Expression of heat shock proteins and subolesin affects stress responses, Anaplasma phagocytophilum infection and questing behaviour in the tick, Ixodes scapularis

We characterized the effects of subolesin and heat shock protein (HSP) expression on Ixodes scapularis Say (Acari: Ixodidae) stress responses to heat shock and feeding, questing behaviour and Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) infection. Ticks and cultured tick cells were analysed before and after subolesin, hsp20 and hsp70 gene knock-down by RNA interference. The results of these studies confirm that HSPs are involved in the tick cell response to heat stress and that subolesin and HSPs are both involved in the tick response to blood-feeding stress and A. phagocytophilum infection. Subolesin and hsp20 are involved in the tick protective response to A. phagocytophilum infection and hsp70 expression may be manipulated by the pathogen to increase infectivity. Importantly, these results demonstrate that subolesin, hsp20 and hsp70 expression also affect tick questing behaviour. Overall, this research demonstrates a relationship between hsp and subolesin expression and tick stress responses to heat shock and blood feeding, A. phagocytophilum infection and questing behaviour, thereby extending our understanding of the tick-host-pathogen interface.     

Comparison of the skeletal effects induced by daily administration of PTHrP (1-36) and PTHrP (107-139) to ovariectomized mice

We here compared the changes induced by subcutaneous injection of PTHrP (1-36) or PTHrP (107-139) (80 μg/kg/day, 5 days/week for 4 or 8 weeks) in bone histology and bone remodeling factors, and in bone marrow cells (BMCs) ex vivo, in ovariectomized (OVX) mice. We also examined the osteogenic effects of these peptides in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro, which recapitulates the effects of OVX. We confirmed that PTHrP (1-36) exerts bone anabolic actions, as assessed by bone histology and osteoblast differentiation markers in the long bones and plasma from OVX mice. PTHrP (107-139) was also efficient in stimulating several bone formation parameters, and it dramatically decreased bone resorption markers. Moreover, both PTHrP peptides modulate DKK-1 and Sost/sclerostin in osteoblast-like UMR-106 cells highly expressing these Wnt pathway inhibitors, related to their osteogenic action in this in vivo scenario. Administration of either PTHrP peptide improved osteogenic differentiation in BMCs from OVX mice ex vivo and in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro. These data demonstrate that PTHrP (1-36) and PTHrP (107-139) can exert similar osteogenic effects in the appendicular skeleton of OVX mice. Our results suggest that these effects might occur in part by modulating the Wnt pathway. These findings lend credence to the notion that the osteogenic action of PTHrP (107-139) is likely a consequence of its anti-resorptive and anabolic features, and further support the usefulness of PTHrP (1-36) as a bone anabolic peptide in the setting of estrogen-depletion.     

Mitochondrial free [Ca(2+)] dynamics measured with a novel low-Ca(2+) affinity aequorin probe

Mitochondria have a very large capacity to accumulate Ca(2+) during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca(2+)](M) (mitochondrial [Ca(2+)]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca(2+)](M) during prolonged stimulation has been previously precluded by the high Ca(2+) affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca(2+)] in the millimolar range for long periods of time without problems derived from aequorin consumption. We show in the present study that addition of Ca(2+) to permeabilized HeLa cells triggers an increase in [Ca(2+)](M) up to an steady state of approximately 2-3 mM in the absence of phosphate and 0.5-1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca(2+)] reaches 0.5-1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca(2+)](M) levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca(2+) uniporter largely reduced the rate of [Ca(2+)](M) increase, but the final steady-state [Ca(2+)](M) reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca(2+)](M) without problems derived from aequorin consumption.     

Estrogen Receptor 1 Gene Expression and Its Combination with Estrogen Receptor 2 or Aromatase Expression Predicts Survival in Non-Small Cell Lung Cancer

The biological roles of estrogen receptor 1 (ERS1), estrogen receptor 2 (ERS2), and aromatase (CYP19A1) genes in the development of non-small cell lung cancer (NSCLC) is unclear, as is the use of their expression as a prognostic factor. The aim of this study was to investigate the prognostic value of estrogen receptors and aromatase mRNA expression, along with aromatase protein concentration, in resected NSCLC patients. Tumor and non-tumor lung tissue samples were analyzed for the mRNA expression of ERS1, ERS2 and CYP19A1 by RT-PCR. Aromatase concentration was measured with an ELISA. A total of 96 patients were included. ERS1 expression was significantly higher in non-tumor tissue than in tumor samples. Two gene expression categories were created for each gene (and protein): high and low. ERS1 high category showed increased overall survival (OS) when compared to the low expression category. Aromatase protein concentration was significantly higher in tumor samples. Higher ERS1 expression in tumor tissues was related to longer overall survival. The analysis of gene expression combinations provides evidence for longer OS when both ERS1 and ERS2 are highly expressed. ESR1, alone or in combination with ERS2 or CYP19A1, is the most determining prognostic factor within the analyzed 3 genes. It seems that ERS1 can play a role in NSCLC prognosis, alone or in combination with other genes such as ERS2 or Cyp19a1. ERS2 in combination with aromatase concentration could have a similar function.     

Lifelong treatment with atenolol decreases membrane fatty acid unsaturation and oxidative stress in heart and skeletal muscle mitochondria and improves immunity and behavior,without changing mice longevity

The membrane fatty acid unsaturation hypothesis of aging and longevity is experimentally tested for the first time in mammals. Lifelong treatment of mice with the β1‐blocker atenolol increased the amount of the extracellular‐signal‐regulated kinase signaling protein and successfully decreased one of the two traits appropriately correlating with animal longevity, the membrane fatty acid unsaturation degree of cardiac and skeletal muscle mitochondria, changing their lipid profile toward that present in much more longer‐lived mammals. This was mainly due to decreases in 22:6n‐3 and increases in 18:1n‐9 fatty acids. The atenolol treatment also lowered visceral adiposity (by 24%), decreased mitochondrial protein oxidative, glycoxidative, and lipoxidative damage in both organs, and lowered oxidative damage in heart mitochondrial DNA. Atenolol also improved various immune (chemotaxis and natural killer activities) and behavioral functions (equilibrium, motor coordination, and muscular vigor). It also totally or partially prevented the aging‐related detrimental changes observed in mitochondrial membrane unsaturation, protein oxidative modifications, and immune and behavioral functions, without changing longevity. The controls reached 3.93 years of age, a substantially higher maximum longevity than the best previously described for this strain (3.0 years). Side effects of the drug could have masked a likely lowering of the endogenous aging rate induced by the decrease in membrane fatty acid unsaturation. We conclude that it is atenolol that failed to increase longevity, and likely not the decrease in membrane unsaturation induced by the drug.     

Cardiac Impairment Evaluated by Transesophageal Echocardiography and Invasive Measurements in Rats Undergoing Sinoaortic Denervation

Background

Sympathetic hyperactivity may be related to left ventricular (LV) dysfunction and baro- and chemoreflex impairment in hypertension. However, cardiac function, regarding the association of hypertension and baroreflex dysfunction, has not been previously evaluated by transesophageal echocardiography (TEE) using intracardiac echocardiographic catheter.

Methods and Results

We evaluated exercise tests, baroreflex sensitivity and cardiovascular autonomic control, cardiac function, and biventricular invasive pressures in rats 10 weeks after sinoaortic denervation (SAD). The rats (n = 32) were divided into 4 groups: 16 Wistar (W) with (n = 8) or without SAD (n = 8) and 16 spontaneously hypertensive rats (SHR) with (n = 8) or without SAD (SHRSAD) (n = 8). Blood pressure (BP) and heart rate (HR) did not change between the groups with or without SAD; however, compared to W, SHR groups had higher BP levels and BP variability was increased. Exercise testing showed that SHR had better functional capacity compared to SAD and SHRSAD. Echocardiography showed left ventricular (LV) concentric hypertrophy; segmental systolic and diastolic biventricular dysfunction; indirect signals of pulmonary arterial hypertension, mostly evident in SHRSAD. The end-diastolic right ventricular (RV) pressure increased in all groups compared to W, and the end-diastolic LV pressure increased in SHR and SHRSAD groups compared to W, and in SHRSAD compared to SAD.

Conclusions

Our results suggest that baroreflex dysfunction impairs cardiac function, and increases pulmonary artery pressure, supporting a role for baroreflex dysfunction in the pathogenesis of hypertensive cardiac disease. Moreover, TEE is a useful and feasible noninvasive technique that allows the assessment of cardiac function, particularly RV indices in this model of cardiac disease.     

A study of Sarcocornia A.J. Scott (Chenopodiaceae) from Western Mediterranean Europe

The latest publications on Sarcocornia taxonomy and phylogeny recognize six taxa in this genus on the Iberian Peninsula: S. perennis, S. fruticosa, S. alpini, S. alpini subsp. carinata, S. hispanica, and S. pruinosa. The present study represents a comprehensive revision of the different taxa in the Sarcocornia genus present in Western Mediterranean Europe by means of morphological, micromorphological and phylogenetic internal transcribed spacer (ITS) analysis. Morphological and micromorphological data were studied from Sarcocornia samples from 113 populations in coastal salt marshes and inland salt pans in Portugal, France, Spain and Italy. Sixteen new ITS sequences were obtained from Mediterranean Sarcocornia species and analysed together with previous reported data. Published karyological, ecological and biogeographical data from Western Mediterranean Europe were also reviewed. The results indicate the presence of a new species, S. lagascae, found growing in coastal Mediterranean areas of the Iberian Peninsula. The species S. fruticosa was found to be absent from the Iberian territories.