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Objectives: This paper examined whether nebivolol protects the heart via nitric oxide (NO) synthase and NO-dependent signaling in an in vivo model of acute myocardial infarction.Background: Beta(3)-adrenergic receptor (AR) activation promotes endothelial nitric oxide synthase (eNOS) activity and NO bioavailability. We hypothesized that specific beta(3)-AR agonists would attenuate myocardial ischemia-reperfusion (MI/R) injury via eNOS activation and increased NO bioavailability.Methods: Mice were subjected to 45 min of myocardial ischemia in vivo followed by 24 h of reperfusion (R). Nebivolol (500 ng/kg), CL 316243 (1 μg/kg), BRL-37344 (1 μg/kg), or vehicle (VEH) was administered at the time of R. Myocardial area-at-risk (AAR) and infarct size (INF)/AAR was measured at 24 h of R. Cardiac tissue and plasma were collected to evaluate eNOS phosphorylation, neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase expression, and nitrite and nitrosothiol levels.Results: Nebivolol (500 ng/kg) reduced INF/AAR by 37% (p < 0.001 vs. VEH) and serum troponin-I levels from 41 ± 4 ng/ml to 25 ± 4 ng/ml (p < 0.05 vs. VEH). CL 316243 and BRL-37344 reduced INF by 39% and 42%, respectively (p < 0.001 vs. VEH). Nebivolol and CL 316243 increased eNOS phosphorylation at Ser-1177 (p < 0.05 vs. VEH) and increased nitrite and total nitrosylated protein levels. Nebivolol and CL 316243 significantly increased myocardial nNOS expression. Nebivolol failed to reduce INF after MI/R in beta(3)-AR (-/-), eNOS(-/-), and in nNOS(-/-) mice.Conclusions: Our results indicate that beta(3)-AR agonists protect against MI/R injury. Furthermore, the cardioprotective effects of beta(3)-AR agonists are mediated by rapid eNOS and nNOS activation and increased NO bioavailability. [ABSTRACT FROM AUTHOR]

Moral injury has emerged as a construct of interest in healthcare workers' (HCW) occupational stress and health. We conducted one of the first multidisciplinary, longitudinal studies evaluating the relationship between exposure to potentially morally injurious events (PMIEs), burnout, and turnover intentions. HCWs (N = 473) completed surveys in May of 2020 (T1) and again in May of 2021 (T2). Generalized Linear Models (robust Poisson regression) were used to test relative risk of turnover intentions, and burnout at T2 associated with PMIE exposure, controlling for T1 covariates. At T1, 17.67% reported they had participated in a PMIE, 41.44% reported they witnessed a PMIE and 76.61% reported feeling betrayed by healthcare or a public health organization. In models including all T1 PMIE exposures and covariates, T2 turnover intentions were increased for those who witnessed a PMIE at T1 (Relative Risk [RR] = 1.66, 95% Confidence Interval [CI] 1.17-2.34) but not those that participated or felt betrayed. T2 burnout was increased for those who participated in PMIE at T1 (RR = 1.38, 95%CI 1.03-1.85) but not those that witnessed or felt betrayed. PMIE exposure is highly prevalent among HCWs, with specific PMIEs associated with turnover intentions and burnout. Organizational interventions to reduce and facilitate recovery from moral injury should account for differences in the type of PMIE exposures that occur in healthcare work environments., (© 2024. The Author(s).)

Purpose: Healthcare workers are at increased risk for mental health problems during disasters such as the COVID-19 pandemic. Identifying resilience mechanisms can inform development of interventions for this population. The current study examined pathways that may support healthcare worker resilience, specifically testing enabling (social support enabled self-efficacy) and cultivation (self-efficacy cultivating support) models., Methods: Healthcare workers (N = 828) in the Rocky Mountain West completed self-report measures at four time points (once per month from April to July of 2020). We estimated structural equation models to explore the potential mediating effects that received social support and coping self-efficacy had (at time 2 and time 3) between traumatic stress symptom severity (at time 1 and time 4). Models included covariates gender, age, minority status, and time lagged co-variations between the proposed mediators (social support and coping self-efficacy)., Results: The full model fit the data well, CFI = .993, SRMR = .027, RMSEA = .036 [90% CIs (0.013, 0.057)]. Tests of sequential mediation supported enabling model dynamics. Specifically, the effects of time 1 traumatic stress severity were mediated through received social support at time 2 and time 3 coping self-efficacy, in sequential order to reduce time 4 traumatic stress severity., Conclusions: Findings show the importance of received social support and coping self-efficacy in mitigating psychopathology risk. Interventions can support mental health by focusing on social resource engagement that facilitates coping empowerment, which may decrease risk for mental health job-related problems among frontline healthcare workers exposed to highly stressful events., (© 2022. The Author(s).)

First responders are at high risk for disorders that arise from repeat exposure to stress and trauma (Post Traumatic Stress Disorder, depression, and problematic alcohol use). Although mental health treatments are available, first responders often do not access them, anchored by barriers that include: lack of knowledge, stigma, negative experience with mental health providers, and time-based burdens. In this study, we designed an intervention to address these barriers, extending a Planned-Action framework. Step 1 involved self-report screening for four mental health risks (PTSD, depression, anxiety, and alcohol use risk), delivered to all personnel electronically, who were free to either consent and participate or opt-out. The detection of risk(s) in Step 1 led to scheduling a Step 2 telehealth appointment with a trained clinician. We report descriptive statistics for participation/attrition/utilization in Steps 1 and 2, rates of risk on four mental health variables, and rate of adherence to follow-up treatment recommendations. Step 1: In total, 53.3% of personnel [229 of 429 full-time employees (221 males; eight females; 95% White; 48% paramedic or Emergency Medical Technician; 25% captain; 19% engineer; 7% other)] initially opted-in by consenting and completing the brief remote screening survey. Among those who opted-in and completed ( n = 229), 43% screened positive for one or more of the following mental health risks: PTSD (7.9%); depression (9.6%); anxiety (13.5%); alcohol use (36.7%). Step 2: A maximum of three attempts were made to schedule "at risk" individuals into Step 2 ( n = 99). Among the 99 who demonstrated a need for mental health treatment (by screening positive for one or more risk), 56 (56.6%) engaged in the telehealth appointment. Of the 56 who participated in Step 2 clinical appointments, 38 were recommended for further intervention (16.6% of full-time personnel who participated). Among the 38 firefighters who were recommended to seek further mental health services, 29 were adherent/followed through (76.3% of those who received recommendations for further services). Taken together, evidence-based, culturally conscious, stepped care models delivered via the virtual/telehealth medium can promote access, utilization, and cost-effective mental health services for first responders. Implications are for informing larger, more rigorous dissemination and implementation efforts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wright, Fuessel-Hermann, Pazdera, Lee, Ridge, Kim, Konopacki, Hilton, Greensides, Langenecker and Smith.)

The mental health of frontline workers is critical to a community's ability to manage crises and disasters. This study assessed risks for mental health problems (traumatic stress, depression, anxiety, alcohol use, insomnia) in association with pandemic-related stressors in a sample of emergency and hospital personnel (N = 571). Respondents completed self-report surveys online from April 1st to May 7th, 2020 in the Rocky Mountain region of the United States. Results showed that roughly fifteen to thirty percent of respondents screened positive for each disorder. Odds of screening positive were similar between groups for probable acute traumatic stress, depressive disorder, anxiety disorder, and alcohol use disorder; emergency personnel reported significantly higher rates of insufficient sleep than healthcare workers. Logistic regressions showed that respondents who reported having an immunocompromised condition had higher odds of acute traumatic stress, anxiety, and depression. Having an immunocompromised household member was associated with higher odds of insufficient sleep and anxiety. Being in a direct care provision role was associated with higher odds of screening positive for risky alcohol use. Being in a management role over direct care providers was associated with higher odds of screening positive for anxiety, risky alcohol use, and insufficient sleep. There was an inverse relationship between number of positive COVID-19 cases and anxiety, such that as positive cases went up, anxiety decreased. Overall, the mental health risks that we observed early in the COVID-19 pandemic are elevated above previous viral outbreaks (SARS) and comparable to rates shown in disasters (9/11 attacks; Hurricane Katrina)., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Modern operational environments can place significant demands on a service member's cognitive resources, increasing the risk of errors or mishaps due to overburden. The ability to monitor cognitive burden and associated performance within operational environments is critical to improving mission readiness. As a key step toward a field-ready system, we developed a simulated marksmanship scenario with an embedded working memory task in an immersive virtual reality environment. As participants performed the marksmanship task, they were instructed to remember numbered targets and recall the sequence of those targets at the end of the trial. Low and high cognitive load conditions were defined as the recall of three- and six-digit strings, respectively. Physiological and behavioral signals recorded included speech, heart rate, breathing rate, and body movement. These features were input into a random forest classifier that significantly discriminated between the low- and high-cognitive load conditions (AUC = 0.94). Behavioral features of gait were the most informative, followed by features of speech. We also showed the capability to predict performance on the digit recall (AUC = 0.71) and marksmanship (AUC = 0.58) tasks. The experimental framework can be leveraged in future studies to quantify the interaction of other types of stressors and their impact on operational cognitive and physical performance., (Copyright © 2020 Massachusetts Institute of Technology.)

Background: Feedback can alter medical student logging practices, although most learners feel feedback is inadequate. A varied case mix in rural and urban contexts offers diverse clinical encounters. Logs are an indicator of these clinical experiences, and contain opportunities for feedback, which can greatly influence learning: we labelled these 'feedback learning opportunities' (FLOs). We asked: How often do FLOs occur? What are the case complexities of rural compared to urban paediatric logs? Do more complex cases result in more FLOs?, Methods: In Western Australia, 25% of medical students are dispersed in a Rural Clinical School (RCSWA) up to 2175 miles (3500 km) from the city. Urban students logged 20 written cases; rural students logged a minimum of 25 paediatric cases electronically. These were reviewed to identify FLOs, using a coding convention. FLO categories provided a structure for feedback: medical, professionalism, insufficient, clinical reasoning, student wellbeing, quality and safety, and sociocultural. Each log was assigned an overall primary, secondary or tertiary case complexity., Results: There were 76 consenting students in each urban and rural group, providing 3034 logs for analysis after exclusions. FLOs occurred in more than half the logs, with significantly more rural (OR 1.35 95% CI 1.17, 1.56; p < 0.0001). Major FLOs occurred in over a third of logs, but with no significant difference between rural and urban (OR 1.10 95% CI 0.94, 1.28; p = 0.24). Medical FLOs were the most common, accounting for 64.0% of rural and 75.2% of urban FLOs (OR 1.71 95% CI 1.37, 2.12; p < 0.0001). Students logged cases with a variety of complexities. Most cases logged by urban students in a tertiary healthcare setting were of primary and secondary complexity. Major medical FLOs increased with increasing patient complexity, occurring in 32.1% of tertiary complexity cases logged by urban students (p < 0.001)., Conclusions: Case logs are a valuable resource for medical educators to enhance students' learning by providing meaningful feedback. FLOs occurred often, particularly in paediatric cases with multiple medical problems. This study strengthens recommendations for regular review and timely feedback on student logs. We recommend the FLOs categories as a framework for medical educators to identify FLOs.

Background: Exposure to a representative case mix is essential for clinical learning, with logbooks established as a way of demonstrating patient contacts. Few studies have reported the paediatric case mix available to geographically distributed students within the same medical school. Given international interest in expanding medical teaching locations to rural contexts, equitable case exposure in rural relative to urban settings is topical. The Rural Clinical School of Western Australia locates students up to 3500 km from the urban university for an academic year. There is particular need to examine paediatric case mix as a study reported Australian graduates felt unprepared for paediatric rotations. We asked: Does a rural clinical school provide a paediatric case mix relevant to future practice? How does the paediatric case mix as logged by rural students compare with that by urban students?, Methods: The 3745 logs of 76 urban and 76 rural consenting medical students were categorised by presenting symptoms and compared to the Australian Institute of Health and Welfare (AIHW) database Major Diagnostic Categories (MDCs)., Results: Rural and urban students logged core paediatric cases, in similar order, despite the striking difference in geographic locations. The pattern of overall presenting problems closely corresponded to Australian paediatric hospital admissions. Rural students logged 91% of cases in secondary healthcare settings; urban students logged 90% of cases in tertiary settings. The top four presenting problems were ENT/respiratory, gastrointestinal/urogenital, neurodevelopmental and musculoskeletal; these made up 60% of all cases. Rural and urban students logged similar proportions of infants, children and adolescents, with a variety of case morbidity., Conclusions: Rural clinical school students logged a mix of core paediatric cases relevant to illnesses of Australian children admitted to public hospitals, with similar order and pattern by age group to urban students, despite major differences in clinical settings. Logged cases met the curriculum learning outcomes of graduates. Minor variations were readily addressed via recommendations about logging. This paper provides evidence of the legitimacy of student logs as useful tools in affirming appropriate paediatric case mix. It validates the rural clinical school context as appropriate for medical students to prepare for future clinical paediatric practice.

: To treat hypertension, combining two or more antihypertensive drugs from different classes is often necessary. β-Blockers and renin-angiotensin-aldosterone system inhibitors, when combined, have been deemed 'less effective' based on partially overlapping mechanisms of action and limited evidence. Recently, the single-pill combination (SPC) of nebivolol (Neb) 5 mg - a vasodilatory β1-selective antagonist/β3 agonist - and valsartan 80 mg, an angiotensin II receptor blocker, was US Food and Drug Administration-approved for hypertension. Pharmacological profiles of Neb and valsartan, alone and combined, are well characterized. In addition, a large 8-week randomized trial in stages I-II hypertensive patients (N = 4161) demonstrated greater blood pressure-reducing efficacy for Neb/valsartan SPCs than component monotherapies with comparable tolerability. In a biomarkers substudy (N = 805), Neb/valsartan SPCs prevented valsartan-induced increases in plasma renin, and a greater reduction in plasma aldosterone was observed with the highest SPC dose vs. valsartan 320 mg/day. This review summarizes preclinical and clinical evidence supporting Neb/valsartan as an efficacious and well tolerated combination treatment for hypertension.

Women engaged in computing career tracks are vastly outnumbered by men and often must contend with negative stereotypes about their innate technical aptitude. Research suggests women's marginalized presence in computing may result in women psychologically disengaging, and ultimately dropping out, perpetuating women's underrepresentation in computing. To combat this vicious cycle, the Computing Research Association's Committee on the Status of Women in Computing Research (CRA-W) runs a multi-day mentorship workshop for women graduate students called Grad Cohort, which consists of a speaker series and networking opportunities. We studied the long-term impact of Grad Cohort on women Ph.D. students' (a) dedication to becoming well-known in one's field, and giving back to the community ( professional goals ), (b) the degree to which one feels computing is an important element of "who they are" ( computing identity) , and (c) beliefs that computing skills are innate ( entity beliefs ). Of note, entity beliefs are known to be demoralizing and can lead to disengagement from academic endeavors. We compared a propensity score matched sample of women and men Ph.D. students in computing programs who had never participated in Grad Cohort to a sample of past Grad Cohort participants. Grad Cohort participants reported interest in becoming well-known in their field to a greater degree than women non-participants, and to an equivalent degree as men. Also, Grad Cohort participants reported stronger interest in giving back to the community than their peers. Further, whereas women non-participants identified with computing to a lesser degree than men and held stronger entity beliefs than men, Grad Cohort participants' computing identity and entity beliefs were equivalent to men. Importantly, stronger entity beliefs predicted a weaker computing identity among students, with the exception of Grad Cohort participants. This latter finding suggests Grad Cohort may shield students' computing identity from the damaging nature of entity beliefs. Together, these findings suggest Grad Cohort may fortify women's commitment to pursuing computing research careers and move the needle toward greater gender diversity in computing.

Combining different classes of antihypertensives is more effective for reducing blood pressure (BP) than increasing the dose of monotherapies. The aims of this phase I study were to investigate pharmacokinetic and pharmacodynamic interactions between nebivolol, a vasodilatory β1-selective blocker, and valsartan, an angiotensin II receptor blocker, and to assess safety and tolerability of the combination. This was a single-center, randomized, open-label, multiple-dose, 3-way crossover trial in 30 healthy adults aged 18-45 years. Participants were randomized into 1 of 6 treatment sequences (1:1:1:1:1:1) consisting of three 7-day treatment periods followed by a 7-day washout. Once-daily oral treatments comprised nebivolol (20 mg), valsartan (320 mg), and nebivolol-valsartan combination (20/320 mg). Outcomes included AUC0-τ,ss, Cmax,ss, Tmax,ss, changes in BP, pulse rate, plasma angiotensin II, plasma renin activity, 24-hour urinary aldosterone, and adverse events. Steady-state pharmacokinetic interactions were observed but deemed not clinically significant. Systolic and diastolic BP reduction was significantly greater with nebivolol-valsartan combination than with either monotherapy. The mean pulse rate associated with nebivolol and nebivolol-valsartan treatments was consistently lower than that associated with valsartan monotherapy. A sharp increase in mean day 7 plasma renin activity and plasma angiotensin II that occurred in valsartan-treated participants was significantly attenuated with concomitant nebivolol administration. Mean 24-hour urine aldosterone at day 7 was substantially decreased after combined treatment, as compared with either monotherapy. All treatments were safe and well tolerated. In conclusion, nebivolol and valsartan coadministration led to greater reductions in BP compared with either monotherapy; nebivolol and valsartan lower BP through complementary mechanisms.

Students in the Rural Clinical School of Western Australia (RCSWA) spend one year of clinical study learning in small groups while embedded in rural or remote communities. This aims to increase the locally trained rural medical workforce. Their learning environment, the clinical context of their learning, and their rural doctor-teachers all contrast with the more traditional learning setting in city hospitals. The RCSWA has succeeded in its outcomes for students and in rural medical workforce impact; it has grown from 4 pilot sites to 14 in 12 years. This reflective piece assimilates observations of the formation of the RCSWA pedagogy and of the strategic alignment of education technologies with learning environment and pedagogy over a seven-year period. Internal and external influences, driving change in the RCSWA, were considered from three observer perspectives in a naturalistic setting. Flexibility in both education technologies and organizational governance enabled education management to actively follow pedagogy. Peter Senge's learning organization (LO) theory was overlaid on the strategies for change response in the RCSWA; these aligned with those of known LOs as well with LO disciplines and the archetypal systems thinking. We contend that the successful RCSWA paradigm is that of an LO., (© 2015 SAGE Publications.)

Introduction: The efficacy of oral pharmacotherapy for erectile dysfunction (ED) (i.e., type 5 phosphodiesterase[PDE5] inhibitors) is significantly reduced in diabetic patients. Nebivolol is a selective β1-blocker used for treatinghy pertension that has been shown to increase the efficacy of sildenafil to reverse ED in diabetic rats., Aim: To evaluate the effects of nebivolol on the efficacy of the PDE5 inhibitors, sildenafil, tadalafil, and vardenafil to relax human corpus cavernosum (HCC) and vasodilate human penile resistance arteries (HPRA) from diabetic patients with ED (DMED). The influence of nebivolol on the capacity of these three PDE5 inhibitors to stimulate cyclic guanosine monophosphate (cGMP) production in HCC was also evaluated., Methods: HCC and HPRA were obtained from organ donors without ED (NEND; n = 18) or patients with diabetes undergoing penile prosthesis implantation (DMED; n = 19). Relaxations of HCC strips and HPRA to sildenafil,tadalafil, and vardenafil were evaluated in organ chambers and wire myographs. cGMP content in HCC was determined by ether extraction and quantification by ELISA., Main Outcome Measures: Effects of nebivolol on PDE5 inhibitor-induced relaxation of HCC, vasodilation ofHPRA and cGMP accumulation in HCC., Results: Treatment with nebivolol (1 μM) significantly potentiated sildenafil-, tadalafil- and vardenafil-induced relaxations of HCC and vasodilations of HPRA from both NEND and DMED. Enhancement of relaxant capacity by nebivolol resulted in reversion of the impairment of PDE5 inhibition-induced responses in DMED and it was accompanied by enhancing the ability of PDE5 inhibitors to increase cGMP in HCC restoring reduced cGMP levelsin HCC from DMED., Conclusions: Nebivolol potentiated the capacity of PDE5 inhibitors to relax vascular structures of erectile tissue from diabetic patients by enhancing the nitric oxide (NO)/cGMP pathway in these tissues. These effects suggest a potential therapeutic utility of nebivolol as an adjunct to PDE5 inhibitors for the treatment of ED associated with diabetes.

We investigated the safety and efficacy of 90 mg/m(2) bendamustine HCL, administered intravenously on days 1 and 2 every 28 days in 10 women with platinum and taxane resistant epithelial ovarian cancer. There were no objective tumor responses observed; 2 patients had stable disease. Plasma samples collected at pre-treatment and end of cycle one were analyzed for changes in circulating total cytokeratin 18 and caspase cleaved cytokeratin 18 as exploratory early biomarkers of bendamustine-induced tumor cell death. All patients had measureable levels of both total and cleaved caspase 3 cytokeratin 18, but no relationship with response was possible due to the lack of clinical benefit in treated patients. Due to the high incidence of adverse events and absence of objective responses, only ten patients were treated as predefined by the Simon Two-Stage Design in the protocol. Overall, the regimen was not well tolerated and was associated with fatigue and a greater number of gastrointestinal side effects as compared to previously reported experiences in different patient populations. However, our study subjects did experience less bone marrow suppression. The lack of tolerability could reflect the degree of tumor burden within the peritoneal cavity as well as the high number of prior regimens (median of 5) received by the patients participating in this study.

The objective of this study was to determine the pharmacokinetic properties and short-term adverse effect profile of single-dose oral rufinamide in healthy dogs. Six healthy adult dogs were included in the study. The pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of 20.0 mg/kg (range 18.6-20.8 mg/kg). Plasma rufinamide concentrations were determined using high-performance liquid chromatography, and pharmacokinetic data were analyzed using commercial software. No adverse effects were observed. The mean terminal half-life was 9.86 ± 4.77 h. The mean maximum plasma concentration was 19.6 ± 5.8 μg/mL, and the mean time to maximum plasma concentration was 9.33 ± 4.68 h. Mean clearance was 1.45 ± 0.70 L/h. The area under the curve (to infinity) was 411 ± 176 μg · h/mL. Results of this study suggest that rufinamide given orally at 20 mg/kg every 12 h in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. Further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted., (© 2011 Blackwell Publishing Ltd.)

Purpose: This phase I trial evaluated intraperitoneal (i.p.) pemetrexed, cisplatin, and paclitaxel in optimally debulked ovarian cancer., Experimental Design: Dose escalation of day 1 i.p. pemetrexed accrued three patients to each of five dose levels (60-1,000 mg/m(2)), along with day 2 i.p. cisplatin (75 mg/m(2)) and day 8 i.p. paclitaxel (60 mg/m(2)). The goals were to determine maximum tolerated dose (MTD), 18-month progression-free survival (PFS), and pharmacokinetics of i.p. pemetrexed., Results: Cycles, given every 21 days, had an 80% 6-cycle completion rate. There was minimal grade III toxicity in the first 4 dose levels and remarkably an almost complete absence of peripheral neuropathy and alopecia. At the highest dose level, two of three patients experienced ≥ grade III and dose-limiting toxicity (DLT; hematologic, infection, gastrointestinal). There was a pharmacokinetic advantage for i.p. pemetrexed with an intraperitoneal:plasma area under the concentration-time curve ratio of 13-fold. Neither analysis of pharmacokinetic nor homocysteine levels explains the unexpected severity of toxicity in those two patients. On the basis of plasma C(24h) levels, the 42 cycles at ≥ 500 mg/m(2) i.p. pemetrexed without DLT, the MTD appears to be 500 mg/m(2). Median PFS is 30.1 months; 18-month PFS is 78.6% (median follow-up 22.4 months)., Conclusions: This i.p.-only regimen in front-line ovarian cancer is feasible with PFS in line with recent literature. We suggest phase II trials of this regimen in this population with i.p. pemetrexed at 500 mg/m(2). The favorable toxicity profile at doses <1,000 mg/m(2), which needs to be confirmed, appears to compare well with standard combination i.v./i.p. platinum/taxane chemotherapy in this disease., (©2012 AACR.)

A method for analysis of lacosamide [(R)-2-acetamido-N-benzyl-3-methoxypropionamide] is needed for both human and veterinary pharmacokinetic investigations. While lacosamide is currently used to manage partial-onset seizures in humans suffering from epilepsy, it is also presently being investigated for use in the treatment of canine epilepsy in veterinary medicine. Currently, no dosing regimen for the drug exists in dogs. A novel and simple high-performance liquid chromatography method was developed for determination of lacosamide in dog serum. Serum proteins (0.1 mL) were precipitated with -20.0°C acetonitrile after addition of the internal standard, daidzein. Separation was achieved with a Phenomenex® Luna® C₁₈ (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 210 nm. The calibration curves were linear ranging from 0.5 to 25 µg/mL. Precision of the assay was <13% (RSD) and was within 12% for all points in the calibration curve. The limit of quantitation for this method was 0.5 µg/mL. The assay was applied successfully to a pre-clinical study of lacosamide pharmacokinetics in dogs., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: To describe the outcome of 3 cases of ivermectin toxicosis in dogs homozygous for the ABCB1-1Δ gene mutation treated with intravenous fat emulsion (IFE)., Series Summary: One Australian Shepherd and 2 Miniature Australian Shepherds were treated for naturally occurring ivermectin toxicosis with IFE. All 3 dogs were homozygous for the ABCB1-1Δ gene mutation. Serum ivermectin concentrations confirmed ivermectin exposure in each case. All 3 dogs exhibited tremors, ptyalism, and central nervous system depression, which progressed over several hours to stupor in 2 dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. A 20% formulation of IFE(a) was administered as an IV bolus (1.5 mL/kg) followed by a slow IV infusion (7.5-15 mL/kg [0.25-0.5 mL/kg/m], over 30 minutes). No change was observed in the neurologic status of any patient. Lipemia visible upon blood sampling persisted for 36 hours in 1 dog however, no other adverse effects were noted. Flumazenil (0.01 mg/kg IV), followed by a constant rate infusion(CRI) of 0.01 mg/kg/h IV was administered in 1 case, without any apparent clinical benefit or adverse effect., New or Unique Information Provided: IFE was ineffective in the treatment of ivermectin toxicosis in these ABCB1-1Δ homozygous mutant dogs. Further investigation is necessary to determine why IFE treatment was unsuccessful in these cases and whether its use can be optimized to yield better results., (© Veterinary Emergency and Critical Care Society 2011.)

The need for completion axillary dissection after a positive sentinel node biopsy continues to be challenged. In the 2 years since we last reviewed this subject, a number of authors have shared their experiences about micrometastatic disease and isolated tumor cells, opining both for and against axillary treatment. Data from the ACOSOG Z0011 trial and other small studies do not appear to support the use of completion axillary dissection even for macro-metastatic disease in patients with clinically node-negative (N0) disease. While existing guidelines still recommend axillary dissection for patients with clinically positive nodes, even when conversion to clinically negative disease following neoadjuvant chemotherapy has occurred, this concept is being questioned in ACOSOG Z1071 and in several other recent small trials. The surgical approach to the treatment of breast cancer continues to move away from the traditional Halstedian concept.

Prerelease reconditioning improves the chance of survival of rehabilitating raptors. Reconditioning may also help to rehabilitate waterfowl, including those that are threatened or endangered, especially if the birds are released during periods of migration. A flying harness, creance, remote-controlled launcher, and portable tower were used to create a means of reconditioning a rehabilitating 5-month-old female wild mallard duck (Anas platyrhynchos) that had been housed in a rehabilitation center for 7 weeks while recovering from an injury. Pre- and postflight serum lactate levels, body condition index scores, and controlled flight distances were used to assess the bird's degree of conditioning. Postflight serum lactate levels never returned to preflight levels and were not deemed a reliable indicator of physical fitness. However, the mallard showed an increase in endurance and strength as well as improved body condition index scores over the course of the reconditioning program.

Purpose: This phase II trial evaluated bevacizumab plus erlotinib in platinum-resistant ovarian cancer; exploratory biomarker analyses, including that of tumor vascular endothelial growth factor A (VEGF-A), were also done., Experimental Design: Forty heavily pretreated patients received erlotinib (150 mg/d orally) and bevacizumab (10 mg/kg i.v.) every 2 weeks until disease progression. Primary end points were objective response rate and response duration; secondary end points included progression-free survival (PFS), toxicity, and correlations between angiogenic protein levels, toxicity, and efficacy., Results: Grade 3 toxicities included skin rash (n = 6), diarrhea (n = 5), fatigue (n = 4), and hypertension (n = 3). Grade 4 toxicities were myocardial infarction (n = 1) and nasal septal perforation (n = 1). Only one grade 3 fistula and one grade 2 bowel perforation were observed. Nine (23.1%) of 39 evaluable patients had a response (median duration, 36.1+ weeks; one complete response), and 10 (25.6%) patients achieved stable disease, for a disease control rate of 49%. Median PFS was 4 months, and 6-month PFS was 30.8%. Biomarker analyses identified an association between tumor cell VEGF-A expression and progression (P = 0.03); for every 100-unit increase in the VEGF-A score, there was a 3.7-fold increase in the odds of progression (95% confidence interval, 1.1-16.6)., Conclusions: Bevacizumab plus erlotinib in heavily pretreated ovarian cancer patients was clinically active and well tolerated. Erlotinib did not seem to contribute to efficacy. Our study raises the intriguing possibility that high levels of tumor cell VEGF-A, capable of both autocrine and paracrine interactions, are associated with resistance to bevacizumab, emphasizing the complexity of the tumor microenvironment., (©2010 AACR.)

Introduction: Traditional beta-blockers have sometimes been associated with erectile dysfunction (ED). Nebivolol is a cardioselective β(1)-adrenoceptor antagonist that promotes vasodilation through a nitric oxide (NO)-dependent mechanism., Aim: We evaluated the effects of nebivolol on the NO/cyclic guanosine monophosphate (cGMP) signaling pathway, on erectile function and dysfunction, and in human penile vascular tissues., Methods: Erectile response to cavernosal nerve electrical stimulation in control and diabetes-induced ED rats were evaluated, along with serum nitrite/nitrate (NOx) concentration and plasma/tissue cGMP levels. Endothelium-dependent and sildenafil-induced relaxation of isolated human corpus cavernosum (HCC) and human penile resistance arteries (HPRA) were also determined., Main Outcome Measures: The effects of nebivolol on erectile function and dysfunction and on NO/cGMP-mediated responses., Results: Treatment with nebivolol significantly potentiated erectile response in control rats, regardless of its effects on blood pressure. Nebivolol increased NOx and plasma cGMP by 3-fold and 2.75-fold, respectively, and significantly augmented the elevation of plasma cGMP produced by sildenafil. Nebivolol enhanced endothelium-dependent and sildenafil-induced relaxations of HCC tissue, and produced endothelium-dependent vasodilation of HPRA. Nebivolol, but not atenolol, significantly improved erectile response in diabetic rats (51.6%, 53.2%, and 87.1% of response at 3 Hz in nondiabetic rats, for vehicle-treated, atenolol-treated, and nebivolol-treated diabetic rats, respectively); after sildenafil administration, ED was completely reversed in nebivolol-treated diabetic rats (69.6% and 112% for diabetic rats treated with sildenafil and nebivolol plus sildenafil, respectively). Accordingly, nebivolol restored systemic NOx levels and cGMP content in penile tissue from these animals., Conclusions: Nebivolol in vivo activated the NO/cGMP pathway, enhanced erectile response and reversed ED in diabetic rats. Moreover, nebivolol in vitro potentiated NO/cGMP-mediated relaxation of human erectile tissues. These effects may account for the low incidence of ED in nebivolol-treated hypertensive patients. Nebivolol therefore may have utility in the treatment of ED, particularly ED associated with diabetes., (© 2010 International Society for Sexual Medicine.)

Ductal carcinoma in situ (DCIS) of the breast historically has been a disease detected by physical examination, diagnosed by open surgical biopsy, and treated by mastectomy and axillary dissection. It is now increasingly detected by screening mammography, diagnosed by needle core biopsy, and treated by lumpectomy, with axillary dissection having been abandoned and sentinel node biopsy being used in axillary staging. However, outcomes related to sentinel node biopsy in DCIS have not been validated in well-controlled clinical trials. Current guideline recommendations are to use sentinel node biopsy when needle core biopsy is highly suspicious for invasive cancer or where there is a high-risk DCIS when lumpectomy identifies invasive breast cancer with the DCIS, or when mastectomy is performed for extensive DCIS. Routine use of sentinel node biopsy for DCIS is not supported.

The increased use of sentinel lymph node (SLN) excision for staging the axilla in women with breast cancer has benefited women by lowering morbidity and at the same time has raised issues related to the extent of treatment needed to the nodal basin. This is of particular concern when micrometastases or isolated tumor cells are found in the sentinel nodes on the final pathology. The probability of finding metastatic disease in non-sentinel lymph nodes (NSLN) ranges from 0 to 20% with only micrometastatic deposits in the SLN. Very low rates (0-3.7%) of axillary recurrence have been reported in selected patients with micrometastases tumor in sentinel nodes who have not had a completion axillary node dissection (ALND). Risk factors for additional positive NSLN include primary tumor size, the presence of lymphovascular invasion and the size of the SN metastatic deposit. Currently, the decision to not complete the ALND when micrometastic disease is found in the SLN should be made on a case-by-case basis. One should consider the tumor characteristics, findings within the SLN, and a multidisciplinary treatment plan. Clinical trial results may help to resolve the dilemma. There appears to be a low risk for axillary recurrence.

Nebivolol (Bystolic) is a cardioselective beta 1 (beta(1))-adrenergic receptor blocker with endothelium-dependent vasodilating properties. The endothelium-dependent relaxation induced by nebivolol is blocked by inhibitors of nitric oxide synthase (NOS) and guanylate cyclase. Nebivolol also increases in vitro and in vivo nitric oxide (NO), which is an essential signaling molecule involved in the maintenance of cardiovascular homeostasis. This review summarizes the data involving nebivolol and NO bioavailability. Endothelium-dependent relaxation of blood vessels, which is impaired in hypertensive animals and humans, is reversed by nebivolol treatment. Animals exhibiting endothelial dysfunction also show an improvement in NO-cyclic guanosine monophosphate (cGMP) signaling and an increase in NO bioavailability when treated with nebivolol. When blood vessel and cultured endothelial cells from hypertensive animals are treated with nebivolol, there is a decrease in superoxide production and an increase in the expression and activity of endothelial NOS (eNOS). As a result of the increased bioavailability of NO, nebivolol also increases in vivo arterial distensibility, glomerular filtration rate, and renal plasma flow. In normotensive volunteers, nebivolol infusion increases the forearm blood flow, an effect that is blocked by inhibitors of NOS and restored by the NOS substrate, L-arginine. In hypertensive patients, chronic treatment with nebivolol improves endothelium-dependent vasodilation induced by acetylcholine and shear stress and reverses endothelium-dependent vasoconstriction. Furthermore, nebivolol displays distinct hemodynamic properties in patients that include improvements in stroke volume and a decrease in peripheral vascular resistance. These studies demonstrate that nebivolol produces endothelium-dependent vasodilation by increasing NO release, decreasing oxidative stress to increase NO bioavailability, or both. The NO-dependent vasodilatory action of nebivolol, coupled with its high beta(1)-adrenergic receptor selectivity, is unique among the clinically available beta-blockers and contributes to its efficacy and improved tolerability (e.g., less fatigue and sexual dysfunction) as an antihypertensive agent.

Background and Purpose: TRPC5 is a mammalian homologue of the Drosophila Transient Receptor Potential (TRP) channel and has expression and functions in the cardiovascular and nervous systems. It forms a calcium-permeable cation channel that can be activated by a variety of signals including carbachol (acting at muscarinic receptors), lanthanides (e.g. Gd3+) and phospholipids (e.g. lysophosphatidylcholine: LPC). Here we report the effects of inhalational (halothane and chloroform) and intravenous (propofol) general anaesthetics upon TRPC5., Experimental Approach: Human TRPC5 channels were expressed in HEK 293 cells and studied using fura-2 and patch-clamp recording to measure intracellular calcium and membrane currents respectively at room temperature. Human TRPM2 channels were studied for comparison., Key Results: TRPC5 activation by carbachol, Gd3+ or LPC was inhibited by halothane and chloroform at > or =0.1 and 0.2 mM respectively. Neither agent inhibited TRPM2. Propofol had an initial stimulatory effect on TRPC5 (evident in patch-clamp recordings only) and an inhibitory effect at > or =10 microM. TRPM2 was not affected by propofol. Propofol inhibited activation of TRPC5 by Gd3+ but not LPC, suggesting the effect was not directly on the channel. Propofol's anti-oxidant property was not necessary for its inhibitory effect because di-isopropyl benzene, a propofol analogue that lacks the hydroxyl group, also inhibited TRPC5., Conclusions and Implications: The data show the sensitivity of TRPC5 channel to general anaesthetics and suggest that some of the effects could have clinical relevance. The effects may be explained in part by the sensitivity of the channel to biophysical properties of the lipid bilayer.

Transthyretin (TTR) is a tetrameric protein involved in the distribution of thyroid hormones in vertebrates. The amino acid sequence of TTR is highly conserved across vertebrates. Hypothetical TTR-like proteins (TLPs) were inferred from the identification of genes in nonvertebrate species. Here, we identified five motifs defining TLPs and three motifs defining both TTRs and TLPs. These motifs were mapped onto structurally conserved and functionally important regions of TTRs. These motifs were used to build hidden Markov models for accurate identification of TLPs in other organisms. TLPs were divided into three main groups based on their N-terminal regions. Most TLPs are cytosolic, but in plants and slime mold, we predict they are peroxisomal. We verified that the TLPs from enterobacteria were periplasmic. We demonstrated that TLP genes are expressed in a bacterium (E. coli), an invertebrate animal (C. elegans), and a plant (A. thaliana). These TLPs have similar subunit molecular weights to TTRs, are tetramers, and are predicted to have similar three-dimensional (3D) structures to TTRs, but do not bind thyroid hormones or similar ligands. We suggest that like TTRs, the N-terminal and C-terminal regions of TLPs are integral in defining the function of TLPs in nonvertebrate species and that the TLP gene duplicated in primitive vertebrates to produce the TTR gene. TLP/TTR has retained its overall structure, but changed function and localization during evolution in bacteria, invertebrates, plants, and vertebrates., ((c) 2006 Wiley-Liss, Inc.)

Transthyretin (TTR) is an extracellular thyroid hormone distributor protein in vertebrates, whose structure has been highly conserved between fish and humans. However, the ligand preferentially bound by TTR has changed during evolution from 3',3,5-L-triiodothyronine (T3) to 3',5',3,5-l-tetraiodothyronine (T4). We identified genes in the genomes of >50 species of nonvertebrates, which could code for TTR-like proteins. Molecular modeling suggested most would have similar 3D structures and electrostatic surface potentials to vertebrate TTRs. We amplified TTR-like genes from a C. elegans cDNA library, demonstrating that it is transcribed. We synthesized recombinant TTR-like proteins from S. dublin and C. elegans. These proteins form tetramers similarly to vertebrate TTRs, but their ligands remain elusive.

The dilemma of diagnosing and possibly treating dentinal cracks continues to present a challenge in endodontics. The purpose of this in vitro study was to compare the effectiveness of transillumination and dyes in identifying root-end dentinal cracks. Fifty maxillary central incisors were decoronated, and the canals were instrumented to an ISO size 50 at the working length. The apical 3 mm of the roots was resected, and cracks were artificially created in the apical dentin. Four independent examiners evaluated the root ends at x8 magnification with a surgical operating microscope using transillumination (group 1), sodium fluorescein dye (group 2), caries detect dye (group 3), methylene blue dye (group 4), and methylene blue plus transillumination (group 5). The examiners' ability to identify root ends correctly with and without cracks was analyzed by comparing the data with the predetermined standard (cracked and noncracked) using logistic regression analysis. All techniques used were shown to be more effective than random chance at diagnosing cracks. The areas under the curve of the different techniques were as follows: transillumination, 0.81 (95% confidence interval [CI], 0.69-0.93); sodium fluorescein, 0.72 (95% CI, 0.58-0.86); caries detector, 0.76 (95% CI, 0.63-0.89); methylene blue, 0.70 (95% CI, 0.55-0.84); and methylene blue plus transillumination, 0.82 (95% CI, 0.70-0.94). Thus, the crack assessment techniques that gave the best discrimination between cracked and noncracked specimens, regardless of rater, was methylene blue plus transillumination. This study emphasizes the usefulness of transillumination along with magnification in detecting dentinal cracks.

This article focuses on the collaboration between the University of Wales College of Medicine (UWCM) libraries and the Welsh National Health Service (NHS) libraries to create a joint library catalogue. The goal was to create an all-Wales resource that gave users one access point to search for the location and availability of health-related material in any Welsh medical library. This venture grew out of the existing collaborative scheme, the All-Wales Health Information and Library Extension Services (AWHILES). Currently, all seven UWCM libraries and 17 NHS libraries contribute to the catalogue. Four more libraries are due to join in late 2003 or early 2004. All UWCM and AWHILES libraries would then be contributing members of the joint catalogue. The article discusses the background and motivation to the creation of the database. It explores the positives and negatives of the project plus what was learnt as the venture progressed. It reviews the establishment and operations of the Welsh Health Voyager User Group (WHVUG) created to involve all contributing members in the running and future development of the library catalogue.

The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor that regulates several crucial biological processes such as adipogenesis, glucose homeostasis, and cell growth. It is also the functional receptor for a new class of insulin-sensitizing drugs, the thiazolidinediones, now widely used in the treatment of type 2 diabetes mellitus. Here we report that PPARgamma protein levels are significantly reduced in adipose cells and fibroblasts in response to specific ligands such as thiazolidinediones. Studies with several doses of different ligands illustrate that degradation of PPARgamma correlates well with the ability of ligands to activate this receptor. However, analyses of PPARgamma mutants show that, although degradation does not strictly depend on the transcriptional activity of the receptor, it is dependent upon the ligand-gated activation function 2 (AF2) domain. Proteasome inhibitors inhibited the down-regulation of PPARgamma and ligand activation enhanced the ubiquitination of this receptor. These data indicate that, although ligand binding and activation of the AF2 domain increase the transcriptional function of PPARgamma, these same processes also induce ubiquitination and subsequent degradation of this receptor by the proteasome.

While searching for natural ligands for the peroxisome proliferator-activated receptor (PPAR) gamma, we identified a synthetic compound that binds to this receptor. Bisphenol A diglycidyl ether (BADGE) is a ligand for PPARgamma with a K(d(app)) of 100 microM. This compound has no apparent ability to activate the transcriptional activity of PPARgamma; however, BADGE can antagonize the ability of agonist ligands such as rosiglitazone to activate the transcriptional and adipogenic action of this receptor. BADGE also specifically blocks the ability of natural adipogenic cell lines such as 3T3-L1 and 3T3-F442A cells to undergo hormone-mediated cell differentiation. These results provide the first pharmacological evidence that PPARgamma activity is required for the hormonally induced differentiation of adipogenic cells.

The gamma isoform of the peroxisome proliferator-activated receptor, PPAR gamma, regulates adipocyte differentiation and has recently been shown to be expressed in neoplasia of the colon and other tissues. We have found four somatic PPAR gamma mutations among 55 sporadic colon cancers: one nonsense, one frameshift, and two missense mutations. Each greatly impaired the function of the protein. c.472delA results in deletion of the entire ligand binding domain. Q286P and K319X retain a total or partial ligand binding domain but lose the ability to activate transcription through a failure to bind to ligands. R288H showed a normal response to synthetic ligands but greatly decreased transcription and binding when exposed to natural ligands. These data indicate that colon cancer in humans is associated with loss-of-function mutations in PPAR gamma.

Adipose differentiation is an important part of the energy homeostasis system of higher organisms. Recent data have suggested that this process is controlled by an interplay of transcription factors including PPARgamma, the C/EBPs, and ADD1/SREBP1. Although these factors interact functionally to initiate the program of differentiation, there are no data concerning specific mechanisms of interaction. We show here that the expression of ADD1/SREBP1 specifically increases the activity of PPARgamma but not other isoforms, PPARalpha, or PPARdelta. This activation occurs through the ligand-binding domain of PPARgamma when it is fused to the DNA-binding domain of Gal4. The stimulation of PPARgamma by ADD1/SREBP1 does not require coexpression in the same cells; supernatants from cultures that express ADD1/SREBP1 augment the transcriptional activity of PPARgamma. Finally, we demonstrate directly that cells expressing ADD1/SREBP1 produce and secrete lipid molecule(s) that bind directly to PPARgamma, displacing the binding of radioactive thiazolidinedione ligands. These data establish that ADD1/SREBP1 can control the production of endogenous ligand(s) for PPARgamma and suggest a mechanism for coordinating the actions of these adipogenic factors.

beta-Hexosaminidases (Hex) A and B promote mitogenesis via airway smooth muscle (ASM) mannose receptor. The objective of this study was to elucidate the contribution of protein kinase C (PKC) in Hex-induced mitogenesis in ASM cells (ASMC). Exposure of ASMC to Hex caused increases in both the calcium-dependent and the calcium-independent PKC activities. Both downregulation of PKC and PKC inhibitors staurosporine and calphostin C diminished Hex-induced DNA synthesis and cell number. Hex-induced DNA synthesis was enhanced by a diacylglycerol kinase inhibitor, R-59022, which was blocked by calphostin C. These data suggest that activation of PKC in part mediates Hex-induced mitogenesis in ASMC.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1-induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1 alpha. ET-1 increased 6-keto-PGF1 alpha formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1 alpha formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF1 alpha production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF1 alpha formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF1 alpha formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.

The purpose of this study was to characterize the receptor(s) and second messenger systems involved in prostacyclin (prostaglandin [PG] I2) synthesis elicited by endothelin (ET)-1 in the rat aorta. PGI2 synthesis, measured as immunoreactive 6-keto-PGF1 alpha, was assessed in aortic rings exposed to endothelin receptor agonists in the presence and absence of selective ETA and ETB receptor antagonists. ET-1, which has equal affinity for both endothelin receptor subtypes, and ET-3, a preferential ETB receptor agonist, enhanced 6-keto-PGF1 alpha synthesis in a time- and concentration-dependent manner. ET-1 was more potent than ET-3 in increasing 6-keto-PGF1 alpha synthesis. Moreover, the selective ETB receptor agonists IRL-1620 and sarafotoxin S6c did not significantly increase 6-keto-PGF1 alpha synthesis. Furthermore, ET-1-induced 6-keto-PGF1 alpha synthesis was attenuated by an ETA receptor antagonist, BQ-123, in a dose-dependent manner but not by an ETB receptor antagonist, BQ-788. Depletion of extracellular Ca2+ or addition of Ca2+ channel blockers (nifedipine, verapamil, SK&F 96365) attenuated ET-1-mediated 6-keto-PGF1 alpha synthesis, while a Ca2+ channel agonist, S(-)-Bay K 8644, potentiated this effect of ET-1. Selective protein kinase C inhibitors (bisindolylmaleimide I, calphostin C) did not alter ET-1-induced 6-keto-PGF1 alpha synthesis. These data suggest that PGI2 synthesis elicited by ET-1 in the rat aorta is mediated primarily through influx of extracellular Ca2+ via activation of an ETA receptor and is independent of protein kinase C.

N-Benzyladriamycin-14-valerate (AD 198) is a highly hydrophobic analogue of Adriamycin (ADR) which can circumvent multidrug resistance (MDR) in various cell lines. Unlike ADR, AD 198 avoids extrusion by P-glycoprotein (P-gp) in AD 198-resistant murine macrophage-like J774.2 cells and localizes in the cytoplasm. To determine the structural modification(s) responsible for these different characteristics, intracellular accumulation and distribution of ADR, AD 198, and the two half-substituted AD 198 congeners. N-benzyladriamycin (AD 288) and adriamycin-14-valerate (AD 48), were analyzed in AD 198-sensitive (J774.2) and -resistant (A300) cells. A300 cells exhibited cross-resistance to and reduced accumulation of ADR, AD 48, and AD 288. ADR and AD 288 rapidly localized in the nuclei of parental and A300 cells, while AD 48 and AD 198 localized in the cytoplasm. AD 48 redistributed into nuclei and cytoplasm of both cell lines, but AD 198 maintained a punctate cytoplasmic distribution in A300 cells. These results suggest that both the N-benzyl and C14-valerate substitutions of AD 198 are required for P-gp circumvention and stable cytoplasmic localization in A300 cells, probably as a result of differing intracellular drug trafficking.

Numerous recombinants arose when protoplasts of S. coelicolor were treated with polyethylene glycol and regenerated on non-selective solid medium. In six-factor crosses, recombination frequencies of more than 10% (up to 17%) were routinely observed. This recombination did not require either of the known sex factors, SCPI and SCP2. The proportion of multiple crossover classes was much higher than amongst recombinants produced by conjugated between mycelia. Analysis of the spatial distribution of crossovers in double and quadruple crossover recombinants showed only a slight tendency for crossovers to occur closer together than randomly on the complete linkage group. This suggests that genomes brought together by protoplast fusion are complete, or nearly so (in conjugation, in contrast, one genome is represented by a comparatively short fragment). Individual colonies arising from fused protoplasts did not contain different parental genomes without recombinants, but recombinants often occurred without parentals. Several recombinant genotypes often occurred in the same colony, showing a segregation of some, only, of the parental alleles. Complementary genotypes, parental or recombinant, did not occur in the same colony. It is postulated that complete genomes of fused protoplasts usually become fragmented and that crossing-over, often repeated, occurs between the fragments, to generate haploid recombinants. Analysis of fusions between propoplasts of four different genotypes indicated that the average number of protoplasts fusing together was low, but nevertheless appreciable numbers of fusions involved three or four genomes. Crossing-over between them produced recombinants inheriting markers from three or four parents. The generation of nearly random populations of recombinants between two or more parent strains by propoplast fusion under the conditions described appears to have simple applications in industrial and academic strain construction.

Discrete delicate fibromuscular structures crossing the cavity of the left ventricle were identified on morphological examination in 329 (48%) of 686 hearts from patients of all ages with congenital heart disease, acquired heart disease, or normal hearts. These structures were also present in 151 (95%) of 159 hearts from animals of six species. Cross sectional echocardiographic findings compatible with these structures were obtained in 39 (21.7%) of 179 children reviewed retrospectively and in three of 800 (0.4%) adults studied prospectively. These structures appear to be a normal anatomical finding.

Streptomyces lividans 66 was shown to harbour two self-transmissible plasmids: SLP2, which acts as a sex factor, and SLP3. Derivatives of this strain which had lost both plasmids were used as host strains to study a range of Streptomyces plasmids for their ability to promote their own transfer and to mobilize chromosomal markers. A linkage map of the S. lividans chromosome containing ten markers was derived from the results of matings using several different sex plasmids, and protoplast fusions. SLP2 was transferred interspecifically to S. parvulus ATCC 12434 and to S. coelicolor A3(2); in the latter it acted as a fertility factor. Interspecific crosses also led to the discovery of a further plasmid, SLP4, from S. coelicolor, SLP2, SLP3 and SLP4 could not be visualized on agarose gels using standard plasmid isolation procedures, but their presence was detected by transformation into S. lividans.

Mutations (cda) leading to non-production of the new calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2) were closely linked on the chromosome. One representative mutation (cda-1) was mapped precisely between nicA and adeC. No cosynthesis of CDA was found in any pairwise combinations of 14 cda mutants. Mutations lacking aerial mycelium (bald mutations), mapping to the four previously described loci (bldA-D), were pleiotropically defective in production of CDA.

Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.

The optimum concentration of polyethylene glycol 1000 (PEG) for the production of recombinants through protoplast fusion in Streptomyces coelicolor was about 50% (w/v). The addition of 14% (v/v) dimethyl sulphoxide to the fusion mixture enhanced recombination frequencies, but only at sub-optimal PEG concentrations. After treatment of protoplasts with 50% PEG for 1 min, the frequency of recombinants in a multi-factor 'cross' sometimes exceeded 20% of the total progeny. The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.