Inactive Counterpart Research Articles

Steroid hormones in systemic sclerosis: associations with disease characteristics and modifications during scleroderma renal crisis.

The renin-angiotensin-aldosterone system (RAAS) and glucocorticoids (GCs) are involved in vascular remodeling and fibrosis, but have not been extensively studied in systemic sclerosis (SSc). Our aim was to investigate the RAAS and GC hormones in SSc patients. Serum levels of renin (dosage and activity), aldosterone and its precursors (DOC, B, 18-OH-DOC, 18-OH-B), and GCs (cortisol, cortisone, 11-deoxycortisol, 18-OH-F) were assessed in 122 SSc patients and 52 healthy controls. After applying stringent inclusion criteria aimed at ensuring accurate hormone assessments (exclusion of interfering drugs, strict sampling conditions), we analyzed RAAS hormones in 61 patients, and GCs in 96 patients. Hormone levels were compared between patients and controls; and associations with disease characteristics were assessed in patients. Regarding RAAS hormones, SSc patients displayed significantly lower aldosterone levels (although within normal range), similar renin levels, and higher B levels than controls. Abnormal RAAS hormone levels were associated with a more severe SSc phenotype (lung and skin fibrosis, heart and pulmonary vascular involvements, inflammation). Regarding GC hormones, SSc patients had higher levels of cortisol, 11-desoxycortisol (precursor) and 18-OH-F (metabolite) but lower levels of cortisone (inactive counterpart) than controls.RAAS hormone levels were assessed in 5 SSc patients before and during scleroderma renal crisis (SRC): concentrations varied considerably between patients, but consistently included normal/increased aldosterone levels and elevated renin levels. RAAS and GC hormones are abnormally produced in SSc patients, especially in patients with severe SSc and during SRC. This could suggest a participation of these hormonal systems in SSc pathogenesis.

Recycling glucocorticoids: therapeutic implications of the 11β-HSD1 enzyme system.

Endogenous glucocorticoids and commonly used oral glucocorticoids have the property of existing in an inactive and active form in vivo. The inactive form can be converted back to the active form, or 'recycled' in cells and tissues that express the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. This recycling provides an important contribution to the action of glucocorticoids. This review examines the literature relating to the importance of 11β-HSD1 activity during glucocorticoid treatment, with an emphasis on studies examining bone and joint disease and the ability of glucocorticoids to suppress inflammatory damage in models of arthritis. Animal models with global or selective deletion of 11β-HSD1 have determined the extent to which this recycling is important in normal physiology and during treatment with oral glucocorticoids. These studies demonstrate that 11β-HSD1-mediated recycling of inactive glucocorticoids has a substantial action and indeed is responsible for the majority of the effects of orally administered glucocorticoids on a range of tissues. Importantly, the anti-inflammatory actions of glucocorticoids appear largely through this mechanism such that mice that lack 11β-HSD1 are resistant to the anti-inflammatory actions of glucocorticoids. The recognition that to a large extent the circulating inactive counterpart of these glucocorticoids is more important to anti-inflammatory effects than the active glucocorticoid presents novel opportunities to more selectively target glucocorticoids to tissues or to reduce the likely side effects.

Relation between derived cardiovascular indices, body surface area, and blood pressure/heart rate recovery among active and inactive Nigerian student

BackgroundThe ease in the computation of derived cardiovascular indices, such as mean arterial pressure (MAP), rate pressure product (RPP), and pulse pressure (PP), makes them attractive for use in making clinical decisions for patient management in resource-deprived environments. This study sought to determine the relationship between these indices and heart rate/blood pressure drops during recovery state among physically active and inactive individuals following submaximal exercise.ResultsThis quasi-experimental study conveniently sampled 105 apparently healthy male subjects aged 18–35 years of the University of Maiduguri. Intergroup categorization was executed by IPAQ. The derived indices were calculated using heart rate and blood pressure measurement while the body surface area (BSA) was determined using height and weight. The subjects performed a submaximal exercise test using a bicycle ergometer. Data analysis includes descriptive statistic, Pearson correlation, Student t-test, analysis of covariance, and multiple linear regression. The data was analyzed using SPSS version 25.0 at a significance of p < 0.05. The mean BSA and resting PP, MAP, and RPP were 1.84 ± 0.16 m2, 41.23 ± 7.57 mmHg, 85.92 ± 9 mmHg, and 8266.45 ± 1404.05 respectively. The resting RPP of the physically inactive subjects was significantly higher than that of the active (8742.71 ± 1496.31 Vs 7790.18 ± 1131.59, p = 0.00); however, the active subject had a higher resting MAP than the inactive (87.91 ± 7.98 Vs 83.93 ± 9.59, p = 0.03). A significant negative relationship was found between the RPP and the Absolute/percent recovery HR (r = -0.23, p = 0.02 Vs r = -0.34, p = 0.00). However, for the PP and absolute recovery SBP it was significantly positive (r = 0.22, p = 0.03) and also that of the MAP and absolute recovery SBP was positive (r = 0.33, p = 0.00). The best negative predictor of recovery HR was the RPP (β = −0.45, p = 0.00) while the MAP was the best positive predictor of recovery HR and SBP.ConclusionsOverall, the physically active subjects coped better during the exercise than their inactive counterpart because of lower cardiac work and better blood perfusion to vital body organs. An inverse relationship was found between the RPP and absolute/percent recovery HR at 1 min post-exercise while a positive relationship was found between the PP and absolute drop in SBP/percent drop in DBP 1 min post-exercise. The best predictor of recovery in HR and blood pressure was the RPP.

Luminal Administration of a Water-soluble Carbon Monoxide-releasing Molecule (CORM-3) Mitigates Ischemia/Reperfusion Injury in Rats Following Intestinal Transplantation.

Background.The protective effects of carbon monoxide (CO) against ischemia/reperfusion (IR) injury during organ transplantation have been extensively investigated. Likewise, CO-releasing molecules (CORMs) are known to exert a variety of pharmacological activities via liberation of controlled amounts of CO in organs. Therefore, we hypothesized that intraluminal administration of water-soluble CORM-3 during cold storage of intestinal grafts would provide protective effects against IR injury.Methods.Orthotopic syngeneic intestinal transplantation was performed in Lewis rats following 6 h of cold preservation in Ringer solution or University of Wisconsin solution. Saline containing CORM-3 (100 µmol/L) or its inactive counterpart (iCORM-3) was intraluminally introduced in the intestinal graft before cold preservation.Results.Histopathological analysis of untreated and iCORM-3–treated grafts revealed a similar erosion and blunting of the intestinal villi. These changes in the mucosa structure were significantly attenuated by intraluminal administration of CORM-3. Intestinal mucosa damage caused by IR injury led to considerable deterioration of gut barrier function 3 h postreperfusion. CORM-3 significantly inhibited upregulation of proinflammatory mRNA levels, ameliorated intestinal morphological changes, and improved graft blood flow and mucosal barrier function. Additionally, CORM-3–treated grafts increased recipient survival rates. Pharmacological blockade of soluble guanylyl cyclase activity significantly reversed the protective effects conferred by CORM-3, indicating that CO partially mediates its therapeutic actions via soluble guanylyl cyclase activation.Conclusions.Our study demonstrates that luminally delivered CORM-3 provides beneficial effects in cold-stored rat small intestinal grafts and could be an attractive therapeutic application of CO in the clinical setting of organ preservation and transplantation.

Protein phosphatase PPP3CA (calcineurin A) down-regulates hypoxia-inducible factor transcriptional activity

Hypoxia-inducible factors (HIF) are master regulators of the response to hypoxia. Although several kinases are known to modify their oxygen sensitive HIF-α subunits or affect indirectly their function, little is known about the role of phosphatases in HIF control. To address this issue, a library containing siRNAs for the 25 known catalytic subunits of human phosphatases was used to screen for their effect on HIF transcriptional activity in HeLa cells. Serine-threonine phosphatase PPP3CA (calcineurin A, isoform a) was identified as the strongest candidate for a negative regulator of HIF activity. Indeed, independent silencing of PPP3CA expression stimulated HIF transcriptional activity under hypoxia, without increasing the protein levels of HIF-1α or HIF-2α. Overexpression of a constitutively active PPP3CA form, but not its catalytically inactive counterpart, inhibited HIF activity and expression of HIF target genes but did not affect HIF-1α or HIF-2α expression. These results were phenocopied by treatment with the ionophore ionomycin, that activates endogenous PPP3CA. The effect of ionomycin was mediated by PPP3CA as it was largely abolished by PPP3CA silencing. Furthermore, ionomycin enhanced the down-regulation of HIF activity by wild-type PPP3CA overexpression. Overall, our results suggest the involvement of PPP3CA in fine-tuning the HIF-dependent transcriptional response to hypoxia.

The polyglutamine protein ataxin-3 enables normal growth under heat shock conditions in the methylotrophic yeast Pichia pastoris

The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation.

Effect of PFOS on glucocorticoid-induced changes in human decidual stromal cells in the first trimester of pregnancy.

Perfluorooctane sulfonate (PFOS) is a surfactant and used in treating products for waterproofing and non-stick applications. PFOS has potential influence on reproductive function but the effect of PFOS exposure on the decidual functions remains unknown. By using primary human decidual stromal cells of early pregnancy we demonstrated that PFOS inhibited decidualization of the stromal cells as well as decidualization-induced upregulation of 11β-HSD1, an enzyme that regenerates biologically active cortisol from its inactive counterpart cortisone. Moreover, PFOS attenuated cortisol-induced decidualization and upregulation of 11β-HSD1 in the stromal cells. Furthermore, PFOS inhibited the reduction of IL-6 and IL-1β, the key proinflammatory cytokines in maternal-fetal immune intolerance, by cortisone in the decidual stromal cells indicating attenuated conversion of cortisone to cortisol. In conclusion, exposure to PFOS may disrupt the regeneration of cortisol in the decidual tissue thereby impairing the decidualization and immune-tolerance environment of early pregnancy.

NT-proBNP in cardiopulmonary resuscitated patients treated with mild therapeutic hypothermia is not independently associated with mortality: a retrospective observational study.

BackgroundIn spite of the introduction of mild therapeutic hypothermia (MTH), mortality rates remain high in patients with return of spontaneous circulation (ROSC) after cardiac arrest (CA). To date, no accurate and independent biomarker to predict survival in these patients exists. B-type natriuretic peptide (BNP) was found to provide both prognostic and diagnostic value in various cardiovascular diseases, including survival to hospital discharge in patients with ROSC. However, the biologically inactive counterpart of BNP, NT-proBNP, was found to be a more stable and accurate analyte. The current retrospective observational study investigates the value of NT-proBNP to predict 28-day mortality in post-CA patients treated with MTH, as well as the dynamics of NT-proBNP during MTH.MethodsNT-proBNP levels were measured in post-CA patients cooled via cold intravenous saline infusion and water-circulating body wraps (Medi-Therm®, Gaymar). Plasma samples were obtained before cooling was started, at the start and end of the maintenance phase and at the end of rewarming.Results250 patients, admitted between 2009 and 2013, had NT-proBNP levels measured on ICU admission and were included for the evaluation of NT-proBNP as a prognostic marker. In the 28 days following ICU admission, 114 patients died (46%). Non-survivors had significantly higher NT-proBNP (median 1448 ng/l, IQR 366–4623 vs median 567 ng/1, IQR 148–1899; P < 0.001) levels on ICU admission. Unadjusted odds ratios for 28-day mortality were 1.7 (95% CI 0.8-3.5), 1.6 (0.8-3.3) and 3.6 (1.7-7.5) for increasing quartiles of NT-proBNP as compared to the lowest quartile. Adjusted odds ratios were 1.1 (95% CI 0.5-2.5), 1.1 (0.5-2.5) and 1.6 (0.7-3.8), respectively. A cut-off value of 834 ng/l achieved a sensitivity of 58% and a specificity of 58% to predict 28-day mortality. Of 113 patients, NT-proBNP values of each MTH phase were available and grouped in decreased or increased levels in time. Both decreases and increases of NT-proBNP values were observed during the MTH phases, but presence of either was not associated with outcome.ConclusionsHigh NT-proBNP plasma concentrations on ICU admission are associated with high 28-day mortality in post-CA patients treated with MTH in a univariate analysis, but not in a multivariate analysis. Increases or decreases of NT-proBNP levels during MTH appear unrelated to 28 day mortality.

Changes in physical activity and cognitive decline in older adults living in the community.

Accumulating evidence suggests that physical activity may be beneficial in preserving cognition in late life. This study examined the association between baseline and changes in physical activity and cognitive decline in community-dwelling older people. Data were from the Korean Longitudinal Study of Aging, with 2605 aged 65 years and older subjects interviewed in 2006 and followed up for 2 years. Cognitive decline was defined by calculating the Reliable Change Index using the Mini-Mental State Examination. Physical activity levels were categorized as sedentary, low, or high. Changes in physical activity were classified as inactive, decreaser, increaser, or active. Logistic regression analysis of baseline and changes in physical activity with cognitive decline was performed. Compared with the sedentary group at baseline, both the low and high activity groups were less likely to experience cognitive decline. The active (odds ratio [OR] = 0.40, 95 % confidence interval [CI] 0.23-0.68) and increaser (OR = 0.45, 95 % CI 0.27-0.74) group, compared with the inactive counterpart, demonstrated a significantly lower likelihood of cognitive decline. Older adults who remained active or increased activity over time had a reduced risk of cognitive decline. Engagement in physical activity in late life may have cognitive health benefits.

Antiproliferative effects of carbon monoxide on pancreatic cancer

Carbon monoxide, the gaseous product of heme oxygenase, is a signalling molecule with a broad spectrum of biological activities. The aim of this study was to investigate the effects of carbon monoxide on proliferation of human pancreatic cancer. In vitro studies were performed on human pancreatic cancer cells (CAPAN-2, BxPc3, and PaTu-8902) treated with a carbon monoxide-releasing molecule or its inactive counterpart, or exposed to carbon monoxide gas (500 ppm/24h). For in vivo studies, pancreatic cancer cells (CAPAN-2/PaTu-8902) were xenotransplanted subcutaneously into athymic mice, subsequently treated with carbon monoxide-releasing molecule (35 mg/kg b.w. i.p./day), or exposed to safe doses of carbon monoxide (500 ppm 1h/day; n = 6 in each group). Both carbon monoxide-releasing molecule and carbon monoxide exposure significantly inhibited proliferation of human pancreatic cancer cells (p<0.05). A substantial decrease in Akt phosphorylation was observed in carbon monoxide-releasing molecule compared with inactive carbon monoxide-releasing molecule treated cancer cells (by 30-50%, p<0.05). Simultaneously, carbon monoxide-releasing molecule and carbon monoxide exposure inhibited tumour proliferation and microvascular density of xenotransplanted tumours (p<0.01), and doubled the survival rates (p<0.005). Exposure of mice to carbon monoxide led to an almost 3-fold increase in carbon monoxide content in tumour tissues (p=0.006). These data suggest a new biological function for carbon monoxide in carcinogenesis, and point to the potential chemotherapeutic/chemoadjuvant use of carbon monoxide in pancreatic cancer.

Abstract 88: High NT-proBNP in Cardiopulmonary Resuscitated Patients Treated With Mild Therapeutic Hypothermia is Associated With Mortality

Introduction: Despite the apparent appropriateness of mild therapeutic hypothermia (MTH) to limit neurological injury and improve survival in patients with return of spontaneous circulation (ROSC) after cardiac arrest (CA), mortality rates remain high. To date, no accurate and independent biomarker to predict survival in these patients exists. B-type natriuretic peptide (BNP) was found to provide both prognostic and diagnostic value in various cardiovascular diseases. However, the biologically inactive counterpart of BNP, NT-proBNP, was found to be more stable and accurate. Objective: Investigate the value of NT-proBNP to predict 28-day mortality in post-CA patients treated with MTH, as well as the influence of MTH on NT-proBNP levels. Methods and Results: NT-proBNP levels were measured in post-CA patients who were non-invasively cooled using body wraps (Medi-Therm ® , Gaymar). Plasma samples were obtained before cooling was started, during 24 hours maintenance and at the end of re-warming. Between 2009 and 2013 327 patients were admitted to the ICU for post-CA MTH treatment. Baseline NT-proBNP values were available for 81 (25%) patients and were subsequently included for the evaluation of NT-proBNP as a prognostic marker. In the 28 days following ICU admission, 38 patients died (47%). Non-survivors had significantly higher baseline NT-proBNP levels (median 1525ng/l, IQR 450-3859 versus median 267ng/l, IQR 99-1766; P &lt;0.001) compared to survivors. Adjusted odds ratios for 28-day mortality were 3.3 (95% CI = 0.8, 13.4), 4.4 (95% CI = 1.1, 17.8) and 9.3 (95% CI = 2.2, 40) for increasing quartiles of NT-proBNP as compared to the lowest quartile. A cut-off value of 643ng/l achieved a sensitivity of 67% and a specificity of 66% to predict 28-day mortality. During the different phases of MTH NT-proBNP values decreased or increased. The presence of either pattern in NT-proBNP expression was not associated with outcome. Conclusions: High NT-proBNP plasma levels on ICU admission are associated with high mortality in post-CA patients, but are of moderate prognostic value as an independent biomarker. Increases or decreases in patterns of NT-proBNP expression during the separate phases of MTH treatment appear unrelated to 28-day mortality.

Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2

HtrA2, a complex trimeric pyramidal mitochondrial serine protease that regulates critical biological functions and diseases, including apoptosis and cancer, is a promising therapeutic target. It promotes apoptosis through multiple pathways, complex mechanisms of which are still elusive. The existing model of activation that emphasizes relative intramolecular movements between C-terminal PDZ and protease domains (PDZ-protease collapse in inactive and resting states) has not been able to unambiguously demonstrate dynamics of its actions. Using structure-guided design, molecular biology and protein biochemistry, we obtained various combinations of HtrA2 domains and mutants. Conformational changes and stability were characterized using molecular dynamics simulation and spectroscopic tools while functional enzymology delineated their roles in regulating enzyme catalysis. Quantitative Förster resonance energy transfer showed lesser intramolecular PDZ-protease distance in trimeric HtrA2 compared to its inactive monomeric counterpart (∼21 and ∼22.3 Å, respectively, at 37°C). Our findings highlight importance of N-terminal region, oligomerization, and intricate intermolecular PDZ-protease interaction in proper active-site formation, enzyme-substrate complex stabilization, and hence HtrA2 functions. These observations redefine the existing activation model and showcase a unique example of how precise interdomain coordination, plasticity, and intermolecular contacts lead to distinct functional properties and hence provide new insights into HtrA2 structure, function, and dynamics.

Expression of 11β-hydroxysteroid dehydrogenase enzymes in human osteosarcoma: potential role in pathogenesis and as targets for treatments

Osteosarcoma (OS) is a primary malignant tumour of bone occurring predominantly in children and young adults. Despite chemotherapy, relapse is common and mortality remains high. Non-transformed osteoblasts are highly sensitive to glucocorticoids, which reduce proliferation and induce apoptosis. Previously, we observed that OS cells, but not normal osteoblasts, express 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). This enzyme inactivates cortisol (active) to cortisone (inactive) and expression of 11β-HSD2 renders OS cells resistant to glucocorticoids. By contrast, the related enzyme 11β-HSD1 converts cortisone to cortisol and reduces OS cell proliferation in vitro. Some synthetic glucocorticoids (e.g. dehydrodexamethasone (DHD), inactive counterpart of dexamethasone (DEX)) have been reported to be activated by 11β-HSD2. We therefore investigated expression and enzymatic activity of 11β-HSD isozymes in human OS tissue, determined whether 11β-HSD expression has prognostic value in the response to therapy, and evaluated the potential use of synthetic glucocorticoids to selectively target OS cells. OS samples expressed both 11β-HSD1 and 11β-HSD2. 11β-HSD1 expression in pretreatment biopsy specimens positively correlated with primary tumour size. Expression and activity of 11β-HSD1 in post-treatment biopsies were unrelated to the degree of tumour necrosis following chemotherapy. However, high 11β-HSD2 expression in post-treatment biopsies correlated with a poor response to therapy. OS cells that expressed 11β-HSD2 inactivated endogenous glucocorticoids; but these cells were also able to generate DEX from DHD. These results suggest that OS treatment response is related to 11β-HSD2 enzyme expression. Furthermore, OS cells expressing this enzyme could be targeted by treatment with synthetic glucocorticoids that are selectively reactivated by the enzyme.

Acute myocardial infarction in streptozotocin-induced hyperglycaemic rats: protection by a carbon monoxide-releasing molecule (CORM-3)

Here, we have studied the effects of a carbon monoxide-releasing molecule (CORM-3, tricarbonylchloro(glycinato)ruthenium(II)) on acute myocardial ischemia/reperfusion (I/R) injury in hyperglycaemic streptozotocin-treated rats (STZ rats). Occlusion of the left descending coronary artery for 25 min followed by a 2-h reperfusion in STZ-induced hyperglycaemic rats was used as the model. CORM-3 and its inactive counterpart (iCORM-3) were administered 1 h prior to ischemia. The parameters measured included myocardial infarct size (IS) and a selection of inflammatory, oxidative markers and endothelial progenitor cells (CD34⁺ and CD117/c-kit⁺. In STZ-induced hyperglycaemic rats, occlusion of the left descending coronary artery caused injury of the myocardial tissue with an IS of ~70%, expressed as fraction of the area at risk. Given intraperitoneally 1 h prior to ischemia, CORM-3 (2-8 mg/kg) afforded significant dose-dependent cardio-protection. Specifically, pre-treatment with CORM-3 reduced infarct size by 14 ± 0.6%, 34 ± 1% and 53 ± 1.6% for doses of 2, 4 and 8 mg/kg, respectively. A negative control (iCORM-3) failed to prevent the cardiac damage induced by I/R. CORM-3 pre-treatment augmented cardiac heme oxygenase-1 (HO-1) protein levels and was associated with an increased number of CD34⁺- and CD117/c-kit⁺-positive immunostaining. Modulation of these markers was associated with augmented cardiac eNOS expression and levels of the cytokines TNF-α and IL-1 beta. CORM-3 afforded significant cardio-protection against acute myocardial infarction in STZ-induced hyperglycaemic rats through liberation of small amounts of CO. Of interest, CORM-3 promoted recruitment of the endogenous endothelial progenitor cells within the myocardium, possibly through modulation of cardiac HO-1 and eNOS expression and/or function.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells

Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.

The additive value of N-terminal pro-B-type natriuretic peptide testing at the emergency department in patients with acute dyspnoea

Background B-type natriuretic peptide (BNP) and its inactive counterpart NT-proBNP can help to identify or rule out heart failure in patients presenting with acute dyspnoea. It is not well known whether measurement of these peptides can be omitted in certain patient groups. Methods We conducted a prospective observational study of 221 patients presenting with acute dyspnoea at the emergency department. The attending physicians estimated the probability of heart failure by clinical judgement. NT-proBNP was measured, but not reported. An independent panel made a final diagnosis of all available data including NT-proBNP level and judged whether and how NT-proBNP would have altered patient management. Results NT-proBNP levels were highest in patients with heart failure, alone or in combination with pulmonary failure. Additive value of NT-proBNP was present in 40 of 221 (18%) of the patients, and it mostly indicated that a more intensive treatment for heart failure would have been needed. Clinical judgement was an independent predictor of additive value of NT-proBNP with a maximum at a clinical probability of heart failure of 36%. Conclusion NT-proBNP measurement has additive value in a substantial number of patients presenting with acute dyspnoea, but can possibly be omitted in patients with a clinical probability of heart failure of > 70%.

Asymmetric and symmetric dimethylarginines are of similar predictive value for cardiovascular risk in the general population

ObjectivesAsymmetric dimethylarginine (ADMA) has raised considerable interest, as it is an endogenous inhibitor of nitric oxide synthesis. While increased plasma levels of ADMA have been reported in different cardiovascular disease states, its association with symmetric dimethylarginine (SDMA) has not been evaluated in a prospective population-based study. Methods and resultsWe performed a mass spectrometry-based analysis of ADMA and SDMA in the plasma of 572 participants of the Bruneck study. Levels of ADMA and SDMA were significantly correlated with each other (r=0.189, p<0.001). Age and parameters of renal function, however, showed a stronger influence on SDMA than on ADMA. Both ADMA and SDMA were predictive of cardiovascular disease in multivariate analysis and the associated hazard ratios over the 5-year observation period were of similar strength: 3.86 (1.36–10.9) and 7.91 (1.94–32.3) for ADMA and SDMA, respectively (p=0.011 and 0.004). Separate analyses focused on quintile groups of SDMA revealed that the increase in cardiovascular risk was mainly confined to the top category (>0.80μmol/L). ConclusionThis study argues against an exclusive ADMA effect in mediating cardiovascular risk. Instead, SDMA, its supposedly inactive counterpart, has similar diagnostic value in this large prospective cohort.

Improved Myocardial Function After Cold Storage With Preservation Solution Supplemented With a Carbon Monoxide–Releasing Molecule (CORM-3)

Carbon monoxide-releasing molecules (CO-RMs) are pharmacologically active as they protect against cardiac graft rejection and cold ischemia-mediated renal dysfunction. We investigated the cardioprotective role of carbon monoxide (CO) released from CORM-3 against cold ischemia-mediated injury in the heart and evaluated its potential application in the clinical setting of cardiac transplantation. Isolated rat hearts underwent cold ischemic storage for 4 or 6 hours using St Thomas Hospital solution that was supplemented with either CORM-3 (50 mumol/liter) or its inactive counterpart (iCORM-3), which does not release CO. Hearts were then reperfused. Both functional parameters and release of cardiac enzymes were assessed. Addition of CORM-3 to the preservation solution resulted in a significant improvement in systolic and diastolic function as well as coronary flow when compared with hearts treated with iCORM-3. In addition, lower levels of the cardiac enzymes creatine kinase and lactate dehydrogenase were measured in the perfusate of hearts stored with CORM-3. The improved functional recovery and reduced enzyme release after cardiac cold storage with CORM-3, but not iCORM-3, indicate that CO is the main mediator of myocardial protection. Thus, CO-RMs can be used as adjuvants to improve the preservation of hearts for transplantation.

Effects of (R,S)/(S,R)-4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazolines and (R,S)/(S,R)-2,3-bis(2-chloro-4-hydroxyphenyl)piperazines on estrogen receptor alpha level and transcriptional activity in MCF-7 cells

4,5-Diaryl-2-imidazolines (Ims) and 2,3-diarylpiperazines (Pips) belong to the type II class of estrogens. These compounds enhance ERα-mediated transcription of ERE-driven reporter genes in MCF-7 cells but do not compete with [3H]estradiol (E2) for receptor binding, because of distinct anchoring modes. The present study examined whether the estrogenic action of Ims and Pips is associated with a down regulation of ERα, as reported for conventional agonists. Im and Pip derivatives displaying a large spectrum of activities in three distinct ERE-dependent transactivation systems were selected for that purpose. ERα immunostaining as well as Western blotting analysis revealed that both classes of compounds down regulated ERα with an efficiency closely related to their transactivation potency. MG-132 abrogated this down regulation, pointing to a proteasomal degradation process. Ims and Pips with strong transactivation potency also altered [3H]E2 binding parameters, leading to a progressive decrease of cellular estrogen binding capacity. This property occurred largely before ERα down regulation and persisted even in presence of MG-132, indicating that it did not result from ERα breakdown but rather from a conformational change of the receptor. The additional finding that the most active agonist tested in this study enhanced the capacity of a purified ERα recombinant to recruit LxxLL co-activators, while its inactive counterpart failed to do so confirmed this hypothesis. Altogether, our data indicate that the association of Ims and Pips with ERα elicits similar responses to conventional agonists, even if they interact with distinct residues of the binding pocket.

Role of racemization in optically active drugs development

U.S. Food and Drug Administration issues certain guidelines for marketing of optically active drugs as some enantiomers racemize into human body, leading to the generation of other antipodes, which may be toxic or ballast to the human beings. Moreover, racemization reduces the administrated dosage concentration as optically active enantiomer converted into its inactive counter part. Therefore, the study of racemization of such type of drugs is an important and urgent need of today. This article describes in vitro and in vivo racemization of optically active drugs. The racemization process of various optically active drugs has been discussed considering the effect of different variables i.e. pH, temperature, concentration of the drug, ionic concentration, etc. Attempts have also been made to discuss the mechanisms of racemization. Besides, efforts have been made to suggest the safe dosages of such type of drugs too.