A rapid and visual loop-mediated isothermal amplification assay to detect Leifsonia xyli subsp. xyli targeting a transposase gene.

UNLABELLED: Leifsonia xyli subsp. xyli (Lxx), causal organism of ratoon stunt (RSD), does not produce any reliable internal or external symptoms on sugarcane. Its detection on a large scale is solely based on microscopic and serological methods. These methods require well-equipped laboratories, are time consuming and are not feasible for near-field detection of Lxx. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of Lxx without the use of sophisticated equipment. To the best of our knowledge, this is the first report on the detection of Lxx in 30 min via an isothermal amplification method at 65°C. A transposase gene, ISLxx5, was used to design a set of six primers specifically targeting eight genomic sequences. The xylem sap was used as template, thus circumventing the need to isolate pure genomic DNA. The positive reactions were visually detected through a colour change of hydroxynaphthol blue (HNB) from violet to light blue, thus, eliminating the need for gel electrophoresis. The LAMP method was 10 times more sensitive than serological detection and as sensitive as immunofluorescence microscopy (IFM). The simplicity and sensitivity of the ISLxx5 LAMP assay makes it suitable for near-field diagnosis of RSD. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection of Leifsonia xyli subsp. xyli (Lxx) on a large scale is based on serological assays such as evaporative-binding enzyme-linked immunoassay (EB-EIA). These methods are time consuming and require well-equipped laboratories. This study presents the development of a loop-mediated isothermal amplification (LAMP) assay which allows detection of Lxx in 30 min at 65°C, using xylem sap as the template. The assay requires minimal laboratory equipment and could be used at near farm conditions, thus saving time and money required to transfer samples from remote areas to diagnostic laboratories. The LAMP method shows potential as an alternative detection method for RSD.

Enhanced expression of the PDGFR/Abl signaling pathway in aromatase inhibitor-resistant breast cancer.

BACKGROUND: We have found that the platelet-derived growth factor receptor (PDGFR)/Abl signaling pathway is up-regulated as a determinant of the acquisition of resistance to estrogen deprivation in vitro. We aimed to determine its clinical relevance in aromatase inhibitor (AI)-resistant breast cancer. PATIENTS AND METHODS: We identified a cohort of 45 patients with estrogen receptor-positive breast cancer who had been treated with an AI, subsequently relapsed and had biopsy material available from both the presentation and post-AI recurrent lesion. PDGFRα, PDGFRß and Abl expression was assessed in formalin-fixed paraffin-embedded sections. RESULTS: Tumor protein expression of PDGFRα (1.39-fold, P=0.0065), PDGFRß (4.32-fold, P=0.006) and Abl (1.8-fold, P=0.001) was increased at the point of relapse. Tumor and stromal expression of PDGFRα as well as PDGFRß was significantly correlated in pre-treatment and relapse samples. High post-treatment tumor and stromal PDGFRß levels were associated with a short time to treatment failure (TTF). Expression of PDGFRα in relapsing tumor specimens was correlated with Abl expression and Ki67 levels. Furthermore, changes in Abl correlated significantly with changes in ER expression. CONCLUSIONS: These clinical data support a role for enhanced PDGF/Abl signaling in AI-resistant disease and provide a rationale for targeting the pathway in endocrine-resistant breast cancer.

Polychlorinated biphenyl congeners that increase the glucuronidation and biliary excretion of thyroxine are distinct from the congeners that enhance the serum disappearance of thyroxine.

Polychlorinated biphenyl (PCB) congeners differentially reduce serum thyroxine (T(4)) in rats, but little is known about their ability to affect biliary excretion of T(4). Thus, male Sprague-Dawley rats were orally administered Aroclor-1254, Aroclor-1242 (32 mg/kg per day), PCB-95, PCB-99, PCB-118 (16 mg/kg per day), PCB-126 (40 µg/kg per day), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (3.9 µg/kg per day), or corn oil for 7 days. Twenty-four hours after the last dose, [(125)I]T(4) was administered intravenously, and blood, bile, and urine samples were collected for quantifying [(125)I]T(4) and in bile [(125)I]T(4) metabolites. Serum T(4) concentrations were reduced by all treatments, but dramatic reductions occurred in response to Aroclor-1254, PCB-99 [phenobarbital (PB)-type congener], and PCB-118 (mixed-type congener). None of the treatments increased urinary excretion of [(125)I]T(4). Aroclor-1254, PCB-118, TCDD, and PCB-126 (TCDD-type congener) increased biliary excretion of T(4)-glucuronide by 850, 756, 710, and 573%, respectively, corresponding to marked induction of hepatic UDP-glucuronosyltransferase (UGT) activity toward T(4). PCB-95 and PCB-99 did not induce UGT activity; therefore, the increased biliary excretion of T(4)-glucuronide was related to the affinity of congeners for the aryl hydrocarbon receptor. The disappearance of [(125)I]T(4) from serum was rapid (within 15-min) and was increased by Aroclor-1254, PCB-99 and PCB-118. Thus, reductions in serum T(4) in response to PCBs did not always correspond with UGT activity toward T(4) or with increased biliary excretion of T(4)-glucuronide. The rapid disappearance of [(125)I]T(4) from the serum of rats treated with PB-like PCBs suggests that increased tissue uptake of T(4) is an additional mechanism by which PCBs may reduce serum T(4).

Therapeutic optimization through goal-oriented prescription in nursing homes.

Background People living in nursing homes are highly vulnerable and frail. Polypharmacy and inappropriate prescription (IP) are also common problems. Objectives The objectives of the study are (i) to study the baseline situation and calculate the frailty index (FI) of the residents, (ii) to assess the results of routine clinical practice to do a pharmacotherapy review (patient-centred prescription (PCP) model) (Molist Brunet et al., Eur Geriatr Med. 2015;6:565-9) and (iii) to study the relationship between IP and frailty, functional dependence, advanced dementia and end-of-life situation. Setting Two nursing homes in the same geographical area in Catalonia (Spain). Method This was a prospective, descriptive and observational study of elderly nursing home residents. Each patient's treatment was analysed by applying the PCP model, which centres therapeutic decisions on the patient's global assessment and individual therapeutic goal. Main outcome measure Prevalence of polypharmacy and IP. Results 103 patients were included. They were characterized by high multimorbidity and frailty. Up to 59.2% were totally dependent. At least one IP was identified in 92.2% of residents. Prior to the pharmacological review, the mean number of chronic medications prescribed per resident was 6.63 (SD 2.93) and after this review it was 4.97 (SD 2.88). Polypharmacy decreased from 72.55% to 52.94% and excessive polypharmacy fell from 18.62% to 5.88%.The highest prevalence of IP was detected in people with a higher FI, in those identified as end-of-life, and also in more highly dependent residents (p < 0.05). Conclusions People who live in nursing homes have an advanced frailty. Establishing individualized therapeutic objectives with the application of the PCP model enabled to detect 92.2% of IP. People who are frailer, are functionally more dependent and those who are end-of-life are prescribed with inappropriate medication more frequently.

Endourological treatment for lumbar ureteral stones. What are the benefits of flexible ureterorenoscopy?

INTRODUCTION AND OBJECTIVES: Currently, there are no established criteria regarding treatment for lumbar ureteral stones. The objective of this work is to present our results in the endourological treatment of this pathology, analyzing the variables associated with the use of the flexible ureterorenoscope. MATERIAL AND METHODS: Retrospective review of 103 patients who underwent retrograde URS with semi-rigid or flexible ureterorenoscope. Proximal location: L2-L3. Medial location: L4-L5. Semirigid URS was the initial treatment, with conversion to flexible URS when it was required to complete the procedure. Success was defined as absence of residual fragments (6 weeks). Demographic, surgical, immediate postoperative variables, and those related to the stone, were analyzed. Their correlation with the use of the flexible ureterorenoscope was evaluated. RESULTS: Mean age: 57.2 years (SD 15.6); there were 73 men (70.9%). Stone size: 8 mm (range 4-30; IQR 4.5). Proximal location: 58 (56.3%). Previous JJ: 44.7%. Previous nephrostomy: 10.7%. Semirigid URS with conversion to flexible URS: 51 (49.5%). Impacted stones: 28.2%. Intraoperative complications: 2 (1.9%). Postoperative JJ: 84.5%. Immediate postoperative complications: 23 (22.3%) (Clavien-Dindo I-II: 91.3%). Postoperative ureteral stricture: 5.8%. Success: 88.4%. Residual fragments: 12 (11.7%). Spontaneous passage: 6 (50%). Greater performance of flexible URS in proximal ureteral stones (p = 0.001) of more than 11 mm (p = 0.02) in univariate analysis, and in proximal stones [OR 3.5; 1.5-8.1; p = 0.004] in multivariate analysis. CONCLUSIONS: Endourological treatment obtained a high success rate in our sample. Size greater than 11 mm and proximal ureteral location in univariate and multivariate analysis, respectively, behaved as predictors of flexible URS.

Endourological treatment for lumbar ureteral stones. What are the benefits of flexible ureterorenoscopy? / Tratamiento endourológico de la litiasis ureteral lumbar. ¿Qué aporta la ureterorrenoscopia flexible?

INTRODUCTION AND OBJECTIVES: Currently, there are no established criteria regarding treatment for lumbar ureteral stones. The objective of this work is to present our results in the endourological treatment of this pathology, analyzing the variables associated with the use of the flexible ureterorenoscope. MATERIAL AND METHODS: Retrospective review of 103 patients who underwent retrograde URS with semi-rigid or flexible ureterorenoscope. Proximal location: L2-L3. Medial location: L4-L5. Semirigid URS was the initial treatment, with conversion to flexible URS when it was required to complete the procedure. Success was defined as absence of residual fragments (6 weeks). Demographic, surgical, immediate postoperative variables, and those related to the stone, were analyzed. Their correlation with the use of the flexible ureterorenoscope was evaluated. RESULTS: Mean age: 57.2 years (SD 15.6); there were 73 men (70.9%). Stone size: 8mm (range 4-30; IQR 4.5). Proximal location: 58 (56.3%). Previous JJ: 44.7%. Previous nephrostomy: 10.7%. Semirigid URS with conversion to flexible URS: 51 (49.5%). Impacted stones: 28.2%. Intraoperative complications: 2 (1.9%). Postoperative JJ: 84.5%. Immediate postoperative complications: 23 (22.3%) (Clavien-Dindo I-II: 91.3%). Postoperative ureteral stricture: 5.8%. Success: 88.4%. Residual fragments: 12 (11.7%). Spontaneous passage: 6 (50%). Greater performance of flexible URS in proximal ureteral stones (P=0.001) of more than 11mm (P=0.02) in univariate analysis, and in proximal stones [OR 3.5; 1.5-8.1; P=0.004] in multivariate analysis. CONCLUSIONS: Endourological treatment obtained a high success rate in our sample. Size greater than 11mm and proximal ureteral location in univariate and multivariate analysis, respectively, behaved as predictors of flexible URS.

EGFR/HER2 inhibitor AEE788 increases ER-mediated transcription in HER2/ER-positive breast cancer cells but functions synergistically with endocrine therapy.

BACKGROUND: Cross-talk between receptor tyrosine kinases and the oestrogen receptor (ER) is implicated in resistance to endocrine therapy. We investigated whether AEE788 (a combined inhibitor of EGFR, HER2 and VEGFR) plus tamoxifen or letrozole enhanced the individual anti-tumour effects of these agents. METHODS: Breast cancer cell lines modelling endocrine-resistant and -sensitive disease were engineered to express aromatase (A) and examined using proliferation, western blotting and ER-alpha transcription assays. RESULTS: AEE788 enhanced the anti-proliferative effect of tamoxifen and letrozole in ER(+) cell lines (MCF-7 2A, ZR75.1 A3 and BT474 A3). This associated with an elevated G1 arrest and nuclear accumulation of p27. It is noteworthy that AEE788 alone or in combination with endocrine therapy increased the expression of progesterone receptor (PGR) and TFF1 in BT474 A3 cells. This may indicate a mechanism of resistance to AEE788 in ER(+)/HER2(+) breast cancers. In a ZR75.1 A3 xenograft, AEE788 alone or in combination with tamoxifen provided no further benefit compared with letrozole. However, letrozole plus AEE788 produced a significantly greater inhibition of tumour growth compared with letrozole alone. CONCLUSION: These data suggest that AEE788 plus letrozole in breast cancer overexpressing HER2 may provide superior anti-tumour activity, compared with single agents.

[Eating behaviour and body image in a sample of adolescents from Sevilla]. / Conducta alimentaria e imagen corporal en una muestra de adolescentes de Sevilla.

OBJECTIVE: This study examined the presence of disordered eating behaviours and the influence that on them could have the degree of body dissatisfaction among adolescents. METHOD: By the Eating Attitudes Test-40 (EAT-40), the Sick Control On Fat Food (SCOFF) and the subscale of body dissatisfaction (BD) of the Eating Disorders Inventory-2 (EDI-2) a total of 841 students, aged 12-19, were studied. Eating behaviours, sex and age differences, and eating attitudes and behaviours related to the degree of body dissatisfaction were analized. RESULTS: We found that 21,29% had significant punctuations in the SCOFF and 7,13% in the EAT-40. There were significant sex-differences (13,93% and 3,23% in SCOFF and EAT-40 for males, 29,38% and 10,70% for women). With regard to previous studies, a decrease of the risk is observed in women and an increase in males. Major body dissatisfaction was observed among the 12 to 17-year-old girls, though sex-differences in eating alterations, can be mostly found between the ages of 14 and 16. Body dissatisfaction correlated positively and significantly to Body Mass Index, EAT-40 and SCOFF. CONCLUSION: In order to implement primary programs in the adolescent population it is necessary to explore the eating behaviours of risky and the degree of body dissatisfaction to be able to raise specifically the interventions to be carried out, involving teachers as primary agents for the work in the school context.

The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling.

Long-term culture of MCF-7 wild-type (wt) cells in steroid-depleted medium (LTED) results in hypersensitivity to oestradiol (E2) coinciding with elevated levels of ERalpha and enhanced growth factor signalling. In this study, we aimed to compare the effects of the pure anti-oestrogen ICI 182,780 (ICI) with the competitive anti-oestrogen tamoxifen (TAM) on oestrogen and IGF signalling in these cells. Wt MCF-7 and LTED cells were treated with a log 7 concentration range of E2, TAM or ICI. Effects on cell growth, ERalpha transactivation, expression of ERalpha, ERbeta and components of the IGF pathway were measured with and without insulin. In the presence of insulin, growth of LTED cells was refractory to TAM but inhibited by ICI and E2. In the absence of insulin, LTED cells showed persistent hypersensitivity to E2, and remained inhibited by ICI but were largely unaffected by TAM. ICI but not TAM inhibited ER-mediated gene transcription and treatment with ICI resulted in a dose-dependent reduction in ERalpha levels whilst having no effect on ERbeta expression. IGF-I receptor and insulin receptor substrate 2 levels were increased in LTED versus the Wt MCF-7 cells, and ICI but not TAM reduced their expression in a dose-dependent fashion. Thus IGF signalling as well as ERalpha expression and function are enhanced during LTED. While the resultant cells are resistant to TAM, ICI down-regulates ERalpha, reducing IGF signalling and cell growth. These results support the use of ICI in women with ER-positive breast cancer who have relapsed on an aromatase inhibitor.

Growth factor signalling and response to endocrine therapy: the Royal Marsden Experience.

De novo resistance to endocrine therapy is a near-universal feature of oestrogen receptor (ER)- negative breast cancer. Although many ER-positive breast cancers also show no response to tamoxifen or aromatase inhibitors on objective clinical grounds the large majority show reduced proliferation indicating that some oestrogen dependence is present in almost all ER-positive breast cancer. In neoadjuvant studies HER2 positivity is associated with poor response rates to tamoxifen but not aromatase inhibitors, consistent with preclinical models. Acquired resistance to tamoxifen is associated with decreases in ER positivity but most recurrent lesions remain ER-positive. A small proportion of these show increased HER2 expression and in these patients increased phospho-p38 may contribute to the tamoxifen-resistant phenotype. There is an unfortunate paucity of clinical and biological data on acquired resistance to aromatase inhibitors.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

Genetic prodrug activation therapy for breast cancer: A phase I clinical trial of erbB-2-directed suicide gene expression.

PURPOSE: This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. PATIENTS AND METHODS: Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2-overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. RESULTS: The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2-positive tumor cells, confirming the selectivity of the approach. CONCLUSION: The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.

Survival comparison of radiosurgery-eligible and -ineligible malignant glioma patients treated with hyperfractionated radiation therapy and carmustine: a report of Radiation Therapy Oncology Group 83-02.

PURPOSE: The purpose is twofold: (1) to identify the malignant glioma patients treated in a trial of hyperfractionated radiotherapy (RT) and carmustine (BCNU) who may have been eligible for a stereotactic radiosurgery (SRS) boost; and (2) to compare survival of such patients with that of those considered SRS-ineligible. PATIENTS AND METHODS: From January 1983 to July 1989, 778 malignant glioma patients were enrolled on Radiation Therapy Oncology Group (RTOG) 83-02, a randomized phase I/II hyperfractionated RT dose-escalation trial with BCNU chemotherapy. The SRS criteria used in a single-institution trial were applied to these patients; they are: Karnofsky performance status (KPS) of greater than 60; well-circumscribed tumor less than 4.0 cm; no subependymal spread; and a location not adjacent to brainstem or optic chiasm. RESULTS: Eighty-nine patients (11.9%) were identified as potentially SRS-eligible. The median survival times (MST) and 18-month survival rates of the 89 eligible and 643 ineligible patients were 14.4 versus 11.7 months and 40% versus 27%, respectively (P = .047). The MST and 18-month survival rate of the 544 SRS-ineligible patients with KPS greater than 60 were 12.1 months and 29%, respectively, and were not statistically inferior to the survival of the SRS-eligible group (P = .21). Multivariate analysis revealed age, KPS, and histopathology to be strongly predictive of survival, and SRS eligibility was also significantly predictive (P = .047). CONCLUSION: SRS-eligible patients enrolled on RTOG 83-02 had survival superior to that of the SRS-ineligible group, and this advantage is mainly due to the selection of a subgroup with a high minimum KPS.

Ecological Genetics and Host Range Expansion by Busseola fusca (Lepidoptera: Noctuidae).

The host plant range of pests can have important consequences for its evolution, and plays a critical role in the emergence and spread of a new pest outbreak. This study addresses the ecological genetics of the indigenous African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae), in an attempt to investigate the evolutionary forces that may be involved in the recent host range expansion and establishment of this species in Ethiopian and southern African sugarcane. We used populations from Ethiopia, Zimbabwe, and South Africa to examine whether the host range expansion patterns shared by the Ethiopian and the southern African populations of B. fusca have evolved independently. Base-pair differences in the cytochrome oxidase I (COI) gene were used to characterize haplotype diversity and phylogenetic relationships. There were seven haplotypes among the 30 sequenced individuals collected on four host plant species from 17 localities in the four countries. Of the seven COI haplotypes identified, the two major ones occurred in both sugarcane and maize. Genetic analyses revealed no detectable genetic differentiation between southern African B. fusca populations from maize and sugarcane (FST = 0.019; P = 0.24). However, there was strong evidence of variation in genetic composition between populations of the pest from different geographic regions (FST = 0.948; P < 0.001). The main implication of these findings is that the B. fusca populations in maize in southern Africa are more likely to shift to sugarcane, suggesting that ecological opportunity is an important factor in host plant range expansion by a pest.

A relationship between cerebellar Purkinje cells and spatial working memory demonstrated in a lurcher/chimera mouse model system.

New emphasis has been placed upon cerebellar research because of recent reports demonstrating involvement of the cerebellum in non-motor cognitive behaviors. Included in the growing list of cognitive functions associated with cerebellar activation is working memory. In this study, we explore the potential role of the cerebellum in spatial working memory using a mouse model of Purkinje cell loss. Specifically, we make aggregation chimeras between heterozygous lurcher (Lc/+) mutant embryos and +/+ (wildtype) embryos and tested them in the delayed matching-to-position (DMTP) task. Lc/+ mice lose 100% of their Purkinje cells postnatally due to a cell-intrinsic gain-of-function mutation. Lc/+<->+/+ chimeras therefore have Purkinje cells ranging from 0 to normal numbers. Through histological examination of chimeric mice and observations of motor ability, we showed that ataxia is dependent upon both the number and distribution of Purkinje cells in the cerebellum. In addition, we found that Lc/+ mice, with a complete loss of Purkinje cells, have a generalized deficit in DMTP performance that is probably associated with their motor impairment. Finally, we found that Lc/+<->+/+ chimeric mice, as a group, did not differ from control mice in this task. Rather, surprisingly, analysis of their total Purkinje cells and performance in the DMTP task revealed a significant negative relationship between these two variables. Together, these findings indicate that the cerebellum plays a minor or indirect role in spatial working memory.

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.

A multicenter study to evaluate a novel assay for quantitation of soluble interleukin 2 receptor in renal transplant recipients.

The clinical utility of monitoring soluble interleukin 2 receptor (sIL-2R) as an indicator of immune stimulation in renal transplant patients was evaluated in a retrospective study at 3 centers. Serum samples (n = 2360) were obtained from 86 (17 living related donor, 69 cadaver) transplant recipients. The patients had received either triple therapy (n = 35) or antilymphocyte antibody induction therapy followed by triple therapy (n = 51). The mean period of postoperative observation was 118 days (range, 6-349 days). Serum sIL-2R concentrations were quantitated by an automated microparticle enzyme immunoassay (MEIA) (Abbott Diagnostics) in which sIL-2R was captured by 7G7/B6 monoclonal antibody-coated microparticles and detected by an immunospecific rabbit antihuman sIL-2R-alkaline phosphatase conjugate. A distinct advantage of the technique was rapid turn-around time: 1-24 results were obtained in less than 50 min. Cyclosporine trough concentrations were determined by radioimmunoassay or high-performance liquid chromatography. Diagnosis of rejection was established by clinical and histological criteria. The mean sIL-2R concentration in patients receiving antilymphocyte antibody induction therapy increased from 3486 +/- 1729 U/ml (+/- SD) at the time of transplant to a maximum of 7395 +/- 7101 U/ml on the third day posttransplant; this increase was not observed in patients receiving triple therapy (P less than 0.0001). By the sixth day of posttransplant, there were no differences in sIL-2R levels in the two groups. Fifty rejection episodes were observed in 29 patients on triple therapy. The mean sIL-2R concentration rose from 3022 U/ml at the data point prior to rejection to 3524 U/ml at the time of rejection. Thirty-four rejection episodes were observed in 26 patients receiving induction therapy. The mean sIL-2R concentration was 3015 U/ml at the data point prior to rejection and 4815 U/ml at the time of rejection. The sIL-2R concentrations began increasing earlier and rose higher in rejecting patients who received induction therapy than in those receiving triple therapy. Early posttransplant sIL-2R levels increased significantly more in cadaver recipients than in LRD recipients, reaching a maximum on day 2 posttransplant (P less than 0.001). Prerejection sIL-2R concentrations were significantly lower in LRD recipients than in cadaver recipients (2248 U/ml vs. 4290 U/ml, P less than 0.02), as were sIL-2R levels at the time of diagnosis of rejection (2800 U/ml vs. 4832 U/ml, P = 0.01). The mean sIL-2R level in stable long-term graft recipients was 2110 U/ml, with approximately 90% of values less than 3000 U/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic facilitation of the occurrence of depolarization-induced suppression of inhibition in rat hippocampus.

Depolarization-induced suppression of inhibition is a transient decrease in GABAergic input to a hippocampal pyramidal cell following a brief depolarization of that cell. When recorded under whole-cell voltage clamp, monosynaptic, bicuculline-sensitive, GABA(A)-mediated currents are suppressed for a period lasting up to 1 min in response to a retrograde signal released by the pyramidal cell. The depolarization-induced suppression of inhibition process affects spontaneous, action-potential-dependent inhibitory postsynaptic currents, but suppression of these currents is seldom observed in the absence of carbachol, a cholinergic agonist. Because of the central roles played by cholinergic and GABAergic transmission in the regulation of hippocampal rhythmic activity, it will be important to understand the mechanism by which carbachol facilitates the appearance of depolarization-induced suppression of inhibition. As preliminary steps in the investigation of cholinergic actions on depolarization-induced suppression of inhibition, it is necessary to determine which cholinergic receptors are involved and the degree to which activation of these receptors is required for depolarization-induced suppression of inhibition. Nicotine did not mimic the effects of carbachol, and mecamylamine, a nicotinic receptor antagonist, did not block them. In contrast, the actions of carbachol were abolished by atropine and other muscarinic receptor antagonists. The actions of antagonists with relative selectivities for various subtypes of muscarinic receptors [4-diphenylacetoxy-N-methylpiperidine methiodide, pirenzepine, 11-([2-1-piperidinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzod iaz epine-6-one] suggested that cholinergic facilitation of the occurrence of depolarization-induced suppression of inhibition is likely to be mediated through muscarinic receptors of the M1 or M3 rather than M2 subtype. Despite its potent facilitation of the occurrence of depolarization-induced suppression of inhibition, muscarinic stimulation was not required for expression of depolarization-induced suppression of inhibition. Occasionally, depolarization-induced suppression of inhibition of spontaneous inhibitory postsynaptic currents occurred in the absence of carbachol and could not be blocked by atropine, and hence was not likely to be mediated by endogenous acetylcholine. Also, depolarization-induced suppression of inhibition of monosynaptically evoked inhibitory postsynaptic currents occurred without carbachol perfusion, and this was also insensitive to atropine. Therefore, the mechanism of depolarization-induced suppression of inhibition is not dependent on muscarinic receptor activation. Nevertheless, in vivo, septal cholinergic input to the hippocampus may provide the necessary activation of interneurons to allow depolarization-induced suppression of inhibition to occur.

NIRCA: a rapid robust method for screening for unknown point mutations.

We describe a method for screening for dispersed point mutations, based on the observation that RNase frequently cleaves both strands of base pair mismatches in duplex RNA targets. The mismatched substrates are generated by in vitro transcription of wild-type and mutant templates amplified by the PCR or reverse transcription (RT)-PCR; bacteriophage promoters are incorporated into the PCR primers to permit both strands of the products to be transcribed into RNA. Complementary wild-type and mutant transcripts are hybridized and treated with RNase, and the cleavage products are separated on agarose gels and detected by visualization of the ethidium-stained sample under UV light. The method is thus non-isotopic, and since the cleavage products remain double-stranded during analysis, the labor-intensive RNase inactivation steps required in the original procedure can be eliminated. Also, nonspecific background cleavage is reduced so that longer target regions (1 kb) can be screened in a single step. The Non-Isotopic RNase Cleavage Assay (NIRCA) achieved a detection rate of 88%-90% in blind studies in a Factor IX model system, and it was also used to detect unknown p53 mutations in breast tumor samples. NIRCA provides a rapid method for sensitive, non-isotopic, high-throughput genetic screening.

Influence of a sampling review process for radiation oncology quality assurance in cooperative group clinical trials--results of the Radiation Therapy Oncology Group (RTOG) analysis.

The Radiation Therapy Oncology Group (RTOG) designed a random sampling process and observed its influence upon radiotherapy review mechanisms in cooperative group clinical trials. The method of sampling cases for review was modeled from sampling techniques commonly used in pharmaceutical quality assurance programs, and applied to the initial (on-study) review of protocol cases. 'In control' (IC) status is defined for a given facility as the ability to meet minimum compliance standards. Upon achieving IC status, activation of the sampling process was linked to the rate of continued patient accrual for each participating institution in a given protocol. The sampling design specified that > or = 30% cases not in compliance would be detected with 80% power. A total of 458 cases was analyzed for initial review findings in four RTOG Phase III protocols. Initial review findings were compared with retrospective (final) review results. Of the 458 cases analyzed, 370 underwent initial review at on-study, while 88 did not require review as they were enrolled from institutions that had demonstrated protocol compliance. In the group that had both initial and final review, 345/370 (93%) were found to have followed the protocol or had a minor variation. Of the exempted cases, 79/88 (90%) were found to be per protocol or a minor variant. The sampling process proved itself to be cost-effective and resulted in a noticeable reduction in the workload, thus providing an improved approach to resource allocation for the group. Continued evaluation of the sampling mechanism is appropriate as study designs and participants vary over time, and as more data become available to study.(ABSTRACT TRUNCATED AT 250 WORDS)