Analysis of the Rit subfamily GTPase-mediated signaling and neuronal differentiation and survival.

The Rit subfamily of GTPases is a founding branch within the Ras family of small G-proteins and preserves unique sequences in the G2 effector loop domain and the C-terminus. Rit proteins regulate a diversity of signal transduction pathways, some of which are similar to and others of which differ from the pathways that are regulated by other Ras family GTPases. Rit proteins have been demonstrated to be essential regulators in neuronal differentiation and survival. Here, we describe the materials and methods utilized to characterize cellular signaling for the Rit subfamily of G-proteins in neuronal differentiation and survival.

Rad GTPase deletion increases L-type calcium channel current leading to increased cardiac contraction.

BACKGROUND: The small GTPase Rad is a negative regulator of voltage-dependent L-type calcium channel current (ICaL); however, the effects of Rad ablation on cardiomyocyte function are unknown. The objective of this study is to test the hypothesis that Rad-depletion causes positive inotropic effects without inducing cardiac hypertrophy. METHODS AND RESULTS: Ventricular myocytes from adult Rad(-/-) mice were isolated and evaluated by patch-clamp recordings for I(Ca,L) and action potentials, Ca(2+) transients, and sarcomere shortening. Maximum I(CaL) is elevated in Rad(-/-) (maximal conductance 0.35 ± 0.04 picoSiemens/picoFarad (pS/pF) wild-type; 0.61 ± 0.14 pS/pF Rad(-/-)), decay kinetics are faster, and I(Ca,L) activates at lower voltages (activation midpoint -7.2 ± 0.6 wild-type; -11.7 ± 0.9 Rad(-/-)) mimicking effects of ß-adrenergic receptor stimulation. Diastolic and twitch calcium are elevated in Rad(-/-) (F340/380: 1.03 diastolic and 0.35 twitch for wild-type; 1.47 diastolic and 0.736 twitch for Rad(-/-)) and sarcomere shortening is enhanced (4.31% wild-type; 14.13% Rad(-/-)) at lower pacing frequencies. Consequentially, frequency-dependence of Ca(2+) transients is less in Rad(-/-), and the frequency dependence of relaxation is also blunted. In isolated working hearts, similar results were obtained; chiefly, +dP/dt was elevated at baseline and developed pressure was relatively nonresponsive to acute ß-adrenergic receptor stimulation. In single cells, at subphysiological frequencies, nonstimulated calmodulin-dependent protein kinase-sensitive calcium release is observed. Remarkably, Rad(-/-) hearts did not show hypertrophic growth despite elevated levels of diastolic calcium. CONCLUSIONS: This study demonstrates that the depletion of Rad GTPase is equivalent to sympathomimetic ß-adrenergic receptor, without stimulating cardiac hypertrophy. Thus, targeting Rad GTPase is a novel potential therapeutic target for Ca(2+)-homeostasis-driven positive inotropic support of the heart.

Impact of ureido/carboxypenicillin resistance on the prognosis of ventilator-associated pneumonia due to Pseudomonas aeruginosa.

INTRODUCTION: Although Pseudomonas aeruginosa is a leading pathogen responsible for ventilator-associated pneumonia (VAP), the excess in mortality associated with multi-resistance in patients with P. aeruginosa VAP (PA-VAP), taking into account confounders such as treatment adequacy and prior length of stay in the ICU, has not yet been adequately estimated. METHODS: A total of 223 episodes of PA-VAP recorded into the Outcomerea database were evaluated. Patients with ureido/carboxy-resistant P. aeruginosa (PRPA) were compared with those with ureido/carboxy-sensitive P. aeruginosa (PSPA) after matching on duration of ICU stay at VAP onset and adjustment for confounders. RESULTS: Factors associated with onset of PRPA-VAP were as follows: admission to the ICU with septic shock, broad-spectrum antimicrobials at admission, prior use of ureido/carboxypenicillin, and colonization with PRPA before infection. Adequate antimicrobial therapy was more often delayed in the PRPA group. The crude ICU mortality rate and the hospital mortality rate were not different between the PRPA and the PSPA groups. In multivariate analysis, after controlling for time in the ICU before VAP diagnosis, neither ICU death (odds ratio (OR) = 0.73; 95% confidence interval (CI): 0.32 to 1.69; P = 0.46) nor hospital death (OR = 0.87; 95% CI: 0.38 to 1.99; P = 0.74) were increased in the presence of PRPA infection. This result remained unchanged in the subgroup of 87 patients who received adequate antimicrobial treatment on the day of VAP diagnosis. CONCLUSIONS: After adjustment, and despite the more frequent delay in the initiation of an adequate antimicrobial therapy in these patients, resistance to ureido/carboxypenicillin was not associated with ICU or hospital death in patients with PA-VAP.

Ethacrynic acid analogues lacking the alpha,beta-unsaturated carbonyl unit--potential anti-metastatic drugs.

A series of ethacrynic acid analogues, lacking the alpha,beta-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, MCF-7/AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.

In vitro anticancer activity of Anemopsis californica.

Three different extract conditions (aqueous, EtOH and EtOAc) of four different parts (bracts, leaves, roots and stems) of the plant Anemopsis californica (A. californica) were evaluated for their effect on the growth and migration of human colon cancer cells, HCT-8, and the breast cancer cell lines Hs 578T and MCF-7/AZ. Our aim was to identify potential anticancer activity in crude A. californica extracts, given that this plant is used by Native Americans to treat a variety of diseases, including cancer. Our results demonstrated that for each of the cell lines tested, the majority of ethyl acetate extracts of all the plant parts are more toxic than the aqueous and ethanol extracts. Furthermore, significant growth inhibitory activity against the three cell lines was found for the ethyl acetate extract of the roots, while the aqueous extract of the roots influenced the migratory capacity of the three cell lines. This study provides evidence for the anticancer properties of A. californica when extracted in water and ethyl acetate, and supports the importance for further purification of the crude extracts and isolation of potential new anticancer compounds through bio-guided fractionation.

Validation of a Pseudomonas aeruginosa porcine model of septic shock.

OBJECTIVES: To develop a standardized bacteraemic porcine model of septic shock with cardiovascular and immunological profiles similar to those observed in human clinical states. METHODS: Sepsis was induced by an intravenous challenge of 18 anaesthetized pigs with live Pseudomonas aeruginosa. The pulmonary arterial pressure was monitored and the bacterial infusion was stopped when the systolic pulmonary arterial pressure reached 45 mmHg. Septic shock was treated with fluid resuscitation and epinephrine infusion. The haemodynamic parameters and the rate of different inflammatory cytokines were recorded during 6 h of observation. RESULTS: The mean+/-SD cardiac output increased from 2.4+/-1.2 to 5.7+/-2.1 L/min while the mean+/-SD systemic vascular resistance index decreased from 1957+/-744 to 709+/-221 dyn/s/cm5/m2. The pharmacokinetic profile of the inflammatory cytokines was similar to the one observed in human studies. CONCLUSIONS: The control of the systolic pulmonary arterial pressure during a P. aeruginosa infusion leads to a hyperdynamic, reproducible cardiovascular profile similar to the one observed in human septic shock. Since the immunological profile of the inflammatory cytokines is also similar to the human one, this standardized porcine model appears to be appropriate for experimental research concerning sepsis.