NO and endogenous angiotensin II interact in the generation of renal sympathetic nerve activity in conscious rats

Nitric oxide (NO) appears to inhibit sympathetic tone in anesthetized rats. However, whether NO tonically inhibits sympathetic outflow, or whether endogenous angiotensin II (ANG II) promotes NO-mediated sympathoinhibition in conscious rats is unknown. To address these questions, we determined the effects of NO synthase (NOS) inhibition on renal sympathetic nerve activity (RSNA) and heart rate (HR) in conscious, unrestrained rats on normal (NS), high-(HS), and low-sodium (LS) diets, in the presence and absence of an ANG II receptor antagonist (AIIRA). When arterial pressure was kept at baseline with intravenous hydralazine, NOS inhibition with l-NAME (10 mg/kg i.v.) resulted in a profound decline in RSNA, to 42 +/- 11% of control (P < 0.01), in NS animals. This effect was not sustained, and RSNA returned to control levels by 45 min postinfusion. l-NAME also caused bradycardia, from 432 +/- 23 to 372 +/- 11 beats/min postinfusion (P < 0.01), an effect, which, in contrast, was sustained 60 min postdrug. The effects of NOS inhibition on RSNA and HR did not differ between NS, HS, and LS rats. However, when LS and HS rats were pretreated with AIIRA, the initial decrease in RSNA after l-NAME infusion was absent in the LS rats, while the response in the HS group was unchanged by AIIRA. These findings indicate that, in contrast to our hypotheses, NOS activity provides a stimulatory input to RSNA in conscious rats, and that in LS animals, but not HS animals, this sympathoexcitatory effect of NO is dependent on the action of endogenous ANG II.     

Testing the success of palaeontological methods in the delimitation of clam shrimp (Crustacea,Branchiopoda) on extant species

Fossil spinicaudatan taxonomy heavily relies on carapace features (size, shape, ornamentation) and palaeontologists have greatly refined methods to study and describe carapace variability. Whether carapace features alone are sufficient for distinguishing between species of a single genus has remained untested. In our study, we tested common palaeontological methods on 481 individuals of the extant Australian genus Ozestheria that have been previously assigned to ten species based on genetic analysis. All species are morphologically distinct based on geometric morphometrics (p ≤ 0.001), but they occupy overlapping regions in Ozestheria morphospace. Linear discriminant analysis of Fourier shape coefficients reaches a mean model performance of 93.8% correctly classified individuals over all possible 45 pairwise species comparisons. This can be further increased by combining the size and shape datasets. Nine of the ten examined species are clearly sexually dimorphic but male and female morphologies strongly overlap within species with little influence on model performance. Ornamentation is commonly species-diagnostic; seven ornamentation types are distinguished of which six are species-specific while one is shared by four species. A transformation of main ornamental features (e.g. from punctate to smooth) can occur among closely related species suggesting short evolutionary timescales. Our overall results support the taxonomic value of carapace features, which should also receive greater attention in the taxonomy of extant species. The extensive variation in carapace shape and ornamentation is noteworthy and several species would probably have been assigned to different genera or families if these had been fossils, bearing implications for the systematics of fossil Spinicaudata.     

Effects of siRNA-dependent knock-down of cardiolipin synthase and tafazzin on mitochondria and proliferation of glioma cells

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function, and cell proliferation. Changes in CL are often paralleled by changes in the lipid environment of mitochondria that may contribute to mitochondrial function and proliferation. This study aimed to separate the effects of CL content and CL composition from cellular free fatty acid distribution on bioenergetics and proliferation in C6 glioma cells. To this end, cardiolipin synthase and the CL remodelling enzyme, tafazzin, were knocked-down by siRNA in C6 cells. After 72?h of cultivation, we analysed CL composition by means of LC/MS/MS, distribution of cellular fatty acids by means of gas chromatography, and determined oxygen consumption and proliferation. Knock-down of cardiolipin synthase affected the cellular CL content in the presence of linoleic acid (LA) in the culture medium. Knock-down of tafazzin had no consequence with respect to the pattern of cellular fatty acids but caused a decrease in cell proliferation. It significantly changed the distribution of molecular CL species, increased CL content, decreased oxygen consumption, and decreased cell proliferation when cultured in the presence of linoleic acid (LA). The addition of linoleic acid to the culture medium caused significant changes in the pattern of cellular fatty acids and the composition of molecular CL species. These data suggest that tafazzin is required for efficient bioenergetics and for proliferation of glioma cells. Supplementation of fatty acids can be a powerful tool to direct specific changes in these parameters.     

The carboxyl-terminal domain of closely related endotoxin-binding proteins determines the target of protein-lipopolysaccharide complexes.

The bactericidal/permeability increasing (BPI) and lipopolysaccharide (LPS)-binding (LBP) proteins are closely related two-domain proteins in which LPS binding is mediated by the NH(2)-terminal domain. To further define the role of the COOH-terminal domain of these proteins in delivery of LPS to specific host acceptors, we have compared interactions of LBP, BPI, LBP(N)-BPI(C) (NH(2)-terminal domain of LBP, COOH-terminal domain of BPI), and BPI(N)-LBP(C) with purified (3)H-LPS and, subsequently, with purified leukocytes and soluble (s)CD14. The COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both polymorphonuclear leukocytes and monocytes resulting in cell activation. In the presence of Ca(2+) and Mg(2+), LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent M(r) > 20 x 10(6) Da), but only LPS associated with LBP and BPI(N)-LBP(C) is disaggregated in the presence of CD14. BPI and LBP(N)-BPI(C) promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these closely related endotoxin-binding proteins dictates the route and host responses to complexes they form with endotoxin.     

Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.     

Forecasting phenology: from species variability to community patterns

Shifts in species' phenology in response to climate change have wide-ranging consequences for ecological systems. However, significant variability in species' responses, together with limited data, frustrates efforts to forecast the consequences of ongoing phenological changes. Herein, we use a case study of three North American plant communities to explore the implications of variability across levels of organisation (within and among species, and among communities) for forecasting responses to climate change. We show how despite significant variation among species in sensitivities to climate, comparable patterns emerge at the community level once regional climate drivers are accounted for. However, communities differ with respect to projected patterns of divergence and overlap among their species' phenological distributions in response to climate change. These analyses and a review of hypotheses suggest how explicit consideration of spatial scale and levels of biological organisation may help to understand and forecast phenological responses to climate change.     

Inhibition of 20-HETE abolishes the myogenic response during NOS antagonism in the ovine fetal pulmonary circulation

Mechanisms that maintain high pulmonary vascular resistance (PVR) and oppose vasodilation in the fetal lung are poorly understood. In fetal lambs, increased pulmonary artery pressure evokes a potent vasoconstriction, suggesting that a myogenic response contributes to high PVR in the fetus. In adult systemic circulations, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to modulate the myogenic response, but its role in the fetal lung is unknown. We hypothesized that acute increases in pulmonary artery pressure release 20-HETE, which causes vasoconstriction, or a myogenic response, in the fetal lung. To address this hypothesis, we studied the hemodynamic effects of N-methylsufonyl-12,12-dibromododec-11-enamide (DDMS), a specific inhibitor of 20-HETE production, on the pulmonary vasoconstriction caused by acute compression of the ductus arteriosus (DA) in chronically prepared fetal sheep. An inflatable vascular occluder around the DA was used to increase pulmonary artery pressure under three study conditions: control, after pretreatment with nitro-L-arginine (L-NA; to inhibit shear-stress vasodilation), and after combined treatment with both L-NA and a specific 20-HETE inhibitor, DDMS. We found that DA compression after L-NA treatment increased PVR by 44 +/- 12%. Although intrapulmonary DDMS infusion did not affect basal PVR, DDMS completely abolished the vasoconstrictor response to DA compression in the presence of L-NA (44 +/- 12% vs. 2 +/- 4% change in PVR, L-NA vs. L-NA + DDMS, P < 0.05). We conclude that 20-HETE mediates the myogenic response in the fetal pulmonary circulation and speculate that pharmacological inhibition of 20-HETE might have a therapeutic role in neonatal conditions characterized by pulmonary hypertension.