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This research extends recent efforts to differentiate communication in crisis negotiations (Taylor, 2002) by examining how negotiators' behavior differs across context. Data were 108 interaction episodes transcribed from 12 simulated crisis negotiations and coded at the level of thought units across 41 behavioral variables. Results of a smallest space analysis supported the hypothesized differentiation of communication behavior over 3 facets: overall orientation (Avoidance, Distributive, Integrative), motivational concern (Identity, Instrumental, Relational), and intensity (High to Low). This solution was used as a framework for identifying differences in behavior across simulated and actual negotiations. Analyses showed a systematic pattern of variations in behavior use, with simulated negotiations involving relatively more avoidance-relational and distributive-instrumental behavior than actual negotiations. Predictable differences were also observed in the purpose or function of behavior, with highlyintense behaviors showing greater uniformity in function across contexts compared to lowintensity behaviors.

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 microL) with added deuterium-labeled d3-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 microL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium formate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --130.2; IS m/z 166.2 --87.2). The assay was linear from 0.5 to 100 microg/L (r0.993, n = 9). The accuracy and imprecision of the method for quality control samples were 87.5% to 113% and10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 microg/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

A structured telephone reference check (STRC) was developed to assess three job-relevant personality/interpersonal dimensions (Conscientiousness, Agreeableness, customer focus) for a family of entry-level, customer-contact jobs within a service organization. The procedure was designed to be brief (a 10–15 minute telephone call with each referee) and easy to administer by untrained recruitment staff. Confirmatory factor analyses on referee ratings for 223 applicants indicated that referees were able to adequately discriminate between dimensions of applicants' past performance. A predictive, criterion-related validation study on those applicants who were hired demonstrated that the STRC predicted supervisory ratings of job performance (r= .25, p < .01, uncorrected; .36 corrected for range restriction and criterion unreliability). No evidence was found that referees with shorter working relationships with applicants provided less valid ratings.

Three sulfur (S) treatements were imposed by applying gypsum to three broccoli cultivars (Claudia, Marathon, and TB-234) known to differ in glucoraphanin content of mature seeds. The S treatments were control (very low added S), low S (23 kg S ha(-)(1)), and high S (92 kg S ha(-)(1)). The gypsum applications during the early vegetative phase of the three broccoli cultivars increased S uptake and the glucoraphanin content in each plant organ. There were significant genotypic differences for the content of both S and glucoraphanin in all plant organs at different growth stages with gypsum applications. A large increase in S and glucoraphanin content was found in the green heads of broccoli and mature seeds. S present in glucoraphanin accounted for only 4-10% of total S content in broccoli heads. However, S present in glucoraphanin in mature seeds accounted for 40-46% of the total S in the seeds of moderate and high glucoraphanin cultivars (Marathon and TB-234). The partitioning of S into glucoraphanin also increased with gypsum applications. Differences in S uptake, S distribution between organs, and partitioning of S into glucoraphanin largely explained the differences in glucoraphanin content in the green heads and mature seeds for the three broccoli cultivars and three S treatments.

BACKGROUND The development of hyperlipidemia after liver transplant is frequently treated with hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) such as atorvastatin. As atorvastatin and the primary immunosuppressant drug, cyclosporine, are metabolized by the same pathway, there is the potential for an interaction. OBJECTIVE To determine the effect of atorvastatin on cyclosporine pharmacokinetics in liver transplant recipients. METHODS Six stable, long-term adult liver transplant recipients from a single center who developed posttransplant dyslipidemia were recruited to participate in a 14-day, open-label study of atorvastatin 10 mg/d coadministered with standard posttransplant immunosuppression using constant oral doses of cyclosporine and corticosteroids. A 10-point pharmacokinetic profile was performed prior to and on day 14 after commencement of atorvastatin therapy. Cyclosporine concentrations were measured by HPLC-electrospray-tandem mass spectrometry. The AUC was calculated by the linear trapezoidal rule, with other parameters determined by visual inspection. RESULTS Atorvastatin coadministration increased the cyclosporine AUC by 9% (range 0–20.6%; 3018 vs 3290 ng•h/mL; p = 0.04). No significant change was evident for other cyclosporine pharmacokinetic parameters. Total cholesterol and low-density lipoprotein cholesterol levels were significantly lower on day 14 than at baseline (p < 0.02). One patient developed a twofold increase in transaminases after 2 weeks of atorvastatin therapy, but no other clinical or biochemical adverse events were recorded. CONCLUSIONS Atorvastatin coadministration increases the cyclosporine AUC by approximately 10% in stable liver transplant recipients. This change in systemic exposure to cyclosporine is of questionable clinical significance. Atorvastatin is effective in reducing cholesterol levels in liver transplant recipients.

The immunosuppressant drug mycophenolic acid (MPA) and its major metabolite, mycophenolic acid glucuronide (MPAG), are highly bound to albumin. An HPLC-tandem-MS (HPLC/MS/MS) and an HPLC-UV assay were developed to measure free (unbound) concentrations of MPA and MPAG, respectively. Ultrafiltrate was prepared from plasma (500 microl) by ultrafiltration at 3000 x g for 20 min (20 degrees C). Both MPA and MPAG were isolated from ultrafiltrate (100 microl) by acidification and C18 solid-phase extraction. Free MPA was measured by electrospray tandem mass spectrometry using selected reactant monitoring (MPA: m/z 338.2--206.9) in positive ionisation mode. Chromatography was performed on a PFPP column (50 mm x 2 mm, 5 microm). Total analysis time was 7 min. The assay was linear over the range 1-200 microg/l with a limit of quantification of 1 microg/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 microg/l) were 94-99% and7%, respectively. Free MPAG was chromatographed on a C18 Nova-Pak column (150 mm x 3.9 mm, 5 microm) using a binary gradient over 20 min. The eluent was monitored at 254 nm. The assay was linear over the range 1-50 mg/l with the limit of quantification at 2.5 mg/l. The inter-day accuracy and imprecision of quality controls (5, 20, 45 mg/l) was 101-107% and8% (n = 4), respectively. For both methods no interfering substances were found in ultrafiltrate from patients not receiving MPA. The methods described have a suitable dynamic linear range to facilitate the investigation of free MPA and MPAG pharmacokinetics in transplant patients. Further, this is the first reported HPLC-UV method to determine free MPAG concentrations.

Although monitoring of tacrolimus blood concentrations is standard clinical practice following liver transplantation, a greater understanding of the relationship between trough concentrations and clinical outcome is required. The aim of this study was to perform a pharmacodynamic investigation of tacrolimus in pediatric liver transplant recipients. A retrospective analysis was performed on 35 pediatric liver recipients who received oral tacrolimus as the primary immunosuppressant. Outcome data were recorded corresponding to the times that tacrolimus trough concentrations had been determined (using a validated high-performance liquid chromatography-tandem mass spectrometry assay). A Mann-Whitney rank sum test was used to investigate differences between median concentrations at which drug toxicity, infection, and organ rejection did and did not occur. Outcome data and trough concentrations were recorded from the immediate post-transplant time to over 3 years (656 concentration-effect measures). Seventy one percent of data was obtained after the first 90 days post-transplant. Patients were identified as having experienced hypomagnesemia (20), hypertension (14), hyperkalemia (12), infection (11), nephrotoxicity (8), diarrhea (6), hyperglycemia (3), neurotoxicity(3), and rejection (3) during retrospective follow-up. Median trough concentrations were significantly higher (P

The current article addresses the ongoing debate about whether individuals can perform as well as actuarial techniques when confronted with real world, consequential decisions. A single experiment tested the ability of participants (N=215) and an actuarial technique to accurately predict the residential locations of serial offenders based on information about where their crimes were committed. Results indicated that participants introduced to a ‘Circle’ or ‘Decay’ heuristic showed a significant improvement in the accuracy of predictions, and that their post-training performance did not differ significantly from the predictions of one leading actuarial technique. Further analysis of individual performances indicated that approximately 50% of participants used appropriate heuristics that typically led to accurate predictions even before they received training, while nearly 75% improved their predictive accuracy once introduced to either of the two heuristics. Several possible explanations for participants’ accurate performances are discussed and the practical implications for police investigations are highlighted. Copyright # 2004 John Wiley & Sons, Ltd.

This study compared the validities of the past-experience and situational structured employment interview questions using a two-group, experimental design. Subjects were 130 social workers, purpose...

Shattering of siliquae causes significant seed loss in canola (Brassica napus) production worldwide. There is little genetic variation for resistance to shatter in canola and, hence, the trait has been studied in B. rapa. Previous studies have shown two randomly segregating recessive genes to be responsible for shatter resistance. Three random amplified polymorphic DNA markers were identified as being linked to shatter resistance using bulked segregant analysis in a F3B. rapa population. The population was derived from a cross between a shatter-susceptible Canadian cultivar and a shatter-resistant Indian line. Of the three markers, RAC-3900 and RX-71000 were linked to recessive sh1 and sh2 alleles, and SAC-201300 was linked to both dominant Sh1 and Sh2 alleles. The common marker for the dominant wild-type allele for the two loci was explained to have resulted from duplication of an original locus and the associated markers through chromosome duplication and rearrangements in the process of evolution of the modern B. rapa from its progenitor that had a lower number of chromosomes. Segregation data from double heterozygous F3 families, although limited, indicated the markers were not linked to each other and provided further evidence for the duplication hypothesis.

SUMMARY A genome linkage map was developed for Ascochyta rabiei (Pass.) Labrousse, (teleomorph) Didymella rabiei (Kovachevski), an important pathogen causing ascochyta blight in chickpea (Cicer arietinum L.). The map was constructed using 96 progeny generated from a single pseudothecium produced from a cross between a USA MAT-2 isolate and an Australian MAT-1 isolate. The map comprised 126 molecular markers of which 69 were random amplified polymorphic DNA (RAPD) markers, 46 were amplified fragment length polymorphic (AFLP) markers, 10 were sequence-tagged microsatellite site (STMS) markers, and one was a sequence characterized amplified region (SCAR) marker. Eighteen large and 10 small linkage groups (LG) were characterized and the mating-type locus was mapped on to LGd. The map spanned 1271 cM with an average spacing between markers of 15.1 cM. The SCAR marker, specific for mating type 2, was designed to amplify a region of the MAT locus and was used to identify the mating type of A. rabiei isolates. One AFLP marker, derived from the MAT-1 parent, was closely linked to the mating-type locus (9.6 cM). The linkage map provides a framework for the future identification of the locations of other important traits such as virulence/avirulence and fungicide resistance. Findings from this study suggest that the MAT-2 isolates of D. rabiei should be renamed to MAT-1 isolates because the alpha-box, specific for MAT-1 from other ascomycetes, was amplified from A. rabiei MAT-2 isolates.

To date, there have been no reports of somatic cell nuclear transfer in llamas. The application of this methodology to the camelid industry could be helpful in the propagation of genetically valuable animals. The objective of this study was to produce nuclear transfer llama embryos comparing the development of these llama embryos cultured in either CR1aa medium (treatment A) or G1.2 medium (treatment B) medium. Llamas were superstimulated by double dominant follicle reduction 12 days apart, followed by pFSH administered in daily descending doses over a 3-day interval (total dose of 200 mg). Animals were ovariectomized by flank laparotomy, follicles were aspirated from excised ovaries and oocytes were in vitro matured for a 30-h period. Adult female llama fibroblasts were used as donor karyoplasts and injected into enucleated llama oocytes. Embryo development was assessed after 2 days of culture. A total of 307 follicles were aspirated from nine treated females, resulting in 298 (97%) oocytes recovered. Of a total of 229 evaluated oocytes, 120 (52%) achieved nuclear maturation. Of a total of 80 reconstructed couplets, 50 (62.5%) were successfully fused. Subsequent cleavage rates were 32 and 40% for treatments A and B, respectively, with no significant difference (p0.05) detected between treatment groups. A total of 11 embryos (8-cell to morula stages) were transferred to synchronized recipient llamas. Ultrasonography at 14 days post-transfer indicated that no pregnancies were established. This study shows that nuclear transfer can be successfully applied to the production of llama embryos. Further research is needed to identify optimal parameters to improve efficiency of nuclear transfer in this species.

Free drug measurement and pharmacodymanic markers provide the opportunity for a better understanding of drug efficacy and toxicity. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) is a powerful analytical technique that could facilitate the measurement of free drug and these markers. Currently, there are very few published methods for the determination of free drug concentrations by HPLC-MS. The development of atmospheric pressure ionisation sources, together with on-line microdialysis or on-line equilibrium dialysis and column switching techniques have reduced sample run times and increased assay efficiency. The availability of such methods will aid in drug development and the clinical use of certain drugs, including anti-convulsants, anti-arrhythmics, immunosuppressants, local anaesthetics, anti-fungals and protease inhibitors. The history of free drug measurement and an overview of the current HPLC-MS applications for these drugs are discussed. Immunosuppressant drugs are used as an example for the application of HPLC-MS in the measurement of drug pharmacodynamics. Potential biomarkers of immunosuppression that could be measured by HPLC-MS include purine nucleoside/nucleotides, drug-protein complexes and phosphorylated peptides. At the proteomic level, two-dimensional gel electrophoresis combined with matrix-assisted laser desorption/ionisation time-of-flight (TOF) MS is a powerful tool for identifying proteins involved in the response to inflammatory mediators. (C) 2003 Elsevier Science B.V. All rights reserved.

Aim To develop a population pharmacokinetic model for mycophenolic acid in adult kidney transplant recipients, quantifying average population pharmacokinetic parameter values, and between- and within-subject variability and to evaluate the influence of covariates on the pharmacokinetic variability. Methods Pharmacokinetic data for mycophenolic acid and covariate information were previously available from 22 patients who underwent kidney transplantation at the Princess Alexandra Hospital. All patients received mycophenolate mofetil 1 g orally twice daily. A total of 557 concentration-time points were available. Data were analysed using the first-order method in NONMEM (version 5 level 1.1) using the G77 FORTRAN compiler. Results The best base model was a two-compartment model with a lag time (apparent oral clearance was 271 h(-1), and apparent volume of the central compartment 981). There was visual evidence of complex absorption and time-dependent clearance processes, but they could not be successfully modelled in this study. Weight was investigated as a covariate, but no significant relationship was determined. Conclusions The complexity in determining the pharmacokinetics of mycophenolic acid is currently underestimated. More complex pharmacokinetic models, though not supported by the limited data collected for this study, may prove useful in the future. The large between-subject and between-occasion variability and the possibility of nonlinear processes associated with the pharmacokinetics of mycophenolic acid raise questions about the value of the use of therapeutic monitoring and limited sampling strategies.

Accessions from Cicer echinospermum, a wild relative of chickpea (Cicer arietinum L.), contain resistance to the fungal disease ascochyta blight, a devastating disease of chickpea. A linkage map was constructed based on an interspecific F(2) population, derived from a cross between a susceptible chickpea cultivar (Lasseter) and a resistant C. echinospermum accession (PI 527930). The linkage map incorporated 83 molecular markers, that included RAPD, ISSR, STMS and RGA markers; eight markers remained unlinked. The map comprised eight linkage groups and covered a map distance of 570 cM. Six out of the eight linkage groups were correlated to linkage groups from the integrated Cicer map using STMS markers. Quantitative trait loci (QTLs) associated with ascochyta blight resistance were detected using interval mapping and single-point analysis. The F(2) population was evaluated for seedling and stem resistance in glasshouse trials. At least two QTLs were identified for seedling resistance, both of which were located within linkage group 4. Five markers were associated with stem resistance, four of which were also associated with seedling resistance. QTLs from previous studies also mapped to LG 4, suggesting that this linkage group is an important region of the Cicer genome for resistance to ascochyta blight.

This paper outlines 4 assumptions behind attempts to explain the sequential organization of communication behavior during conflict. These assumptions were supported by an analysis of behavioral sequences coded from 9 hostage negotiations and 20 divorce mediations. Analyses showed that negotiators use only a small proportion of available responses to other party's behavior, and that this proportion rapidly decreases as sequence length increases. Critical to this channeling in behavior was the triple‐interact (i.e., cue‐response‐cue‐response), which represents the maximum sequence length required to enable accurate prediction of negotiators' future behavior. More detailed analysis showed that the triple‐interact reduced uncertainty in behavior by over 70%, which compares to less than 1% from knowledge of negotiation context and approximately 10% from knowledge of individual differences.

An aberrant random amplified polymorphic DNA (RAPD) marker in genomic DNA of tissue culture plantlets was frequently observed during a comparison of DNA fingerprints derived from potato germplasm grown in tissue culture and the field. The RAPD marker was cloned, sequenced and determined to be of bacterial origin. A bacterial contaminant was isolated from the tissue culture plants and identified as a Bacillus pumilus. A set of sequence characterised amplified region (SCAR) primers were designed from the sequence of the cloned fragment and tested for the specific detection of B. pumilus. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) were also used to generate B. pumilus profiles specific to our isolate in order to test and confirm the sequence homology of amplified markers generated from a range of DNA samples isolated from tissue culture plants and pure isolates of B. pumilus-like bacteria.

Patient outcomes in transplantation would improve if dosing of immunosuppressive agents was individualized. The aim of this study is to develop a population pharmacokinetic model of tacrolimus in adult liver transplant recipients and test this model in individualizing therapy. Population analysis was performed on data from 68 patients. Estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (V/F) using the nonlinear mixed effects model program (NONMEM). Factors screened for influence on these parameters were weight, age, sex, transplant type, biliary reconstructive procedure, postoperative day, days of therapy, liver function test results, creatinine clearance, hematocrit, corticosteroid dose, and interacting drugs. The predictive performance of the developed model was evaluated through Bayesian forecasting in an independent cohort of 36 patients. No linear correlation existed between tacrolimus dosage and trough concentration (r(2) = 0.005). Mean individual Bayesian estimates for CL/F and V/F were 26.5 +/- 8.2 (SD) L/hr and 399 +/- 185 L, respectively. CL/F was greater in patients with normal liver function. V/F increased with patient weight. CL/F decreased with increasing hematocrit. Based on the derived model, a 70-kg patient with an aspartate aminotransferase (AST) level less than 70 U/L would require a tacrolimus dose of 4.7 mg twice daily to achieve a steady-state trough concentration of 10 ng/mL. A 50-kg patient with an AST level greater than 70 U/L would require a dose of 2.6 mg. Marked interindividual variability (43% to 93%) and residual random error (3.3 ng/mL) were observed. Predictions made using the final model were reasonably nonbiased (0.56 ng/mL), but imprecise (4.8 ng/mL). Pharmacokinetic information obtained will assist in tacrolimus dosing; however, further investigation into reasons for the pharmacokinetic variability of tacrolimus is required.

This article examines the effects of role on terrorists' use of power and affiliative strategies in negotiation as a function of terrorist ideology, incident type, and the outcome that is achieved. Data were scores on eight behavioral scales designed to reflect the dynamics of 186 terrorist negotiations, as reported in detailed chronological accounts. Results supported the hypothesized one-down effect with terrorists' use of power-oriented strategies complemented by authority's use of affiliation-oriented strategies. The extent to which terrorists used aggressive strategies was related to the resolution of the incident, with attenuated outcomes more likely for those using more aggressive strategies. These dynamics differed across incident type, with aerial hijackings involving more overt power strategies than barricade-siege incidents, which were more likely to involve bargaining for certain outcomes. Finally, terrorist ideology and the associated identity concerns magnified the one-down effect, with religious fundamentalists engaging in more violence and less compromising strategies than terrorists with other ideological backgrounds.

In order to determine genetically diverse parents for the generation of mapping populations segregating for resistance to ascochyta blight in wild Cicer species, the genetic diversity between a selection of resistant and susceptible accessions was assessed using molecular markers. Twenty Cicer accessions - comprising eight C. reticulatum accessions, six C. echinospermum accessions, five C. bijugum accessions, and one C. arietinum accession - were compared using a combination of seven RAPD primers and seven ISSR primers. A total of 231 polymorphic bands were scored and used to determine the genetic distances between accessions using Jaccard similarity coefficients. The most genetically diverse parents for the generation of intraspecific and interspecific populations segregating for resistance to ascochyta blight are reported.

The Response to Offender Personality Disorder Consultation was released in October 2011. For some this is a welcome step in the right direction due to its therapeutic optimism, however for practitioners operating in the secure estate there are significant challenges ahead. This aim of this article is to discuss the increasing convergence of health and criminal justice and their inherent ideological and practical difficulties. It does so with reference to the consultation on offender personality disorder pathways and in particular the implications regarding multi-disciplinary and cross agency approaches to risk, public protection and personality disorder respectfully. It concludes that before embarking on a new wave of determining and responding to those with personality disorder, offender or otherwise, a more in-depth and empirically informed critical reflection is warranted.

Distribution of sinigrin (2-propenylglucosinolate) and glucoraphanin (4-methylsulphinylbutylglucosinolate) concentration was determined in seeds and seedlings of Brassica species: B. oleracea, B. nigra, B. juncea, B. rapa and B. napus using a simple and reliable extraction method. Glucosinolates were extracted from seeds and seedlings using boiling water and were analysed by reverse phase ion-pair chromatography with UV detection. High concentrations of sinigrin were found in B. nigra, B. juncea var. rugosa (mustard greens) and B. oleracea (Brussels sprouts, cabbage, cauliflower, and Chinese broccoli). Sinigrin was absent from B. rapa and B. napus and was present in negligible concentrations in B. oleracea var. italica (broccoli). The concentration of sinigrin in the seed was similar to that in the cotyledon and hypocotyl tissues (7-day-old seedling) in most Brassica species tested. Glucoraphanin was found at high concentrations in B. oleracea var. italica and moderate concentrations in B. oleracea var. capitata (cabbage), whilst was absent in B. nigra, B. juncea, B. rapa and B. napus. Glucoraphanin concentration was between 44.2 and 274.1 μmol g−1 DW among broccoli genotypes. Kalibrini®, a new hybrid line between broccoli and Chinese broccoli (kailan) contained a moderate concentration of glucoraphanin (145.3 μmol g−1 DW) compared to its parent lines (274.1 μmol g−1 DW in broccoli but only 1.2 μmol g−1 DW in kailan). Glucoraphanin concentration in seedlings was significantly lower than that in the seed of broccoli while there was no significant difference in sinigrin concentration in seedlings of most Brassica species tested. The study has shown the extensive variability in sinigrin and glucoraphanin concentrations among Brassica species and genotypes that is available for selection and breeding. New elite lines can be produced for use as commercial crops in the health food or nutraceutical industries.

The aims of this study were to investigate the population pharmacokinetics of tacrolimus in adult kidney transplant recipients and to identify factors that explain variability.Population analysis was performed on retrospective data from 70 patients who received oral tacrolimus twice daily. Morning blood trough concentrations were measured by liquid chromatography-tandem mass spectrometry. Maximum likelihood estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (V/F), with the use of NONMEM (GloboMax LLC, Hanover, Md). Factors screened for influence on these parameters were weight, age, gender, postoperative day, days of tacrolimus therapy, liver function tests, creatinine clearance, hematocrit fraction, corticosteroid dose, and potential interacting drugs.CL/F was greater in patients with abnormally low hematocrit fraction (data from 21 patients only), and it decreased with increasing days of therapy and AST concentrations (P.01). Average parameter estimates were as follows: CL/F = 31.8 L/h (hematocrit0.33), CL/F = 24.2 L/h (hematocrit0.33), and V/F = 2080 L. Marked interindividual variability (42% to 111%) and residual random error (3.7 ng/mL) were observed. On the basis of the derived model, a patient with normal AST (20 U/L) or high AST (200 U/L) concentrations 7 days after commencement of therapy would require a tacrolimus dose of 4.6 mg or 4.0 mg, respectively, to achieve a steady-state trough concentration of 10 ng/mL.The population pharmacokinetics of tacrolimus in adult kidney transplant recipients showed wide variability. Thus it is not possible to use a standard tacrolimus dose as an empiric predictor of concentration in this population. An understanding of factors that influence the pharmacokinetics of tacrolimus may assist in drug dosage decisions.

Hydrolysis products of sinigrin (2-propenylglucosinolate) and glucoraphanin (4-methylsulphinylbutylglucosinolate) have been shown to protect against the development of cancers. However, there was limited information available on the variation of these two glucosinolates throughout the plant cycle. The objective of this study was to evaluate sinigrin and glucoraphanin levels within Brassica plants during development. Sinigrin concentration in B. juncea and B. nigra decreased from seedling to early flowering stage, increased in the late flowering stage and then decreased again during seed maturation. The lowest concentration of sinigrin occurred at the early flowering stage except in one genotype of B. juncea (PI 179858). Sinigrin concentration also increased in maturing seeds while the concentration in pods decreased. The concentration of glucoraphanin in B. oleracea var. italica decreased from the start of seed germination to the flowering stages. The lowest concentration was also found at the flowering stage. A higher concentration of glucoraphanin was detected in the green broccoli heads and flower heads than in other reproductive tissues. However, the highest content of glucoraphanin occurred at the green head stage and then declined as flowering was initiated. These results have shown that green and brown seeds of mustards contained the highest concentration of sinigrin while the highest concentration of glucoraphanin occurred in young broccoli seedlings and seeds. This information should be useful for the development of those compounds as nutraceuticals.

The effects of post-harvest and packaging treatments on glucoraphanin (4-methylsulfinylbutyl glucosinolate), the glucosinolate precursor of anticancer isothiocyanate sulforaphane [4-methylsulfinylbutyl isothiocyanate], were examined in broccoli (Brassica oleracea var. italica) during storage times. The results showed that at 20 degrees C, 55% loss of glucoraphanin concentration occurred in broccoli stored in open boxes during the first 3 days of the treatment and 56% loss was found in broccoli stored in plastic bags by day 7. Under both air and controlled atmosphere (CA) storage, glucoraphanin concentration appeared to fluctuate slightly during 25 days of storage and the concentrations under CA was significantly higher than those stored under air treatment. In modified atmosphere packaging (MAP) treatments, glucoraphanin concentration in air control packaging decreased significantly whereas there were no significant changes in glucoraphanin concentration in MAP with no holes at 4 degrees C and two microholes at 20 degrees C for up to 10 days. Decreases in glucoraphanin concentration occurred when the broccoli heads deteriorated. In the present study, the best method for preserving glucoraphanin concentration in broccoli heads after harvest was storage of broccoli in MAP and refrigeration at 4 degrees C. This condition maintained the glucoraphanin concentration for at least 10 days and also maintained the visual quality of the broccoli heads.

This study compared an enzyme-linked immunosorbent assay (ELISA) to a liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique for measurement of tacrolimus concentrations in adult kidney and liver transplant recipients, and investigated how assay choice influenced pharmacokinetic parameter estimates and drug dosage decisions. Tacrolimus concentrations measured by both ELISA and LC/MS/MS from 29 kidney (n = 98 samples) and 27 liver (n = 97 samples) transplant recipients were used to evaluate the performance of these methods in the clinical setting. Tacrolimus concentrations measured by the two techniques were compared via regression analysis. Population pharmacokinetic models were developed independently using ELISA and LC/MS/MS data from 76 kidney recipients. Derived kinetic parameters were used to formulate "typical dosing" regimens for concentration targeting. Dosage recommendations for the two assays were compared. The relation between LC/MS/MS and ELISA measurements was best described by the regression equation ELISA = 1.02. (LC/MS/MS) + 0.14 in kidney recipients, and ELISA = 1.12. (LC/MS/MS) - 0.87 in liver recipients. ELISA displayed less accuracy than LC/MS/MS at lower tacrolimus concentrations. Population pharmacokinetic models based on ELISA and LC/MS/MS data were similar with residual random errors of 4.1 ng/mL and 3.7 ng/mL, respectively. Assay choice gave rise to dosage prediction differences ranging from 0% to 30%. ELISA measurements of tacrolimus are not automatically interchangeable with LC/MS/MS values. Assay differences were greatest in adult liver recipients, probably reflecting periods of liver dysfunction and impaired biliary secretion of metabolites. While the majority of data collected in this study suggested assay differences in adult kidney recipients were minimal, findings of ELISA dosage underpredictions of up to 25% in the long term must be investigated further.

Criterion-related validities and inter-rater reliabilities for structured employment interview studies using situational questions (e.g. “Assume that you were faced with the following situation … what would you do?”) were compared meta-analytically with studies using past behaviour questions (e.g. “Can you think of a time when … what did you do?”). Validities and reliabilities were further analysed in terms of whether descriptively-anchored rating scales were used to judge interviewees' answers, and validities for each question type were also assessed across three levels of job complexity. While both question formats yielded high validity estimates, studies using past behaviour questions, when used with discriptively anchored answer rating scales, yielded a substantially higher mean validity estimate than studies using the situational question format with descriptively-anchored answer rating scales (.63 versus .47). Question type (situational versus past behaviour) was found to moderate interview validity, after controlling for whether studies used answer rating scales. No support was found for the hypothesis that situational questions are less valid for predicting job performance in high-complexity jobs. Sample-weighted mean inter-rater reliabilities were similar for both situational and past behaviour questions, provided that descriptively-anchored rating scales were used (.79 and .77, respectively), although they were slightly lower (.73) for past behaviour question studies lacking such rating scales.

This paper examines whether patterns in communication behavior over time can predict the outcome of crisis negotiations. A sample of 189 interaction episodes was transcribed from 9 resolved negotiations and coded according to differences in the degree and type of behavior. Partial order scalogram analysis (POSAC) was used to produce a graphical representation of the similarities and differences among episodes while simultaneously uncovering the role of each behavior in shaping the negotiation process. Results showed that episodes could be represented along a partially ordered scale of competitiveness, which was structured by the occurrence of two types of behavior: Distributive‐Expressive and Integrative‐Instrumental. The likelihood of negotiation success reduced with movement up the competitive scale, and negotiations involving episodes that passed a threshold of extreme competition on the scale inevitably ended unsuccessfully regardless of future developments. As negotiations developed over time, behavior alternated between periods of increasing cooperation and periods of increasing competition, with unsuccessful negotiations associated with a concluding trend of increasing competitive behavior.

Pisum sativum specific sequence tagged microsatellite site primers were used to amplify genomic profiles from 15accessions of P. sativum L. that represented the genetic base of the Australian field pea-breeding program and five accessions of the wild related species P. fulvum. The STMS primers were used to assess genetic relationships among the Pisum accessions in two ways. Firstly, to produce RAPD-like multiple banding marker profiles using an adapted RAMS method, for intra- and interspecific diversity analysis. From the 14 flanking primer pairs assessed, 133 markers were obtained. Conservation and reproducibility of markers among individuals within accessions was demonstrated. The largest distance observed among P. sativumaccessions was 22% and among P.fulvum accessions was 40%, similar to that revealed with other PCR-based methods. The maximum distance between P.sativum and P. fulvum accessions was 46%. Phylogenetic clustering of P. sativum accessions, using the neighbour joining method and based on simple matching distances, was distinct and distant to P. fulvum. Secondly, PCR with a higher annealing temperature and fluorescent labeling identified simple and allelic loci markers useful for creating agenotype/fingerprint database for P. sativum cultivars. This is the first report to demonstrate the use of Pisum specific STMS sequences for both diversity analysis and genotype identification.

Udgivelsesdato: 2002-Jan-10 The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

This article integrates existing theoretical perspectives on message content and negotiator motivation to formulate a comprehensive definitional model of the interrelationships among communication behaviors in crisis negotiation. A sample of 189 nuclear dialogue spans were transcribed from 9 resolved cases of hostage negotiation and each utterance coded at the level of thought units across 41 behavioral variables. Results of a nonmetric, multidimensional scaling solution provided clear support for the hypothesized cylindrical structure of communication behavior, revealing 3 dominant levels of suspect-negotiator interaction (Avoidance, Distributive, Integrative). At each level of the structure, interactions were found to modulate around 3 thematic styles of communication (Identity, Instrumental, Relational), which reflected the underlying motivational emphasis of individuals’ dialogue. Finally, the intensity of communication was found to play a polarizing role in the cylinder, with intense, functionally discrete behaviors occurring toward the boundary of the structure.

A number of molecular marker techniques have been applied to improve the quality of cultivated lentil, aiding in the breeding and release of better adapted varieties and in quality assurance of export product. Continued increase in lentil production is dependent on the identification of present and potential limiting factors to the progression of lentil breeding. In particular, this chapter is concerned with limitations related to the threat from disease, limitations on the availability of genetic resources and quality assurance issues. The application of molecular markers in the development of strategies to help combat such threats is discussed.

Reversible protein-tyrosine phosphorylation is catalyzed by the antagonistic actions of protein-tyrosine kinases (PTKs) and phosphatases (PTPs), and represents a major form of cell regulation. Acute myeloid leukemia (AML) is an aggressive hematological malignancy that results from the acquisition of multiple genetic alterations, which in some instances are associated with deregulated protein-phosphotyrosine (pY) mediated signaling networks. However, although individual PTKs and PTPs have been linked to AML and other malignancies, analysis of protein-pY networks as a function of activated PTKs and PTPs has not been done. In this study, MS was used to characterize AML proteomes, and phospho-proteome-subsets including pY proteins, PTKs, and PTPs. AML proteomes resolved into two groups related to high or low degrees of maturation according to French-American-British classification, and reflecting differential expression of cell surface antigens. AML pY proteomes reflect canonical, spatially organized signaling networks, unrelated to maturation, with heterogeneous expression of activated receptor and nonreceptor PTKs. We present the first integrated analysis of the pY-proteome, activated PTKs, and PTPs. Every PTP and most PTKs have both positive and negative associations with the pY-proteome. pY proteins resolve into groups with shared PTK and PTP correlations. These findings highlight the importance of pY turnover and the PTP phosphatome in shaping the pY-proteome in AML.

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies.

A novel set of microsatellite markers were developed and employed for geographical and temporal population analyses of Stagonosporopsis tanaceti, the cause of ray blight of pyrethrum in Australia. Genotyping of 407 isolates, using 13 markers, suggested an asexual mode of reproduction with significant linkage disequilibrium and high levels of clonality. Low geographical differentiation and widespread distribution of a few multilocus genotypes (MLGs), in the absence of airborne ascospores, suggested the role of human-mediated movement of seed as a major means of long-distance pathogen dispersal. The genetic composition of S. tanaceti was stable for a decade then changed rapidly in only 2 years. Bayesian clustering analyses and minimum spanning networks determined only two major clonal lineages in and prior to 2010. However, in 2012, a previously unobserved cluster of MLGs was detected, which significantly increased in frequency and displaced the historically dominant MLGs by 2013. This rapid change in the genetic composition of S. tanaceti could indicate a second introduction then a selective sweep, or strong selection pressures from recently introduced fungicides or pyrethrum varieties. These results may have serious implications for durability of management strategies for this disease.

Coiled-coil-helix-coiled-coil-helix domain-containing 2, a mitochondrial protein, encoded by CHCHD2 is located at chromosome 7p11.2 and proximal to the EGFR gene. Here, bioinformatic analyses revealed that CHCHD2 is consistently coamplified with EGFR in non–small cell lung carcinoma (NSCLC). In addition, CHCHD2 and EGFR protein expression levels were positively correlated and upregulated relative to normal lung in NSCLC tumor-derived xenografts. Knockdown of CHCHD2 expression in NSCLC cells attenuated cell proliferation, migration, and mitochondrial respiration. CHCHD2 protein–protein interactions were assessed by the complementary approaches of affinity purification mass spectrometry and in vivo proximity ligation. The CHCHD2 interactome includes the apparent hub proteins C1QBP (a mitochondrial protein) and YBX1 (an oncogenic transcription factor), and an overlapping set of hub-associated proteins implicated in cell regulation. Implications: CHCHD2 influences mitochondrial and nuclear functions and contributes to the cancer phenotype associated with 7p11.2 amplification in NSCLC. Mol Cancer Res; 13(7); 1119–29. ©2015 AACR.

Failure of aldosterone suppression by sodium loading during fludrocortisone suppression testing (FST) or saline suppression testing (SST) confirms primary aldosteronism (PA). We previously found recumbent SST (RSST) to lack sensitivity. Aldosterone levels can be higher upright (e.g. seated) than recumbent in patients with PA and upright levels are used during FST. We therefore hypothesized that seated SST (SSST) is more sensitive than RSST, especially for posture-responsive PA.Of 66 patients who underwent FST (upright plasma aldosterone levels measured at 10am basally and after 4 days fludrocortisone 0.1 mg 6-hourly and oral salt loading), 31 underwent SST (aldosterone levels measured basally at 8am and after infusion of 2 L normal saline over 4h) both recumbent and seated in randomized order and at least 2 weeks apart.FST confirmed PA in 23 of 31 patients (day 4 upright aldosterone level165 pmol/L), excluded PA in three and was originally "inconclusive" in five. However, one with "inconclusive" FST had PA confirmed by lateralizing AVS and was reclassified "unilateral PA". Of 24 with confirmed PA (eight unilateral, 11 bilateral, and five undetermined subtype), 23 (96%) tested positive by SSST (4-h aldosterone level165 pmol/L) compared with 8 (33%) by RSST (4-h plasma aldosterone level140 pmol/L) (P.001). RSST missed one unilateral, all bilateral, and four with as-yet undetermined subtype. RSST was positive in 7 of 10 (70%) posture-unresponsive vs one of 14 (7.1%) posture-responsive patients (P.005).These preliminary results suggest that seated SST may be superior to recumbent SST in terms of sensitivity for detecting PA, especially posture-responsive forms, and may represent a reliable alternative to FST.

Stagonosporopsis chrysanthemi, S. inoxydabilis, and S. tanaceti are closely related Ascomycetes associated with ray blight of the Asteraceae. To date, only S. tanaceti has been identified in Australia, incurring substantial losses to the pyrethrum industry. In contrast to the homothallic S. chrysanthemi and S. inoxydabilis, a sexual state has not been observed for S. tanaceti. The MAT1 locus in S. tanaceti was identified through de novo assembly of shotgun reads, and was further used to develop primers for amplification of the full-length MAT1/2 locus in S. chrysanthemi and S. inoxydabilis. As expected, S. chrysanthemi and S. inoxydabilis possessed a MAT1/2 locus typical of homothallic Dothideomycetes with two adjacent MAT1-1 and MAT1-2 idiomorphs. However, only MAT1-1 could be detected in the assembled genome of S. tanaceti. Although a sexual mode of reproduction cannot be ruled out for S. tanaceti, evidence so far suggests this is absent or occurring at very low frequency in Australian pyrethrum fields.

We report the validation of a quantitative method for paraquat in plasma and urine using high-performance liquid chromatography (HPLC) with ultraviolet detection (260 nm). Furthermore, we illustrate the use of this method in the clinic (over five years), in conjunction with a qualitative urine paraquat screen. Urine or plasma sample (1 mL) preparation was performed in duplicate using C18 solid-phase extraction. Chromatographic separation was achieved on a Zorbax RX-Silica column (250 x 4.6-mm i.d.). The mobile phase consisted of 96% sodium chloride (5 g/L) and 4% acetonitrile (pH 2.2) pumped at 1.0 mL/min. Using a single-point calibration (1.0 mg/L), the method was found to be linear from 0.1 to 5.0 mg/L. The accuracy and imprecision of the method, over the linear range and for plasma and urine, were 94.7-104.9% and12.2%, respectively. The limit of quantitation for both matrices was 0.1 mg/L. The absolute recovery of paraquat from plasma and urine was 79.9 +/- 5.3% and 88.2 +/- 5.3%, respectively. From January 1995 to February 2000, 47 qualitative urine paraquat screens were requested throughout Australia. Nine screens were positive, and eight were confirmed to have paraquat present by our HPLC method. One sample was not analyzed by HPLC because the patient died prior to analysis. Thus, no false-positive results were reported for the qualitative urine screen. An additional 11 samples were referred for patients with positive screens from other sites for HPLC confirmation. The presence of paraquat was confirmed in nine of these samples. In conclusion, a qualitative urine screen combined with our validated HPLC confirmation is an effective protocol for assessing suspected cases of paraquat poisoning.

The effects of storage time (0-8 days), temperature (4 degrees C and 30 degrees C in dark and light), and freeze-thaw cycles on the stability of sirolimus in blood were examined. Sirolimus quantification was undertaken using HPLC-electrospray-tandem mass spectrometry. Whole blood samples supplemented with sirolimus (5.0, 15.0, and 30.0 microg/L) and pooled renal and heart transplant samples were found to be stable during the 8 days under all conditions (10% decrease in concentration). No significant difference was observed in sirolimus concentration between freshly collected patient samples and sirolimus-supplemented samples (5.0, 15.0, and 30.0 microg/L) after three freeze-thaw cycles (p0.198). In conclusion, blood samples can be transported with or without cooling for up to 8 days without sirolimus results being compromised. The reanalysis of sirolimus samples, which may entail freeze-thaw cycles, can be undertaken if the number of cycles is three or less.

Sirolimus, an immunosuppressive agent, is undergoing clinical trials in the prophylaxis of organ rejection.The aim of this study was to compare the performance of the semi-automated prototype (mode IA) microparticle enzyme immunoassay (MEIA) against a validated high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for measuring sirolimus concentrations. A secondary objective was to identify potential factors that may influence sirolimus measurement.The comparison was based on predose samples (n = 841) from 74 renal transplant patients receiving sirolimus therapy. Samples were collected up to 12 months after transplantation.The mean (+/- SD) overestimation by MEIA was 42.5%+/-16.9%. Several variables were investigated to determine potential contributors to the observed overestimation. Stratification of the data based on the mean sirolimus concentrations determined by both assays yielded no statistically significant differences in bias between concentration subgroups within the clinically relevant range. Multiple linear regression analysis identified HPLC-MS sirolimus concentration (P = 0.03), hemoglobin concentration (P0.001), and time after transplantation (P0.001) as significant variables in the prediction of overestimation by MEIA. Analysis of the effect of time after transplantation on overestimation yielded a statistically significant difference up to 6 months after transplantation (35.6% to 46.4%) compared with 9 (23.9%) and 12 months (24.4%). A relationship between hemoglobin concentration and time after transplantation may explain the reduction in bias observed after 6 months.The MEIA overestimates sirolimus concentrations in renal transplant patients compared with HPLC-MS. The clinical importance of this observed overestimation requires further investigation.

Puccinia menthae is highly variable, with several varieties and variants within varieties. Two principal groups of races, nominated spearmint rust and peppermint rust, have been recognized according to their host range on commercially-important Mentha species, yet both belong to the same variety, P. menthae var. menthae. Ten collections of P. menthae teliospores from four Mentha species in Victoria, Australia, were examined using light microscopy and SEM. The teliospores from M. spicata, M.×cordifolia and M. suaveolens, all hosts to spearmint rust, were verrucose with two equally-sized cells, whereas those from M.×piperita, host to peppermint rust, were generally smooth-walled, and the apical cell was larger and thicker-walled than the basal cell. Fifteen isolates of P. menthae collected from four Mentha species in Victoria were assessed for genomic variation using RAPD analysis and PCR-generated length polymorphism of the ITS region of rDNA. Six out of 113 RAPD primers amplified reproducible marker profiles, 58 polymorphic bands were scored and simple matching distances were computed between the isolates. UPGMA cluster analysis was used to produce a dendrogram and non-metric multi-dimensional scaling to produce a two-dimensional map of the isolates. The spearmint rust and peppermint rust isolates clustered into two non-overlapping groups, providing good evidence that there is limited gene flow between them. All isolates had the same sized ITS fragments. The taxonomic implications of these results are discussed and it is suggested that the peppermint and spearmint rusts should be accorded separate varietal or specific status.

Background: Mycophenolic acid kinetics have been reported to vary after renal transplantation, and mycophenolic acid area under the concentration-time curve (AUC) is the best predictor of suppression of graft rejection. Methods: To determine whether mycophenolic acid kinetics vary after renal transplantation and to examine the potential role of enterohepatic recirculation, we investigated the kinetics of mycophenolic acid and mycophenolic acid glucuronide on days 2, 5, and 28 after transplantation in 10 kidney transplant recipients (male/female ratio, 1.5; mean age, 41.7 +/- 5.0 years) given 1 g mycophenolate mofetil twice a day. To facilitate therapeutic drug monitoring, we examined a limited sampling strategy for estimating 12-hour mycophenolic acid [AUC(0-12)]. Results: The mean +/- SE AUC(0-12) for mycophenolic acid on day 28 was 38.5 +/- 1.6 mg . h/L, with a secondary peak 4 to 8 hours after dosing that was attributable to enterohepatic recirculation. Marked variability was shown in the kinetic profile of mycophenolic acid among patients across the three sampling days. Mycophenolic acid AUC(0-12) was positively predicted by both serum creatinine (P = .01) and serum albumin (P = .03) but not by time after transplantation, body weight, or trough concentration. Limited sampling (at 0, 1, 3, and 6 hours) accounted for 84.1% of the variability in the mycophenolic acid AUC(0-12) data and predicted the AUC(0-12) closely (r(2) = 0.954) when evaluated in 10 different kidney transplant recipients. Conclusions: Mycophenolic acid AUC(0-12) is predicted by serum albumin and creatinine after kidney transplantation, and the AUC(0-12) may be determined during the early posttransplant period while the patient remains hospitalized with use of a limited sampling strategy to facilitate therapeutic drug monitoring.

A longitudinal evaluation was conducted on the effects of introducing a performance management system (PMS), which featured merit-based bonus pay, on subsequent employee attitudes and self-reported work effort in a small, government organization. Additionally, employees' targets of blame for receiving lower-than-expected ratings were explored. A significant change in employees' organizational commitment occurred over the time that the PMS was implemented, with a substantial increase occurring within the performance planning/goal-setting phase, followed by a slight decay over the following year, but still ending higher than the pre-PMS baseline level. Substantial increases in ratings of satisfaction and cooperation with one's supervisor were found with the introduction of the PMS for low performers (particularly following the performance planning/goal-setting phase). In contrast, however, high performers had high base-line levels of these attitudes toward supervision, followed by substantial drops immediately after receiving appraisal and bonus pay distributions. As anticipated, most participants in this study had expected a performance rating higher than they actually received, and most of these individuals made external attributions for the rating discrepancy, blaming either their supervisor, the organization, or the PMS itself. However, neither having received a lower-than-expected appraisal rating nor having made external attributions for a lower-than-expected rating were related to changes in attitudes or self-reported effort.

Background: The measurement of renin activity is complicated by difficulties in the quantification of angiotensin 1 (Ang1), the product of the renin-catalyzed reaction. We report an HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of Ang1 as a measure of plasma renin activity (PRA).Methods: After incubation (37 °C for 3 or 18 h), samples were prepared using C18 solid-phase extraction. [Val]5Ang1 was used as the internal standard (IS). Chromatography was performed on a C18 column, using 200 mL/L ammonium acetate buffer–800 mL/L methanol as the mobile phase. The flow rate was 150 μL/min, with a chromatographic run time of 5 min/sample. Mass spectrometric detection was in the positive ionization mode with selected reaction monitoring (Ang1 m/z 649.0→784.0; IS m/z 641.9→770.4).Results: The assay was linear over the range 2.5–500 ng Ang1/mL, which corresponded to a limit of detection (signal-to-noise ratio of 3:1) of PRA of 0.14 ng Ang1 · mL−1 · h−1. The imprecision (CV) of the assay at PRA values of 26.1, 13.5, 3.2, and 0.78 ng Ang1 · mL−1 · h−1 was 7.0%, 7.0%, 15%, and 11%, respectively. Absolute recoveries were 92.3% (Ang1) and 87.4% (IS). Incubation times of 3 h vs 18 h in the PRA assay gave good agreement at PRA Conclusion: The HPLC-ESI-MS/MS method allows sensitive and specific measurement of PRA. The higher activities measured with the RIA method highlight its potential for overestimation of PRA.

Foliar resistance to Ascochyta lentis is controlled at a single major locus by a dominant gene (AbR 1 ) in the lentil accession ILL5588 (cv ‘Northfield’). Flanking RAPD markers that are closely linked to the resistance locus in coupling phase were identified by bulked segregant analysis. Out of 261 decanucleotide primers screened 7 produced a polymorphic marker that segregated with the resistance locus, and all markers were found to exist within a single linkage group. Five of the seven RAPD markers were within 30 cM of the resistance locus. Log likelihood analysis for detecting QTL associated with the foliar resistance revealed that a single narrow peak accounted for almost 90% of the variance of resistance between the bulks. Preliminary mapping in an F3 population revealed that the closest flanking markers were approximately 6 and 14 centiMorgans (cM) away from the resistance locus. These markers should be useful for the discrimination of resistant germplasm through marker-assisted selection in future breeding programmes and represent the first essential step towards the map-based cloning of this resistance gene.

Eight Lathyrus sativus L. accessions from a variety of geographic origins were used to study intraspecific genetic diversity using RAPD analysis. Fourteen decamer primers produced 64 amplification products, 50% of which were polymorphic between the samples. Jaccard's coefficient of genetic similarity was calculated between samples and a dendrogram was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). The dendrogram showed that most of the L. sativus plants clustered into accessions or common geographical areas. The average genetic similarity coefficient within accessions was 0.12 and between accessions was 0.20, indicating a low level of intraspecific genetic variation. Interspecific genetic diversity and phylogenetic relationships of eight Lathyrus species, including L. sativus and Pisum sativum L. (field pea) were examined using 14 decamer primers which produced 283 amplification products. All amplification products were polymorphic across the nine species. In the dendrogram the Lathyrus species clustered into three distinct groups which correlated with the Sections Lathyrus, Clymenum and Linearicarpus. This supports traditional taxonomic classifications of the genus Lathyrus which are based on morphological traits. Of the species from Section Lathyrus, L. gorgoni and L. cicera were the most similar to L. sativus. The results suggest that a strategy of breeding for producing lines of L. sativus with increased genetic variation would be effectively achieved through hybrid production between accessions from wide geographic areas particularly the Mediterranean area and the Indian subcontinent. However, the most effective method would be introgression of germplasm from other species in Section Lathyrus.

Bacterial biofilms have been observed and reported on food and food-processing surfaces and can contribute to increased risks for product quality and food safety. The colonization of fruit and vegetables by pectynolitic bacteria like Pseudonomas fluorescens attributable to conditions such as soft rot, can also manifest as biofilms. A developed biofilm structure can provide a protective environment for pathogens such as Listeria monocytogenes reducing the effectiveness of sanitisers and other inhibitory agents. Understanding the colonization of bacteria on leaf surfaces is essential to the development of a better understanding of the leaf ecology of vegetable products. Studies of microbial colonization of leaf surfaces have been conducted using SEM and more recently using confocal microsocpy techniques. In the current study, a Leica TCS NT laser scanning confocal microscope was used to investigate biofilm formation using vital fluorescence staining on intact vegetable leaves. Reflection contrast and fluorescence three-dimensional imaging successfully delineated bacterial and biofilm morphology without disturbing the bacterial or leaf surface structure. The results demonstrate the presence and development of biofilm on the surface of lettuce. The biofilms appeared to originate on the cuticle in distinct micro-environments such as in the natural depression of the stomata, or in the intercellular junction. Bacteria also adhered to and developed biofilm colonies within an hour of contact and with clean stainless steel surfaces. Our study investigates the progression of biofilm formation from leaf colonization, and will assist in characterising the critical mechanisms of plant/host interaction and facilitate the development of improved preservation, sanitising and packaging strategies for minimally processed vegetable products.

The authors report the use of high-performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) for the quantification of indomethacin (IND) in plasma with microscale sample preparation. Plasma samples (100 microL) and mefanamic acid (internal standard [IS]), buffered to pH 3.5, were prepared using solid phase extraction and chromatographed using a C8 column. The mobile phase composition was 80% methanol to 20% ammonium acetate buffer (40 mM, pH 5.1). A flow rate of 300 microL per minute was used with a 1-to-12 postcolumn split into the mass spectrometer. Selected reaction monitoring with mass transitions m/z 357.9-->139.0 and m/z 242-->209.0 were used for IND and IS, respectively. The chromatographic analysis time was 4 minutes. The assay was linear from 5 microg/L to 2000 microg/L with interday imprecision (n=5) over the analytic range (5%). At four concentrations (10 microg/L, 25 microg/L, 250 microg/L, 1500 microg/L), assay imprecision was 9% (total coefficient of variation [CV]) and accuracy ranged between 96.5% and 102.8% (n=16). The absolute recovery of IND and IS was 74% (n=8) and 95% (n=24), respectively. This method was developed and validated in less than 10 working days, had a lower limit of quantification than reported HPLC-ultraviolet (UV) methods, and uses small sample volumes. These factors illustrate the power of HPLC-ESI-MS/MS for drug analysis. Furthermore, the ability of this method to measure IND over a wide concentration range makes it suitable for therapeutic drug monitoring and pharmacokinetic studies.