance, there was a significant group difference in response to pharmacologic challenge. Riluzole increased the prefrontal cortex inhibitory index in ASD but decreased it in controls. There was also a significant group difference in prefrontal functional connectivity at baseline, which was abolished by riluzole within the ASD group. Our results also show, for we believe the first time in ASD, that E-I flux can be 'shifted' with a pharmacologic challenge, but that responsivity is significantly different from controls. Further, our initial evidence suggests that abnormalities in functional connectivity can be 'normalised' by targeting E-I, even in adults.

Assessment and Optimisation of Lung Cancer Patients for Treatment with Curative Intent.

Over the past decade the field of lung cancer management has seen many developments. Coupled with an ageing population and increasing rates of comorbid illness, the work-up for treatments with curative intent has become more complex and detailed. As well as improvements in imaging and staging techniques, developments in both surgery and radiotherapy may now allow patients who would previously have been considered unfit or not appropriate for treatment with curative intent to undergo radical therapies. This overview will highlight published studies relating to investigation and staging techniques, together with assessments of fitness, with the aim of helping clinicians to determine the most appropriate treatments for each patient. We also highlight areas where further research may be required.

The RNA helicase p68 (DDX5) is selectively required for the induction of p53-dependent p21 expression and cell-cycle arrest after DNA damage.

The RNA helicase p68 (DDX5) is an established co-activator of the p53 tumour suppressor that itself has a pivotal role in orchestrating the cellular response to DNA damage. Although several factors influence the biological outcome of p53 activation, the mechanisms governing the choice between cell-cycle arrest and apoptosis remain to be elucidated. In the present study, we show that, while p68 is critical for p53-mediated transactivation of the cell-cycle arrest gene p21(WAF1/CIP1), it is dispensable for induction of several pro-apoptotic genes in response to DNA damage. Moreover, p68 depletion results in a striking inhibition of recruitment of p53 and RNA Pol II to the p21 promoter but not to the Bax or PUMA promoters, providing an explanation for the selective effect on p21 induction. Importantly, these findings are mirrored in a novel inducible p68 knockout mouse model in which p68 depletion results in a selective inhibition of p21 induction in several tissues. Moreover, in the bone marrow, p68 depletion results in an increased sensitivity to γ-irradiation, consistent with an increased level of apoptosis. These data highlight a novel function of p68 as a modulator of the decision between p53-mediated growth arrest and apoptosis in vitro and in vivo.

Soil-test N recommendations augmented with PEST-optimized RZWQM simulations.

Improved understanding of year-to-year late-spring soil nitrate test (LSNT) variability could help make it more attractive to producers. We test the ability of the Root Zone Water Quality Model (RZWQM) to simulate watershed-scale variability due to the LSNT, and we use the optimized model to simulate long-term field N dynamics under related conditions. Autoregressive techniques and the automatic parameter calibration program PEST were used to show that RZWQM simulates significantly lower nitrate concentration in discharge from LSNT treatments compared with areas receiving fall N fertilizer applications within the tile-drained Walnut Creek, Iowa, watershed (>5 mg NL(-1) difference for the third year of the treatment, 1999). This result is similar to field-measured data from a paired watershed experiment. A statistical model we developed using RZWQM simulations from 1970 to 2005 shows that early-season precipitation and early-season temperature account for 90% of the interannual variation in LSNT-based fertilizer N rates. Long-term simulations with similar average N application rates for corn (Zea mays L.) (151 kg N ha(-1)) show annual average N loss in tile flow of 20.4, 22.2, and 27.3 kg N ha(-1) for LSNT, single spring, and single fall N applications. These results suggest that (i) RZWQM is a promising tool to accurately estimate the water quality effects of LSNT; (ii) the majority of N loss difference between LSNT and fall applications is because more N remains in the root zone for crop uptake; and (iii) year-to-year LSNT-based N rate differences are mainly due to variation in early-season precipitation and temperature.

Bone grafting, orthopaedic biomaterials, and the clinical need for bone engineering.

As the population ages, the number of operations performed on bone is expected to increase. Diseases such as arthritis, tumours, and trauma can lead to defects in the skeleton requiring an operation to replace or restore the lost bone. Surgeons can use autografts, allografts, and/or bone graft substitutes to restore areas of bone loss. Surgical implants are also used in addition or in isolation to replace the diseased bone. This review considers the application of available bone grafts in different clinical settings. It also discusses recently introduced bioactive biomaterials and highlights the clinical difficulties and technological deficiencies that exist in our current surgical practice.

Outcome of palliative esophageal stenting for malignant dysphagia: a retrospective analysis.

Greater than 50% of patients with esophageal carcinoma are found to be incurable at the time of diagnosis, leaving only palliative options. Self-expanding metal stents (SEMs) are effective for relieving symptoms and complications associated with esophageal carcinoma and improving quality of life. We undertook a retrospective analysis to evaluate the experience of palliative esophageal stenting for symptomatic malignant dysphagia in our institution over a period of 7 years. Between January 1999 and January 2006, 126 patients who received SEMs for malignant dysphagia were identified using an upper gastrointestinal specialist nurse clinician database. Data were obtained from patient case notes, endoscopy, histopathology, radiology, and external agency databases. Of the 126 identified, 36 patients were excluded from the analysis. A number of variables including age, sex, presenting complaints, type of stent, indications of stenting, success or failure of stent insertion, survival rate, and complication rate were analyzed. Of the 90 patients, 55 (61%) were male and 35 (39%) were female. The mean age of patients was 70.79 (range 40-97) years. The predominant presenting complaints were dysphagia (n = 81) and weight loss (n = 48). The indication for stenting was worsening dysphagia in all patients. Tumors were confined to the distal esophagus and esophagogastric junction in 73 patients (81%), and the mid-esophagus in 17 (19%). Adenocarcinoma was identified in 61 patients (67.8%) and squamous cell carcinoma in 29 (32.2%). Stenting numbers were comparable in endoscopic and radiologic groups (47 vs. 43), with successful stent deployment in 89 patients. The 7- and 30-day mortality was 9% (n = 8) and 28% (n = 25), respectively. Comparable numbers of early deaths were seen in both radiologic (n = 13) and endoscopic (n = 12) groups. Causes of early inpatient death included hemorrhage (n = 5), pneumonia (n = 7), exhaustion (n = 2), cardiac causes (n = 3), perforation (n = 1), and sepsis (n = 1). The number of patients with complications was 41 (45.6%), 25 in the surgical group and 15 in the radiologic group; the difference was not significant (P = 0.13). The mean survival time was 92.5 (0-638) days and median survival time was 61 days. A subgroup of patients with complete dysphagia (score 4) gained a mean survival of 59 days. Those patients receiving adjuvant chemotherapy or radiotherapy survived significantly longer than those receiving stenting alone (152.8 days vs. 71.8 days). There is no significant difference in complications or survival when using endoscopic or radiologic methods to deploy SEMs in patients with inoperable esophageal cancer. Mortality is low; however, the morbidity rate is significant. Patients receiving adjuvant chemotherapy or radiotherapy, in addition to stenting, survived significantly longer than those with a stent only.

Incidence of acquired demyelination of the CNS in Canadian children.

BACKGROUND: The incidence of acquired demyelination of the CNS (acquired demyelinating syndromes [ADS]) in children is unknown. It is important that physicians recognize the features of ADS to facilitate care and to appreciate the future risk of multiple sclerosis (MS). OBJECTIVE: To determine the incidence, clinical features, familial autoimmune history, and acute management of Canadian children with ADS. METHODS: Incidence and case-specific data were obtained through the Canadian Pediatric Surveillance Program from April 1, 2004, to March 31, 2007. Before study initiation, a survey was sent to all pediatric health care providers to determine awareness of MS as a potential outcome of ADS in children. RESULTS: Two hundred nineteen children with ADS (mean age 10.5 years, range 0.66-18.0 years; female to male ratio 1.09:1) were reported. The most common presentations were optic neuritis (ON; n = 51, 23%), acute disseminated encephalomyelitis (ADEM; n = 49, 22%), and transverse myelitis (TM; n = 48, 22%). Children with ADEM were more likely to be younger than 10 years, whereas children with monolesional ADS (ON, TM, other) were more likely to be older than 10 years (p < 0.001). There were 73 incident cases per year, leading to an annual incidence of 0.9 per 100,000 Canadian children. A family history of MS was reported in 8%. Before study initiation, 65% of physicians indicated that they considered MS as a possible outcome of ADS in children. This increased to 74% in year 1, 81% in year 2, and 87% in year 3. CONCLUSION: The incidence of pediatric acquired demyelinating syndromes (ADS) is 0.9 per 100,000 Canadian children. ADS presentations are influenced by age.

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis.

Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2beta causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1-Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found that CK2beta participates in PLK1-Wee1 complex formation whereas its cellular depletion leads to disruption of PLK1-Wee1 interaction and reduced Wee1 phosphorylation at Ser53 and 121. The data reported here reinforce the notion that CK2beta has functions that are independent of its role as the CK2 regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.

Transcription factor TAFII250 promotes Mdm2-dependent turnover of p53.

The p53 tumour suppressor is regulated mainly by Mdm2, an E3 ubiquitin ligase that promotes the ubiquitylation and proteasome-mediated degradation of p53. Many agents that induce p53 are inhibitors of transcription, suggesting that the p53 pathway can detect a signal(s) arising from transcriptional malfunction. Mdm2 associates with TAFII250, a component of the general transcription factor TFIID. Inactivation of TAFII250 in ts13 cells, which express a temperature-sensitive mutant of TAFII250, leads to the induction of p53 and cell cycle arrest. In the present study, we show that TAFII250 stimulates the ubiquitylation and degradation of p53 in a manner that is dependent upon Mdm2 and requires its acidic domain. Mechanistically, TAFII250 downregulates Mdm2 auto-ubiquitylation, leading to Mdm2 stabilization, and promotes p53-Mdm2 association through a recently defined second binding site in the acidic domain of Mdm2. These data provide a novel route through which TAFII250 can directly influence p53 levels and are consistent with the idea that the maintenance of p53 turnover is coupled to the integrity of RNA polymerase II transcription.

c-Abl phosphorylates Hdm2 at tyrosine 276 in response to DNA damage and regulates interaction with ARF.

The p53 tumour-suppressor protein is tightly regulated through its association with the Hdm2 E3 ligase. Activation of p53 by DNA strand breaks is orchestrated by the ataxia-telangiectasia mutated (ATM) protein kinase and involves interruption of Hdm2-mediated p53 degradation. As part of this mechanism ATM itself, and the ATM-activated protein tyrosine kinase, c-Abl, inhibit Hdm2 function through phosphorylation of serine 395 and tyrosine 394 (Y394), respectively. In the present study, we have identified a novel target of c-Abl in the Hdm2 protein, tyrosine 276 (Y276). We show that c-Abl phosphorylates this residue in vitro and confirm that Y394 is a target of c-Abl. We also show that Y276 is phosphorylated in a c-Abl-dependent manner in cultured cells and provide evidence that Y276 is phosphorylated in response to DNA damage coincident with the activation of c-Abl. Finally, we show that Y276 phosphorylation stimulates interaction with ARF, leading to increased levels of nucleolar Hdm2 and decreased turnover of p53. These data establish Y276 as a physiological target of c-Abl that contributes functionally to the induction of p53.

Agricultural practices influence flow regimes of headwater streams in western Iowa.

Agricultural tillage influences runoff and infiltration, but consequent effects on watershed hydrology are poorly documented. This study evaluated 25 yr (1971-1995) hydrologic records from four first-order watersheds in Iowa's loess hills. Two watersheds were under conventional tillage and two were under conservation (ridge) tillage, one of which was terraced. All four watersheds grew corn (Zea mays L.) every year. Flow-frequency statistics and autoregressive modeling were used to determine how conservation treatments influenced stream hydrology. The autoregressive modeling characterized variations in discharge, baseflow, and runoff at multi-year, annual, and shorter time scales. The ridge-tilled watershed (nonterraced) had 47% less runoff and 36% more baseflow than the conventional watershed of similar landform and slope. Recovery of baseflow after drought was quicker in the conservation watersheds, as evidenced by 365-d moving average plots, and 67% greater baseflow during the driest 2 yr. The two conventional watersheds were similar, except the steeper watershed discharged more runoff and baseflow during short (<30 d), wet periods. Significant multi-year and annual cycles occurred in all variables. Under ridge-till, seasonal (annual-cycle) variations in baseflow had greater amplitude, showing the seasonality of subsurface contaminant movement could increase under conservation practices. However, deviations from the modeled cycles of baseflow were also more persistent under conservation practices, indicating baseflow was more stable. Indeed, flow-frequency curves showed wet-weather discharge decreased and dry-weather discharge increased under conservation practices. Although mean discharge increased in the conservation watersheds, variance and skewness of daily values were smaller. Ridge tillage with or without terraces increased stream discharge but reduced its variability.

Nitrate leaching to subsurface drains as affected by drain spacing and changes in crop production system.

Subsurface drainage is a beneficial water management practice in poorly drained soils but may also contribute substantial nitrate N loads to surface waters. This paper summarizes results from a 15-yr drainage study in Indiana that includes three drain spacings (5, 10, and 20 m) managed for 10 yr with chisel tillage in monoculture corn (Zea mays L.) and currently managed under a no-till corn-soybean [Glycine max (L.) Merr.] rotation. In general, drainflow and nitrate N losses per unit area were greater for narrower drain spacings. Drainflow removed between 8 and 26% of annual rainfall, depending on year and drain spacing. Nitrate N concentrations in drainflow did not vary with spacing, but concentrations have significantly decreased from the beginning to the end of the experiment. Flow-weighted mean concentrations decreased from 28 mg L(-1) in the 1986-1988 period to 8 mg L(-1) in the 1997-1999 period. The reduction in concentration was due to both a reduction in fertilizer N rates over the study period and to the addition of a winter cover crop as a "trap crop" after corn in the corn-soybean rotation. Annual nitrate N loads decreased from 38 kg ha(-1) in the 1986-1988 period to 15 kg ha(-1) in the 1997-1999 period. Most of the nitrate N losses occurred during the fallow season, when most of the drainage occurred. Results of this study underscore the necessity of long-term research on different soil types and in different climatic zones, to develop appropriate management strategies for both economic crop production and protection of environmental quality.

Using the late spring nitrate test to reduce nitrate loss within a watershed.

Excessive nitrate leaching from the U.S. Corn Belt has created serious water quality problems and contributed to the expansion of the hypoxic zone in the Gulf of Mexico. We evaluated the effect of implementing the late spring nitrate test (LSNT) for corn (Zea mays L.) grown within a 400-ha, tile-drained subbasin in central Iowa. Surface water discharge and NO3 concentrations from the treated subbasin and two adjacent subbasins receiving primarily fall-applied, anhydrous ammonia were compared. In two of four years, the LSNT method significantly reduced N fertilizer applications compared with the farmers' standard practices. Average corn yield from LSNT fields and nonlimiting N fertilizer check strips was not significantly different. Autoregressive (AR) models using weekly time series in surface water NO3 concentration differences between the LSNT and control subbasins indicated no consistent significant differences during the pre-LSNT (1992-1996) period. However, by the second year (1998) of the treatment period (1997-2000), NO3 concentrations in surface water from the treated subbasin were significantly lower than the concentrations coming from both control basins. Annual average flow-weighted NO3 concentrations for the last two years (1999-2000) were 11.3 mg N L(-1) for the LSNT and subbasin and 16.0 mg N L(-1) for the control subbasins. Based on these values and the AR models, widespread adoption of the LSNT program for managing N fertilizer where fall N application is typically practiced could result in a > or = 30% decrease for NO3 concentrations in surface water.

Evaluation of nitrate nitrogen fluxes from a tile-drained watershed in central Iowa.

Nitrate N fluxes from tile-drained watersheds have been implicated in water quality studies of the Mississippi River basin, but actual NO3-N loads from small watersheds during long periods are poorly documented. We evaluated discharge and NO3-N fluxes passing the outlet of an Iowa watershed (5134 ha) and two of its tile-drained subbasins (493 and 863 ha) from mid-1992 through 2000. The cumulative NO3-N load from the catchment was 168 kg ha(-1), and 176 and 229 kg ha(-1) from the subbasins. The outlet had greater total discharge (1831 mm) and smaller flow-weighted mean NO3-N concentration (9.2 mg L(-1)) than the subbasins, while the larger subbasin had greater discharge (1712 vs. 1559 mm) and mean NO3-N concentration (13.4 vs. 11.3 mg L(-1)) than the smaller subbasin. Concentrations exceeding 10 mg L(-1) were common, but least frequent at the outlet. Nitrate N was generally not diluted by large flows, except during 1993 flooding. The outlet showed smaller NO3-N concentrations at low flows. Relationships between discharge and NO3-N flux showed log-log slopes near 1.0 for the subbasins, and 1.2 for the outlet, considering autocorrelation and measurement-error effects. We estimated denitrification of subbasin NO3-N fluxes in a hypothetical wetland using published data. Assuming that temperature and NO3-N supply could limit denitrification, then about 20% of the NO3-N would have been denitrified by a wetland constructed to meet USDA-approved criteria. The low efficiency results from the seasonal timing and NO3-N content of large flows. Therefore, agricultural and wetland best management practices (BMPs) are needed to achieve water quality goals in tile-drained watersheds.

Critical roles for the serine 20, but not the serine 15, phosphorylation site and for the polyproline domain in regulating p53 turnover.

The p53 tumour suppressor protein is a short-lived transcription factor that becomes stabilized in response to a wide range of cellular stresses. Ubiquitination and the targeting of p53 for degradation by the proteasome are mediated by Mdm2 (mouse double minute clone 2), a negative regulatory partner of p53. Previous studies have suggested that DNA-damage-induced phosphorylation of p53 at key N-terminal sites has a pivotal role in regulating the interaction with Mdm2 but the precise role of phosphorylation of serines 15 and 20 is still unclear. Here we show that replacement of serine 15 and a range of other key N-terminal phosphorylation sites with alanine, which cannot be phosphorylated, has little effect on the ubiquitination and degradation of full-length human p53. In contrast, replacement of serine 20 makes p53 highly sensitive to Mdm2-mediated turnover. These results define distinct roles for serines 15 and 20, two sites previously demonstrated to be dependent on phosphorylation through mechanisms mediated by DNA damage and ATM (ataxia telangiectasia mutated). We also show that the polyproline region of p53, a domain that has a key role in p53-induced apoptosis, exerts a critical influence over the Mdm2-mediated turnover of p53.

Nitrate loss in subsurface drainage as affected by nitrogen fertilizer rate.

The relationships between N fertilizer rate, yield, and NO3 leaching need to be quantified to develop soil and crop management practices that are economically and environmentally sustainable. From 1996 through 1999, we measured yield and NO3 loss from a subsurface drained field in central Iowa at three N fertilizer rates: a low (L) rate of 67 kg ha(-1) in 1996 and 57 kg ha(-1) in 1998, a medium (M) rate of 135 kg ha(-1) in 1996 and 114 kg ha(-1) in 1998, and a high (H) rate of 202 kg ha(-1) in 1996 and 172 kg ha(-1) in 1998. Corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] were grown in rotation with N fertilizer applied in the spring to corn only. For the L treatment, NO3 concentrations in the drainage water exceeded the 10 mg N L(-1) maximum contaminant level (MCL) established by the USEPA for drinking water only during the years that corn was grown. For the M and H treatments, NO3 concentrations exceeded the MCL in all years, regardless of crop grown. For all years, the NO3 mass loss in tile drainage water from the H treatment (48 kg N ha(-1)) was significantly greater than the mass losses from the M (35 kg N ha(-1)) and L (29 kg N ha(-1)) treatments, which were not significantly different. The economically optimum N fertilizer rate for corn was between 67 and 135 kg ha(-1) in 1996 and 114 and 172 kg ha(-1) in 1998, but the net N mass balance indicated that N was being mined from the soil at these N fertilizer levels and that the system would not be sustainable.

Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.

Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteasome. Stress signals which impinge on p53, leading to its activation, promote disruption of the p53-MDM2 complex, as in the case of ionizing radiation, or block MDM2 synthesis and thereby reduce cellular MDM2 levels, as in the case of UV radiation. It is therefore likely that MDM2, which is known to be modified by ubiquitination, SUMOylation and multi-site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser(267), which lies within the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser(267) protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser(267)-->Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser(267) is a cellular target for phosphorylation. Ser(267) mutants are still able to direct the degradation of p53, but in a slightly reduced capacity. These data highlight a potential route by which one of several physiological modifications occurring within the central acidic domain of the MDM2 protein can occur.