Effects of dietary starch and ruminally undegraded protein on glucogenic precursors in lactating dairy cows.

Gluconeogenesis is a large contributor to the blood supply of glucose carbons. The impact of varying dietary starch and ruminally degraded protein (RDP) on glucose entry, and the contributions of propionate and lactate to total plasma glucose entry were evaluated. Six cannulated, lactating, Holstein cows were fed one of four treatment diets arranged as a 2 × 2 factorial within a 4 × 4 partially replicated Latin Square design: (1) 8% RDP (LRDP) and 16% starch (LSt), (2) LRDP and 30% starch (HSt), (3) 11% RDP (HRDP) and LSt, or (4) HRDP and HSt. On d 12 of each period, 2-[13C]-sodium propionate (0.15 g/h) was ruminally infused for 4 h; on d 13, 1,2-[13C2]-glucose (0.2 g/h) was infused into the jugular vein for 1 h followed by 1-[13C]-lactate (0.1 g/h) for 1 h. Blood samples were serially collected starting prior to the infusions, and analyzed for plasma glucose, propionate, and lactate isotopic ratios. A one-compartment, glucose carbon model with inputs from lactate, propionate, and other glucogenic precursors (Oth, primarily absorbed glucose plus amino acids) was fitted to the isotope ratio data to derive glucose entry rates and conversion of the precursors to glucose. Milk protein production additively increased when HSt and HRDP were fed (P = 0.05 and P = 0.02, respectively). Plasma glucose and propionate concentrations increased with HSt (P = 0.04 and P = 0.01, respectively) and LRDP (P = 0.02 and P < 0.01, respectively). Total glucose and Oth entry increased (P = 0.03 and P = 0.03, respectively) with HSt, indicating greater glucose absorption from the small intestine or conversion of amino acids to glucose in the liver. However, neither entry rate was affected by RDP. The lack of an RDP effect suggests the increase in microbial outflow in response to RDP did not significantly alter glucose precursor supplies. Entry rates of propionate and lactate carbon to glucose carbon were not affected by treatment suggesting that neither starch nor RDP significantly affected fermentation or lactate production. Derivation of absolute entry rates and contributions to glucose using isotopic tracers is complicated by single carbon removals in the pentose phosphate (PPP), tri-carboxylic acid (TCA), and gluconeogenic pathways, and label randomization with the PPP and TCA pathways. Multiple tracers must be used to avoid assumptions regarding the proportional entries. These results provide insights on glucose supply and contributors, and draw attention to significant label cycling when utilizing isotope techniques.

Surgical Outcome and Oncological Survival of Osteofibrous Dysplasia-Like and Classic Adamantinomas: An International Multicenter Study of 318 Cases.

BACKGROUND: Osteofibrous dysplasia-like adamantinoma (OFD-AD) and classic adamantinoma (AD) are rare, neoplastic diseases with only limited data supporting current treatment protocols. We believe that our retrospective multicenter cohort study is the largest analysis of patients with adamantinoma to date. The primary purpose of this study was to describe the disease characteristics and evaluate the oncological outcomes. The secondary purpose was to identify risk factors for local recurrence after surgical treatment and propose treatment guidelines. METHODS: Three hundred and eighteen confirmed cases of OFD-AD and AD for which primary treatment was carried out between 1985 and 2015 were submitted by 22 tertiary bone tumor centers. Proposed clinical risk factors for local recurrence such as size, type, and margins were analyzed using univariable and multivariate Cox regression analysis. RESULTS: Of the 318 cases, 128 were OFD-AD and 190 were AD. The mean age at diagnosis was 17 years (median, 14.5 years) for OFD-AD and 32 years (median, 28 years) for AD; 53% of the patients were female. The mean tumor size in the OFD-AD and AD groups combined was 7.8 cm, measured histologically. Sixteen percent of the patients sustained a pathological fracture prior to treatment. Local recurrence was recorded in 22% of the OFD-AD cases and 24% of the AD cases. None of the recurrences in the OFD-AD group progressed to AD. Metastatic disease was found in 18% of the AD cases and fatal disease, in 11% of the AD cases. No metastatic or fatal disease was reported in the OFD-AD group. Multivariate Cox regression analysis demonstrated that uncontaminated resection margins (hazard ratio [HR] = 0.164, 95% confidence interval [CI] = 0.092 to 0.290, p < 0.001), pathological fracture (HR = 1.968, 95% CI = 1.076 to 3.600, p = 0.028), and sex (female versus male: HR = 0.535, 95% CI = 0.300 to 0.952, p = 0.033) impacted the risk of local recurrence. CONCLUSIONS: OFD-AD and AD are parts of a disease spectrum but should be regarded as different entities. Our results support reclassification of OFD-AD into the intermediate locally aggressive category, based on the local recurrence rate of 22% and absence of metastases. In our study, metastatic disease was restricted to the AD group (an 18% rate). We advocate wide resection with uncontaminated margins including bone and involved periosteum for both OFD-AD and AD. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Individual risk evaluation for local recurrence and distant metastasis in Ewing sarcoma: A multistate model: A multistate model for Ewing sarcoma.

BACKGROUND: We investigated the effects of surgical margins, histological response, and radiotherapy on local recurrence (LR), distant metastasis (DM), and survival in Ewing sarcoma. PROCEDURE: Disease evolution was retrospectively studied in 982 patients with Ewing sarcoma undergoing surgery after chemotherapy using a multistate model with initial state surgery, intermediate states LR, pulmonary metastasis (DMpulm), other DM ± LR (DMother), and final state death. Effect of risk factors was estimated using Cox proportional hazard models. RESULTS: The median follow-up was 7.6 years (95% CI, 7.2-8.0). Risk factors for LR are pelvic location, HR 2.04 (1.10-3.80), marginal/intralesional resection, HR 2.28 (1.25-4.16), and radiotherapy, HR 0.52 (0.28-0.95); for DMpulm the risk factors are <90% necrosis, HR 2.13 (1.13-4.00), and previous pulmonary metastasis, HR 4.90 (2.28-8.52); for DMother are 90% to 99% necrosis, HR 1.56 (1.09-2.23), <90% necrosis, HR 2.66 (1.87-3.79), previous bone/other metastasis, HR 3.08 (2.03-4.70); and risk factors for death without LR/DM are pulmonary metastasis, HR 8.08 (4.01-16.29), bone/other metastasis, HR 10.23 (4.90-21.36), and <90% necrosis, HR 6.35 (3.18-12.69). Early LR (0-24 months) negatively influences survival, HR 3.79 (1.34-10.76). Once DMpulm/DMother arise only previous bone/other metastasis remain prognostic for death, HR 1.74 (1.10-2.75). CONCLUSION: Disease extent and histological response are risk factors for progression to DM or death. Tumor site and surgical margins are risk factors for LR. If disease progression occurs, previous risk factors lose their relevance. In case of isolated LR, time to recurrence is important for decision-making. Radiotherapy seems protective for LR especially in pelvic/axial. Low percentages of LR in extremity tumors and associated toxicity question the need for radiotherapy in extremity Ewing sarcoma.

Surgical Treatment of Localized-Type Tenosynovial Giant Cell Tumors of Large Joints: A Study Based on a Multicenter-Pooled Database of 31 International Sarcoma Centers.

BACKGROUND: Localized-type tenosynovial giant cell tumor (TGCT) is a rare, neoplastic disease with only limited data supporting treatment protocols. We describe treatment protocols and evaluate their oncological outcome, complications, and functional results in a large multicenter cohort of patients. A secondary study aim was to identify factors associated with local recurrence after surgical treatment. METHODS: Patients with histologically proven localized TGCT of a large joint were included if they had been treated between 1990 and 2017 in 1 of 31 tertiary sarcoma centers. Of 941 patients with localized TGCT, 62% were female. The median age at initial treatment was 39 years, and the median duration of follow-up was 34 months. Sixty-seven percent of the tumors affected the knee, and the primary treatment at the tertiary center was 1-stage open resection in 73% of the patients. Proposed factors for predicting a first local recurrence after treatment in the tertiary center were tested in a univariate analysis, and those that demonstrated significance were subsequently included in a multivariate analysis. RESULTS: The localized TGCT recurred in 12% of all cases, with local-recurrence-free rates at 3, 5, and 10 years of 88%, 83%, and 79%, respectively. The strongest factor for predicting recurrent disease was a prior recurrence (p < 0.001). Surgical treatment decreased pain and swelling in 71% and 85% of the patients, respectively, and such treatment was associated with complications in 4% of the patients. Univariate and multivariate analyses of the patients who had not undergone therapy previously yielded positive associations between local recurrence and a tumor size of ≥5 cm versus <5 cm (hazard ratio [HR] = 2.50; 95% confidence interval [CI] = 1.32 to 4.74; p = 0.005). Arthroscopy (versus open surgery) was significantly associated with tumor recurrence in the univariate analysis (p = 0.04) but not in the multivariate analysis (p = 0.056). CONCLUSIONS: Factors associated with recurrence after resection of localized-type TGCT were larger tumor size and initial treatment with arthroscopy. Relatively low complication rates and good functional outcomes warrant an open approach with complete resection when possible to reduce recurrence rates in high-risk patients. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Easy-to-use clinical tool for survival estimation in Ewing sarcoma at diagnosis and after surgery.

Accurate survival estimations in Ewing sarcoma are necessary to develop risk- and response adaptive treatment strategies allowing for early decision-making. We aim to develop an easy-to-use survival estimation tool from diagnosis and surgery. A retrospective study of 1314 Ewing sarcoma patients was performed. Associations between prognostic variables at diagnosis/surgery and overall survival (OS), were investigated using Kaplan-Meier and multivariate Cox models. Predictive accuracy was evaluated by cross-validation and Harrell C-statistics. Median follow-up was 7.9 years (95%CI 7.6-8.3). Independent prognostic factors at diagnosis were age, volume, primary tumor localization and disease extent. 5 risk categories (A-E) were identified with 5-year OS of 88% (86-94), 69% (64-74), 57% (50-64), 51% (42-60) and 28% (22-34) respectively. Harrell C-statistic was 0.70. Independent prognostic factors from surgery were age, volume, disease extent and histological response. In categories A-B, 5y OS increased to 92% (87-97) and 79% (71-87) respectively for 100% necrosis and decreased to 76% (67-85) and 62% (55-69) respectively for <100% necrosis. In categories C-E, 5y OS increased to 65% (55-75), 65% (52-78) and 52% (38-66) respectively for ≥90% necrosis and decreased to 38% (22-54), 11% (0-26) and 7% (0-19) respectively for <90% necrosis. We present an easy-to-use survival estimation tool from diagnosis in Ewing sarcoma based on age, volume, primary tumor localization and disease extent. Histological response is a strong additional prognostic factor for OS.

Dynamic prediction of overall survival for patients with high-grade extremity soft tissue sarcoma.

PURPOSE: There is increasing interest in personalized prediction of disease progression for soft tissue sarcoma patients. Currently, available prediction models are limited to predictions from time of surgery or diagnosis. This study updates predictions of overall survival at different times during follow-up by using the concept of dynamic prediction. PATIENTS AND METHODS: Information from 2232 patients with high-grade extremity soft tissue sarcoma, who underwent surgery at 14 specialized sarcoma centers, was used to develop a dynamic prediction model. The model provides updated 5-year survival probabilities from different prediction time points during follow-up. Baseline covariates as well as time-dependent covariates, such as status of local recurrence and distant metastases, were included in the model. In addition, the effect of covariates over time was investigated and modelled accordingly in the prediction model. RESULTS: Surgical margin and tumor histology show a significant time-varying effect on overall survival. The effect of margin is strongest shortly after surgery and diminishes slightly over time. Development of local recurrence and distant metastases during follow-up have a strong effect on overall survival and updated predictions must account for their occurrence. CONCLUSION: The presence of time-varying effects, as well as the effect of local recurrence and distant metastases on survival, suggest the importance of updating predictions during follow-up. This newly developed dynamic prediction model which updates survival probabilities over time can be used to make better individualized treatment decisions based on a dynamic assessment of a patient's prognosis.

Incidence, outcomes and prognostic factors during 25 years of treatment of chondrosarcomas.

BACKGROUND: There are few studies detailing the incidence, patient outcomes and prognostic factors for chondrosarcomas (CS). Those that do exist have small sample sizes and/or use older datasets. The purpose of this study was to determine the incidence, overall survival (OS) and prognostic factors for OS of CS patients, as well as investigate the efficacy of curettage. METHODS: We analyzed data of 2186 patients diagnosed with chondrosarcomas between '89-'13 from the Netherlands Cancer Registry. The effect of risk factors on OS was assessed with a multivariate Cox regression. Median Follow-up was determined with reversed Kaplan-Meier. OS was estimated using Kaplan-Meier method. RESULTS: The relative incidence of CS was 2.88 per million citizens between '89-'96, 4.15 between '96-'04 and 8.78 between '05-'13. Most of the increase in incidence came from atypical cartilaginous tumours/grade I chondrosarcoma (ACT/CS I). The 3-, 5- and 10-years survival were, respectively, 96%, 93% and 88% for ACT/CS I, 82%, 74% and 62% for grade II CS and 38%, 31% and 26% for grade III CS. Prognostics factors significantly associated with OS were age, histological grade, year of diagnosis, tumour location and size. CONCLUSION: The incidence of CS, and especially ACT/CS I, has increased over time, which could be driven by both an ageing population and increased diagnostic imaging. With the increased number of diagnosed ACT/CS I, the number of preventative curettages of this tumour has also increased. Despite the supposed preventative character of this treatment, the incidence of high-grade CS did not decrease.

[Parental Information and Consent in Neonatology]. / Aufklärung in der Neonatologie.

Careful analysis of current adjudication reveals increasing demand of adequate record-keeping as well as meticulously documented informed consent forms regarding all aspects of medicine. Although standardized informed consent forms or explicit guidelines for obtaining procedural consent already exist in surgical disciplines there is strong evidence that, however, in neonatology (and paediatric intensive care) these processes are still incomplete and qualitatively insufficiently implemented. Therefore the author discussed all existing information prescriptions with the legal department and quality management of a large German clinic group especially in terms of relevant legislation, recent case law and specialist literature in order to obtain potential for improvement. Based on the results of this audit of expert opinions improved recommendations could be implemented in the daily practise of a department of neonatology and paediatric intensive care on a tertiary level.

Structural and functional characterization of a winter malting barley.

The development of winter malting barley (Hordeum vulgare L.) varieties is emerging as a worldwide priority due to the numerous advantages of these varieties over spring types. However, the complexity of both malting quality and winter hardiness phenotypes makes simultaneous improvement a challenge. To obtain an understanding of the relationship between loci controlling winter hardiness and malt quality and to assess the potential for breeding winter malting barley varieties, we structurally and functionally characterized the six-row accession "88Ab536", a cold-tolerant line with superior malting quality characteristics that derives from the cross of NE76129/Morex//Morex. We used 4,596 SNPs to construct the haplotype structure of 88Ab536 on which malting quality and winter hardiness loci reported in the literature were aligned. The genomic regions determining malting quality and winter hardiness traits have been defined in this founder germplasm, which will assist breeders in targeting regions for marker-assisted selection. The Barley1 GeneChip array was used to functionally characterize 88Ab536 during malting. Its gene expression profile was similar to that of the archetypical malting variety Morex, which is consistent with their similar malting quality characteristics. The characterization of 88Ab536 has increased our understanding of the genetic relationships of malting quality and winter hardiness, and will provide a genetic foundation for further development of more cold-tolerant varieties that have malt quality characteristics that meet or exceed current benchmarks.

Psychodynamic vulnerability factors in the development of panic disorders--a prospective trial in patients after vestibular neuritis.

BACKGROUND: In our 2-year prospective study of 80 patients admitted consecutively to our clinic with an episode of acute vestibular neuritis, a total of 8 patients later developed a panic disorder according to DSM-III-R criteria. The goal of our analysis was to determine whether certain conflict patterns (e.g. in the area of autonomy vs. dependence) or deficient psychological structure could predict later panic disorder, as might be expected based on psychodynamic theory. SAMPLING AND METHODS: Between 4 and 8 weeks after the acute vestibular episode, we evaluated all patients using operationalized psychodynamic diagnostics (OPD). With the different axes of the OPD system, we were able to assess patients' experience of illness (Axis I), potential conflicts (Axis III), and psychological structure (Axis IV) in a semiquantitative manner. RESULTS AND CONCLUSIONS: Poor psychosocial integration, a lack of social support, a high burden of suffering, and moderate to severe impairment of self-experience were able to account for 32.1% (Nagelkerkes R(2)=0.321) of variance in the development of panic disorder over the course of 2 years. However, contrary to what might have been expected based on psychodynamic theory, patients who later developed a panic disorder did not exhibit any differences in their Axis III or IV scores compared to patients who remained psychologically healthy.

Fine mapping of a malting-quality QTL complex near the chromosome 4H S telomere in barley.

Malting quality has long been an active objective in barley (Hordeum vulgare L.) breeding programs.However, it is difficult for breeders to manipulate malting-quality traits because of inheritance complexity and difficulty in evaluation of these quantitative traits. Quantitative trait locus (QTL) mapping provides breeders a promising basis with which to manipulate quantitative trait genes. A malting-quality QTL complex, QTL2, was mapped previously to a 30-cM interval in the short-arm telomere region of barley chromosome 4H in a "Step-toe"/"Morex" doubled haploid population by the North American Barley Genome Project, using an interval mapping method with a relatively low-resolution genetic map. The QTL2 complex has moderate effects on several malting-quality traits, including malt extract percentage(ME), a-amylase activity (AA), diastatic power (DP), malt 13-glucan content (BG), and seed dormancy, which makes it a promising candidate gene source in malting barley-cultivar development. Fine mapping QTL2 is desirable for precisely studying barley malting-quality trait inheritance and for efficiently manipulating QTL2 in breeding. A reciprocal-substitution mapping method was employed to fine map QTL2. Molecular marker-assisted backcrossing was used to facilitate the generation of isolines. Fourteen different types of "Steptoe" isolines, including regenerated "Steptoe" and 13 different types of "Morex" isolines,including regenerated "Morex", were made within a 41.5-cM interval between MWG634 and BCD265B on chromosome 4H. Duplicates were identified for 12 "Steptoe" and 12 "Morex" isoline types. The isolines together with "Steptoe" and "Morex" were grown variously at three locations in 2 years for a total of five field environments.Four malting-quality traits were measured: ME, DP, AA,and BG. Few significant differences were found between duplicate isolines for these traits. A total of 15 putative QTLs were mapped; three for ME, four for DP, six for AA,and two for BG. Background genotype seemed to make a difference in expression/detection of QTLs. Of the 15 QTLs identified, ten were from the "Morex" and only five from the "Steptoe" background. By combining the results from different years, field environments, and genetic backgrounds and taking into account overlapping QTLsegments, six QTLs can be conservatively estimated: two each for ME and AA and one each for DP and BG with chromosome segments ranging from 0.7 cM to 27.9 cM. A segment of 15.8 cM from the telomere (MWG634-CDO669) includes all or a portion of all QTLs identified. Further study and marker-assisted breeding should focus on this 15.8-cM chromosome region.

Effect of reducing and oxidizing agents and pH on malt endoproteolytic activities and brewing mashes.

The activities of the four endoproteinase classes of malted barley are known to vary with pH, and it seemed likely that the cysteine enzyme activities could be altered by redox agents. This study determined how altering the pH and adding redox agents to mashes influenced the worts that were produced during the brewing process. The reducing agents cysteine.HCl, dithiothreitol, and beta-mercaptoethanol increased the proteolysis that occurred in malt extracts and mashes. This increased proteolysis was negated by the addition of the oxidizing agents diamide or hydrogen peroxide. The addition of reducing agents to mashes increased the soluble protein, free amino nitrogen (FAN), and extract values of their resultant worts, and this effect was abolished by the concomitant addition of oxidizing agents. Raising the pH values of the mashes strongly reduced their proteolytic activities, soluble protein, FAN, and extract values, but not their beta-glucan levels. These results show that several of the major aspects of malting and brewing quality can be adjusted by varying the pH and redox qualitites of mashes, which could be helpful to brewers. These results also strengthen the previous proposal made by Buchanan et al. that the redox status of plants may play a significant part in controlling their physiology.

Characterization of the Ustilago maydis sid2 gene, encoding a multidomain peptide synthetase in the ferrichrome biosynthetic gene cluster.

Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation of L-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase. sid1 encodes L-ornithine N(5)-oxygenase, which catalyzes hydroxylation of L-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. In this report we characterize sid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream of sid1 and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulated sid2 transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption of urbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis in Ustilago. Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1 of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation of sid1, altered the differential regulation of sid2 by iron.

Two histone fold proteins, CHRAC-14 and CHRAC-16, are developmentally regulated subunits of chromatin accessibility complex (CHRAC).

The ISWI ATPase of Drosophila is a molecular engine that can drive a range of nucleosome remodelling reactions in vitro. ISWI is important for cell viability, developmental gene expression and chromosome structure. It interacts with other proteins to form several distinct nucleosome remodelling machines. The chromatin accessibility complex (CHRAC) is a biochemical entity containing ISWI in association with several other proteins. Here we report on the identification of the two smallest CHRAC subunits, CHRAC-14 and CHRAC-16. They contain histone fold domains most closely related to those found in sequence-specific transcription factors NF-YB and NF-YC, respectively. CHRAC-14 and CHRAC-16 interact directly with each other as well as with ISWI, and are associated with functionally active CHRAC. The developmental expression profiles of both subunits suggest specialized roles in chromatin remodelling reactions in the early embryo for both histone fold subunits.

Dielectric changes in membrane properties and cell interiors of human mesothelial cells in vitro after crocidolite asbestos exposure.

Asbestos induces cytogenetic and genotoxic effects in cultured cell lines in vitro. For further investigations of the fiber-induced cellular changes, electrorotation (ROT) measurements can be used to determine early changes of surface properties and dielectric cellular changes. In the present study, human mesothelial cells (HMC) were exposed to nontoxic concentrations of crocidolite asbestos (1 microg/cm(2)) for 12, 24, 30, 50, and 72 hr, and were investigated for changes in dielectric properties, morphologic and biochemical changes using ROT measurements, electron microscopy, and flow cytometry, respectively. The results of ROT measurements revealed slightly increased internal conductivity and decreased membrane conductance of HMC during the first 12 hr of exposure to crocidolite. This may be due to functional changes of ion channels of the cellular membrane. However, after exposures of >= 30 hr, reduced internal conductivity and increased membrane conductance of HMC occurred. These effects may be caused by permeabilization of the cell membrane and the leakage of ions into the surrounding medium. The membrane capacitance of HMC is always decreased during exposure of cells to crocidolite fibers. This decreased membrane capacitance may result from the observed reduction in the number of microvilli and from the shrinkage of cells as observed by electron microscopy and flow cytometry. Changes in composition of the plasma membrane were also observed after the labeling of phosphatidylserines (PS) on the cell surface. These observed changes can be related to apoptotic events. Whereas during the first 50 hr of exposure only a small number of HMC with increased exposure of PS on the cell surface was detected by flow cytometry, the dielectric properties of HMC showed marked changes during this time. Our results show that surface property changes of the cellular membrane of HMC as well as interior dielectric changes occur after the exposure of cells to crocidolite fibers. The observed changes are discussed in terms of complex combined cellular effects after amphibole asbestos exposure.

p53 represses ribosomal gene transcription.

Induction of the tumor suppressor protein p53 restricts cellular proliferation. Since actively growing cells require the ongoing synthesis of ribosomal RNA to sustain cellular biosynthesis, we studied the effect of p53 on ribosomal gene transcription by RNA polymerase I (Pol I). We have measured rDNA transcriptional activity in different cell lines which either lack or overexpress p53 and demonstrate that wild-type but not mutant p53 inhibits cellular pre-rRNA synthesis. Conversely, pre-rRNA levels are elevated both in cells which express mutant p53 and in fibroblasts from p53 knock-out mice. Transient transfection assays with a set of rDNA deletion mutants demonstrate that intergenic spacer sequences are dispensable and the minimal rDNA promoter is sufficient for p53-mediated repression of Pol I transcription. However, in a cell-free transcription system, recombinant p53 does not inhibit rDNA transcription, indicating that p53 does not directly interfere with the basal Pol I transcriptional machinery. Thus, repression of Pol I transcription by p53 may be a consequence of p53-induced growth arrest.

A method for overcoming the ceiling effect of bounded pain scales.

OBJECTIVE: The Verbal Numerical Scale (VNS) for rating pain is bounded between 0 (= no pain) and 10 (= worst pain imaginable). We hypothesized that the limitations inherent to this boundary when rating extremely painful stimuli may be identified by integrating the VNS with an unbounded score such as magnitude estimation of relative change. METHODS: Volunteers received stimuli of increasing current via cutaneous electrodes until they rated >5 on the VNS scale. This stimulus, termed S, was arbitrarily assigned a magnitude estimate of 100%. Then, stimuli of varying currents were delivered; two were 10 mA and 20 mA higher than S (S(+10) and S(+20)), two were 1/2 of the current for the S stimulus (S(1/2)), and one was at the original current (Srepeat). The pain elicited by each stimulus was scored in proportion to the S stimulus. The extrapolated VNS score (VNSext) was determined by multiplying this magnitude estimate (%) by the VNS score for S. MAIN RESULTS: Seventy percent of the stimuli with higher intensity than S generated a VNSext score above 10. The mean magnitude estimations for S(+10) and S(+20) were 186% and 242%: they generated mean (median) VNSext values of 12.4 and 16.2, respectively (p = 0.019 for the difference between them by Wilcoxon signed rank test). CONCLUSIONS: The combined use of VNS and magnitude estimation confirmed that the ceiling of the bounded pain scale may significantly limit a patient's ability to describe a new pain stimulus. VNSext may provide a means of overcoming this limitation.